[Effects of immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice].

Objective: To explore the effects of the immune responses mediated by topological structures of three-dimensional bioprinted scaffolds on hair follicle cycle in mice. Methods: The study was an experimental research. The alginate-gelatin composite hydrogels were printed into scaffolds using a three-dimensional bioprinter and named T45 scaffolds, T60 scaffolds, and T90 scaffolds according to the 3 topological structures of the scaffolds (the rotation angles of the printhead during printing were 45°, 60°, and 90°, respectively), and the morphology of the three scaffolds was observed after cross-linking by naked eyes. Nine 8-week-old female C57BL/6J mice were divided into T45 group, T60 group, and T90 group, according to the random number table, with three mice in each group, and the T45, T60, and T90 scaffolds were subcutaneously implanted on the back of mice, respectively. On post implantation day (PID) 7, the hair growth in the dorsal depilated area of mice was observed, the thickness of the fiber capsule around the scaffolds was observed by hematoxylin-eosin staining, and the expression levels of CD68, bone morphogenetic protein-2 (BMP-2), and tumor necrosis factor (TNF) protein in the tissue surrounding the scaffolds were observed by immunofluorescence staining. The samples of the above experiments were all 3. Results: The topological structures of the three scaffolds were all clear with high fidelity after cross-linking. On PID 7, the hair growth was obvious in the dorsal depilated area of mice in T45 group and T90 group, while hair growth was slow in the scaffold implantation area of mice in T60 group, which was significantly different from that of the unimplanted area. On PID 7, compared with (18±4) µm in T90 group, the thickness of both the fiber capsule around the scaffolds ((39±4) and (55±8) µm) of mice in T45 group and T60 group was significantly increased (P<0.05); the thickness of the fiber capsule around the scaffolds of mice in T60 group was also significantly increased compared with that in T45 group (P<0.05). On PID 7, the expression level of CD68 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05). The expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T60 group was significantly higher than the levels in T45 group and T90 group (with both P values <0.05), and the expression level of BMP-2 protein in the tissue surrounding the scaffolds of mice in T45 group was significantly higher than that in T90 group (P<0.05). The expression level of TNF protein in the tissue surrounding the scaffolds of mice in T60 group was significantly lower than the levels in T45 group and T90 group (with both P values <0.05). Conclusions: Three-dimensional bioprinted scaffolds with different topological structures mediate different degrees of immune responses after being implanted in mice. A moderate immune response promotes hair growth in depilated area of mice, while an excessive immune response results inhibits the hair follicle entering into the anagen phase.

[Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice].

Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 µL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 µg/mL GO (without GO solution, the same as below) group, 0.1 µg/mL GO group, 1.0 µg/mL GO group, 5.0 µg/mL GO group, and 10.0 µg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 µg/mL GO group, 0.1 µg/mL GO group, 1.0 µg/mL GO group, and 5.0 µg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 µg/mL GO composite hydrogel group, 0.1 µg/mL GO composite hydrogel group, 1.0 µg/mL GO composite hydrogel group, and 5.0 µg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 µg/mL GO composite hydrogel group, 0.1 µg/mL GO composite hydrogel group, 1.0 µg/mL GO composite hydrogel group, and 5.0 µg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 µg/mL GO composite hydrogel group and 0.1 µg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 µm and the length of about 50 µm. The absorbance value of HSFs in 10.0 µg/mL GO group was significantly lower than that in 0 µg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 µg/mL GO group was significantly higher than that in 0 µg/mL GO group, 1.0 µg/mL GO group, and 5.0 µg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 µg/mL GO group was significantly higher than that in 0 µg/mL GO group, 1.0 µg/mL GO group, and 5.0 µg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 µg/mL GO group was significantly lower than that in 0 µg/mL GO group and 1.0 µg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 µg/mL GO group was significantly higher than that in 0 µg/mL GO group and 5.0 µg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 µg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 µg/mL GO composite hydrogel group was significantly higher than that in 0 µg/mL GO composite hydrogel group, 1.0 µg/mL GO composite hydrogel group, and 5.0 µg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 µg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 µg/mL GO composite hydrogel group, 1.0 µg/mL GO composite hydrogel group, and 5.0 µg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 µg/mL GO composite hydrogel group and 5.0 µg/mL GO composite hydrogel group was significantly higher than that in 0 µg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 µg/mL GO composite hydrogel group was significantly more than that in 0 µg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 µg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 µg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 µg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.

A clinical investigation into the usefulness of fractional exhaled nitric oxide in guiding glucocorticoid therapy in children with bronchial asthma.

The present study aims to investigate the value of fractional exhaled nitric oxide (FeNO) combined with pulmonary function in guiding the dose adjustment of inhaled glucocorticosteroids (ICSs) in children with asthma. A total of 133 children aged 6-12 years with newly diagnosed asthma were enrolled as the study subjects and randomly divided into the experimental group (n=68) and the control group (n=65). After three months of ICS treatment, in the experimental group, the dose of ICSs was adjusted based on the control status of the children and the results of the pulmonary function tests and FeNO assays, and in the control group, the dose was adjusted based on the control status of the children and the results of the pulmonary function tests. After another three months of treatment, the number of acute asthma attacks and the Childhood Asthma Control Test (C-ACT) scores were compared between the two groups, and the outcome of pulmonary function tests and FeNO assays during treatment were analyzed. When examining pulmonary function and FeNO levels, when compared with before treatment, there were no statistically significant differences in either group or between the groups after three months of ICS treatment (P>0.05). After dose adjustment and another three months of treatment, when compared with the control group, the improvement in pulmonary function in the experimental group was greater, the reduction in FeNO levels was greater, the incidence of acute asthma attacks was lower, and the C-ACT score was higher (P<0.05). We concluded that the combination of FeNO assays and pulmonary function tests to guide the ICS dose adjustment in children with asthma could improve asthma control and reduce the risk of acute asthma attacks.

The development of immunoassays to four biological threat agents in a bidiffractive grating biosensor.

A critical need exists for a field deployable biosensor to detect environmental infectious agents in collected air samples rapidly, with sensitivity and specificity approaching that of standard laboratory procedures. The ideal sensor would analyze unknown samples in minutes, have programmable operation for unattended sample analysis, and be capable of multiple agent analysis for a number of agents. The goal of this project was to further the development of the bidiffractive grating biosensor (BDG) created through collaboration between Battelle Memorial Institute (BMI), Hoffman LaRoche (HLR), and the Naval Medical Research Command (NMRC). This manuscript details the development, optimization, and evaluation of this device as a potential field deployable biosensor. Well-characterized immunochemical reagents developed by the Biological Defense Research Department (BDRD) at NMRI were employed to develop assays in the BDG. These results were compared to those obtained with antigen capture enzyme linked immunosorbent assays (ELISAs). Four separate antigens were evaluated: Staphylococcus aureus enterotoxin B (SEB), ricin (RIC), Francisella tularensis (FT), and Clostridium botulinum toxin (BOT).

Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties.

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony-stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage-like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the "reticuloendothelial" phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.