[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.

Modeling human hypertrophic scars with 3D preformed cellular aggregates bioprinting.

The therapeutic interventions of human hypertrophic scars (HHS) remain puzzle largely due to the lack of accepted models. Current HHS models are limited by their inability to mimic native scar architecture and associated pathological microenvironments. Here, we create a 3D functional HHS model by preformed cellular aggregates (PCA) bioprinting, firstly developing bioink from scar decellularized extracellular matrix (ECM) and alginate-gelatin (Alg-Gel) hydrogel with suitable physical properties to mimic the microenvironmental factors, then pre-culturing patient-derived fibroblasts in this bioink to preform the topographic cellular aggregates for sequent printing. We confirm the cell aggregates preformed in bioink displayed well defined aligned structure and formed functional scar tissue self-organization after bioprinting, hence showing the potential of creating HHS models. Notably, these HHS models exhibit characteristics of early-stage HHS in gene and protein expression, which significantly activated signaling pathway related to inflammation and cell proliferation, and recapitulate in vivo tissue dynamics of scar forming. We also use the in vitro and in vivo models to define the clinically observed effects to treatment with concurrent anti-scarring drugs, and the data show that it can be used to evaluate the potential therapeutic target for drug testing. The ideal humanized scar models we present should prove useful for studying critical mechanisms underlying HHS and to rapidly test new drug targets and develop patient-specific optimal therapeutic strategies in the future.

A comparative study of sagittal spinal-pelvic parameters between patients with adolescent idiopathic scoliosis and healthy controls / Un estudio comparativo de los parámetros sagitales de la columna vertebral-pélvic entre pacientes adolescentes con escoliosis idiopática y controles sanos

This study aimed to investigate the sagittal spinal-pelvic morphological changes, as well as the relationship between pelvic anatomical changes and the spinal-pelvic plane in patients with adolescent idiopathic scoliosis (AIS), in order to provide guidelines for orthopedic surgery in AIS. X-ray data were collected for retrospective analysis from 30 patients diagnosed as AIS in the Departments of Radiology at the Second Affiliated Hospital of Inner Mongolia Medical University and the Inner Mongolia International Mongolian Medical Hospital from April 2014 to November 2018, along with 30 normal adolescents as control. Pelvic parameters, including pelvic incidence (PI), pelvic tilt (PT), and sacral slope (SS), a spinal parameter, lumbar lordosis (LL), and anatomical parameters, including sacral width (SW) and femoral head- sacrum distance (FH-S), were measured. The spinal-pelvic parameters were compared between AIS patients and normal controls and also between male and female AIS patients. Pearson correlation was performed to analyze correlation between spinal-pelvic parameters and between spinal-pelvic parameters and anatomical parameters in both AIS patients and normal controls. PT was significantly lower in AIS patients than in normal controls (P < 0.05), whereas no significant difference was found in the other spinal-pelvic parameters, i. e. , LL, PI, and SS. There was a significant difference in PT between sexes in AIS patients. SS was significantly correlated with LL in EIA patients (P < 0.05, r > 0.5). SS was significantly correlated with LL and PI, and PT with LL, PI, and SS in normal controls (all P < 0.05), and there was no significant correlation between the other sagittal spinal-pelvic parameters (P > 0.05). FH-S was significantly correlated with LL, PI, SS, and PT in AIS patients (all P < 0.05). AIS affects some of the sagittal spinalpelvic parameters and anatomical parameters. In AIS, there is a significant correlation between the spinal-pelvic parameters, and the anatomical parameter is significantly correlated with multiple spinal-pelvic parameters.

Este estudio tuvo como objetivo investigar los cambios morfológicos sagitales de la columna vertebral-pélvica, así como la relación entre los cambios anatómicos pélvicos y el plano espinal-pélvico en pacientes con escoliosis idiopática adolescente (EIA), con el fin de proporcionar pautas para la cirugía ortopédica en AIS. Se obtuvieron los datos de rayos X para el análisis retrospectivo de 30 pacientes diagnosticados como EIA en los Departamentos de Radiología del hospital Second Affiliated Hospital of Inner Mongolia Medical University y el hospital Inner Mongolia International Mongolian Medical Hospital, desde abril de 2014 hasta noviembre del 2018, junto con 30 adolescentes normales como control. Se midieron los parámetros pélvicos, que incluyeron incidencia pélvica (IP), inclinación pélvica (P) y pendiente sacra (PS), un parámetro espinal, lordosis lumbar (LL) y parámetros anatómicos, que incluyeron el ancho sacro (AS) y la distancia del sacro cabeza femoral (FH-S). Los parámetros espinalpélvicos se compararon entre los pacientes con EIA y los controles normales, como también entre pacientes con EIA masculinos y femeninos. La correlación de Pearson se realizó para analizar la correlación entre los parámetros espinal-pélvicos y entre los parámetros espinal-pélvicos y los parámetros anatómicos tanto en pacientes con EIA como en controles normales. PT fue significativamente menor en pacientes con EIA que en los controles normales (P <0,05), mientras que no se encontraron diferencias significativas en los otros parámetros espinal-pélvicos, i. mi. , LL, PI y SS. Hubo una diferencia significativa en PT en pacientes de ambos sexos con EIA. SS se correlacionó significativamente con LL en pacientes con EIA (P <0,05, r> 0,5). SS se correlacionó significativamente con LL y PI, y PT con LL, PI y SS en controles normales (todos P <0,05), y no hubo correlación significativa entre los otros parámetros sagitales de la columna vertebral-pélvica (P> 0,05) FH-S se correlacionó significativamente con LL, PI, SS y PT en pacientes con EIA (todos P <0,05). EIA afecta algunos de los parámetros sagitales de la columna vertebral-pélvica y los parámetros anatómicos. En EIA, existe una correlación significativa entre los parámetros espinal-pélvicos, y el parámetro anatómico se correlaciona significativamente con múltiples parámetros espinales-pélvicos.

Mesenchymal stem cells delivered in a microsphere-based engineered skin contribute to cutaneous wound healing and sweat gland repair.

BACKGROUND: Bone-marrow-derived mesenchymal stem cells (BM-MSCs) can contribute to wound healing after skin injury. However, the role of BM-MSCs on repairing skin appendages in renewal tissues is incompletely explored. Moreover, most preclinical studies suggest that the therapeutic effects afforded by BM-MSCs transplantation are short-lived and relatively unstable. OBJECTIVE: To assess whether engrafted bone-marrow-derived mesenchymal stem cells via a delivery system can participate in cutaneous wound healing and sweat-gland repair in mice. METHODS: For safe and effective delivery of BM-MSCs to wounds, epidermal growth factor (EGF) microspheres were firstly developed to both support cells and maintain appropriate stimuli, then cell-seeded microspheres were incorporated with biomimetic scaffolds and thus fabricated an engineered skin construct with epithelial differentiation and proliferative potential. The applied efficacy was examined by implanting them into excisional wounds on both back and paws of hind legs in mice. RESULTS: After 3 weeks, BM-MSC-engineered skin (EGF loaded) treated wounds exhibited accelerated healing with increased re-epithelialization rates and less skin contraction. Furthermore, histological and immunofluorescence staining analysis revealed sweat glands-like structures became more apparent in BM-MSC-engineered skin (EGF loaded) treated wounds but the number of implanted BM-MSCs were decreased gradually in later phases of healing progression. CONCLUSIONS: Our study suggests that BM-MSCs delivered by this EGF microspheres-based engineered skin model may be a promising strategy to repair sweat glands and improve cutaneous wound healing after injury and success in this study might provide a potential benefit for BM-MSCs administration clinically.