Multifaceted Characterization of Human Embryonic Stem Cell-Derived Mesenchymal Stem/Stromal Cells Revealed Amelioration of Acute Liver Injury in NOD-SCID Mice.

Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future.

Exosomal IRF1-loaded rat adipose-derived stem cell sheet contributes to wound healing in the diabetic foot ulcers.

BACKGROUND: Cell-based therapy has been recognized as a novel technique for the management of diabetic foot ulcers, and cell-sheet engineering leads to improved efficacy in cell transplantation. This study aims to explore the possible molecular mechanism of the rat adipose-derived stem cell (ASC) sheet loaded with exosomal interferon regulatory factor 1 (IRF1) in foot wound healing. METHODS: Rats were rendered diabetic with streptozotocin, followed by measurement of miR-16-5p expression in wound tissues. Relationship between IRF1, microRNA (miR)-16-5p, and trans-acting transcription factor 5 (SP5) was analyzed using luciferase activity, RNA pull-down, and chromatin immunoprecipitation assays. IRF1 was overexpressed in rat ASCs (rASCs) or loaded onto the rASC sheet, and then exosomes were extracted from rASCs. Accordingly, we assessed the effects of IRF1-exosome or IRF1-rASC sheet on the proliferation and migration of the fibroblasts along with endothelial cell angiogenesis. RESULTS: miR-16-5p was poorly expressed in the wound tissues of diabetic rats. Overexpression of miR-16-5p promoted fibroblast proliferation and migration as well as endothelial cell angiogenesis, thus expediting wound healing. IRF1 was an upstream transcription factor that could bind to the miR-16-5p promoter and increase its expression. In addition, SP5 was a downstream target gene of miR-16-5p. IRF1-exosome from rASCs or the IRF1-rASC sheet facilitated the foot wound healing in diabetic rats through miR-16-5p-dependent inhibition of SP5. CONCLUSION: The present study demonstrates that exosomal IRF1-loaded rASC sheet regulates miR-16-5p/SP5 axis to facilitate wound healing in diabetic rats, which aids in development of stem cell-based therapeutic strategies for diabetic foot wounds.

Effects of the establishment of trauma centres on the mortality rate among seriously injured patients: a propensity score matching retrospective study.

BACKGROUND: Little evidence suggests that trauma centres are associated with a lower risk of mortality in severely injured patients (Injury Severity Score (ISS) ≥16) with multiple injuries in China. The objective of this study was to determine the association between the establishment of trauma centres and mortality among severely injured patients with multiple injuries and to identify some risk factors associated with mortality. METHODS: A retrospective single-centre study was performed including trauma patients admitted to the First Affiliated Hospital of Nanchang University (FAHNU) between January 2016 and December 2021. To determine whether the establishment of a trauma centre was an independent predictor of mortality, logistic regression analysis and propensity score matching (PSM) were performed. RESULTS: Among 431 trauma patients, 172 were enrolled before the trauma centre was built, while 259 were included after the trauma centre was built. A higher frequency of older age and traffic accident injury was found in patients diagnosed after the trauma centre was built. The times for the completion of CT examinations, emergency operations and blood transfusions in the "after trauma centre" group were shorter than those in the "before trauma centre" group. However, the total expenditure of patients was increased. In the overall group, univariate and multivariate logistic regression analyses showed that a higher ISS was an independent predictor for worse mortality (OR = 17.859, 95% CI, 8.207-38.86, P < 0.001), while the establishment of a trauma centre was favourable for patient survival (OR = 0.492), which was also demonstrated by PSM. After determining the cut-off value of time for the completion of CT examination, emergency operation and blood transfusion, we found that the values were within the "golden one hour", and it was better for patients when the time was less than the cut-off value. CONCLUSION: Our study showed that for severely injured patients, the establishment of a trauma centre was favourable for a lower mortality rate. Furthermore, the completion of a CT examination, emergency surgery and blood transfusion in a timely manner and a lower ISS were associated with a decreased mortality rate.

Highly Adhesive Antibacterial Bioactive Composite Hydrogels With Controllable Flexibility and Swelling as Wound Dressing for Full-Thickness Skin Healing.

Polyzwitterionic hydrogels as skin wound dressings have been extensively studied owing to their superior antibacterial properties and skin adhesiveness, but their practical applications still suffer from a low adhesion strength and a high swelling ratio, which hinder the application of hydrogel for cutaneous healing. Here, we developed a novel biocompatible poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (PolySBMA) composite hydrogel with high stretchability, low swelling, strong skin adhesiveness, and antibacterial effect for enhancing wound healing. Naturally rigid polymers including quaternized chitosan methacrylate (QCSMA) and gelatin methacrylate (GelMA) are used as bioactive cross-linkers to endow PolySBMA/QCSMA/GelMA (SQG) hydrogel with a low swelling ratio and high bioactivity. The optimized hydrogel has excellent mechanical flexibility, with the ultimate tensile strength, tensile strain, modulus, and toughness of up to 344.5 kPa, 364%, 14.7 kPa, and 33.4 kJ m-3, respectively. The adhesiveness of the hydrogel to the skin tissue is as high as 38.2 kPa, which is critical for stopping the bleeding from the wound. The synergistic contributions from the PolySBMA and QCSMA endow hydrogel with good antibacterial properties against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Moreover, the natural polymer cross-linked polyzwitterionic hydrogel shows good cell activity, hemocompatibility, and histocompatibility. The in vivo full-thickness skin defect model demonstrates that the SQG hydrogel efficiently improves the granulation tissue formation and collagen deposition. In summary, such superiorly skin-adhesive antibacterial biocompatible hydrogel with controllable flexibility and swelling holds great promise as wound dressings for acute wounds.

Extracellular vesicle-enclosed miR-486-5p mediates wound healing with adipose-derived stem cells by promoting angiogenesis.

Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing.

Biological function evaluation and effects of laser micro-pore burn-denatured acellular dermal matrix.

OBJECTIVE: In the field of burns repairs, many problems exist in the shortage of donor skin, the expense of allograft or xenograft skin, temporary substitution and unsatisfactory extremity function after wound healing. Previous studies showed that burn-denatured skin could return to normal dermis formation and function. This study investigates the application of laser micro-pore burn-denatured acellular dermis matrix (DADM) from an escharotomy in the repair of burn wounds and evaluates the biological properties and wound repair effects of DADM in implantation experiments in Kunming mice. METHODS: Specific-pathogen-free (SPF) Kunming mice were used in this study. A deep II° burn wound was created on the dorsum of the mice by an electric heated water bath. The full-thickness wound tissue was harvested. The necrotic tissue and subcutaneous tissue were removed. The denatured dermis was preserved and treated with 0.25% trypsin, 0.5% Triton X-100. The DADM was drilled by laser micro-pore. The biological properties and grafting effects of laser micro-pore burn-DADM were evaluated by morphology, cytokine expression levels and subcutaneous implantation experiments in Kunming mice. RESULTS: We found statistical significance (P<0.05) of the elastic modulus (MPa), maximum load force (N) and contraction measurement (CM) of the laser micro-pore burn-DADM (experimental group) compared to the control group (no laser micro-pore burn-DADM). Cytokine expression level was different in the dermal matrixes harvested at various time points after burn (24h, 48h, 72h and infected wound group). Comparing the dermal matrix from 24h burn tissue to infected wound tissue, the expression level of IL-6, MMP-24, VE-cadherin and VEGF were decreased. We found no inflammatory cells infiltration in the dermal matrix were observed in both experimental and control groups (24h burn group), while the obviously vascular infiltration and fiber fusion were observed in the experimental group after subcutaneous implantation experiments. CONCLUSION: There was better bio-performance, low immunogenicity and better dermal incorporation after treated by laser micro-pore drilling and decellularized deep II° burn-DADM, which may be considered as a better substitute for dermal matrix. Furthermore, the earlier harvested DADM after burn (24h) shows the better transplantation effect.

[Research progress of presepsin in sepsis diagnosis].

OBJECTIVE: Sepsis is a frequently met syndrome with complex clinical symptoms and high mortality in emergency department. Early diagnosis of sepsis and timely treatment can improve survival. In recent years, the application of biomarkers [such as procalcitonin (PCT), C-reactive protein (CRP), interleukin-6 (IL-6)] are commonly used in the early diagnosis of sepsis, but their specificity and sensitivity are limited because of the long lag of report time. Soluble leukocyte differentiation antigen 14 sub type (sCD14-ST, namely presepsin) is a kind of novel biomarkers. Presepsin has a high specificity and sensitivity in the diagnosis of sepsis. It has some value to evaluate the severity of sepsis, antibiotic treatment of antibiotics, and prognosis of the patients with sepsis, and its latest detection method is fast and accurate, so it has the feasibility of clinical application.

Standard-b-Value Versus Low-b-Value Diffusion-Weighted Imaging in Hepatic Lesion Discrimination: A Meta-analysis.

OBJECTIVE: We sought to determine the comparative diagnostic performance of standard-b-value (500-1000s/mm) versus low-b-value (≤500 s/mm) diffusion-weighted imaging (DWI) in the discrimination of hepatic lesions. METHODS: A total of 1775 hepatic malignant lesions and 1120 benign hepatic lesions from 21 studies were included. RESULTS: (1) The global sensitivity was 0.86 (95% confidence interval [CI], 0.847-0.879), the specificity was 0.82 (95% CI, 0.797-0.842), the positive likelihood ratio (PLR) was 6.234 (95% CI, 4.260-9.123), the negative likelihood ratio (NLR) was 0.175 (95% CI, 0.135-0.227), and diagnostic odds ratio (DOR) was 42.836 (95% CI, 24.134-76.031). The area under the curve (AUC) and Q* index were 0.93 and 0.87. Publication bias was not present (P > 0.05). (2)The sensitivity of a subgroup meta-analysis of standard-b-value DWI was 0.858 (95% CI, 0.835-0.880), the specificity was 0.836 (95% CI, 0.807-0.863), the PLR was 6.527 (95% CI, 3.857-11.046), the NLR was 0.168 (95% CI, 0.123-0.239), and the DOR was 49.716 (95% CI, 22.897-107.98). The AUC and Q* index were 0.941 and 0.88. (3)The sensitivity of a subgroup meta-analysis of low-b-value DWI was 0.87 (95% CI, 0.84-0.89), the specificity was 0.80 (95% CI, 0.76-0.83), the PLR was 6.22 (95% CI, 3.29-11.76), the NLR was 0.19 (95% CI, 0.12-0.29), and the DOR was 37.14 (95% CI, 14.80-93.18). The AUC and Q* index were 0.922 and 0.86. CONCLUSIONS: Hepatic DWI is useful in differentiating between malignant and benign hepatic lesions. Standard-b-value DWI displayed an overall superior diagnostic accuracy over low-b-value DWI. Further trials needed to determine whether increasing b values beyond 1000 s/mm affects the diagnostic accuracy of hepatic lesion discrimination.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds.

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

[Effects of microporous porcine acellular dermal matrix combined with bone marrow mesenchymal cells of rats on the regeneration of cutaneous appendages cells in nude mice].

OBJECTIVE: To observe the effects of microporous porcine acellular dermal matrix (ADM) combined with bone marrow mesenchymal cells (BMMCs) population containing bone mesenchymal stem cells (BMSCs) of rats on the regeneration of cutaneous appendages cells in nude mice. METHODS: Split-thickness dermal grafts, 20 cm×10 cm in size and 0.3 mm in thickness, were prepared from a healthy pig which was sacrificed under sanitary condition. Laser microporous porcine ADM (LPADM) was produced by laser punching, hypertonic saline solution acellular method, and crosslinking treatment, and nonporous porcine ADM (NPADM) was produced by the latter two procedures. Then the appearance observation, histological examination and scanning electron microscope observation were conducted. BMMCs were isolated and cultured from tibia and femur after sacrifice of an SD rat. Osteogenic and adipogenic differentiation experiments were conducted among the adherent cells in the third passage. Then they were inoculated to LPADM and NPADM to construct BMMCs-LPADM and BMMCs-NPADM materials. Twenty-one healthy nude mice were divided into BMMCs-LPADM+NPADM group (A, n = 6), LPADM+split-thickness skin graft group (B, n = 6), BMMCs-LPADM+split-thickness skin graft group (C, n = 6), BMMCs-NPADM+split-thickness skin graft group (D, n= 3) according to randomized block. After anesthesia, a 2 cm×2 cm full-thickness skin defect reaching deep fascia was reproduced in the middle of the back of each nude mouse, and a split-thickness skin graft of the same size was obtained, and then prepared skin grafts were transplanted to cover the wounds respectively. On post transplantation day (PTD) 5, 7, and 14, local condition and adverse effects observation was conducted; one nude mouse was sacrificed each time to harvest all the transplant for tissue structure observation with HE staining. On PTD 7 and 14, neonatal skin appendages in corresponding composite materials were observed with transmission electron microscope. RESULTS: (1) LPADM and NPADM appeared to be porcelain white, soft, and flexible. No cellular component was observed in acellular dermal matrix. Scanning electron microscope showed that the collagen fibers were orderly arranged. LPADM had microporous structure. (2) Cells in the third passage were orderly arranged with the shape similar to fibroblasts with high growth speed. (3) Induced differentiation experiments showed that cells could differentiate into osteoblasts and adipocytes. (4) On PTD 5, the NPADM in group A was dry in part; skin grafts in group D were dry and necrotic, and there was no infection and inflammation in groups A and D; skin grafts in groups B and C survived. On PTD 7 and 14, the overlaying material in group A was black, dry, and hard in part; the skin grafts in group D turned to be completely black, dry, and necrotic, and pale yellow clear exudate was found in subcutaneous area; there was no obvious purulent discharge in groups A and D; the appearance of skin grafts in groups B and C was close to the surrounding skin. (5) On PTD 5 and 7, in groups A, B, and C, vascularization was apparent in the pores of dermal matrix, and red blood cells could be found. In group D, skin grafts were dry and necrotic. On PTD 14, in groups A, B, and C, the pore structure of dermal matrix was fully vascularized in which a large number of red blood cells were visible. In group A, the microporous dermal matrix survived, but the overlaying NPADM was not attached closely. In groups B and C, the skin grafts were closely connected to the dermal matrix, and no cutaneous appendages were observed. In group C, special monolayer cells were found at the junction between skin graft and dermal matrix. (6) Skin grafts in group D failed to survive; they were not observed with the electron microscope. On PTD 7, there were no significant differences among groups A, B, and C. On PTD 14, no sebaceous gland-like cell or sweat gland-like cell and no newborn nerve ending were observed in skin grafts in groups A and B, in spite of the immigration of fibroblasts. In group C, a large number of new capillaries were observed at the junction between the skin graft and dermal matrix; rough endoplasmic reticulum of fibroblasts proliferated exuberantly; newborn unmyelinated nerve endings were observed; single free sweat gland-like cells and sebaceous gland-like cells were observed in superficial dermal matrix. CONCLUSIONS: LPADM, which provides a "cell niche-like" micro-environment for the migration and differentiation of the BMMCs population, when combining with the split-thickness skin graft, can induce exogenous differentiation of BMSCs in vivo, thus achieving the reconstruction of skin appendages.

[Study on Acinetobacter baumannii plasmid with 3 types of beta-lactamase genes in a burn ward].

OBJECTIVE: To study the transferrable character of Acinetobacter baumannii (AB) plasmids with 3 types of beta-lactamase gene. METHODS: The plasmid of multi-drug resistant AB (donor) isolated from burn wound were transferred to E. coil ATCC25922 (receptor) through conjugation, and drug sensitivity was also observed. Drug-resistant gene and stability of filial generation and zygote were analyzed by PCR. RESULTS: The drug-resistance of donor plasmids to Sulfamethoxazole, Ampicillin, Cefalotin, Cefpodoxime, Cefuroxime, Imipenem/Cilastatin and Ampicillin/Sulbactam, and three types of beta-lactamase gene were transferred to the receptor, and were also stably transmitted for passages. The minimum inhibitor concentration of receptor to Sulfamethoxazole was > 2 mg/L after conjugation with donor, and inhibitory character could be transferred to next generation. CONCLUSION: bla(TEM-1), bla(PER-1) and bla(OXA-23) genes carried in the plasmid of AB can be transferred through conjugation and stably transmitted for passages, and it is one of the molecular mechanisms for AB with multi-drug resistance after burn infections.

Molecular mechanism of osteochondroprogenitor fate determination during bone formation.

Osteoblasts and chondrocytes, which derive from a common mesenchymal precursor (osteochondroprogenitor), are involved in bone formation and remodeling in vivo. Determination of osteochondroprogenitor fate is under the control of complex hormonal and local factors converging onto a series of temporospatial dependent transcription regulators. Sox9, together with L-Sox5 and Sox6, of the Sox family is required for chondrogenic differentiation commitment, while Runx2/Cbfa 1, a member of runt family and Osterix/Osx, a novel zinc finger-containing transcription factor play a pivotal role in osteoblast differentiation decision and hypertrophic chondrocyte maturation. Recent in vitro and in vivo evidence suggests beta-catenin, a transcriptional activator in the canonical Wnt pathway, can act as a determinant factor for controlling chondrocyte and osteoblast differentiation. Here we focus on several intensively studied transcription factors and Wnt/beta-catenin signal molecules to illustrate the regulatory mechanism in directing commitment between osteoblast and chondrocyte, which will eventually allow us to properly manipulate the mesenchymal progenitor cell differentiation on bone and regeneration of cartilage tissue engineering.