A sustainable strategy for generating highly stable human skin equivalents based on fish collagen.

Tissue engineered skin equivalents are increasingly recognized as potential alternatives to traditional skin models such as human ex vivo skin or animal skin models. However, most of the currently investigated human skin equivalents (HSEs) are constructed using mammalian collagen which can be expensive and difficult to extract. Fish skin is a waste product produced by fish processing industries and identified as a cost-efficient and sustainable source of type I collagen. In this work, we describe a method for generating highly stable HSEs based on fibrin fortified tilapia fish collagen. The fortified fish collagen (FFC) formulation is optimized to enable reproducible fabrication of full-thickness HSEs that undergo limited contraction, facilitating the incorporation of human donor-derived skin cells and formation of biomimetic dermal and epidermal layers. The morphology and barrier function of the FFC HSEs are compared with a commercial skin model and validated with immunohistochemical staining and transepithelial electrical resistance testing. Finally, the potential of a high throughput screening platform with FFC HSE is explored by scaling down its fabrication to 96-well format.

Design of hydrogel-based scaffolds for in vitro three-dimensional human skin model reconstruction.

In vitro three-dimensional (3D) skin tissue models are critical tools in advancing our understanding of basic skin physiology and function as well as in specific applications such as toxicity testing of dermatological compounds. However, the utilization of such skin models is often limited by the structural instability of the construct, lack of physiologically relevant features and weak barrier function. In this review, we highlight the current research efforts in hydrogel biomaterial selection and scaffold design that allow for maturation of engineered skin in vitro, with special emphasis on matured full-thickness (including epidermal and dermal compartments) skin. The different types of scaffold biomaterials, broadly categorized as natural, synthetic, or composite will also be discussed. At the same time, we will outline strategies for next-generation biomimetic skin templates incorporating skin appendages or perfusion systems that can more closely reflect the native skin environment. STATEMENT OF SIGNIFICANCE: In vitro 3D human skin models are critical tools in advancing our understanding of skin physiology and function. Many of the existing reconstructed models are limited in terms of structure and complexity, thus failing to recapitulate native human skin. In order to address this, hydrogels have been identified as useful scaffold materials for fabricating the dermal equivalent of 3D skin models, allowing for greater flexibility and control in scaffold properties and cellular incorporation. This review aims to provide a critical discussion of the biomaterial selection and design strategies in the construction of hydrogel-based full-thickness skin equivalents. At the same time, we will offer insights into the future developments and technological advances which can accelerate the progress in this field.

Improving hand hygiene in a medical ward: a multifaceted approach.

BACKGROUND: Hand hygiene is a fundamental action which is simple, inexpensive and an effective tool in reducing hospital-acquired infections, yet compliance remains low in healthcare settings. In 2014, Changi General Hospital embarked on a pilot project to improve hand hygiene compliance in a pilot ward with the intention to eventually spread a multifaceted set of interventions hospital wide. METHODS: A before and after interventional study of a pilot project. Hand hygiene data collection was through direct observations by auditors using WHO monitoring standards and techniques based on the five-moment model. SETTING: A medical ward in an acute hospital in Singapore. RESULTS: Overall hand hygiene compliance improved from a median of 53% in 2015 to 80% by end of 2017. Hand hygiene compliance of doctors increased from 43% to 60% (p=0.00), nurses from 62% to 89% (p=0.014) and allied health staff from 67% to 83% (p=0.002). CONCLUSIONS: A multifaceted set of interventions developed by the project team was effective in improving hand hygiene compliance of doctors, nurses and allied health staff.

SYBR® Green RT-qPCR for the Universal Detection of Citrus Viroids.

Quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR have now become the gold standard for molecular diagnostics because of its sensitivity, specificity, and reproducibility. In addition, qPCR diagnostics are flexible because they can be scaled for high- or low-throughput applications. Here we describe an optimized assay and workflow for the universal detection of eight citrus viroid species and their variants by RT-qPCR. The assay allows for quick and efficient molecular detection of viroids without the need to run RT-qPCR for each individual viroid species.

Recent Advances in the Design of Three-Dimensional and Bioprinted Scaffolds for Full-Thickness Wound Healing.

Three-dimensional (3D) printed scaffolds have recently emerged as an innovative treatment option for patients with critical-sized skin wounds. Current approaches to managing life-threatening wounds include skin grafting and application of commercially sourced skin substitutes. However, these approaches are not without several challenges. Limited donor tissue and donor site morbidity remain a concern for tissue grafting, while engineered skin substitutes fail to fully recapitulate the complex native environment required for wound healing. The implementation of 3D printed dermal scaffolds offers a potential solution for these shortcomings. Spatial control over scaffold structure, the ability to incorporate multiple materials and bioactive ingredients, enables the creation of conditions specifically optimized for wound healing. Three-dimensional bioprinting, a subset of 3D printing, allows for the replacement of lost cell populations and secreted active compounds that contribute to tissue repair and recovery. The replacement of damaged and lost cells delivers beneficial effects directly, or synergistically, supporting injured tissue to recover its native state. Despite encouraging results, the promise of 3D printed scaffolds has yet to be realized. Further improvements to current material formulations and scaffold designs are required to achieve the goal of clinical adoption. Herein, we provide an overview of 3D printing techniques and discuss several strategies for healing of full-thickness wounds by using 3D printed acellular scaffolds or bioprinted cellular scaffolds, aimed at translating this technology to the clinical management of skin lesions. We identify the challenges associated with designing and optimizing printed tissue replacements, and discuss the future perspectives of this emerging option for managing patients who present with critical-sized life-threatening cutaneous wounds. Impact statement Chronic wounds and burn injuries often present with the full-thickness loss of skin, threatening the life of the patient and generating significant socioeconomic burden for these patients, their treating clinicians, and the wider community in which these patients live. Effective clinical management that permits damaged skin tissue to repair and restore its native functional state reduces the strain on health care systems. Three-dimensional (3D) printed scaffolds have been proposed as a potential solution and could be instrumental in facilitating the recovery and healing process. In this review, we will summarize the current research approaches, technologies, and limitations of 3D printed scaffolds as an efficient and effective approach to managing cutaneous wound healing.

Thymosin ß4 increases cardiac cell proliferation, cell engraftment, and the reparative potency of human induced-pluripotent stem cell-derived cardiomyocytes in a porcine model of acute myocardial infarction.

Rationale: Previous studies have shown that human embryonic stem cell-derived cardiomyocytes improved myocardial recovery when administered to infarcted pig and non-human primate hearts. However, the engraftment of intramyocardially delivered cells is poor and the effectiveness of clinically relevant doses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in large animal models of myocardial injury remains unknown. Here, we determined whether thymosin ß4 (Tb4) could improve the engraftment and reparative potency of transplanted hiPSC-CMs in a porcine model of myocardial infarction (MI). Methods: Tb4 was delivered from injected gelatin microspheres, which extended the duration of Tb4 administration for up to two weeks in vitro. After MI induction, pigs were randomly distributed into 4 treatment groups: the MI Group was injected with basal medium; the Tb4 Group received gelatin microspheres carrying Tb4; the CM Group was treated with 1.2 × 108 hiPSC-CMs; and the Tb4+CM Group received both the Tb4 microspheres and hiPSC-CMs. Myocardial recovery was assessed by cardiac magnetic resonance imaging (MRI), arrhythmogenesis was monitored with implanted loop recorders, and tumorigenesis was evaluated via whole-body MRI. Results: In vitro, 600 ng/mL of Tb4 protected cultured hiPSC-CMs from hypoxic damage by upregulating AKT activity and BcL-XL and promoted hiPSC-CM and hiPSC-EC proliferation. In infarcted pig hearts, hiPSC-CM transplantation alone had a minimal effect on myocardial recovery, but co-treatment with Tb4 significantly enhanced hiPSC-CM engraftment, induced vasculogenesis and the proliferation of cardiomyocytes and endothelial cells, improved left ventricular systolic function, and reduced infarct size. hiPSC-CM implantation did not increase incidence of ventricular arrhythmia and did not induce tumorigenesis in the immunosuppressed pigs. Conclusions: Co-treatment with Tb4-microspheres and hiPSC-CMs was safe and enhanced the reparative potency of hiPSC-CMs for myocardial repair in a large-animal model of MI.

Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

Early Regenerative Capacity in the Porcine Heart.

BACKGROUND: The adult mammalian heart has limited ability to repair itself after injury. Zebrafish, newts, and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown whether hearts of young large mammals can regenerate. METHODS: We examined the regenerative capacity of the pig heart in neonatal animals (ages 2, 3, or 14 days postnatal) after myocardial infarction or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry, and Western blotting were performed to study cell proliferation, sarcomere dynamics, and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed. RESULTS: After myocardial infarction, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day-old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls, and did not recover function. Genome-wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day-old animals that was absent in 2-day-old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the myocardial infarction showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly. CONCLUSIONS: Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

Maturation of Pluripotent Stem Cell-Derived Cardiomyocytes: a Critical Step for Drug Development and Cell Therapy.

Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are emerging as an invaluable alternative to primarily sourced cardiomyocytes. The potentially unlimited number of hPSC-derived cardiomyocytes (hPSC-CMs) that may be obtained in vitro facilitates high-throughput applications like cell transplantation for myocardial repair, cardiotoxicity testing during drug development, and patient-specific disease modeling. Despite promising progress in these areas, a major disadvantage that limits the use of hPSC-CMs is their immaturity. Improvements to the maturity of hPSC-CMs are necessary to capture physiologically relevant responses. Herein, we review and discuss the different maturation strategies undertaken by others to improve the morphology, contractility, electrophysiology, and metabolism of these derived cardiomyocytes.