The treatment of hypertrophic scars (HSs) is considered to be the most challenging task in wound rehabilitation. Conventional silicone sheet therapy has a positive effect on the healing process of HSs. However, the dimensions of the silicone sheet are typically larger than those of the HS itself which may negatively impact the healthy skin that surrounds the HS. Furthermore, the debonding and displacement of the silicone sheet from the skin are critical problems that affect treatment compliance. Herein, we propose a bespoke HS treatment design that integrates pressure sleeve with a silicone sheet and use of silicone gel using a workflow of three-dimensional (3D) printing, 3D scanning and computer-aided design, and manufacturing software. A finite element analysis (FEA) is used to optimize the control of the pressure distribution and investigate the effects of the silicone elastomer. The result shows that the silicone elastomer increases the amount of exerted pressure on the HS and minimizes unnecessary pressure to other parts of the wrist. Based on this treatment design, a silicone elastomer that perfectly conforms to an HS is printed and attached onto a customized pressure sleeve. Most importantly, unlimited scar treating gel can be applied as the means to optimize treatment of HSs while the silicone sheet is firmly affixed and secured by the pressure sleeve.

ABSTRACTIntroductionRisk factors of adverse outcomes in COVID-19 are defined but stratification of mortality using non-laboratory measured scores, particularly at the time of pre-hospital SARS-CoV-2 testing, is lacking.MethodsMultivariate regression with bootstrapping was used to identify independent mortality predictors in a derivation cohort of COVID-19 patients. Predictions were externally validated in a large random sample of the ISARIC cohort (N=14,231) and a smaller cohort from Aintree (N=290).Results983 patients (median age 70, IQR 53-83; in-hospital mortality 29.9%) were recruited over an 11-week study period. Through sequential modelling, a 5-predictor score termed SOARS (SpO2, Obesity, Age, Respiratory rate, Stroke history) was developed to correlate COVID-19 severity across low, moderate and high strata of mortality risk. The score discriminated well for in-hospital death, with area under the receiver operating characteristic values of 0.82, 0.80 and 0.74 in the derivation, Aintree and ISARIC validation cohorts respectively. Its predictive accuracy (calibration) in both external cohorts was consistently higher in patients with milder disease (SOARS 0-1), the same individuals who could be identified for safe outpatient monitoring. Prediction of a non-fatal outcome in this group was accompanied by high score sensitivity (99.2%) and negative predictive value (95.9%).ConclusionThe SOARS score uses constitutive and readily assessed individual characteristics to predict the risk of COVID-19 death. Deployment of the score could potentially inform clinical triage in pre-admission settings where expedient and reliable decision-making is key. The resurgence of SARS-CoV-2 transmission provides an opportunity to further validate and update its performance.

Introduction We aimed to develop and validate a prediction table for a simplified measure of rightward shift of the fetal oxyhaemoglobin saturation (SpO 2 ) versus inspired oxygen pressure ( P I O 2 ) curve as an objective marker of lung disease severity in very preterm infants, independent of unit altitude or oxygen prescribing policies. Methods Very preterm infants (n=219) had an oxygen reduction test at median (IQR) test age of 35 4 (34 5 –36 0 ) weeks’ postmenstrual age (PMA). Shift was derived from at least three paired SpO 2 versus P I O 2 measurements using a computer algorithm, using the fetal oxyhaemoglobin dissociation curve as the reference. Linear regression of resultant shift values enabled construction of a table to predict shift using a single paired SpO 2 versus P I O 2 measurement, validated subsequently in a separate infant cohort using Bland-Altman analysis. Receiver operating curve analysis provided threshold values equating to a clinical diagnosis of mild bronchopulmonary dysplasia (BPD) or moderate to severe BPD. Results The median (IQR) age of 63 infants in the validation cohort was 36 0 (35 6 –36 2 ) weeks’ PMA. Mean difference (95% CI) between predicted and measured shift was 2.1 (−0.8% to 4.9%) with wide limits of agreement (−20.7% to 24.8%). Predicted shift >10.1 kPa identified mild BPD with 71% sensitivity and 88% specificity while values>13.0 kPa identified moderate to severe BPD with 81% sensitivity and 100% specificity. Discussion Shift predicted from a single paired SpO 2 versus P I O 2 measurement using our validated table enables objective bedside screening of lung disease severity in very preterm infant cohorts at 36 weeks’ PMA.

Current treatments for severe skin damage involve the grafting of extremely limited autogenic skin or the use of synthetic skin grafts that do not fully recapitulate the biological properties of native skin. In this study we developed a novel bi-layer scaffold that provides the microenvironmental cues favorable to promoting skin healing and regeneration. The scaffold is composed of a superficial chitosan/PCL nanofibrous mat (CP-nano mat) and an underlying PLLA microporous disc (PLLA-micro disc). The porous structure of the scaffold permits the interaction of biomolecules released from two types of cells distributed, respectively, throughout the two layers of the scaffold, but the nanofibers prevent the direct intermingling of the cell types. The CP-nano mat and PLLA-micro disc were fabricated by electrospinning and thermally induced phase separation, respectively, and host keratinoctyes as an epidermal equivalent and fibroblasts as a dermal equivalent, respectively, present in the native skin. The potential of this bi-layer scaffold to serve as a skin equivalent was evaluated by co-culture of keratinocytes and fibroblasts and subsequent assessment of cell proliferation, cell morphology, gene transcription, and protein expression. The cell proliferation was found to be greatest in co-culture on bi-layer scaffolds. The gene and protein expression analyses further confirmed that the bi-layer scaffold provided a micro-environment similar to those present in the native extracellular matrix during initial wound healing. Our study suggested that the bi-layer scaffold has great potential to serve as a skin equivalent in tissue engineering.

Skeletal muscle injury can lead to severe motor deficits that adversely affect movement and quality of life. Current surgical treatments for skeletal muscle are hindered by the poor formation of organized myotube bundles at the wound site. Tissue-engineered skeletal muscle constructs to date have been unable to generate high degrees of myotube density and alignment. Generating a suitable in vitro tissue-engineered skeletal muscle construct requires the design of a scaffold that recapitulates the structural combination of nanoscale collagen fibrils and aligned microscale basal lamina tracks present in the native extracellular matrix (ECM). We hypothesized that a 3D aligned tubular porous scaffold containing aligned nanofibers inside the pores can mimic the native muscle tissue environment. We constructed a laminar section of the hypothesized scaffold with aligned chitosan-PCL nanofibers arranged co-axially with the aligned microscale chitosan scaffold bands to mimic the required myogenic environment. A 6-day study of C2C12 mouse myoblast cells cultured on this hybrid scaffold indicated that the nanofibers and scaffold bands in the scaffold played a synergetic role in directing cell orientation, interaction, migration and organization. Our results showed that aligned nanofibers mediated cell alignment and the aligned scaffold bands induced the formation of a more compact assembly of myotube cells as compared to various control substrates including chitosan films, nanofibers, and chitosan bands. The expression levels of both early and late-stage myogenic differentiation genes associated with myogenin and myosin heavy chain, respectively, were higher on the hybrid substrate than on control substrates. Our study suggests that the combination of nano and microscale topological features in the ECM can direct myogenic differentiation, and the hybrid material has the potential to improve the outcome of skeletal tissue engineering.

Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.

There is an urgent need for a rationally-designed, cellularized skin graft capable of reproducing the micro-environmental cues necessary to promote skin healing and regeneration. To address this need, we developed a composite scaffold, namely, CA/C-PEG, composing of a porous chitosan-alginate (CA) structure impregnated with a thermally reversible chitosan-poly(ethylene glycol) (C-PEG) gel to incorporate skin cells as a bi-layered skin equivalent. Fibroblasts were encapsulated in C-PEG to simulate the dermal layer while the keratinocytes were seeded on the top of CA/C-PEG composite scaffold to mimic the epidermal layer. The CA scaffold provided mechanical support for the C-PEG gel and the C-PEG gel physically segregated the keratinocytes from fibroblasts in the construct. Three different tissue culture micro-environments were tested: CA scaffolds without C-PEG cultured in cell culture medium without air-liquid interface (-gel-interface), CA scaffolds impregnated with C-PEG and cultured in cell culture medium without air-liquid interface (-gel-interface), and CA scaffolds impregnated with C-PEG cultured in cell culture medium with air-liquid interface (-gel- interface). We found that the presence of C-PEG increased the cellular proliferation rates of both keratinocytes and fibroblasts, and the air-liquid interface induced keratinocyte maturation. This CA/C-PEG composite scaffold design is able to recapitulate micro-environments relevant to skin tissue engineering, and may be a useful tool for future skin tissue engineering applications.

Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We present a novel approach to effectively enhance myogenic differentiation of human embryonic stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a protein. The myogenic differentiation was assessed by cell morphology, gene activities, and protein expression. hESCs grown on C-PCL uniaxially aligned nanofibers in media containing Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber orientation, with increased expressions of marker genes and proteins associated with myogenic differentiation as compared to control substrates. The combination of Wnt3a signaling and aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system for embryonic myogenesis and muscle regeneration.

In vitro models that accurately mimic the microenvironment of invading glioblastoma multiform (GBM) cells will provide a high-throughput system for testing potential anti-invasion therapies. Here, the ability of chitosan-polycaprolactone polyblend nanofibers to promote a migratory phenotype in human GBM cells by altering the nanotopography of the nanofiber membranes is investigated. Fibers are prepared with diameters of 200 nm, 400 nm, and 1.1 μm, and are either randomly oriented or aligned to produce six distinct nanotopographies. Human U-87 MG GBM cells, a model cell line commonly used for invasion assays, are cultured on the various nanofibrous substrates. Cells show elongation and alignment along the orientation of aligned fibers as early as 24 h and up to 120 h of culture. After 24 h of culture, human GBM cells cultured on aligned 200 nm and 400 nm fibers show marked upregulation of invasion-related genes including β-catenin, Snail, STAT3, TGF-β, and Twist, suggesting a mesenchymal change in these migrating cells. Additionally, cells cultured on 400 nm aligned fibers show similar migration profiles as those reported in vivo, and thus these nanofibers should provide a unique high-throughput in vitro culture substrate for developing anti-migration therapies for the treatment of GBM.

This study investigated the use of three-dimensional porous chitosan-alginate (CA) scaffolds for critical size calvarial defect (diameter, 5.0 mm) repair in Sprague-Dawley rats. CA scaffolds have been used for in vitro culture of many cell types and demonstrated osteogenesis in ectopic locations in vivo, but have yet to be evaluated for functional bone tissue engineering applications. CA scaffolds demonstrated the ability to support undifferentiated mesenchymal stem cells (MSCs) in culture for 14 days in vitro and promoted spherical morphology. In vivo tests were performed using CA scaffolds and CA scaffolds with treatments including undifferentiated MSCs, bone marrow aspirate, and bone morphogenetic protein-2 (BMP-2) growth factor in comparison to unfilled bone defect used as a control. The samples were analyzed with MicroCT, histology, and immunohistochemical staining at 4 and 16 weeks. Partial defect closure was observed in all experimental groups at 16 weeks, with the greatest defect closure (71.56 ± 19.74%) in the animal group treated with CA scaffolds with BMP-2 (CA + BMP-2). The experimental samples demonstrated osteogenesis in histology and immunohistochemical staining, with the CA + BMP-2 group, showing the greatest level of osteogenesis. Tissue engineered CA scaffolds show promise in reconstruction of critical size bone defects.

Tendon injury occurs frequently and tendon repair is limited by its poor self-healing. The current tissue engineering approach for treating tendon injuries has showed limited success, largely due to the lack of scaffolds with suitable structural and biological properties, and suitable growth factors for differentiation of stem cells into tendon cells. This study investigated if the combination of environmental and biological cues from aligned chitosan-poly-caprolactone (C-PCL) combined with TGF-β3 growth factor can efficiently and rapidly direct the tenogenic differentiation of primary human bone marrow stem cells (BMSCs). C-PCL nanofibers were prepared to have the anisotropic nanostructure, and mechanical and biological properties similar to those of the native tendon extracellular matrix (ECM). The tenogenic commitment of BMSCs was assessed using cell morphology, and gene and protein expressions. BMSCs grown on uniaxially aligned C-PCL nanofibers in a medium containing TGF-β3 displayed an elongated morphology along nanofiber orientation, upregulated expressions of marker genes, and increased collagen production associated with tenogenic differentiation as compared to control substrates. Significantly, this tenogenic microenvironment induced the transcription of tenogenic markers in 5 days and production of a large amount of Collagen I in 10 days, more effective and faster than existing scaffolds combined with growth factors. This research reveals that a combinative effect of aligned C-PCL nanofibers and TGF-β3, as environmental and biological cues, can lead to rapid, effective BMSC differentiation into tenogenic progenitors, offering a potential strategy for managing tendon disorders.

Chitosan, a biodegradable naturally occurring polymer, has drawn considerable attention in recent years as a scaffolding material in tissue engineering and regenerative medicine. Despite its favorable biological properties, the weak mechanical strength of scaffolds produced from chitosan has limited the scope of their application. Here we fabricated 3D pristine porous chitosan scaffolds with unprecedented mechanical strength and investigated the regulatory role of chitosan and acidic concentrations on the crystallinity and thus on the mechanical and biological properties of produced scaffolds. Chitosan scaffolds of varying mechanical properties were prepared from solutions with chitosan concentrations of 4-12 wt%. The produced scaffolds showed no apparent shape change after immersion in Dulbecco's Modified Eagle Medium (DMEM), phosphate buffered saline, and simulated body fluid for two weeks. We showed that the crystallinity of the scaffold increased with increasing chitosan concentration or decreasing solution acidity, and the maximum compressive mechanical strength and modulus of 1.74 +/- 0.01 MPa and 17.99 +/- 0.11 MPa, respectively, were achieved at a chitosan concentration of 12 wt%. MG-63 osteoblast cells demonstrated improved adhesion, proliferation and osteogenic activity on chitosan scaffolds of increased chitosan concentration or mechanical strength. The ability to produce high-strength chitosan scaffolds and engineer their mechanical properties can substantially expand the applicability of chitosan in tissue engineering as well as other engineering applications.

Increasing cell seeding efficiency in a tissue engineering construct can enhance cellular activity and tissue formation in vivo. Here, we demonstrate the use of alginate gel as a secondary phase material in 3D porous β-tricalcium phosphate scaffolds to improve cell seeding and provide controlled release of growth factors for bone tissue engineering. Cells were seeded in scaffolds in three ways: conventional seeding (CS), alginate gel-assisted seeding (GS), and alginate GS with bone morphogenetic protein-2 (BMP-2, GSB). In vitro study with MG-63 cells showed that cell seeding efficiency and cell population 1 week after seeding were significantly elevated in GS and GSB samples compared to CS samples. The GSB system demonstrated a sustained, steady release of BMP-2 over 2 weeks. In vivo, scaffolds seeded with rat mesenchymal stem cells were implanted ectopically into Sprague-Dawley rats for 8 weeks. GS and GSB samples exhibited improved osteogenic activity, with the GSB samples inducing the greatest osteocalcin and osteoid deposition. This study suggests that the alginate gel-assisted cell seeding increases seeding efficiency and allows for sustained release of growth factors. The use of the secondary phase polymer bolsters bone formation in vivo and has the potential for improving outcome in other tissue engineering applications.

Chitosan-based fibrous matrices are prepared to mimic the ECM architecture and elucidate substrate-mediated hESC differentiation due to topographical scale and anisotropy without exogenic morphogens. Fibrous matrices support fewer pluripotent hESCs than films but enable topography-mediated hESC differentiation. Matrices composed of 400 nm and 1.1 µm diameter fibers support increased expression of neural markers indicative of ectodermal commitment while matrices of 200 nm diameter fibers increase expression of osteogenic and hepatic markers indicative of endodermal and mesodermal commitment. The fibrous-mediated hESC differentiation highlights the significant implication of tailored ECM-like substrates for hESC-based therapies.

Chitosan and alginate are two natural and accessible polymers that are known to be biocompatible and biodegradable. When combined, they exhibit desirable characteristics and can be created into a scaffold for cell culture. Growing cells in a 3D scaffold is more conducive to natural cell morphology than traditional 2D dish cultures. These chitosan-alginate scaffolds can be used for in vitro cell culture, but many aspects have yet to be studied and optimized. Cell recovery is essential for analysis of growth and viability. Combination of different chitosan and alginate scaffolds were tested in different degradation solutions. A solution of 100mM EDTA and 100mM Na2HPO4 was found to have the best results. This solution was non-toxic to HEPg2 cells. Cell viability in the scaffolds was also studied. Results show that cells can survive in scaffolds and can be recovered, making chitosan-alginate scaffolds a viable means of in vitro cell culture.

Dr Fraser's letter follows his review on the topic of muscle volume regulation (Usher-Smith et al. 2009) in which the authors take the view that muscle cells do not regulate their volume in response to changes in the tonicity of their extracellular environment. This view is puzzling in view of the fact that most animal cells studied to date display volume regulation characteristics (O'Neill, 1999; Lang, 2007). The fact that mammalian skeletal muscle cells also regulate volume has also been demonstrated (Sitdikov et al. 1989; Urazaev, 1998; Lindinger et al. 2011). To refute the likelihood that mammalian skeletal muscle cells regulate cell volume, Dr Fraser presents some interesting results on amphibian muscle cells, and raises some good points regarding experimental design. He shows that in single Rana pectoris muscle fibres the volume responses occurring after addition of either 100 mm DMSO or 100 mm glycerol result from an initial osmotic loss of volume by muscle, followed by volume recovery. The mechanism by which the cells recover volume is not mentioned, but it may be implied that volume recovery is due to the influx of DMSO or glycerol into the cell interior, and that volume recovery is solely due to achieving complete chemical equilibration of the solute across the sarcolemma. There is no evidence presented that the vacuolation is not an occurrence that occurs with fibres under various non-DMSO conditions in his experimental set-up. It is also unknown if factors other than DMSO, or in combination with DMSO, contributed to the vacuolation or, indeed, if this is a normal response of muscle (Launikonis & Stephenson, 2004). It also appears that some of the methodology and results presented in our paper (Lindinger et al. 2011) were misinterpreted. Regarding methodology, we did not simultaneously add NaCl and remove DMSO as these tasks, perhaps fortunately, cannot be performed simultaneously. In his letter, Fraser referred to procedures performed using single muscle fibres. However, Fig. 4 is in reference to experiments performed on intact muscle. Even when using single fibres, the ‘50 s delay’ is the time after completion of the triple rinsing procedure and the acquisition of new images. The total time from first removal of the bumetanide-containing solution was closer to 5 min. Regardless, when baseline images were obtained prior to imposing the osmotic challenge, no volume changes occurred. When performing experiments in which muscle was incubated with bumetanide with DMSO, the solution containing bumetanide was first removed and flushed, and replaced with solution that was free of bumetanide and DMSO. This required about 5 min; baseline measurements were then obtained over the next 5–10 min – during this period no volume changes occurred. Then extracellular osmolarity was increased, resulting in cell shrinkage, followed by volume recovery. The observed responses cannot be explained by a DMSO-induced phenomenon. We have never observed volume transients during baseline acquisition periods following bumetanide treatments. Furthermore, we have observed the same volume responses reported in these experiments in hundreds of single cells treated with step increases in extracellular osmolarity that have not been exposed to DMSO, i.e. not calcein loaded and not incubated in bumetanide (M. Leung, J Moynes & MI Lindinger, unpublished and presented at several meetings). Dr Fraser's pilot data do raise concerns regarding use of DMSO in physiological experiments and the timing of experiments when using muscle treated with DMSO (also see Santos et al. 2003; Velasco et al. 2003). Further investigation is required to definitively and rigorously demonstrate whether a vacuolation response to DMSO treatment occurs in all such treated frog muscle fibres, as well as cells of other species using the experimental conditions employed by other labs, and then the physiological impact can be determined.

Lack of safe and effective carriers for delivery of RNA therapeutics remains a barrier to its broad clinical application. We report the development of a cell tanscytosing magnetic nanovector engineered as an siRNA carrier. Iron oxide nanoparticles were modified with poly(ethylene glycol) (PEG), small interfering RNA (siRNA), and a cationic polymer layer. Three nanovector formulations with cationic polymer coatings of poly-arginine (pArg), polylysine (pLys), and polyethylenimine (PEI), respectively, were prepared. The three nanovector formulations where evaluated for safety and ability to promote gene silencing in three types of cancer cells C6/GFP(+), MCF7/GFP(+), and TC2/GFP(+), mimicking human cancers of the brain, breast, and prostate, respectively. Cell viability and fluorescence quantification assays revealed that pArg-coated nanovectors were most effective in promoting gene knockdown and least toxic of the three nanovector formulations tested. Transmission electron microscopy (TEM) imaging of nanovector treated cells further demonstrated that pArg-coated nanovectors enter cells through cell transcytosis, while pLys and PEI coated nanovectors enter cells endocytosis. Our findings suggest that NPs engineered to exploit the cell transcytosis intracellular trafficking pathway may offer a more safe and efficient route for siRNA delivery.

Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the degree of change in extracellular osmolarity, is muscle specific, and is dependent on the functioning of the NKCC and Na+, K+-ATPase.