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Radiotherapy (RT), a common cancer treatment, unintentionally harms surrounding tissues, including the skin, and hinders wound healing years after treatment. This study aims to understand the mechanisms behind these late-onset adverse effects. We compare skin biopsies from previously irradiated (RT + ) and non-irradiated (RT - ) sites in breast cancer survivors who underwent RT years ago. Here we show that the RT + skin has compromised healing capacity and fibroblast functions. Using ATAC-seq, we discover altered chromatin landscapes in RT + fibroblasts, with THBS1 identified as a crucial epigenetically primed wound repair-related gene. This is further confirmed by single-cell RNA-sequencing and spatial transcriptomic analysis of human wounds. Notably, fibroblasts in both murine and human post-radiation wound models show heightened and sustained THBS1 expression, impairing fibroblast motility and contractility. Treatment with anti-THBS1 antibodies promotes ex vivo wound closure in RT + skin from breast cancer survivors. Our findings suggest that fibroblasts retain a long-term radiation memory in the form of epigenetic changes. Targeting this maladaptive epigenetic memory could mitigate RT's late-onset adverse effects, improving the quality of life for cancer survivors., (© 2024. The Author(s).)

The Coronavirus Disease 2019 (COVID-19), caused by virus SARS-CoV-2, is characterized by massive inflammation and immune system imbalance. Despite the implementation of vaccination protocols, the accessibility of treatment remains uneven. Furthermore, the persistent threat of new variants underscores the urgent need for expanded research into therapeutic options for SARS-CoV-2. Mesenchymal stem cells (MSCs) are known for their immunomodulatory potential through the release of molecules into the extracellular space, either as soluble elements or carried by extracellular vesicles (EVs). The aim of this study was to evaluate the anti-inflammatory potential of EVs obtained from human adipose tissue (ASC-EVs) against SARS-CoV-2 infection. ASC-EVs were purified by size-exclusion chromatography, and co-culture assays confirmed that ASC-EVs were internalized by human lung cells and could colocalize with SARS-CoV-2 into early and late endosomes. To determine the functionality of ASC-EVs, lung cells were infected with SARS-CoV-2 in the presence of increasing concentrations of ASC-EVs, and the release of cytokines, chemokines and viruses were measured. While SARS-CoV-2 replication was significantly reduced only at the highest concentrations tested, multiplex analysis highlighted that lower concentrations of ASC-EV sufficed to prevent the production of immune modulators. Importantly, ASC-EVs did not contain detectable inflammatory cytokines, nor did they trigger inflammatory mediators, nor affect cellular viability. In conclusion, this work suggests that ASC-EVs have the potential to attenuate inflammation by decreasing the production of pro-inflammatory cytokines in lung cells following SARS-CoV-2 infection., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Long-term diabetes often leads to chronic wounds refractory to treatment. Cell-based therapies are actively investigated to enhance cutaneous healing. Various cell types are available to produce biological dressings, such as adipose-derived stem/stromal cells (ASCs), an attractive cell source considering their abundancy, accessibility, and therapeutic secretome. In this study, we produced human ASC-based dressings under a serum-free culture system using the self-assembly approach of tissue engineering. The dressings were applied every 4 days to full-thickness 8-mm splinted skin wounds created on the back of polygenic diabetic NONcNZO10/LtJ mice and streptozotocin-induced diabetic K14-H2B-GFP mice. Global wound closure kinetics evaluated macroscopically showed accelerated wound closure in both murine models, especially for NONcNZO10/LtJ; the treated group reaching 98.7% ± 2.3% global closure compared to 76.4% ± 11.8% for the untreated group on day 20 ( p =0.0002). Histological analyses revealed that treated wounds exhibited healed skin of better quality with a well-differentiated epidermis and a more organized, homogeneous, and 1.6-fold thicker granulation tissue. Neovascularization, assessed by CD31 labeling, was 2.5-fold higher for the NONcNZO10/LtJ treated wounds. We thus describe the beneficial impact on wound healing of biologically active ASC-based dressings produced under an entirely serum-free production system facilitating clinical translation., Competing Interests: The authors declare that no competing interests exist., (Copyright © 2024 Meryem Safoine et al.)

Calcific aortic valve disease (CAVD) is characterized by the fibrosis and mineralization of the aortic valve, which leads to aortic stenosis and heart failure. At the cellular level, this is due to the osteoblastic-like differentiation of valve interstitial cells (VICs), resulting in the calcification of the tissue. Unfortunately, human VICs are not readily available to study CAVD pathogenesis and the implicated mechanisms in vitro; however, adipose-derived stromal/stem cells (ASCs), carrying the patient's specific genomic features, have emerged as a promising cell source to model cardiovascular diseases due to their multipotent nature, availability, and patient-specific characteristics. In this study, we describe a comprehensive transcriptomic analysis of tissue-engineered, scaffold-free, ASC-embedded mineralized tissue sheets using bulk RNA sequencing. Bioinformatic and gene set enrichment analyses revealed the up-regulation of genes associated with the organization of the extracellular matrix (ECM), suggesting that the ECM could play a vital role in the enhanced mineralization observed in these tissue-engineered ASC-embedded sheets. Upon comparison with publicly available gene expression datasets from CAVD patients, striking similarities emerged regarding cardiovascular diseases and ECM functions, suggesting a potential link between ECM gene expression and CAVDs pathogenesis. A matrisome-related sub-analysis revealed the ECM microenvironment promotes the transcriptional activation of the master gene runt-related transcription factor 2 ( RUNX2 ), which is essential in CAVD development. Tissue-engineered ASC-embedded sheets with enhanced mineralization could be a valuable tool for research and a promising avenue for the identification of more effective aortic valve replacement therapies.

Tissue-engineered skin substitutes (TESs) are used as a treatment for severe burn injuries. Their production requires culturing both keratinocytes and fibroblasts. The methods to grow these cells have evolved over the years, but bovine serum is still commonly used in the culture medium. Because of the drawbacks associated with the use of serum, it would be advantageous to use serum-free media for the production of TESs. In a previous study, we developed a serum-free medium (Surge SFM) for the culture of keratinocytes. Herein, we tested the use of this medium, together with a commercially available serum-free medium for fibroblasts (Prime XV), to produce serum-free TESs. Our results show that serum-free TESs are macroscopically and histologically similar to skin substitutes produced with conventional serum-containing media. TESs produced with either culture media expressed keratin 14, Ki-67, transglutaminase 1, filaggrin, type I and IV collagen, and fibronectin comparably. Mechanical properties, such as contraction and tensile strength, were comparable between TESs cultured with and without serum. Serum-free TESs were also successfully grafted onto athymic mice for a six-month period. In conclusion, Surge SFM and Prime XV serum-free media could be used to produce high quality clinical-grade skin substitutes.

Hydrogels are widely used as cell scaffolds in several biomedical applications. Once implanted in vivo, cell scaffolds must often be visualized, and monitored overtime. However, cell scaffolds appear poorly contrasted in most biomedical imaging modalities such as magnetic resonance imaging (MRI). MRI is the imaging technique of choice for high-resolution visualization of low-density, water-rich tissues. Attempts to enhance hydrogel contrast in MRI are performed with "negative" contrast agents that produce several image artifacts impeding the delineation of the implant's contours. In this study, a magnetic ink based on ultra-small iron oxide nanoparticles (USPIONs; <5 nm diameter cores) is developed and integrated into biocompatible alginate hydrogel used in cell scaffolding applications. Relaxometric properties of the magnetic hydrogel are measured, as well as biocompatibility and MR-visibility (T 1 -weighted mode; in vitro and in vivo). A 2-week MR follow-up study is performed in the mouse model, demonstrating no image artifacts, and the retention of "positive" contrast overtime, which allows very precise delineation of tissue grafts with MRI. Finally, a 3D-contouring procedure developed to facilitate graft delineation and geometrical conformity assessment is applied on an inverted template alginate pore network. This proof-of-concept establishes the possibility to reveal precisely engineered hydrogel structures using this USPIONs ink high-visibility approach., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

The importance of the inert environment in the transmission of pathogens has been reassessed in recent years. To reduce cross-contamination, new biocidal materials used in high touch surfaces (e.g., stair railings, door handles) have been developed. However, their impact on skin remains poorly described. The present study aimed to evaluate the antibacterial properties and the risk of skin irritation of two materials based on hard-anodized aluminum (AA) impregnated with quaternary ammonium compound solutions (QAC#1 or QAC#2). The QAC#1 or QAC#2 solutions vary in composition, QAC#2 being free of dioctyl dimethyl ammonium chloride (Dio-DAC) and octyl decyl dimethyl ammonium chloride (ODDAC). Unlike AA used as a control, both AA-QAC#1 and AA-QAC#2 had excellent and rapid antibacterial efficacy, killing 99.9 % of Staphylococcus aureus and Escherichia coli bacteria, in 15 s and 1 min, respectively. The impregnation solutions (QAC#1 and QAC#2) did not show any skin sensitizing effect on transformed human keratinocytes. Nevertheless, these solutions as well as the materials (AA-QAC#1, AA-QAC#2), and the liquid extracts derived from them, induced a very rapid cytotoxicity on L929 murine fibroblasts (>70 % after 1 h of contact) as shown by LDH, MTS and neutral red assays. This cytotoxicity can be explained by the fast QACs release occurring when AA-QAC#1 and AA-QAC#2 were immersed in aqueous medium. To overcome the limitation of assays based on liquid condition, an in vitro skin irritation assay on reconstructed human epidermis (RHE) was developed. The effect of the materials upon their direct contact with the epidermis grown at the liquid-air interface was determined by evaluating tissue viability and quantifying interleukin-1 alpha (IL-1α) which is released in skin during injury or infection. AA-QAC#1 induced a significant decrease in RHE viability, close to OECD and ISO 10993-10 acceptability thresholds and enhanced the pro-inflammatory IL-1α secretion compared with AA-QAC#2. Finally, these results were corroborated by in vivo assays on mice using erythema and edema visual scores, histological observations, and epidermal thickness measurement. AA had no effect on the skin, while a stronger irritation was induced by AA-QAC#1 compared with AA-QAC#2. Hence, these materials were classified as moderate and slight irritants, respectively. In summary, this study revealed that AA-QAC#2 without Dio-DAC and ODDAC could be a great candidate for high touch surface applications, showing an extremely effective and rapid bactericidal activity, without inducing adverse effects for skin tissue., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests. MAG (A3Surfaces) provided the materials that were tested independently by JJ under the supervision of LCF and NF as described above. The authors declare that they have no personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2023. Published by Elsevier B.V.)

Background: The Skin Investigation Network of Canada (SkIN Canada) is a new national skin research network. To shape the research landscape and ensure its value to patient care, research priorities that are important to patients, caregivers, and health care providers must be identified., Objectives: To identify the Top Ten research priorities for 9 key skin conditions., Methods: We first surveyed health care providers and researchers to select the top skin conditions for future research within the categories of inflammatory skin disease, skin cancers (other than melanoma), and wound healing. For those selected skin conditions, we conducted scoping reviews to identify previous priority setting exercises. We combined the results of those scoping reviews with a survey of patients, health care providers, and researchers to generate lists of knowledge gaps for each condition. We then surveyed patients and health care providers to create preliminary rankings to prioritize those knowledge gaps. Finally, we conducted workshops of patients and health care providers to create the final Top Ten lists of research priorities for each condition., Results: Overall, 538 patients, health care providers, and researchers participated in at least one survey or workshop. Psoriasis, atopic dermatitis and hidradenitis suppurativa (inflammatory skin disease); chronic wounds, burns and scars (wound healing); and basal cell, squamous cell and Merkel cell carcinoma (skin cancer) were selected as priority skin conditions. Top Ten lists of knowledge gaps for inflammatory skin conditions encompassed a range of issues relevant to patient care, including questions on pathogenesis, prevention, non-pharmacologic and pharmacologic management., Conclusions: Research priorities derived from patients and health care providers should be used to guide multidisciplinary research networks, funders, and policymakers in Canada and internationally.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Background: For dermatology to effectively address the ever-growing medical needs, longstanding communication barriers across investigators working in different research pillars and practicing clinicians must be improved. To address this problem, trainee-specific programs are now evolving to align their educational landscape across basic science, translational and clinical research programs., Objectives: To establish a Skin Investigation Network of Canada (SkIN Canada) training roadmap for the career and skill development of future clinicians, clinican scientists and basic scientists in Canada. This Working Group aims to strengthen and harmonize collaborations and capacity across the skin research community., Methods: The Working Group conducted a search of established international academic societies which offered trainee programs with mandates similar to SkIN Canada. Societies' program items and meetings were evaluated by use of an interview survey and/or the collection of publicly available data. Program logistics, objectives and feedback were assessed for commonalities and factors reported or determined to improve trainee experience., Results: Through the various factors explored, the Working Group discovered the need for increasing program accessibility, creating opportunities for soft skill development, emphasizing the importance of current challenges, collecting and responding to feedback, and improving knowledge sharing to bridge pillars of skin research., Conclusions: Although improvements have been made to trainee education in recent years, a plurality of approaches exist and many of the underlying roadblocks remain unresolved. To establish fundamental clinician-basic scientist collaboration and training efforts, this Working Group highlights important factors to include and consider in building a trainee program and emphasizes the importance of trainee education.

Urologic patients may be affected by pathologies requiring surgical reconstruction to re-establish a normal function. The lack of autologous tissues to reconstruct the urethra led clinicians toward new solutions, such as tissue engineering. Tridimensional tissues were produced and characterized from a clinical perspective. The balance was optimized between increasing the mechanical resistance of urethral-engineered tissue and preserving the urothelium's barrier function, essential to avoid urine extravasation and subsequent inflammation and fibrosis. The substitutes produced using a mix of vesical (VF) and dermal fibroblasts (DF) in either 90%:10% or 80%:20% showed mechanical resistance values comparable to human native bladder tissue while maintaining functionality. The presence of mature urothelium markers such as uroplakins and tight junctions were documented. All substitutes showed similar histological features except for the noticeable decrease in polysaccharide globules for the substitutes made with a higher proportion of DF. The degree of maturation evaluated with electron microscopy was positively correlated with the increased concentration of VF in the stroma. Substitutes produced with VF and at least 10% of DF showed sufficient mechanical resistance to withstand surgeon manipulation and high functionality, which may improve long-term patients' quality of life, representing a great future alternative to current treatments., (© 2022. The Author(s).)

Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.

The increasing need for tissue substitutes in reconstructive surgery spurs the development of engineering methods suited for clinical applications. Cell culture and tissue production traditionally require the use of fetal bovine serum (FBS) which is associated with various complications especially from a translational perspective. Using the self-assembly approach of tissue engineering, we hypothesized that all important parameters of tissue reconstruction can be maintained in a production system devoid of FBS from cell extraction to tissue reconstruction. We studied two commercially available serum-free medium (SFM) and xenogen-free serum-free medium (XSFM) for their impact on tissue reconstruction using human adipose-derived stem/stromal cells (ASCs) in comparison to serum-containing medium. Both media allowed higher ASC proliferation rates in primary cultures over five passages compared with 10% FBS supplemented medium while maintaining high expression of mesenchymal cell markers. For both media, we evaluated extracellular matrix production and deposition necessary to engineer manipulatable tissues using the self-assembly approach. Tissues produced in SFM exhibited a significantly increased thickness (up to 6.8-fold) compared with XSFM and FBS-containing medium. A detailed characterization of tissues produced under SFM conditions showed a substantial 50% reduction of production time without compromising key tissue features such as thickness, mechanical resistance and pro-angiogenic secretory capacities (plasminogen activator inhibitor 1, hepatocyte growth factor, vascular endothelial growth factor, angiopoietin-1) when compared to tissues produced in the control FBS-containing medium. Furthermore, we compared ASCs to the frequently used human dermal fibroblasts (DFs) in the SFM culture system. ASC-derived tissues displayed a 2.4-fold increased thickness compared to their DFs counterparts. In summary, we developed all-natural human substitutes using a production system compatible with clinical requirements. Under culture conditions devoid of bovine serum, the resulting engineered tissues displayed similar and even superior structural and functional properties over the classic FBS-containing culture conditions with a considerable 50% shortening of production time., (Creative Commons Attribution license.)

Every year, thousands of aortic valve replacements must take place due to valve diseases. Tissue-engineered heart valves represent promising valve substitutes with remodeling, regeneration, and growth capabilities. However, the accurate reproduction of the complex three-dimensional (3D) anatomy of the aortic valve remains a challenge for current biofabrication methods. We present a novel technique for rapid fabrication of native-like tricuspid aortic valve scaffolds made of an alginate-based hydrogel. Using this technique, a sodium alginate hydrogel formulation is injected into a mold produced using a custom-made sugar glass 3D printer. The mold is then dissolved using a custom-made dissolving module, revealing the aortic valve scaffold. To assess the reproducibility of the technique, three scaffolds were thoroughly compared. CT (computed tomography) scans showed that the scaffolds respect the complex native geometry with minimal variations. The scaffolds were then tested in a cardiac bioreactor specially designed to reproduce physiological flow and pressure (aortic and ventricular) conditions. The flow and pressure profiles were similar to the physiological ones for the three valve scaffolds, with small variabilities. These early results establish the functional repeatability of this new biofabrication method and suggest its application for rapid fabrication of ready-to-use cell-seeded sodium alginate scaffolds for heart valve tissue engineering.

High-intensity laser therapy (HILT) has been gaining popularity in the treatment of chronic musculoskeletal pain, including vulvodynia. The objective of this study was to critically appraise and synthesize the available evidence on the efficacy of HILT for reducing pain and improving function in vulvodynia and other chronic primary musculoskeletal pain conditions. Electronic databases and the grey literature were searched. Effects on pain intensity, function, and adverse events were assessed. One study investigating HILT in the treatment of vulvodynia and 13 studies on the treatment of chronic musculoskeletal pain were selected. The study assessing vulvodynia showed favorable results for reducing pain. Regarding chronic musculoskeletal pain, 12 out of the 13 studies selected consistently showed that HILT was more effective than the placebo/active comparator for reducing pain and improving function. The available effect sizes for pain showed large to huge effects. Similar effects were observed for function except for two studies showing moderate effects. The GRADE score was moderate. Conclusions: There are insufficient data to support the use of HILT in vulvodynia, but the promising results encourage further research. HILT appears to be effective in musculoskeletal pain conditions. More high-quality studies are needed to identify effective laser protocols.

Fibrillin-1 is an extracellular glycoprotein present throughout the body. Mutations in fibrillin-1 cause a wide spectrum of type I fibrillinopathies, including Marfan syndrome characterized by clinical manifestations in adipose tissues, among others. This study addresses the hypothesis that fibrillin-1 regulates adipocyte development and plays a vital role in adipose tissue homeostasis. We employed two mouse models - Fbn1 mgR/mgR (20-25% of normal fibrillin-1) and Fbn1 C1041G/+ (missense mutation in fibrillin-1) to examine the role of fibrillin-1 in adipose tissue development and homeostasis. Fibrillin-1 was detected around mature adipocytes in both mouse and human white adipose tissues. As expected, Fbn1 mgR/mgR mice displayed a significant reduction of fibrillin-1 in white adipose tissue, and no change was observed for Fbn1 C1041G/+ mice, each compared to their respective littermates. Male Fbn1 mgR/mgR mice had more white and brown adipose tissues, whereas female Fbn1 mgR/mgR and both male and female Fbn1 C1041G/+ showed no difference compared to their respective wild-type littermates. Consistent with this data, male Fbn1 mgR/mgR mice displayed hyperinsulinemia and an insulin resistance phenotype with higher levels of cholesterol and high-density lipoproteins in the serum. Fibrillin-1 deficiency in male Fbn1 mgR/mgR mice also promoted adipogenic gene expression and led to hypertrophic expansion of mature adipocytes. To further elucidate the fibrillin-1-dependent adipogenic mechanisms in cell culture, we used primary bone marrow derived mesenchymal stem/stromal cells (MSCs) from Fbn1 mgR/mgR , Fbn1 C1041G/+ and wild-type mice. Increased lipid content, adipogenic differentiation and pAKT levels were observed when MSCs from both male and female Fbn1 mgR/mgR mice were differentiated. Furthermore, a recombinant fragment spanning the C-terminal half of fibrillin-1 significantly reduced adipocyte differentiation i) by binding to MSCs and inhibiting adipogenic commitment, and ii) by sequestering insulin, together suppressing the AKT signaling pathway. This fibrillin-1 fragment also rescued enhanced adipogenic differentiation of MSCs derived from Fbn1 mgR/mgR mice. Overall, this study shows that altered adipose tissue homeostasis observed in fibrillin-1 deficient mice depends on the type of fibrillin-1 deficiency and the biological sex, and it shows that fibrillin-1 is a negative regulator of adipogenesis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Historically, non-small-cell lung cancer (NSCLC) has been regarded as a nonimmunogenic tumor; however, recent studies have shown that NSCLCs are among the most responsive cancers to monoclonal antibody immune checkpoint inhibitors (ICIs). ICIs have dramatically improved clinical outcomes for a subset of patients (∼20%) with locally advanced and metastatic NSCLC, and they have also demonstrated promise as neoadjuvant therapy for early-stage resectable disease. Nevertheless, the majority of patients with NSCLC are refractory to ICIs for reasons that are poorly understood. Thus, major questions are: how do we initially identify the patients most likely to derive significant clinical benefit from these therapies; how can we increase the number of patients benefiting; what are the mechanisms of primary and acquired resistance to immune-based therapies; are there additional immune checkpoints besides PD-1/PD-L1 and CTLA-4 that can be targeted to provide greater clinical benefit to patients; and how do we best combine ICI therapy with surgery, radiotherapy, chemotherapy, and targeted therapy? To answer these questions, we need to deploy the latest technologies to study tumors and their microenvironment and how they interact with components of the innate and adaptive immune systems. There is also a need for new preclinical model systems to investigate the molecular mechanisms of resistance to treatment and identify novel therapeutic targets. Recent advances in technology are beginning to shed new light on the immune landscape of NSCLC that may uncover biomarkers of response and maximize the clinical benefit of immune-based therapies. Identification of the mechanisms of resistance should lead to the identification of novel targets and the generation of new therapeutic strategies that improve outcomes for a greater number of patients. In the sections below, we discuss the results of studies examining the immune microenvironment in NSCLC, summarize the clinical experience with immunotherapy for NSCLC, and review candidate biomarkers of response to these agents in NSCLC., (Copyright © 2022 Cold Spring Harbor Laboratory Press; all rights reserved.)

The success of dental implant treatment after tooth extraction is generally maximized by preserving the alveolar ridge using cell-free biomaterials. However, these treatments can be associated with inflammatory reactions, leading to additional bone volume loss hampering dental implant positioning. Our group developed a self-assembled bone-like substitute constituted of osteogenically induced human adipose-derived stromal/stem cells (hASCs). We hypothesized that a bone morphogenetic protein (BMP) supplementation could improve the in vitro osteogenic potential of the bone-like substitute, which would subsequently translate into enhanced alveolar bone healing after tooth extraction. ASCs displayed a better osteogenic response to BMP-9 than to BMP-2 in monolayer cell culture, as shown by higher transcript levels of the osteogenic markers RUNX2 , osterix ( OSX / SP7 ), and alkaline phosphatase after three and six days of treatment. Interestingly, BMP-9 treatment significantly increased OSX transcripts and alkaline phosphatase activity, as well as pro-angiogenic angiopoietin-1 gene expression, in engineered bone-like substitutes after 21 days of culture. Alveolar bone healing was investigated after molar extraction in nude rats. Microcomputed tomography and histological evaluations revealed similar, or even superior, global alveolar bone preservation when defects were filled with BMP-9-treated bone-like substitutes for ten weeks compared to a clinical-grade biomaterial, with adequate gingival re-epithelialization in the absence of resorption.

Polyunsaturated fatty acids (PUFAs) play an important role in the establishment and the maintenance of the skin barrier function. However, the impact of their derived lipid mediators remains unclear. Skin substitutes were engineered according to the self-assembly method with a culture medium supplemented with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA). The supplementation with ALA and LA decreased testosterone absorption through a tissue-engineered reconstructed skin model, thus indicating an improved skin barrier function following supplementation. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes. Indeed, the dual supplementation increased the levels of eicosapentaenoic acid (EPA) (15-fold), docosapentaenoic acid (DPA) (3-fold), and LA (1.5-fold) in the epidermal phospholipids while it increased the levels of ALA (>20-fold), DPA (3-fold) and LA (1.5-fold) in the epidermal triglycerides. The bioactive lipid mediator profile of the skin substitutes, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols, was next analyzed using liquid chromatography-tandem mass spectrometry. The lipid supplementation further modulated bioactive lipid mediator levels of the reconstructed skin substitutes, leading to a lipid mediator profile more representative of the one found in normal human skin. These findings show that an optimized supply of PUFAs via culture media is essential for the establishment of improved barrier function in vitro. STATEMENT OF SIGNIFICANCE: Supplementation of the culture medium with 10 μM of both α-linolenic acid (ALA) and linoleic acid (LA) improved the skin barrier function of a tissue-engineered skin model. The exogenously provided fatty acids were incorporated into the phospholipid and triglyceride fractions of the skin substitutes and further modulated bioactive lipid mediator levels, including prostaglandins, hydroxy-fatty acids, N-acylethanolamines and monoacylglycerols. These findings highlight the important role of ALA and LA in skin homeostasis and show that an optimized supply of polyunsaturated fatty acids via culture media is essential for the establishment of improved barrier function in vitro., Competing Interests: Declaration of Competing Interest The authors declare no competing interests, (Copyright © 2021. Published by Elsevier Ltd.)

The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions. Current therapeutic strategies are challenged by a growing shortage of adequate tissues. Therefore, new options must be considered for the treatment of patients, with the use of stem cells (SCs) being attractive. Two different strategies can be derived from stem cell use: Cell therapy and tissue therapy, mainly through tissue engineering. The recent advances using these approaches are described in this review, with a focus on stromal/mesenchymal cells found in adipose tissue. Indeed, the accessibility, high yield at harvest as well as anti-fibrotic, immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients. Finally, an innovative technique allowing tissue reconstruction without exogenous material, the self-assembly approach, will be presented. Despite advances, more studies are needed to translate such approaches from the bench to clinics in urology. For the 21 st century, cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine., Competing Interests: Conflict-of-interest statement: The authors declare no conflict of interests for this article., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Clinical studies have shown that diets enriched with omega-3 (also know as n-3) polyunsaturated fatty acids could relieve the symptoms of patients with psoriasis. However, the mechanisms involved remain poorly understood. The aim of this study was to investigate the effects of α-linolenic acid (ALA) on the proliferation and differentiation of psoriatic keratinocytes in a three-dimensional skin model. Skin models featuring healthy (healthy substitute) or psoriatic (psoriatic substitute) cells were engineered by the self-assembly method of tissue engineering using a culture medium supplemented with 10 μM ALA in comparison with the regular unsupplemented medium. ALA decreased keratinocyte proliferation and improved psoriatic substitute epidermal differentiation, as measured by decreased Ki67 staining and increased protein expression of FLG and loricrin. The added ALA was notably incorporated into the epidermal phospholipids and metabolized into long-chain n-3 polyunsaturated fatty acids, mainly eicosapentaenoic acid and n-3 docosapentaenoic acid. ALA supplementation led to increased levels of eicosapentaenoic acid derivatives (15-hydroxyeicosapentaenoic acid and 18-hydroxyeicosapentaenoic acid) as well as a decrease in levels of omega-6 (also know as n-6) polyunsaturated fatty acid lipid mediators (9-hydroxyoctadecadienoic acid, 12-hydroxyeicosatetraenoic acid, and leukotriene B 4 ). Furthermore, the signal transduction mediators extracellular signal‒regulated kinases 1 and 2 were the kinases most activated after ALA supplementation. Taken together, these results show that ALA decreases the pathologic phenotype of psoriatic substitutes by normalizing keratinocyte proliferation and differentiation in vitro., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: Chronic pain is a highly prevalent and debilitating condition, and there is a pressing need to find safe, effective and affordable treatments to tackle this public health issue. This pilot study aimed to assess whether therapeutic exercises supplemented by transcutaneous electrical nerve stimulation induces a greater hypoalgesic effect than therapeutic exercises supplemented by sham transcutaneous electrical nerve stimulation, in an elderly population suffering from chronic pain., Materials and Methods: Eighteen elderly participants suffering from chronic pain completed a therapeutic exercise program consisting of 45-min group sessions administered twice a week for 4 weeks. Half of the participants received real transcutaneous electrical nerve stimulation during the exercise sessions, while the others received sham transcutaneous electrical nerve stimulation. Participants completed pain questionnaires (McGill Pain Questionnaire, Brief Pain Inventory, Beck Depression Index) before and after the intervention, and recorded their pain levels on an 11-point numerical rating scale before and after each session (Clinical Trial.Gov ID: NCT02445677)., Results and Conclusion: Our results suggest that supplementing exercise sessions with transcutaneous electrical nerve stimulation does not improve the long-term outcomes of elderly patients suffering from chronic pain, but does induce short-term hypoalgesia during exercise sessions. Our study also offers valuable guidelines for the implementation of a future and adequately powered study looking at this research question.Implications for rehabilitationThe application of transcutaneous electrical nerve stimulation during exercises is well tolerated by elderly individuals suffering from chronic pain.Supplementing exercises with transcutaneous electrical nerve stimulation does not seem to improve general outcome in elderly suffering from chronic pain.Notwithstanding, the addition of transcutaneous electrical nerve stimulation tends to produce a marked hypoalgesic effect during the exercise sessions, an effect that could prompt indirect benefits for pain patients.

In vitro prevascularization has the potential to address the challenge of maintaining cell viability at the core of engineered constructs, such as bone substitutes, and to improve the survival of tissue grafts by allowing quicker anastomosis to the host microvasculature. The self-assembly approach of tissue engineering allows the production of biomimetic bone-like tissue constructs including extracellular matrix and living human adipose-derived stromal/stem cells (hASCs) induced towards osteogenic differentiation. We hypothesized that the addition of endothelial cells could improve osteogenesis and biomineralization during the production of self-assembled human bone-like tissues using hASCs. Additionally, we postulated that these prevascularized constructs would consequently improve graft survival and bone repair of rat calvarial bone defects. This study shows that a dense capillary network spontaneously formed in vitro during tissue biofabrication after two weeks of maturation. Despite reductions in osteocalcin levels and hydroxyapatite formation in vitro in prevascularized bone-like tissues (35 days of culture), in vivo imaging of prevascularized constructs showed an improvement in cell survival without impeding bone healing after 12 weeks of implantation in a calvarial bone defect model (immunocompromised male rats), compared to their stromal counterparts. Globally, these findings establish our ability to engineer prevascularized bone-like tissues with improved functional properties.

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitro human 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Vincent Roy et al.)

Reconstructed human adipose tissues represent novel tools available to perform in vitro pharmaco-toxicological studies. We used adipose-derived human stromal/stem cells to reconstruct, using tissue engineering techniques, such an adipose tridimensional model. To determine to what extent the in vitro model is representative of its native counterpart, adipogenic differentiation, triglycerides accumulation and phospholipids profiles were analysed. Ingenuity Pathway Analysis software revealed pathways enriched with differentially-expressed genes between native and reconstructed human adipose tissues. Interestingly, genes related to fatty acid metabolism were downregulated in vitro, which could be explained in part by the insufficient amount of essential fatty acids provided by the fetal calf serum used for the culture. Indeed, the lipid profile of the reconstructed human adipose tissues indicated a particular lack of linoleic acid, which could interfere with physiological cell processes such as membrane trafficking, signaling and inflammatory responses. Supplementation in the culture medium was able to influence the lipid profile of the reconstructed human adipose tissues. This study demonstrates the possibility to directly modulate the phospholipid profile of reconstructed human adipose tissues. This reinforces its use as a relevant physiological or pathological model for further pharmacological and metabolic studies of human adipose tissue functions., Competing Interests: The authors have declared that no competing interests exist.

Skin models with efficient skin barrier function are required for percutaneous absorption studies. The contribution of media supplementation with n-3 and n-6 polyunsaturated fatty acids (PUFAs) to the development of the skin barrier function of in vitro skin models remains incompletely understood. To investigate whether PUFAs, alpha-linolenic acid (ALA, n-3 PUFA) and linoleic acid (LA, n-6 PUFA), could enhance the impermeability of a three-dimensional reconstructed human skin model, skin substitutes were produced according to the self-assembly method using culture media supplemented with either 10 μM ALA or 10 μM LA. The impact of PUFAs on skin permeability was studied by using a Franz cell diffusion system to assess the percutaneous absorption of testosterone and benzoic acid. Our findings showed that ALA supplementation induced a decrease in the absorption of testosterone, while LA supplementation did not significantly influence the penetration of testosterone and benzoic acid under present experimental conditions. Both ALA and LA were incorporated into phospholipids of the skin substitutes, resulting in an increase in n-3 total PUFAs or n-6 total PUFAs. Collectively, these results revealed the under-estimated impact of n-3 PUFA supplementation as well as the importance of the n-6 to n-3 ratio on the formation of the skin barrier of in vitro reconstructed human skin models.

Primary endothelial cells are needed for angiogenesis studies, and more particularly in the field of tissue engineering, to engineer pre-vascularized tissues. Investigations often use human umbilical vein endothelial cells due to their extensive characterization, but also because they are easy to obtain and isolate. An alternative is the use of human dermal microvascular endothelial cells, more representative of adult skin angiogenesis and vascularization processes. This chapter presents a detailed methodology to isolate and culture microvascular endothelial cells from skin biopsies based on enzymatic digestion and mechanical extraction.

Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adipocytes and an abundant matrix in addition to the presence of host capillaries within the grafts. The substitutes thickness and volume were not significantly different between groups over the short-term time course of 14 days (d). For the microvascularized adipose tissues, human CD31 staining revealed a human capillary network connecting with the host microvasculature as early as 3 d after grafting. The detection of murine red blood cells within human CD31+ structures confirmed the functionality of the human capillary network. By analyzing the extent of the global vascularization achieved, a tendency towards increased total capillary network surface and volume was revealed for prevascularized tissues over 14 d. Therefore, applying this strategy on thicker reconstructed adipose tissues with rate-limiting oxygen diffusion might procure added benefits and prove useful to provide voluminous substitutes for patients suffering from adipose tissue loss or defects.

While being the rarest skin cancer, melanoma is also the deadliest. To further drug discovery and improve clinical translation, new human cell-based in vitro models are needed. Our work strives to mimic the melanoma microenvironment in vitro as an alternative to animal testing. We used the self-assembly method to produce a 3D human melanoma model exempt of exogenous biomaterial. This model is based on primary human skin cells and melanoma cell lines while including a key feature for tumor progression: blood and lymphatic capillaries. Major components of the tumor microenvironment such as capillaries, human extracellular matrix, a stratified epidermis (involucrin, filaggrin) and basement membrane (laminin 332) are recapitulated in vitro. We demonstrate the persistence of CD31 + blood and podoplanin + /LYVE-1 + lymphatic capillaries in the engineered tissue. Chronic treatment with vemurafenib was applied to the model and elicited a dose-dependent response on proliferation and apoptosis, making it a promising tool to test new compounds in a human-like environment.

Population studies have linked insulin resistance to systemic low-grade chronic inflammation and have reported elevated levels of inflammatory cytokines such as TNFα, IL-1β and IL-6, individually or in certain combinations, in adipose tissues or in the serum. We undertook this comprehensive study to simultaneously evaluate the expression of several pro-inflammatory and anti-inflammatory cytokines in serum and in the visceral and subcutaneous adipose tissues from obese patients undergoing bariatric surgery. We observed that several inflammatory cytokines implicated in obesity-associated inflammation showed no significant difference in protein or gene expression between obese patients with or without diabetes and control groups. IL1B gene expression was significantly elevated in the visceral adipose tissues of obese patients, but did not correlate with their diabetes status. Despite the significant increase in IL1B expression in the obese group, a significant proportion of obese patients did not express TNFA, IL1B or IL6 in visceral adipose tissues. Certain inflammatory cytokines showed correlation with the chemokine CCL2 and VEGF-A in visceral adipose tissues. Our findings suggest that the inflammatory cytokine profile in metabolic syndrome is more complex than what is currently perceived and that chronic inflammation in obese patients likely results from incremental contribution from different cytokines and possibly other inflammatory mediators from within and outside the adipose tissues. It is possible that this obesity associated chronic inflammation is not predicted by a single mediator, but rather includes a large spectrum of possible profiles., (Copyright © 2018 German Center for Neurodegenerative Diseases (DZNE). Published by Elsevier B.V. All rights reserved.)