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Abstract Background Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. Methods To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. Results Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. Conclusions Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Patients with cardiovascular disease often need replacement or bypass of a diseased blood vessel. With disadvantages of both autologous blood vessels and synthetic grafts, tissue engineering is emerging as a promising alternative of advanced therapy medicinal products for individualized blood vessels. By reconditioning of a decellularized blood vessel with the recipient’s own peripheral blood, we have been able to prevent rejection without using immunosuppressants and prime grafts for efficient recellularization in vivo. Recently, decellularized veins reconditioned with autologous peripheral blood were shown to be safe and functional in a porcine in vivo study as a potential alternative for vein grafting. In this study, personalized tissue engineered arteries (P-TEA) were developed using the same methodology and evaluated for safety in a sheep in vivo model of carotid artery transplantation. Five personalized arteries were transplanted to carotid arteries and analyzed for safety and patency as well as with histology after four months in vivo. All grafts were fully patent without any occlusion or stenosis. The tissue was well cellularized with a continuous endothelial cell layer covering the luminal surface, revascularized adventitia with capillaries and no sign of rejection or infection. In summary, the results indicate that P-TEA is safe to use and has potential as clinical grafts.

Abstract Percutaneous implants are frequently affected by bacterial growth at the skin-implant interface. Integration between implant and surrounding skin is important to prevent bacteria from spreading to the underlying tissue. The standard method to evaluate skin-implant integration is by histomorphometry on samples which have been placed in tissue grown in vivo or ex vivo. In this study, a biomechanical method was developed and evaluated. The integration of implants into porcine skin was studied in an ex vivo model, where pig skin samples were cultivated in a nutrient solution. Cylindrical shaped implants, consisting of polyether ether ketone (PEEK) and titanium (Ti) with different surface treatments, were implanted in the skin tissue and the skin was grown in nutrient solution for 2 weeks. The implants were then extracted from the implantation site and the mechanical force during extraction was measured as a quantitative assessment of skin-implant integration. Implants from each group were also processed for histomorphometry and the degree of epidermal downgrowth (ED) and tissue to implant contact (TIC) was measured. A higher mean pullout force was observed for the PEEK implants compared to the Ti implants. Applying nanosized hydroxyapatite (HA) on Ti and PEEK increased the pullout force compared to uncoated controls, 24% for machined and 70% for blasted Ti, and 51% for machined PEEK. Treatment of Ti and PEEK with nanosized zirconium phosphate (ZrP) did not increase the pullout force. The histomorphometry analysis showed correlation between ED and pullout force, where the pullout force was inversely proportional to ED. For TIC, no significant differences were observed between the groups of same material (i.e. Ti, Ti+HA, Ti+ZrP, and PEEK, PEEK + HA, PEEK + ZrP), but it was significantly higher for PEEK compared to Ti. Scanning electron microscopy analysis was done on samples before and after the pullout tests, showing that the ZrP coating was unaffected by the 2 week ex vivo implantation and pullout procedure, no dissolution or detachment of the coating was observed. For the HA coating, a loss of coating was seen on approximately 5% of the total surface area of the implant. Graphical abstract

Breast cancer is the most common cancer among women, and even though treatments are available, efficiency varies with the patients. In vitro 2D models are commonly used to develop new treatments. However, 2D models overestimate drug efficiency, which increases the failure rate in later phase III clinical trials. New model systems that allow extensive and efficient drug screening are thus required. Three-dimensional printed hydrogels containing active components for cancer cell growth are interesting candidates for the preparation of next generation cancer cell models. Macromolecules, obtained from marine- and land-based resources, can form biopolymers (polysaccharides such as alginate, chitosan, hyaluronic acid, and cellulose) and bioactive components (structural proteins such as collagen, gelatin, and silk fibroin) in hydrogels with adequate physical properties in terms of porosity, rheology, and mechanical strength. Hence, in this study attention is given to biofabrication methods and to the modification with biological macromolecules to become bioactive and, thus, optimize 3D printed structures that better mimic the cancer cell microenvironment. Ink formulations combining polysaccharides for tuning the mechanical properties and bioactive polymers for controlling cell adhesion is key to optimizing the growth of the cancer cells.

Abstract Background Colorectal cancer is the second most common cause of cancer‐related death worldwide and standardized therapies often fail to treat the more aggressive and progressive types of colorectal cancer. Tumor cell heterogeneity and influence from the surrounding tumor microenvironment (TME) contribute to the complexity of the disease and large variability in clinical outcomes. Methods To model the heterogeneous nature of colorectal cancer, we used patient‐derived scaffolds (PDS), which were obtained via decellularization of surgically resected tumor material, as a growth substrate for standardized cell lines. Results After confirmation of native cell absence and validation of the structural and compositional integrity of the matrix, 89 colorectal PDS were repopulated with colon cancer cell line HT29. After 3 weeks of PDS culture, HT29 cells varied their gene and protein expression profiles considerably compared to 2D‐grown HT29 cells. Markers associated with proliferation were consistently decreased, while markers associated with pluripotency were increased in PDS‐grown cells compared to their 2D counterparts. When comparing the PDS‐induced changes in HT29 cells with clinically relevant tumor information from individual patients, we observed significant associations between stemness/pluripotency markers and tumor location, and between epithelial‐to‐mesenchymal transition (EMT) markers and cancer mortality. Kaplan–Meier analysis revealed that low PDS‐induced EMT correlated with worse cancer‐specific survival. Conclusions The colorectal PDS model can be used as a simplified personalized tool that can potentially reveal important diagnostic and pathophysiological information related to the TME.

Introduction: A disc herniation has traditionally been considered as disc tissue that has slipped out from an intervertebral disc. However, it was recently suggested that the disc herniation mass is a product of bioactive substances from the disc and that the disc hernia would more likely be scar tissue than herniated disc material. In this study, we aimed to analyze the structural components of experimentally induced disc herniations and compare with scar tissue and nucleus pulposus, in the rat. Methods: Twenty-eight rats had their L4-5 discs punctured. After three weeks, the nodule that had been formed over the puncture site, scar tissue from the spine musculature, and normal nucleus pulposus were harvested and processed for further analysis. Results: Proteomics analysis demonstrated that the formed nodule was more similar to scar tissue than to nucleus pulposus. Gene expression analysis showed that there was no resemblance between any tissues when looking at inflammatory genes but that, there was a clear resemblance between the nodule and scar tissue when analyzing extracellular matrix-related genes. Analysis of the GAG and polysaccharide size distribution revealed that only the nodule and scar tissue contained the shorter versions, potentially short chain hyaluronic acid that is known to induce inflammatory responses. The hematoxylin and eosin stained sections of the nodule, disc tissue, and scar tissue indicated that the morphology of the nodule and scar tissue was very similar. Conclusions: The nodule formed after experimental disc puncture, and that resembles a disc hernia, has a more structural resemblance to scar tissue than disc tissue. The nodule is, therefore, more likely to be induced by disc-derived bioactive substances than being formed by herniated disc material.

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial–mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

Three-dimensional (3D) printing has emerged as a highly valuable tool to manufacture porous constructs. This has major advantages in, for example, tissue engineering, in which 3D scaffolds provide a microenvironment with adequate porosity for cell growth and migration as a simulation of tissue regeneration. In this study, we assessed the suitability of three cellulose nanofibrils (CNF) that were obtained through 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO)-mediated oxidation. The CNFs were obtained by applying three levels of carboxylation, i.e., 2.5, 3.8, and 6.0 mmol sodium hypochlorite (NaClO) per gram of cellulose. The CNFs exhibited different nanofibrillation levels, affecting the corresponding viscosity and 3D printability of the CNF gels (0.6 wt%). The scaffolds were manufactured by micro-extrusion and the nanomechanical properties were assessed with nanoindentation. Importantly, fibroblasts were grown on the scaffolds and the expression levels of the marker genes, which are relevant for wound healing and proliferation, were assessed in order to reveal the effect of the 3D-scaffold microenvironment of the cells.

Patient-derived scaffolds (PDSs) generated from primary breast cancer tumors can be used to model the tumor microenvironment in vitro. Patient-derived scaffolds are generated by repeated detergent washing, removing all cells. Here, we analyzed the protein composition of 15 decellularized PDSs using liquid chromatography-mass spectrometry/mass spectrometry. One hundred forty-three proteins were detected and their relative abundance was calculated using a reference sample generated from all PDSs. We performed heatmap analysis of all the detected proteins to display their expression patterns across different PDSs together with pathway enrichment analysis to reveal which processes that were connected to PDS protein composition. This protein dataset together with clinical information is useful to investigators studying the microenvironment of breast cancers. Further, after repopulating PDSs with either MCF7 or MDA-MB-231 cells, we quantified their gene expression profiles using RNA sequencing. These data were also compared to cells cultured in conventional 2D conditions, as well as to cells cultured as xenografts in immune-deficient mice. We investigated the overlap of genes regulated between these different culture conditions and performed pathway enrichment analysis of genes regulated by both PDS and xenograft cultures compared to 2D in both cell lines to describe common processes associated with both culture conditions. Apart from our described analyses of these systems, these data are useful when comparing different experimental model systems. Downstream data analyses and interpretations can be found in the research article “Patient-derived scaffolds uncover breast cancer promoting properties of the microenvironment” [1].

Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical in vitro, ex vivo, and in vivo antimicrobial activity, as well as the potential to cause skin irritation, of human kininogen-derived antimicrobial peptide DPK-060 in different formulations designed for topical delivery. We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro (minimum microbicidal concentration

Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls ( p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Diabetic retinopathy and retinopathy of prematurity are diseases caused by pathological angiogenesis in the retina as a consequence of local hypoxia. The underlying mechanism for epiretinal neovascularization (tuft formation), which contributes to blindness, has yet to be identified. Neural cell adhesion molecule (N-CAM) is expressed by Müller cells and astrocytes, which are in close contact with the retinal vasculature, during normal developmental angiogenesis.Notably, during oxygen induced retinopathy (OIR) N-CAM accumulated on astrocytes surrounding the epiretinal tufts. Here, we show that N-CAM ablation results in reduced vascular tuft formation due to reduced endothelial cell proliferation despite an elevation in VEGFA mRNA expression, whereas retinal developmental angiogenesis was unaffected.We conclude that N-CAM exhibits a regulatory function in pathological angiogenesis in OIR. This is a novel finding that can be of clinical relevance in diseases associated with proliferative vasculopathy.