ECM Composition Research Articles

Photobiomodulation Therapy on Myocardial Infarction in Rats: Transcriptional and Posttranscriptional Implications to Cardiac Remodeling.

Induction of myocardial infarction (MI) in rats by occlusion of the left anterior descending coronary artery is an experimental model used in research to elucidate functional, structural, and molecular modifications associated with ischemic heart disease. Photobiomodulation therapy (PBMT) has become a therapeutic alternative by modulating various biological processes eliciting several effects, including anti-inflammatory and pro-proliferative actions. The main objective of this work was to evaluate the effect of PBMT in the modulation of transcriptional and post-transcriptional changes that occurred in myocardium signal transduction pathways after MI. Continuous wave (CW) non-thermal laser parameters were: 660 nm wavelength, power 15 mW, with a total energy of 0.9 J, fluence of 1.15 J/cm2 , spot size of 0.785 cm2 , and time of 60 seconds. Using in silico analysis, we selected and then, quantified the expression of messenger RNA (mRNA) of 47 genes of 9 signaling pathways associated with MI (angiogenesis, cell survival, hypertrophy, oxidative stress, apoptosis, extracellular matrix, calcium kinetics, cell metabolism, and inflammation). Messenger RNA expression quantification was performed in myocardial samples by polymerase chain reaction real-time array using TaqMan customized plates. Our results evidenced that MI modified mRNA expression of several well-known biomarkers related to detrimental cardiac activity in almost all signaling pathways analyzed. However, PBMT reverted most of these transcriptional changes. More expressively, PBMT provoked a robust decrease in mRNA expression of molecules that participate in post-MI inflammation and ECM composition, such as IL-6, TNF receptor, TGFb1, and collagen I and III. Global microRNA (miRNA) expression analysis revealed that PBMT decreased miR-221, miR-34c, and miR-93 expressions post-MI, which are related to deleterious effects in cardiac remodeling. Thus, the identification of transcriptional and post-transcriptional changes induced by PBMT may be used to interfere in the molecular dynamics of cardiac remodeling post-MI.

Formation and characterization of biofilms formed by salt-tolerant yeast strains in seawater-based growth medium.

Yeast whole cells have been widely used in modern biotechnology as biocatalysts to generate numerous compounds of industrial, chemical, and pharmaceutical importance. Since many of the biocatalysis-utilizing manufactures have become more concerned about environmental issues, seawater is now considered a sustainable alternative to freshwater for biocatalytic processes. This approach plausibly commenced new research initiatives into exploration of salt-tolerant yeast strains. Recently, there has also been a growing interest in possible applications of microbial biofilms in the field of biocatalysis. In these complex communities, cells demonstrate higher resistance to adverse environmental conditions due to their embedment in an extracellular matrix, in which physical, chemical, and physiological gradients exist. Considering these two topics, seawater and biofilms, in this work, we characterized biofilm formation in seawater-based growth media by several salt-tolerant yeast strains with previously demonstrated biocatalytic capacities. The tested strains formed both air-liquid-like biofilms and biofilms on silicone surfaces, with Debaryomyces fabryi, Schwanniomyces etchellsii, Schwanniomyces polymorphus, and Kluyveromyces marxianus showing the highest biofilm formation. The extracted biofilm extracellular matrices mostly consisted of carbohydrates and proteins. The latter group was primarily represented by enzymes involved in metabolic processes, particularly the biosynthetic ones, and in the response to stimuli. Specific features were also found in the carbohydrate composition of the extracellular matrix, which were dependent both on the yeast isolate and the nature of formed biofilms. Overall, our findings presented herein provide a unique data resource for further development and optimization of biocatalytic processes and applications employing seawater and halotolerant yeast biofilms.Key points• Ability for biofilm formation of some yeast-halotolerant strains in seawater medium• ECM composition dependent on strain and biofilm-forming surface• Metabolic enzymes in the ECM with potential applications for biocatalysis.

Dissecting the anti-biofilm potency of kappa-carrageenan capped silver nanoparticles against Candida species

Global antimicrobial crisis and advent of drug resistant fungal strains has substantially distressed disease management for clinicians. Biodegradable silver nanoparticles (AgNps) emerge as an excellent alternative remedial option. In the current study, the anti-biofilm activity of microwave irradiated kappa-carrageenan (CRG) capped AgNps against Candida albicans, and Candida glabrata was investigated in terms of their effect on reactive oxygen species (ROS) generation, cellular morphology, biochemical composition, and the activity of enzymes of extracellular matrix. Minimum inhibitory concentration and fungicidal concentration value of CRG-AgNps against both Candida spp. ranged between 400 and 500 μg/mL. The 80% of Candida biofilm was inhibited and eradicated by CRG-AgNps at a concentration of ~300 μg/mL. Microscopic studies indicate that CRG-AgNps caused morphological damage through membrane disruption and pore formation. Further, CRG-AgNps generated ROS in a concentration-dependent manner and modulated the composition of Candida biofilm ECM by increasing the carbohydrate and eDNA content. CRG-AgNps also significantly inactivated the hydrolytic enzymes, thus hindering the biofilm forming ability. In conclusion, all these results suggest that the CRG-AgNps are potential antifungal agents against Candida biofilms, and they inhibit/eradicate the fungal biofilms through multiple signalling mechanisms.

A developmental basis for the anatomical diversity of dermis in homeostasis and wound repair.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re‐enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Abstract PO-065: Utilization of optimized extracellular matrix substratum for inclusive capture of circulating tumor cells in stage IV colorectal cancer

Abstract Current methods to recover circulating tumour cells (CTCs) from the peripheral blood circulation of carcinoma patients rely principally on the expression of epithelial markers such as EpCAM, physical characteristics, and the absence of hematopoietic biomarkers. However, this strategy favours recovery of a homogeneous cell population with primarily differentiated epithelial characteristics. It fails to fully include those subpopulations that do not fit predictions and in particular does not properly capture cells that have stem-like characteristics or have undergone epithelial-mesenchymal transition (EMT). Yet these are often the cells that are most likely to give rise to successful metastasis and contribute to disease progression. We favour an approach that anticipates cellular heterogeneity, includes cells that have poor expression of differentiated characteristics, and relies on the very functional properties that are involved in successful negotiation of the route that culminates in metastasis. We therefore use a ‘tuned ECM’ (tECM) approach in which a complex ECM substratum is designed to extract and recover heterogeneous CTCs from the blood of patients with the cancer of interest, allowing subsequent single-cell analysis of cell behaviours to inform on features that have been shown to predict disease progression and metastasis. In the work described here we first used model colorectal cancer (CRC) cell lines, including those made resistant to drugs used in CRC regimens, to establish the best composition of ECM and full procedure to maximally capture cells for functional evaluation. For CRC we identified a defined mixture of collagen I and human plasma fibronectin as optimal for cell capture, providing a total recovery equivalent to complex ECM derived from tumor sources. In addition, this mixture accentuated the recovery of CRC cells made resistant to SN-38, the active metabolite of irinotecan. We then applied this approach to the recovery of CTCs from the blood from patients with stage IV CRC, using density centrifugation for prefractionation of nucleated blood cells followed by incubation on the tECM substratum. The captured cells could be immunostained in situ for both cytoplasmic and cell-surface markers and showed suitability for future assessment of cell behaviours in unfixed preparations. Optimisation of the capture-surface coating concentrations and subsequent fixation approach led to a 128-fold improvement in the CTC capture and identification from CRC patient blood. This optimised tECM approach offers promise for future inclusive recovery of heterogeneous CTC samples based on functional behaviours that relate to the metastastic process of the particular carcinoma, permitting further analyses including of chemokine pathways that are believed to be important in CRC metastasis. Citation Format: Deep Patel, Mala Bahl, Mario Valdes, Jonathan Blay. Utilization of optimized extracellular matrix substratum for inclusive capture of circulating tumor cells in stage IV colorectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-065.

Progressive and Prognostic Performance of an Extracellular Matrix-Receptor Interaction Signature in Gastric Cancer.

The role of an extracellular matrix- (ECM-) receptor interaction signature has not been fully clarified in gastric cancer. This study performed comprehensive analyses on the differentially expressed ECM-related genes, clinicopathologic features, and prognostic application in gastric cancer. The differentially expressed genes between tumorous and matched normal tissues in The Cancer Genome Atlas (TCGA) and validation cohorts were identified by a paired t-test. Consensus clusters were built to find the correlation between clinicopathologic features and subclusters. Then, the least absolute shrinkage and selection operator (lasso) method was used to construct a risk score model. Correlation analyses were made to reveal the relation between risk score-stratified subgroups and clinicopathologic features or significant signatures. In TCGA (26 pairs) and validation cohort (134 pairs), 25 ECM-related genes were significantly highly expressed and 11 genes were downexpressed in gastric cancer. ECM-based subclusters were slightly related to clinicopathologic features. We constructed a risk score model = 0.081∗log2 (CD36) + 0.043∗log2 (COL5A2) + 0.001∗log2 (ITGB5) + 0.039∗log2 (SDC2) + 0.135∗log2 (SV2B) + 0.012∗log2 (THBS1) + 0.068∗log2 (VTN) + 0.023∗log2 (VWF). The risk score model could well predict the outcome of patients with gastric cancer in both training (n = 351, HR: 1.807, 95% CI: 1.292-2.528, P = 0.00046) and validation (n = 300, HR: 1.866, 95% CI: 1.347-2.584, P = 0.00014) cohorts. Besides, risk score-based subgroups were associated with angiogenesis, cell adhesion molecules, complement and coagulation cascades, TGF-beta signaling, and mismatch repair-relevant signatures (P < 0.0001). By univariate (1.845, 95% CI: 1.382-2.462, P < 0.001) and multivariate (1.756, 95% CI: 1.284-2.402, P < 0.001) analyses, we regarded the risk score as an independent risk factor in gastric cancer. Our findings revealed that ECM compositions became accomplices in the tumorigenesis, progression, and poor survival of gastric cancer.

Microstructure and resident cell-types of the feline optic nerve head resemble that of humans

The lamina cribrosa (LC) region of the optic nerve head (ONH) is considered a primary site for glaucomatous damage. In humans, biology of this region reflects complex interactions between retinal ganglion cell (RGC) axons and other resident ONH cell-types including astrocytes, lamina cribrosa cells, microglia and oligodendrocytes, as well as ONH microvasculature and collagenous LC beams. However, species differences in the microanatomy of this region could profoundly impact efforts to model glaucoma pathobiology in a research setting. In this study, we characterized resident cell-types, ECM composition and ultrastructure in relation to microanatomy of the ONH in adult domestic cats (Felis catus). Longitudinal and transverse cryosections of ONH tissues were immunolabeled with astrocyte, microglia/macrophage, oligodendrocyte, LC cell and vascular endothelial cell markers. Collagen fiber structure of the LC was visualized by second harmonic generation (SHG) with multiphoton microscopy. Fibrous astrocytes form glial fibrillary acidic protein (GFAP)-positive glial columns in the pre-laminar region, and cover the collagenous plates of the LC region in lamellae oriented perpendicular to the axons. GFAP-negative and alpha-smooth muscle actin-positive LC cells were identified in the feline ONH. IBA-1 positive immune cells and von Willebrand factor-positive blood vessel endothelial cells are also identifiable throughout the feline ONH. As in humans, myelination commences with a population of oligodendrocytes in the retro-laminar region of the feline ONH. Transmission electron microscopy confirmed the presence of capillaries and LC cells that extend thin processes in the core of the collagenous LC beams. In conclusion, the feline ONH closely recapitulates the complexity of the ONH of humans and non-human primates, with diverse ONH cell-types and a robust collagenous LC, within the beams of which, LC cells and capillaries reside. Thus, studies in a feline inherited glaucoma model have the potential to play a key role in enhancing our understanding of ONH cellular and molecular processes in glaucomatous optic neuropathy.

Biofabrication of cell-laden allografts of goat urinary bladder scaffold for organ reconstruction/regeneration

IntroductionBladder dysfunction has been considered as one of the most critical health conditions with no proper treatment. Current therapeutic approaches including enterocystoplasty have several limitations. Hence, biofabrication of cell-laden biological allografts using decellularized Goat urinary bladder scaffolds for organ reconstruction/regeneration was major objective of this study. Materials and methodsAn efficient method for decellularization of Goat urinary bladder (N = 3) was developed by perfusion of gradient change of detergents through ureter. The retention of organ architecture, extracellular matrix composition, mechanical properties and removal of cellular components was characterized using histological, cellular and molecular analysis. Further, mesenchymal stem cells (MSCs) from human umbilical cord blood (UCB) were used for preparing biological construct of decellularized urinary bladder (DUB) scaffolds to augment the urinary bladder reconstruction/regeneration. ResultsThe decellularization method adopted in this study generated completely DUB scaffolds within 10 h at 100 mm Hg pressure and constant flow rate of 1 mL/min. The DUB scaffold retains organ architecture, ECM composition, and mechanical strength. No significant amount of residual nucleic acid was observed post-decellularization. Furthermore, MSCs derived from human UCB engrafted and proliferated well on DUB scaffolds in highly aligned manner under xeno-free condition. ConclusionBiofabricated humanized urinary bladder constructs provides xeno-free allografts for future application in augmenting urinary bladder reconstruction/regeneration with further development.

Ultrastructure study of skin fibroblasts in patients with Ehlers-Danlos Syndrome (EDS): preliminary results.

To investigate, in vivo and in vitro, the fibroblast-to-myofibroblast transition in patients with hypermobile Ehlers-Danlos Syndrome (EDS). To analyze the dermis of patients with classical form of EDS (cEDS) and with hEDS, to identify qualitative and/or quantitative differences in ECM component and ultrastructural changes in collagen. Seven subjects, aged over 18, two with cEDS and five with hEDS underwent two skin biopsy. One sample was prepared for transmission electron microscopy (TEM), the other for immunofluorescence. The diameter of collagen fibers was measured with TEM. Fibrils were analyzed in four patients: the two with cEDS and two with hEDS. For each patient, the diameter of n=250 collagen fibrils was measured. αSMA was used as specific marker for myofibroblast to highlight their presence in vivo in the skin of patients with hEDS. IF observation could not assess an increased expression of αSMA in hEDS patients, which showed no statistical difference compared to classic form patients. The major result from the analysis of TEM images is the clear difference in ECM composition between the two forms of EDS: ECM in hEDS is optically more dense and more prominently composed of elastic fibers. Our study provides the following important evidence: 1) the absence in vivo of dermal fibroblasts in patients with hEDS, demonstrated by αSMA negativity; 2) the presence of statistically significant changes in the diameter of collagen fibrils between the classic and the hypermobile forms.

1965-P: The Extracellular Matrix Modulates Adipocyte Thermogenic Capacity

Obesity is characterized by an increase in adipose mass and is the leading risk factor for type 2 diabetes. Increasing adipose thermogenic capacity, by activation of brown or beige fat, could be a treatment for metabolic diseases. Nevertheless, many approaches fail in maintaining transformed adipose tissue in vivo, in part, due to the limited understanding of how environmental factors control cell fate and maintenance. We have shown that mice with a null mutation in the laminin α4 exhibit resistance to obesity, enhanced expression of thermogenic fat markers (UCP1) in subcutaneous white adipose tissue (sWAT), increased energy expenditure and enhanced insulin sensitivity. The knockout (KO) of Lama4 results in complex changes in overall ECM composition. We used liquid chromatography/tandem mass spectrometry (LC/MS) to evaluate ECM composition in sWAT from wild type (WT) and KO mice. In addition to the expected absence of Lama4, collagen 1A1 (Col1A1) and collagen 3A1 (Col3A1) were significantly lower in KO mice. Also, integrins α7 (ITα7) and β1 (ITβ1) were dramatically reduced. Immunohistochemical staining confirmed lower levels of Itα7 in KO compared to WT mice (P&amp;lt;0.0001). Knocking down Itα7 with siRNA promoted UCP1 expression (P=0.003) in vitro. In addition, culture of ADSC on Lama4, Col3A1 and Col1A1 coated surfaces had lower UCP1 expression (P&amp;lt;0.0001). Finally, mRNA levels of ITα7 and Lama4 decreased following 12 days of beige differentiation in human ADSC (P&amp;lt;0.0001). These results demonstrate that ECM can regulate adipocyte thermogenic capacity. Specifically, Lama4, Itα7, Col1A1 and Col3A1 are involved in the modulation of metabolic function in adipocytes. A better understanding of the mechanisms underlying these interactions allows for the potential to specifically manipulate these cells to improve systemic energy metabolism and glucose homeostasis. Disclosure M.A. Gonzalez Porras: None. K. Stojkova: None. M.K. Vaicik: None. A.A. Goddi: None. R.N. Cohen: None. E. Brey: None.

451-P: Proteome Profiling in Rat Cardiomyoblasts: Understanding Diabetic Cardiomyopathy

Introduction: Diabetic cardiomyopathy is characterized by oversupply of nutrients with concurrent loss of metabolic flexibility. Even in excess substrate availability the heart suffers energy depletion as metabolic fluxes are diminished. We established a stably GLUT4 overexpressing cell line derived from H9C2 that presents a diabetic cardiomyopathy like phenotype. Protein expression patterns are analyzed by proteomic approaches. Material and Methods: Rat cardiomyoblasts stably overexpressing GLUT4 (H9C2-GLUT4) were cultured under hyperglycemic conditions (30mM glucose) either short time (3 days) or long time (more than 3 months). Expression profile was compared to non-transfected H9C2 cells (H9C2), using same culture conditions. Cell culture samples were prepared for mass spectrometric analysis by sample lysis followed by tryptic digestion. Peptides were analyzed on an Orbitrap Elite (ThermoFisher, Scientific) and resulting files were taken for the identification and quantification of proteins using MaxQuant and Perseus. DAVID Bioinformatics Resources 6.8 functioned as Pathway and GO term enrichment tool for further in depth analysis of the data set. Results: We were able to quantify 2,841 proteins of whose 881 were found to be significantly regulated (5% FDR; ANOVA corrected p-value). Further analysis of long time and short time exposure of either WT or GLUT4 overexpressing cells lead to the identification of 135 significantly expressed proteins in the WT and 112 significantly expressed proteins in the H9C2-GLUT4 model (5% FDR; ANOVA corrected p-value). In depth pathway and GO term enrichment analysis support effects on metabolism, protein biosynthesis and handling, and ECM composition. Discussion: Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycemia resulted in a metabolic induced proteome profile change. These models may thus increase our knowledge of biological effects of chronic hyperglycemia on the diabetic heart. Disclosure B. Stratmann: Speaker’s Bureau; Self; AstraZeneca, Neoplas Tools GmbH. B. Eggers: None. Y. Mattern: None. K. Marcus: None. D. Tschoepe: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH. Research Support; Self; AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Neoplas tools GmbH.

PDGFRα ablation leads to alteration of ECM and immune cell composition in the lung

Abstract Fibroblasts are the major cells responsible for tissue homeostasis, repair, and remodeling. In particular, PDGFRα is expressed by mesenchymal progenitors in the lung that constitute the myofibroblast population which transiently express α-SMA during alveologenesis. In the fibrotic lungs, PDGFRα+ lineages can differentiate into subpopulation of myofibroblasts which regulate tissue repair and remodeling. Although the contribution of PDGFRα+lineage to lung development and fibrosis are particularly well-known, their roles in the lung parenchyma at baseline are less characterized. To explore how PDGFRα+ fibroblasts affect extracellular matrix in baseline lung, we used genetic ablation of PDGFRα+ fibroblasts using Pdgfrα-CreERT2-driven expression of diphtheria toxin A. Histologically, thin mesenchyme was observed in fibroblast-ablated adult lungs. A majority of PDGFRα+ lineages are Col1a1-GFP+ cells, but PDGFRα-ablated lungs showed a significant decrease in the number of PDGFRα lineage and Col1a1-GFP+ cells. In addition, decreased expressions of ECM genes, such as Col1a1, Col1a2, and fibronectin-1 were observed in PDGFRα-ablated lungs. These data indicated that PDGFRα+ cells are a major source of extracellular proteins and PDGFRα ablation leads to alteration of ECM homeostasis in the baseline lung. Flow cytometry analysis of the lung tissues revealed that PDGFRα ablation resulted in elevated numbers of leukocytes and neutrophils. However, fibroblast loss did not affect macrophage numbers. These data suggested that fewer PDGFRα+ cells or loss of ECM or both can affect the recruitment of immune cells in the lung. In summary, changes in ECM environment by PDGFRα ablation leads to altered immune cell composition in the lung.

Optimising micro-hydroxyapatite reinforced poly(lactide acid) electrospun scaffolds for bone tissue engineering.

HA-mineralised composite electrospun scaffolds have been introduced for bone regeneration due to their ability to mimic both morphological features and chemical composition of natural bone ECM. Micro-sized HA is generally avoided in electrospinning due to its reduced bioactivity compared to nano-sized HA due to the lower surface area. However, the high surface area of nanoparticles provides a very high surface energy, leading to agglomeration. Thus, the probability of nanoparticles clumping leading to premature mechanical failure is higher than for microparticles at higher filler content. In this study, two micron-sized hydroxyapatites were investigated for electrospinning with PLA at various contents, namely spray dried HA (HA1) and sintered HA (HA2) particles to examine the effect of polymer concentration, filler type and filler concentration on the morphology of the scaffolds, in addition to the mechanical properties and bioactivity. SEM results showed that fibre diameter and surface roughness of 15 and 20 wt% PLA fibres were significantly affected by incorporation of either HA. The apatite precipitation rates for HA1 and HA2-filled scaffolds immersed in simulated body fluid (SBF) were similar, however, it was affected by the fibre diameter and the presence of HA particles on the fibre surface. Degradation rates of HA2-filled scaffolds in vitro over 14 days was lower than for HA1-filled scaffolds due to enhanced dispersion of HA2 within PLA matrix and reduced cavities in PLA/HA2 interface. Finally, increasing filler surface area led to enhanced thermal stability as it reduced thermal degradation of the polymer.

Proteomic Profiling of the Ecm in Health and Disease

Purpose: The ECM is a complex meshwork of proteins providing architectural support to cells and conferring biomechanical properties to tissues. In addition, it provides chemical cues that orchestrate cellular functions such as proliferation and survival, adhesion, and migration. Degradation, hyper-production or alteration of the composition of the ECM cause or accompany numerous pathologies such as musculo-skeletal and cardio-vascular diseases, cancers, and fibroses. Thus, a better characterization of ECM composition, metabolism, and biology can lead to the identification of novel prognostic and diagnostic markers and offer novel therapeutic opportunities.

Lung developmental arrest caused by PDGF-A deletion: consequences for the adult mouse lung.

PDGF-A is a key contributor to lung development in mice. Its expression is needed for secondary septation of the alveoli and deletion of the gene leads to abnormally enlarged alveolar air spaces in mice. In humans, the same phenotype is the hallmark of bronchopulmonary dysplasia (BPD), a disease that affects premature babies and may have long lasting consequences in adulthood. So far, the knowledge regarding adult effects of developmental arrest in the lung is limited. This is attributable to few follow-up studies of BPD survivors and lack of good experimental models that could help predict the outcomes of this early age disease for the adult individual. In this study, we used the constitutive lung-specific Pdgfa deletion mouse model to analyze the consequences of developmental lung defects in adult mice. We assessed lung morphology, physiology, cellular content, ECM composition and proteomics data in mature mice, that perinatally exhibited lungs with a BPD-like morphology. Histological and physiological analyses both revealed that enlarged alveolar air spaces remained until adulthood, resulting in higher lung compliance and higher respiratory volume in knockout mice. Still, no or only small differences were seen in cellular, ECM and protein content when comparing knockout and control mice. Taken together, our results indicate that Pdgfa deletion-induced lung developmental arrest has consequences for the adult lung at the morphological and functional level. In addition, these mice can reach adulthood with a BPD-like phenotype, which makes them a robust model to further investigate the pathophysiological progression of the disease and test putative regenerative therapies.

Elastic and surgeon friendly electrospun tubes delivering PDGF-BB positively impact tendon rupture healing in a rabbit Achilles tendon model

A major problem after tendon laceration is the low mechanical strength of the repaired tissue. One viable strategy for improving the functional and biomechanical properties of ruptured and repaired tendons is the delivery of growth factors at the injury site. Here, bioactive and reversibly expandable double-layered emulsion and coaxially electrospun tubes made from biodegradable DegraPol® (DP) (polyester urethane), delivering platelet-derived growth factor BB (PDGF-BB), are explored as implants to improve tendon healing in a rabbit Achilles tendon full laceration model. In vitro studies showed that both emulsion and coaxially electrospun scaffolds allow sustained delivery of bioactive PDGF-BB with similar release kinetics (150–190 pg PDGF-BB/mg of DP scaffold) over a period of 30 days. In vivo assessment after three weeks showed that PDGF-BB delivery through the bioactive DP tubes increased the tensile strength of the treated tendons 2-fold without additional pro-fibrotic effects, i.e., cell hyperproliferation or increase in α-smooth muscle actin expression at the wound site. While no major differences in ECM composition at the wound site were observed for ± PDGF-BB treated samples, collagen I and III were upregulated and fibronectin was downregulated compared to native tendons. In areas away from the wound, increased fibronectin expression was observed qualitatively in regions with lower collagen I and III expression. Both types of bioactive DP tubes provided surgeon-friendly and stable implants to deliver bioactive molecules and positively affected the strength of the repaired tendons after 3 weeks, thus presenting promising bioactive implants for clinical applications in the tendon repair field.

Multilayered microcasting of agarose–collagen composites for neurovascular modeling

The in vitro fabrication of vascular networks is one of the most complex challenges currently faced in tissue engineering. We describe a method to create multi-layered, cell-laden hydrogel microstructures with coaxial geometries and heterogeneous elastic moduli. The technique can be used to build in vitro vascular structures that are fully embedded in physiologically realistic hydrogels. Our technique eliminates rigid polymeric surfaces from the vicinity of the cells—overcoming a limitation of many microfluidic models—and allows layers of multiple cell types to be defined with tailored ECM composition and stiffness, and in direct contact with each other. We demonstrate channels with internal diameters as small as 175 ​μm, and agarose–collagen (AC) gels whose Young’s moduli range from 1.4–8.3 ​kPa. We also show co-axial geometries with layer thicknesses as small as 125 ​μm. One potential application of such structures is to simulate brain microvasculature. Towards this goal, the composition and mechanical properties of the composite AC hydrogels are optimized for cell viability and biological performance in both 2D and 3D culture. Seven-day viability of human microvascular endothelial cells (HMECs) and SY5Y glial cells is found to be maximized with a collagen content of 0.05% (w/v) when agarose content ranges between 0.25% and 1% (w/v). Additionally, we quantify the roles of type I bovine and rat-tail collagen, Matrigel, and poly-d-lysine–collagen–Matrigel coatings in promoting HMEC spreading, proliferation and confluence. 3D triple-layer vascular constructs have been fabricated, composed of a cannular monolayer of HMECs surrounded by two regions of SY5Ys with differing spatial densities. The endothelia are confluent and maintain trans-endothelial electrical resistance (TEER) values around 300 ​Ω ​cm2 over 11.5 days. This prototype opens the way for intricate multi-luminal blood vessels to be fabricated in vitro.

The Effect of Hapln4 Link Protein Deficiency on Extracellular Space Diffusion Parameters and Perineuronal Nets in the Auditory System During Aging.

Hapln4 is a link protein which stabilizes the binding between lecticans and hyaluronan in perineuronal nets (PNNs) in specific brain regions, including the medial nucleus of the trapezoid body (MNTB). The aim of this study was: (1) to reveal possible age-related alterations in the extracellular matrix composition in the MNTB and inferior colliculus, which was devoid of Hapln4 and served as a negative control, (2) to determine the impact of the Hapln4 deletion on the values of the ECS diffusion parameters in young and aged animals and (3) to verify that PNNs moderate age-related changes in the ECS diffusion, and that Hapln4-brevican complex is indispensable for the correct protective function of the PNNs. To achieve this, we evaluated the ECS diffusion parameters using the real-time iontophoretic method in the selected region in young adult (3 to 6-months-old) and aged (12 to 18-months-old) wild type and Hapln4 knock-out (KO) mice. The results were correlated with an immunohistochemical analysis of the ECM composition and astrocyte morphology. We report that the ECM composition is altered in the aged MNTB and aging is a critical point, revealing the effect of Hapln4 deficiency on the ECS diffusion. All of our findings support the hypothesis that the ECM changes in the MNTB of aged KO animals affect the ECS parameters indirectly, via morphological changes of astrocytes, which are in direct contact with synapses and can be influenced by the ongoing synaptic transmission altered by shifts in the ECM composition.

Laminin-driven Epac/Rap1 regulation of epithelial barriers on decellularized matrix

Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. Statement of significanceEfforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM.These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.

Dissecting and rebuilding the glioblastoma microenvironment with engineered materials.

Glioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Several decades of research have provided great insight into GBM progression; however, the prognosis remains poor with a median patient survival time of ~ 15 months. The tumour microenvironment (TME) of GBM plays a crucial role in mediating tumour progression and thus is being explored as a therapeutic target. Progress in the development of treatments targeting the TME is currently limited by a lack of model systems that can accurately recreate the distinct extracellular matrix composition and anatomic features of the brain, such as the blood-brain barrier and axonal tracts. Biomaterials can be applied to develop synthetic models of the GBM TME to mimic physiological and pathophysiological features of the brain, including cellular and ECM composition, mechanical properties, and topography. In this Review, we summarize key features of the GBM microenvironment and discuss different strategies for the engineering of GBM TME models, including 2D and 3D models featuring chemical and mechanical gradients, interfaces and fluid flow. Finally, we highlight the potential of engineered TME models as platforms for mechanistic discovery and drug screening as well as preclinical testing and precision medicine.