Neutrophil extracellular traps promoting fibroblast activation and aggravating limb ischemia through Wnt5a pathway.

Although the formation of NETs contributes to cancer cell invasion and distant metastasis, its role in the pathological progression of limb ischemia remains unknown. This study investigated the functional significance of NETs in cell-cell crosstalk during limb ischemia. The changes of cell subsets in lower limb ischemia samples were detected by single-cell RNA sequencing. The expression of neutrophil extracellular traps (NETs) related markers in lower limb ischemia samples was detected by immunohistochemistry and Western blotting. The signaling pathway of NETs activation in fibroblasts was verified by immunofluorescence, PCR and Western blotting. Through single-cell RNA sequencing (scRNA-seq), we identified 9 distinct cell clusters, with significantly upregulated activation levels in fibroblasts and neutrophils and phenotypic transformation of smooth muscle cells (SMCs) into a proliferative state in ischemic tissue. At the same time, the interaction between fibroblasts and smooth muscle cells was significantly enhanced in ischemic tissue. NETs levels rise and fibroblast activation is induced in ischemic conditions. Mechanistically, activated fibroblasts promote smooth muscle cell proliferation through the Wnt5a pathway. In ischemic mice, inhibition of Wnt5a mitigated vascular remodeling and subsequent ischemia. These findings highlighting the role of cell-cell crosstalk in ischemia and vascular remodeling. We found that the NETs-initiated fibroblast-SMC interaction is a critical regulator of limb ischemia via Wnt5a pathway, a potential therapeutic target for the treatment.

Neutrophil extracellular traps induced by IL-1ß promote endothelial dysfunction and aggravate limb ischemia.

Vascular inflammation and endothelial dysfunction contribute to vascular diseases. While neutrophil extracellular traps (NETs) participate in some vascular pathologies, their roles in lower limb ischemia remain poorly defined. This study investigated the functional significance of NETs in vascular inflammation and remodeling associated with limb ischemia. Single-cell RNA sequencing (scRNA-seq) and flow cytometry revealed neutrophil activation and upregulated NETs formation in human limb ischemia, with immunofluorescence confirming IL-1ß-induced release of NETs for vascular inflammation. Endothelial cell activation was examined via scRNA-seq and western blotting, indicating enhanced proliferation, expression of adhesion molecules (VCAM-1, ICAM-1), inflammatory cytokines (IL-1ß, IL-6) and decreased expression of VE-cadherin, that could be mediated by NETs to exacerbate endothelial inflammation. Mechanistically, NETs altered endothelial cell function via increased pSTAT1/STAT1 signaling. Vascular inflammation and subsequent ischemia were alleviated in vivo by NETosis or IL-1ß inhibition in ischemic mice. IL-1ß-NETs induce endothelial activation and inflammation in limb ischemia by stimulating STAT1 signaling. Targeting NETs may thus represent a novel therapeutic strategy for inflammatory vascular diseases associated with limb ischemia. Graphical abstract of NETs regulation of the development of vascular inflammation in lower limb ischemia via pSTAT1/STAT1 signaling pathway.

Peripheral vascular remodeling during ischemia.

About 230 million people worldwide suffer from peripheral arterial disease (PAD), and the prevalence is increasing year by year. Multiple risk factors, including smoking, dyslipidemia, diabetes, and hypertension, can contribute to the development of PAD. PAD is typically characterized by intermittent claudication and resting pain, and there is a risk of severe limb ischemia, leading to major adverse limb events, such as amputation. Currently, a major progress in the research field of the pathogenesis of vascular remodeling, including atherosclerosis and neointima hyperplasia has been made. For example, the molecular mechanisms of endothelial dysfunction and smooth muscle phenotype switching have been described. Interestingly, a series of focused studies on fibroblasts of the vessel wall has demonstrated their impact on smooth muscle proliferation and even endothelial function via cell-cell communications. In this review, we aim to focus on the functional changes of peripheral arterial cells and the mechanisms of the pathogenesis of PAD. At the same time, we summarize the progress of the current clinical treatment and potential therapeutic methods for PAD and shine a light on future perspectives.

Electrospun Biodegradable α-Amino Acid-Substituted Poly(organophosphazene) Fiber Mats for Stem Cell Differentiation towards Vascular Smooth Muscle Cells.

Mesenchymal stem cells, derived from human-induced pluripotent stem cells (iPSC), are valuable for generating smooth muscle cells (SMCs) for vascular tissue engineering applications. In this study, we synthesized biodegradable α-amino acid-substituted poly(organophosphazene) polymers and electrospun nano-fibrous scaffolds (~200 nm diameter) to evaluate their suitability as a matrix for differentiation of iPSC-derived mesenchymal stem cells (iMSC) into mature contractile SMCs. Both the polymer synthesis approach and the electrospinning parameters were optimized. Three types of cells, namely iMSC, bone marrow derived mesenchymal stem cells (BM-MSC), and primary human coronary artery SMC, attached and spread on the materials. Although L-ascorbic acid (AA) and transforming growth factor-beta 1 (TGF-ß1) were able to differentiate iMSC along the smooth muscle lineage, we showed that the electrospun fibrous mats provided material cues for the enhanced differentiation of iMSCs. Differentiation of iMSC to SMC was characterized by increased transcriptional levels of early to late-stage smooth muscle marker proteins on electrospun fibrous mats. Our findings provide a feasible strategy for engineering functional vascular tissues.

Bioactive fluorescent hybrid microparticles as a stand-alone osteogenic differentiation inducer.

Osteogenic differentiation of stem cells is one of the essential steps in bone regeneration. While supplementing exogenous factors using differentiation media is the established method to differentiate stem cells into osteoblasts on biomaterials, designing biomaterials that can act as a stand-alone differentiation inducer and promote bone regeneration is preferred for clinical translation. In this work, we report dexamethasone-loaded organic-inorganic hybrid microparticles synthesized from an intrinsically fluorescent poly (ester amide) and tertiary bioactive glass (PEA-BG) as a stand-alone osteogenic differentiation inducer. The mechanical properties data indicated that the compressive modulus of fluorescent hybrid microparticles could be modulated by its composition. The hybrid fluorescent microparticles supported the adhesion and proliferation of 10T1/2 â€‹cells in culture for up to seven days. Both pristine and dexamethasone-loaded PEA-BG microparticles were able to induce osteogenic differentiation of 10T1/2 â€‹cells in the absence of any media supplement, to a level even higher than standard osteogenic media, as evidenced by the expression of osteogenic markers on gene and protein levels and matrix mineralization. Taken together, the fluorescent PEA-BG hybrid microparticles have the potential to be used as a stand-alone biomaterial for osteogenic differentiation and bone regeneration.

Immobilization of Jagged1 Enhances Vascular Smooth Muscle Cells Maturation by Activating the Notch Pathway.

In Notch signaling, the Jagged1-Notch3 ligand-receptor pairing is implicated for regulating the phenotype maturity of vascular smooth muscle cells. However, less is known about the role of Jagged1 presentation strategy in this regulation. In this study, we used bead-immobilized Jagged1 to direct phenotype control of primary human coronary artery smooth muscle cells (HCASMC), and to differentiate embryonic multipotent mesenchymal progenitor (10T1/2) cell towards a vascular lineage. This Jagged1 presentation strategy was sufficient to activate the Notch transcription factor HES1 and induce early-stage contractile markers, including smooth muscle α-actin and calponin in HCASMCs. Bead-bound Jagged1 was unable to regulate the late-stage markers myosin heavy chain and smoothelin; however, serum starvation and TGFß1 were used to achieve a fully contractile smooth muscle cell. When progenitor 10T1/2 cells were used for Notch3 signaling, pre-differentiation with TGFß1 was required for a robust Jagged1 specific response, suggesting a SMC lineage commitment was necessary to direct SMC differentiation and maturity. The presence of a magnetic tension force to the ligand-receptor complex was evaluated for signaling efficacy. Magnetic pulling forces downregulated HES1 and smooth muscle α-actin in both HCASMCs and progenitor 10T1/2 cells. Taken together, this study demonstrated that (i) bead-bound Jagged1 was sufficient to activate Notch3 and promote SMC differentiation/maturation and (ii) magnetic pulling forces did not activate Notch3, suggesting the bead alone was able to provide necessary clustering or traction forces for Notch activation. Notch is highly context-dependent; therefore, these findings provide insights to improve biomaterial-driven Jagged1 control of SMC behavior.

The therapeutic potential of BRD4 in cardiovascular disease.

Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extra terminal (BET) protein family that has gained wide attention in the field of cancer due to its role in the formation of super enhancers (SEs) and the regulation of oncogene expression. However, there is increasing evidence that BRD4 also plays a pivotal role in a variety of cardiovascular diseases, suggesting that understanding the mechanisms of BRD4 in these diseases is important to advance studies and clinical treatment. In this article, we summarize the mechanisms of BRD4 in cardiovascular diseases, including pulmonary arterial hypertension, heart failure, atherosclerosis, and hypertension. In addition, we discuss small molecule inhibitors of BRD4 as novel therapeutic strategies for cardiovascular diseases.

Embryonic Mesenchymal Multipotent Cell Differentiation on Electrospun Biodegradable Poly(ester amide) Scaffolds for Model Vascular Tissue Fabrication.

Vascular differentiation of stem cells and matrix component production on electrospun tubular scaffolds is desirable to engineer blood vessels. The mouse embryonic multipotent mesenchymal progenitor cell line (10T1/2) provides an excellent tool for tissue engineering since it shares similar differentiation characteristics with mesenchymal stem cells. Although 10T1/2 cells have been widely studied in the context of skeletal tissue engineering, their differentiation to smooth muscle lineage is less known. In this study, we fabricated tubular electrospun poly(ester amide) (PEA) fibers from L-phenylalanine-derived biodegradable biomaterials and investigated cell-scaffold interactions as well as their differentiation into vascular smooth muscle cell and subsequent elastin expression. PEA scaffolds fabricated under different collector speeds did not have an impact on the fiber directionality/orientation. 10T1/2 cytocompatibility and proliferation studies showed that PEA fibres were not cytotoxic and were able to support proliferation for 14 days. Furthermore, cells were observed infiltrating the fibrous scaffolds despite the small pore sizes (~ 5 µm). Vascular differentiation studies of 10T1/2 cells using qPCR, Western blot, and immunostaining showed a TGFß1-induced upregulation of vascular smooth muscle cell (VSMC)-specific markers smooth muscle alpha-actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). Differentiated 10T1/2 cells produced both elastin and fibrillin-1 suggesting the potential of fibrous PEA scaffolds to fabricate model vascular tissues.

Preparation and characterization of electrospun rGO-poly(ester amide) conductive scaffolds.

Tissue engineering scaffolds should support tissue maturation through exposure to relevant stimuli and through successful cell infiltration. External electrical stimulation is particularly relevant for cardiac and neural applications, and requires conductive scaffolds to propagate electrical signals; cell infiltration is only possible with scaffolds that have sufficient porosity. The aim of this study was to impart conductivity and increased porosity of electrospun poly(ester amide) (PEA) scaffolds. Reduced graphene oxide (rGO) was incorporated into PEA and PEA-chitosan fibrous scaffolds, which increased scaffold conductivity and supported cardiac differentiation. The novel combination of ultrasonication and leaching of a sacrificial polymer was used to modify scaffold porosity, and resulted in an increase in pore area evaluated through image analysis. This approach aims to potentially promote tissue maturation with electrospun PEA scaffolds, by modifying both scaffold conductivity and porosity. This extends the relevance of electrospun PEA scaffolds to cardiac tissue engineering for the first time.

Porous and biodegradable polycaprolactone-borophosphosilicate hybrid scaffolds for osteoblast infiltration and stem cell differentiation.

The composition and microstructure of bone tissue engineering scaffolds play a significant role in regulating cell infiltration, proliferation, differentiation, and extracellular matrix production. While boron is an essential trace element for bone formation, growth, and health, boron-containing biomaterials are poorly studied. Specifically, the effect of boron in hybrid scaffolds on stem cell differentiation is unknown. We have previously reported the synthesis and characterization of class II hybrid biomaterials from polycaprolactone and borophosphosilicate glass (PCL/BPSG). In this study, PCL/BPSG hybrid porous scaffolds were fabricated by a solvent-free casting and particulate leaching method having consistent pore-size distribution, controlled porosity, and pore interconnectivity. The mechanical properties with respect to porogen loading and degradation time demonstrated that these scaffolds were competent for bone tissue engineering applications. In cell culture experiments, significant number of cells infiltrated and adhered into the scaffolds interior. Induced pluripotent stem cells (iPSCs) differentiation to osteogenic lineage was dependent on the amount of boron incorporated into the hybrid scaffolds. Consistent with this, scaffolds containing 2-mol% boron (calculated as % of the inorganic component) had an optimum effect on lineage expressions for alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN). These results suggest that PCL/BPSG hybrid scaffolds with optimum-level boron may enhance bone formation.

Bioreactor-induced mesenchymal progenitor cell differentiation and elastic fiber assembly in engineered vascular tissues.

In vitro maturation of engineered vascular tissues (EVT) requires the appropriate incorporation of smooth muscle cells (SMC) and extracellular matrix (ECM) components similar to native arteries. To this end, the aim of the current study was to fabricate 4mm inner diameter vascular tissues using mesenchymal progenitor cells seeded into tubular scaffolds. A dual-pump bioreactor operating either in perfusion or pulsatile perfusion mode was used to generate physiological-like stimuli to promote progenitor cell differentiation, extracellular elastin production, and tissue maturation. Our data demonstrated that pulsatile forces and perfusion of 3D tubular constructs from both the lumenal and ablumenal sides with culture media significantly improved tissue assembly, effectively inducing mesenchymal progenitor cell differentiation to SMCs with contemporaneous elastin production. With bioreactor cultivation, progenitor cells differentiated toward smooth muscle lineage characterized by the expression of smooth muscle (SM)-specific markers smooth muscle alpha actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). More importantly, pulsatile perfusion bioreactor cultivation enhanced the synthesis of tropoelastin and its extracellular cross-linking into elastic fiber compared with static culture controls. Taken together, the current study demonstrated progenitor cell differentiation and vascular tissue assembly, and provides insights into elastin synthesis and assembly to fibers. STATEMENT OF SIGNIFICANCE: Incorporation of elastin into engineered vascular tissues represents a critical design goal for both mechanical and biological functions. In the present study, we seeded porous tubular scaffolds with multipotent mesenchymal progenitor cells and cultured in dual-pump pulsatile perfusion bioreactor. Physiological-like stimuli generated by bioreactor not only induced mesenchymal progenitor cell differentiation to vascular smooth muscle lineage but also actively promoted elastin synthesis and fiber assembly. Gene expression and protein synthesis analyses coupled with histological and immunofluorescence staining revealed that elastin-containing vascular tissues were fabricated. More importantly, co-localization and co-immunoprecipitation experiments demonstrated that elastin and fibrillin-1 were abundant throughout the cross-section of the tissue constructs suggesting a process of elastin protein crosslinking. This study paves a way forward to engineer elastin-containing functional vascular substitutes from multipotent progenitor cells in a bioreactor.

Xenogeneic Decellularized Scaffold: A Novel Platform for Ovary Regeneration.

Women younger than 40 years may face early menopause because of premature ovarian failure (POF). The cause of POF can be idiopathic or iatrogenic, especially the cancer-induced oophorectomy and chemo- or radiation therapy. The current treatments, including hormone replacement therapy (HRT) and cryopreservation techniques, have increased risk of ovarian cancer and may reintroduce malignant cells after autografting. Decellularization technique has been regarded as a novel regenerative medicine strategy for organ replacement, wherein the living cells of an organ are removed, leaving the extracellular matrix (ECM) for cellular seeding. This study aimed to produce a xenogeneic decellularized ovary (D-ovary) scaffold as a platform for ovary regeneration and transplantation. We have developed a novel decellularization protocol for porcine ovary by treatment with physical, chemical, and enzymatic methods. Using hematoxylin and eosin (H&E) staining, DAPI staining, scanning electron microscopy (SEM), and quantitative analysis, this approach proved effective in removing cellular components and preserving ECM. Furthermore, the results of biological safety evaluation demonstrated that the D-ovary tissues were noncytotoxic for rat ovarian cells in vitro and caused only a minimal immunogenic response in vivo. In addition, the D-ovary tissues successfully supported rat granulosa cell penetration ex vivo and showed an improvement in estradiol (E2) hormone secretion.

Establishment and Validation of GV-SAPS II Scoring System for Non-Diabetic Critically Ill Patients.

BACKGROUND AND AIMS: Recently, glucose variability (GV) has been reported as an independent risk factor for mortality in non-diabetic critically ill patients. However, GV is not incorporated in any severity scoring system for critically ill patients currently. The aim of this study was to establish and validate a modified Simplified Acute Physiology Score II scoring system (SAPS II), integrated with GV parameters and named GV-SAPS II, specifically for non-diabetic critically ill patients to predict short-term and long-term mortality. METHODS: Training and validation cohorts were exacted from the Multiparameter Intelligent Monitoring in Intensive Care database III version 1.3 (MIMIC-III v1.3). The GV-SAPS II score was constructed by Cox proportional hazard regression analysis and compared with the original SAPS II, Sepsis-related Organ Failure Assessment Score (SOFA) and Elixhauser scoring systems using area under the curve of the receiver operator characteristic (auROC) curve. RESULTS: 4,895 and 5,048 eligible individuals were included in the training and validation cohorts, respectively. The GV-SAPS II score was established with four independent risk factors, including hyperglycemia, hypoglycemia, standard deviation of blood glucose levels (GluSD), and SAPS II score. In the validation cohort, the auROC values of the new scoring system were 0.824 (95% CI: 0.813-0.834, P< 0.001) and 0.738 (95% CI: 0.725-0.750, P< 0.001), respectively for 30 days and 9 months, which were significantly higher than other models used in our study (all P < 0.001). Moreover, Kaplan-Meier plots demonstrated significantly worse outcomes in higher GV-SAPS II score groups both for 30-day and 9-month mortality endpoints (all P< 0.001). CONCLUSIONS: We established and validated a modified prognostic scoring system that integrated glucose variability for non-diabetic critically ill patients, named GV-SAPS II. It demonstrated a superior prognostic capability and may be an optimal scoring system for prognostic evaluation in this patient group.

Platelet-to-Lymphocyte Ratio: A Novel Prognostic Factor for Prediction of 90-day Outcomes in Critically Ill Patients With Diabetic Ketoacidosis.

Diabetic ketoacidosis (DKA) is a life-threatening acute complication of diabetes mellitus and the novel systemic inflammation marker platelet-to-lymphocyte ratio (PLR) may be associated with clinical outcome in patients with DKA. This study aimed to investigate the utility of PLR in predicting 90-day clinical outcomes in patients with DKA. Patient data exacted from the Multiparameter Intelligent Monitoring in Intensive Care II (MIMIC II) database was analyzed. A cutoff value for PLR of 267.67 was determined using Youden index (P < 0.05) and used to categorize subjects into a high PLR group and a low PLR group. The hazard ratios (HRs) and 95% confidence intervals (CIs) for DKA were calculated across PLR. Clinical outcomes in our study were defined as intensive care unit (ICU) 90-day readmission and all-cause mortality. A total of 278 ICU admissions were enrolled and stratified by cutoff value of PLR. The incidence of readmission and mortality was 17.8% in the high PLR group, significantly higher than 7.4% in the low PLR group. In the multivariable model, after adjusting for known confounding variables including clinical parameters, comorbidities, laboratory parameters, the HRs for DKA were 2.573 (95% CI 1.239-5.345; P = 0.011), 2.648 (95% CI 1.269-5.527; P = 0.009), and 2.650 (95% CI 1.114-6.306; P = 0.028), respectively. The Kaplan-Meier survival curve showed that a high PLR level was associated with a higher risk for 90-day outcomes in patients with DKA. The authors report that higher PLR presents a higher risk for 90-day incidence of readmission and mortality in patients with DKA. It appears to be a novel independent predictor of 90-day outcomes in critically ill DKA patients in ICU units.

Activation of Transcription Factor GAX and Concomitant Downregulation of IL-1ß and ERK1/2 Modulate Vascular Smooth Muscle Cell Phenotype in 3D Fibrous Scaffolds.

Since vascular smooth muscle cells (VSMCs) display phenotypic plasticity in response to changing environmental cues, understanding the molecular mechanisms underlying the phenotypic modulation mediated by a three-dimensional (3D) scaffold is important to engineer functional vasculature. Following cell seeding into 3D scaffolds, the synthetic phenotype is desired to enable cells to expand rapidly and produce and assemble extracellular matrix components, but must revert to a quiescent contractile phenotype after tissue fabrication to impart the contractile properties found in native blood vessels. This study shows that 3D electrospun fibrous scaffolds regulate human coronary artery smooth muscle cells (HCASMCs) toward a more synthetic phenotype characterized by reduced contractile markers, such as smooth muscle alpha-actin and calponin. The reduction in contractile markers expression was mediated by endogenously expressed proinflammatory cytokine interleukin-1ß (IL-1ß). 3D topography transiently induces concomitant upregulation of IL-1ß and MAPK ERK1/2 through nuclear factor-κB-dependent signaling pathway. An early burst of expression of IL-1ß is essential for suppression of the homeobox transcription factor Gax and related cyclin-dependent kinase inhibitor p21(Cip1), which are key regulators for cells exiting from cell cycle. Our findings provide new insights for understanding signaling mechanisms of HCASMCs in electrospun 3D fibrous scaffolds, which have considerable value for application in vascular tissue engineering.

Regulation of vascular smooth muscle cell phenotype in three-dimensional coculture system by Jagged1-selective Notch3 signaling.

The modulation of vascular smooth muscle cell (VSMC) phenotype is an essential element to fabricate engineered conduits of clinical relevance. In vivo, owing to their close proximity, endothelial cells (ECs) play a role in VSMC phenotype switching. Although considerable progress has been made in vascular tissue engineering, significant knowledge gaps exist on how the contractile VSMC phenotype is induced at the conclusion of the tissue fabrication process. The objectives of this study were as follows: (1) to establish ligand presentation modes on transcriptional activation of VSMC-specific genes, (2) to develop a three-dimensional (3D) coculture model using human coronary artery smooth muscle cells (HCASMCs) and human coronary artery endothelial cells (HCAECs) on porous synthetic scaffolds and, (3) to investigate EC-mediated Notch signaling in 3D cultures and the induction of the HCASMC contractile phenotype. Whereas transcriptional activation of VSMC-specific genes was not induced by presenting soluble Jagged1 and Jagged1 bound to protein G beads, a direct link between HCAEC-bound Jagged1 and HCASMC differentiation genes was observed. Our 3D culture results showed that HCASMCs seeded to scaffolds and cultured for up to 16 days readily attached, infiltrated the scaffold, proliferated, and formed dense confluent layers. HCAECs, seeded on top of an HCASMC layer, formed a distinct, separate monolayer with cell-type partitioning, suggesting that HCAEC growth was contact inhibited. While we observed EC monolayer formation with 200,000 HCAECs/scaffold, seeding 400,000 HCAECs/scaffold revealed the formation of cord-like structures akin to angiogenesis. Western blot analyses showed that 3D coculture induced an upregulation of Notch3 receptor in HCASMCs and its ligand Jagged1 in HCAECs. This was accompanied by a corresponding induction of the contractile HCASMC phenotype as demonstrated by increased expression of smooth muscle-α-actin (SM-α-actin) and calponin. Knockdown of Jagged1 with siRNA showed a reduction in SM-α-actin and calponin in cocultures, identifying a link between Jagged1 and the expression of contractile proteins in 3D cocultures. We therefore conclude that the Notch3 signaling pathway is an important regulator of VSMC phenotype and could be targeted when fabricating engineered vascular tissues.

Fibrous biodegradable l-alanine-based scaffolds for vascular tissue engineering.

In vascular tissue engineering, three-dimensional (3D) biodegradable scaffolds play an important role in guiding seeded cells to produce matrix components by providing both mechanical and biological cues. The objective of this work was to fabricate fibrous biodegradable scaffolds from novel poly(ester amide)s (PEAs) derived from l-alanine by electrospinning, and to study the degradation profiles and its suitability for vascular tissue-engineering applications. In view of this, l-alanine-derived PEAs (dissolved in chloroform) were electrospun together with 18-30% w/w polycaprolactone (PCL) to improve spinnability. A minimum of 18% was required to effectively electrospin the solution while the upper value was set in order to limit the influence of PCL on the electrospun PEA fibres. Electrospun fibre mats with average fibre diameters of ~0.4 µm were obtained. Both fibre diameter and porosity increased with increasing PEA content and solution concentration. The degradation of a PEA fibre mat over a period of 28 days indicated that mass loss kinetics was linear, and no change in molecular weight was found, suggesting a surface erosion mechanism. Human coronary artery smooth muscle cells (HCASMCs) cultured for 7 days on the fibre mats showed significantly higher viability (p < 0.0001), suggesting that PEA scaffolds provided a better microenvironment for seeded cells compared with control PCL fibre mats of similar fibre diameter and porosity. Furthermore, elastin expression on the PEA fibre mats was significantly higher than the pure PEA discs and pure PCL fibre mat controls (p < 0.0001). These novel biodegradable PEA fibrous scaffolds could be strong candidates for vascular tissue-engineering applications.

Role of bioactive 3D hybrid fibrous scaffolds on mechanical behavior and spatiotemporal osteoblast gene expression.

Three-dimensional (3D) bioactive organic-inorganic (O/I) hybrid fibrous scaffolds are attractive extracellular matrix (ECM) surrogates for bone tissue engineering. With the aim of regulating osteoblast gene expression in 3D, a new class of hybrid fibrous scaffolds with two distinct fiber diameters (260 and 600 nm) and excellent physico-mechanical properties were fabricated from tertiary (SiO2-CaO-P2O5) bioactive glass (BG) and poly (ε-caprolactone) (PCL) by in situ sol-gel and electrospinning process. The PCL/BG hybrid fibrous scaffolds exhibited accelerated wetting properties, enhanced pore sizes and porosity, and superior mechanical properties that were dependent on fiber diameter. Contrary to control PCL fibrous scaffolds that were devoid of bonelike apatite particles, incubating PCL/BG hybrid fibrous scaffolds in simulated body fluid (SBF) revealed bonelike apatite deposition. Osteoblast cells cultured on PCL/BG hybrid fibrous scaffolds spread with multiple attachments and actively proliferated suggesting that the low temperature in situ sol-gel and electrospinning process did not have a detrimental effect. Targeted bone-associated gene expressions by rat calvarial osteoblasts seeded on these hybrid scaffolds demonstrated remarkable spatiotemporal gene activation. Transcriptional-level gene expressions for alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) were significantly higher on the hybrid fibrous scaffolds (p < 0.001) that were largely dependent on fiber diameter compared. Taken together, our results suggest that PCL/BG fibrous scaffolds may accelerate bone formation by providing a favorable microenvironment.

The role of Ras-ERK-IL-1ß signaling pathway in upregulation of elastin expression by human coronary artery smooth muscle cells cultured in 3D scaffolds.

Incorporation of endogenous elastin, a key structural component of the vascular extracellular matrix (ECM), is an important requirement for engineered vascular tissues. In addition to providing elastic recoil of the tissue, elastin influences cell function and promotes cell signaling by interacting with specific cell surface receptors. Although progress has been made in understanding the mechanisms of in vivo elastin expression and incorporation into fibers, it is notably absent from engineered vessels. Recently we showed that the three-dimensional (3D) scaffold topography was able to upregulate elastin synthesis by human coronary artery smooth muscle cells (HCASMC). The present study was undertaken to explore the molecular mechanisms responsible for 3D scaffold-induced elastin gene transcription. Here, we show several lines of evidence that signal transduction pathway leading to elastin gene expression by HCASMC cultured in synthetic 3D scaffolds to be strikingly different from two-dimensional (2D) surfaces. In 3D scaffolds, α5ß1 integrin engagement by HCASMC was significantly reduced and the putative focal adhesion kinase (FAK) was poorly phosphorylated concomitant with FAK and protein tyrosine kinase Pyk2 downregulation. FAK-associated adhesion proteins vinculin and paxillin were also significantly downregulated by the 3D scaffold topography. Furthermore, contrary to 2D cultures, HCASMC cultured on 3D scaffolds had no Rho activation suggesting pliability of the elastomeric synthetic scaffold. Elastin expression in 3D cultures followed Ras-ERK1/2 signal transduction pathway and was further dependent on endogenously expressed interleukin-1ß (IL-1ß). Blocking of ERK1/2 activation using a pharmacologic inhibitor reduced both elastin and IL-1ß gene expressions in 3D cultures. Transient transfection of IL-1ß using siRNA, however, did not affect ERK1/2 activation but downregulated elastin gene expression suggesting that endogenous IL-1ß acts downstream from ERK1/2. Taken together, results of the present study provide evidence that endogenous IL-1ß play a role in elastin gene upregulation and, that this upregulation is mediated by the Ras-ERK1/2 pathway in 3D cultures.