Extracellular Vesicular Analysis of Glypican 1 mRNA and Protein for Pancreatic Cancer Diagnosis and Prognosis.

Detecting pancreatic duct adenocarcinoma (PDAC) in its early stages and predicting late-stage patient prognosis undergoing chemotherapy is challenging. This work shows that the activation of specific oncogenes leads to elevated expression of mRNAs and their corresponding proteins in extracellular vesicles (EVs) circulating in blood. Utilizing an immune lipoplex nanoparticle (ILN) biochip assay, these findings demonstrate that glypican 1 (GPC1) mRNA expression in the exosomes-rich (Exo) EV subpopulation and GPC1 membrane protein (mProtein) expression in the microvesicles-rich (MV) EV subpopulation, particularly the tumor associated microvesicles (tMV), served as a viable biomarker for PDAC. A combined analysis effectively discriminated early-stage PDAC patients from benign pancreatic diseases and healthy donors in sizable clinical from multiple hospitals. Furthermore, among late-stage PDAC patients undergoing chemotherapy, lower GPC1 tMV-mProtein and Exo-mRNA expression before treatment correlated significantly with prolonged overall survival. These findings underscore the potential of vesicular GPC1 expression for early PDAC screenings and chemotherapy prognosis.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

An immunogold single extracellular vesicular RNA and protein (Au SERP) biochip to predict responses to immunotherapy in non-small cell lung cancer patients.

Conventional PD-L1 immunohistochemical tissue biopsies only predict 20%-40% of non-small cell lung cancer (NSCLC) patients that will respond positively to anti-PD-1/PD-L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (Au SERP) as a non-invasive alternative. With only 20 µl of purified serum, PD-1/PD-L1 proteins on the surface of extracellular vesicles (EVs) and EV PD-1/PD-L1 messenger RNA (mRNA) cargo were detected at a single-vesicle resolution and exceeded the sensitivities of their bulk-analysis conventional counterparts, ELISA and qRT-PCR, by 1000 times. By testing a cohort of 27 non-responding and 27 responding NSCLC patients, Au SERP indicated that the single-EV mRNA biomarkers surpass the single-EV protein biomarkers in predicting patient responses to immunotherapy. Dual single-EV PD-1/PD-L1 mRNA detection differentiated responders from non-responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for Au SERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.

Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVµBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. The diameter and cellular occupancy of the EVµBRs could be precisely tailored to various configurations, supporting the formation of breast cancer tumor spheroids. To immobilize the EVµBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. Breast cancer tumor spheroid-derived EVs were harvested with as little as 20 µL from the microfluidic system and quantified by single-EV immunofluorescence for CD63 and CD81. Despite the low-volume extraction, differences in biomarker expression and coexpression of the tetraspanins on single EVs were observed. Furthermore, the EVµBRs were capable of recapitulating heterogeneity at a cellular and vesicular degree, indicating the utility and robustness of the microfluidic system to investigate physiologically relevant EVs in breast cancer and other disease models.

Liposome interaction with macrophages and foam cells for atherosclerosis treatment: effects of size, surface charge and lipid composition.

Liposomes are potential drug carriers for atherosclerosis therapy due to low immunogenicity and ease of surface modifications that allow them to have prolonged circulation half-life and specifically target atherosclerotic sites to increase uptake efficiency. However, the effects of their size, charge, and lipid compositions on macrophage and foam cell behaviour are not fully understood. In this study, liposomes of different sizes (60 nm, 100 nm and 180 nm), charges (-40 mV, -20 mV, neutral, +15 mV and +30 mV) and lipid compositions (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, L-a-phosphatidylcholine, and egg sphingomyelin) were synthesized, characterized and exposed to macrophages and foam cells. Compared to 100 nm neutral 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes, flow cytometry and confocal imaging indicated that cationic liposomes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DSPC) liposomes were internalized more by both macrophages and foam cells. Through endocytosis inhibition, phagocytosis and clathrin-mediated endocytosis were identified as the dominant mechanisms of uptake. Anionic and DSPC liposomes induced more cholesterol efflux capacity in foam cells. These results provide a guide for the optimal size, charge, and lipid composition of liposomes as drug carriers for atherosclerosis treatment.

Keratin-Alginate Sponges Support Healing of Partial-Thickness Burns.

Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin-alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin-alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in Sus scrofa pigs. Keratin-alginate composite sponges supported the enhanced proliferation of human dermal fibroblasts compared to alginate-only sponges and exhibited decreased contraction in vitro when compared to keratin only sponges. As dermal substitutes in vivo, the sponges supported the expression of keratin 14, alpha-smooth muscle actin, and collagen IV within wound sites, comparable to collagen sponges. Keratin-alginate composite sponges supported the regeneration of basement membranes in the wounds more than in collagen-treated wounds and non-grafted controls, suggesting the subsequent development of pathological scar tissues may be minimized. Results from this study indicate that crosslinked keratin-alginate sponges are suitable alternative dermal substitutes for clinical applications in wound healing and skin regeneration.

Anti-inflammatory potential of simvastatin loaded nanoliposomes in 2D and 3D foam cell models.

Atherosclerosis is a multifactorial disease triggered and sustained by risk factors such as high cholesterol, high blood pressure and unhealthy lifestyle. Inflammation plays a pivotal role in atherosclerosis pathogenesis. In this study, we developed a simvastatin (STAT) loaded nanoliposomal formulation (LIPOSTAT) which can deliver the drug into atherosclerotic plaque, when administered intravenously. This formulation is easily prepared, stable, and biocompatible with minimal burst release for effective drug delivery. 2D and 3D in vitro models were examined towards anti-inflammatory effects of STAT, both free and in combination with liposomes. LIPOSTAT induced greater cholesterol efflux in the 2D foam cells and significantly reduced inflammation in both 2D and 3D models. LIPOSTAT alleviated inflammation by reducing the secretion of early and late phase pro-inflammatory cytokines, monocyte adherence marker, and lipid accumulation cytokines. Additionally, the 3D foam cell spheroid model is a convenient and practical approach in testing various anti-atherosclerotic drugs without the need for human tissue.

Analyzing Inter-Leukocyte Communication and Migration In Vitro: Neutrophils Play an Essential Role in Monocyte Activation During Swarming.

Neutrophils are known to be the first responders to infection or injury. However, as inflammation progresses, other leukocytes become increasingly important in inflammation propagation, tissue reconstruction, and inflammation resolution. In recent years, there has been an increase in publications that analyze neutrophil behavior in vitro, but there remains a gap in the literature for in vitro technologies that enable quantitatively measuring interactions between different types of human leukocytes. Here, we used an in vitro platform that mimics inflammation by inducing neutrophil swarming to analyze the behavior of various leukocytes in a swarming setting. Using human peripheral blood leukocytes isolated directly from whole blood, we found that myeloid cells and lymphoid cells had different migratory behaviors. Myeloid cells, which are predominately neutrophils, exhibited swarming behavior. This behavior was not seen with lymphoid cells. We perturbed the peripheral blood leukocyte system by adding exogenous leukotriene B4 (LTB4) to the medium. Notably, only the myeloid cell compartment was significantly changed by the addition of LTB4. Additionally, LTB4 had no significant impact on myeloid cell migration during the recruitment phase of swarming. To further investigate the myeloid cell compartment, we isolated neutrophils and monocytes to analyze their interaction on the platform. We found that neutrophils increase monocyte migration toward the bioparticle clusters, as measured through speed, chemotactic index, track straightness, and swarm size. These results were confirmed with in vivo mouse experiments, where monocyte accumulation only occurred when neutrophils were present. Additionally, we found that both neutrophils and monocytes release the monocyte chemoattractant proteins CCL2 and CCL3 in the presence of Staphylococcus aureus bioparticles. Furthermore, extracellular vesicles from swarming neutrophils caused monocyte activation. These findings suggest that neutrophils play an essential role in the onset of inflammation not only by sealing off the site of infection or injury, but also by recruiting additional leukocytes to the site.

Immunomagnetic sequential ultrafiltration (iSUF) platform for enrichment and purification of extracellular vesicles from biofluids.

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5-100 mL) can be significantly enriched (up to 1000 times), with up to 99% removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% ± 3.6%, 96.0% ± 2.0% and 94.0% ± 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads to target EV subpopulations. Serum from a cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

Liposomal Nanotherapy for Treatment of Atherosclerosis.

Atherosclerosis is a chronic disease that can lead to life-threatening events such as myocardial infarction and stroke, is characterized by the build-up of lipids and immune cells within the arterial wall. It is understood that inflammation is a hallmark of atherosclerosis and can be a target for therapy. In support of this concept, an injectable nanoliposomal formulation encapsulating fluocinolone acetonide (FA), a corticosteroid, is developed that allows for drug delivery to atherosclerotic plaques while reducing the systemic exposure to off-target tissues. In this study, FA is successfully incorporated into liposomal nanocarriers of around 100 nm in size with loading efficiency of 90% and the formulation exhibits sustained release up to 25 d. The anti-inflammatory effect and cholesterol efflux capability of FA-liposomes are demonstrated in vitro. In vivo studies carried out with an apolipoprotein E-knockout (Apoe-/- ) mouse model of atherosclerosis show accumulation of liposomes in atherosclerotic plaques, colocalization with plaque macrophages and anti-atherogenic effect over 3 weeks of treatment. This FA-liposomal-based nanocarrier represents a novel potent nanotherapeutic option for atherosclerosis.

Surface engineering within a microchannel for hydrodynamic and self-assembled cell patterning.

The applications of cell patterning are widespread due to the high-throughput testing and different resolutions offered by these platforms. Cell patterning has aided in deconvoluting in vivo experiments to better characterize cellular mechanisms and increase therapeutic output. Here, we present a technique for engineering an artificial surface via surface chemistry to form large-scale arrays of cells within a microchannel by employing microstamping. By changing the approach in surface chemistry, H1568 cells were patterned hydrodynamically using immunoaffinity, and neutrophils were patterned through self-assembly via chemotaxis. The high patterning efficiencies (93% for hydrodynamic patterning and 68% for self-assembled patterning) and the lack of secondary adhesion demonstrate the reproducibility of the platform. The interaction between H1568 and neutrophils was visualized and quantified to determine the capability of the platform to encourage cell-cell interaction. With the introduction of H1568 cells into the self-assembled patterning platform, a significant hindrance in the neutrophils' ability to swarm was observed, indicating the important roles of inflammatory mediators within the nonsmall cell lung cancer tumor microenvironment.

Extracellular vesicles as mediators of in vitro neutrophil swarming on a large-scale microparticle array.

Neutrophils combat infections and promote healing of damaged tissues while protecting the surrounding healthy tissue through a process called swarming. Swarming neutrophils release soluble factors that recruit additional neutrophils and shape the inflammation response. Additionally, neutrophils release extracellular vesicles (EVs), which are gaining attention as important intercellular mediators. We developed a large-scale array of bioparticles on a glass substrate that triggers neutrophil swarming in vitro in a spatially and temporally controlled manner that facilitates the analysis of neutrophil migration. Our platform can generate 30 000 neutrophil swarms on a glass slide in a highly reproducible manner (98% patterning efficiency), which produces an EV-rich supernatant that enables quantitative characterization of inflammation-specific EVs. Healthy neutrophils were able to form uniform swarms across the bioparticle array, which demonstrates a high degree of intercellular coordination. However, neutrophils swarming on the bioparticle array tended to have a lower radial velocity than neutrophils swarming toward a single target. After collecting and isolating EVs released by swarming and non-swarming neutrophils, we found that neutrophils constitutively release exosomes and microvesicles. Furthermore, EVs released by swarming neutrophils cause neutrophil activation and contain the proinflammatory mediator galectin-3, suggesting that EVs have an active role during neutrophil swarming. Ultimately, understanding EVs' role in intercellular communication during swarming will improve understanding of the complex signaling pathways involved in the regulation of inflammation.

The Potential of Fluocinolone Acetonide to Mitigate Inflammation and Lipid Accumulation in 2D and 3D Foam Cell Cultures.

Inflammation plays an important role in all stages of atherosclerosis development. Therefore, the use of anti-inflammatory drugs could reduce the risk of major adverse cardiovascular events due to atherosclerosis. Herein, we explored the capacity of fluocinolone acetonide (FA), a glucocorticoid (GC), in modulating foam cell formation and response. Human THP-1 derived foam cells were produced using 100 µg/mL oxidized low-density lipoproteins (OxLDL) and fetal bovine serum (1 and 10%). 2D cultures of these cells were treated with FA (0.1, 1, 10, and 50 µg/mL) in comparison with dexamethasone (Dex). Results showed that treatment with 0.1 and 1 µg/mL FA and Dex improved foam cell survival. FA and Dex also inhibited inflammatory cytokine (CD14, M-CSF, MIP-3α, and TNF-α) secretion. Notably, at the concentration of 1 µg/mL, both FA and Dex reduced cholesteryl ester accumulation. Compared to Dex, FA was significantly better in reducing lipid accumulation at the therapeutic concentrations of 1 and 10 µg/mL. In a novel 3D foam cell spheroid model, FA was shown to be more effective than Dex in diminishing lipid accumulation, at the concentration of 0.1 µg/mL. Taken together, FA was demonstrated to be effective in preventing both lipid accumulation and inflammation in foam cells.

Mesenchymal Stem Cell Secretome Improves Tendon Cell Viability In Vitro and Tendon-Bone Healing In Vivo When a Tissue Engineering Strategy Is Used in a Rat Model of Chronic Massive Rotator Cuff Tear.

BACKGROUND: Massive rotator cuff tears (MRCTs) represent a major clinical concern, especially when degeneration and chronicity are involved, which highly compromise healing capacity. PURPOSE: To study the effect of the secretome of mesenchymal stem cells (MSCs) on tendon cells (TCs) followed by the combination of these activated TCs with an electrospun keratin-based scaffold to develop a tissue engineering strategy to improve tendon-bone interface (TBi) healing in a chronic MRCT rat model. STUDY DESIGN: Controlled laboratory study. METHODS: Human TCs (hTCs) cultured with the human MSCs (hMSCs) secretome (as conditioned media [CM]) were combined with keratin electrospun scaffolds and further implanted in a chronic MRCT rat model. Wistar-Han rats (N = 15) were randomly assigned to 1 of 3 groups: untreated lesion (MRCT group, n = 5), lesion treated with a scaffold only (scaffold-only group, n = 5), and lesion treated with a scaffold seeded with hTCs preconditioned with hMSCs-CM (STC_hMSC_CM group, n = 5). After sacrifice, 16 weeks after surgery, the rotator cuff TBi was harvested for histological analysis and biomechanical testing. RESULTS: The hMSCs secretome increased hTCs viability and density in vitro. In vivo, a significant improvement of the tendon maturing score was observed in the STC_hMSC_CM group (mean ± standard error of the mean, 15.6 ± 1.08) compared with the MRCT group (11.0 ± 1.38; P < .05). Biomechanical tests revealed a significant increase in the total elongation to rupture (STC_hMSC_CM, 11.99 ± 3.30 mm; scaffold-only, 9.89 ± 3.47 mm; MRCT, 5.86 ± 3.16 mm; P < .05) as well as a lower stiffness (STC_hMSC_CM, 6.25 ± 1.74 N/mm; scaffold-only, 6.72 ± 1.28 N/mm; MRCT, 11.54 ± 2.99 N/mm; P < .01). CONCLUSION: The results demonstrated that hMSCs-CM increased hTCs viability and density in vitro. Clear benefits also were observed when these primed cells were integrated into a tissue engineering strategy with an electrospun keratin scaffold, as evidenced by improved histological and biomechanical properties for the STC_hMSC_CM group compared with the MRCT group. CLINICAL RELEVANCE: This work supports further investigation into the use of MSC secretome for priming TCs toward a more differentiated phenotype, and it promotes the tissue engineering strategy as a promising modality to help improve treatment outcomes for chronic MRCTs.

Evaluating the antioxidant effects of human hair protein extracts.

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC50 = 16.22 µM). As a supplement in cell culture media, the extracts protected HDFs from H2O2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.

Human Hair Keratin for Biocompatible Flexible and Transient Electronic Devices.

Biomaterials have been attracting attention as a useful building block for biocompatible and bioresorbable electronics due to their nontoxic property and solution processability. In this work, we report the integration of biocompatible keratin from human hair as dielectric layer for organic thin-film transistors (TFTs), with high performance, flexibility, and transient property. The keratin dielectric layer exhibited a high capacitance value of above 1.27 µF/cm2 at 20 Hz due to the formation of electrical double layer. Fully solution-processable TFTs based on p-channel poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-b:5,4-b]dithiophen-2-yl)-alt[1,2,5]thiadiazolo[3,4-c]-pyridine] (PCDTPT) and keratin dielectric exhibited high electrical property with a saturation field-effect mobility of 0.35 cm2/(Vs) at a low gate bias of -2 V. We also successfully demonstrate flexible TFTs, which exhibited good mechanical flexibility and electrical stability under bending strain. An artificial electronic synaptic PCDTPT/keratin transistor was also realized and exhibited high-performance synaptic memory effects via simple operation of proton conduction in keratin. An added functionality of using keratin as a substrate was also presented, where similar PCDTPT TFTs with keratin dielectric were built on top of keratin substrate. Finally, we observed that our prepared devices can be degraded in ammonium hydroxide solution, establishing the feasibility of keratin layer as various components of transient electrical devices, including as a substrate and dielectric layer.

Engineered nanoparticles for the detection, treatment and prevention of atherosclerosis: how close are we?

Atherosclerosis is one of the leading causes of morbidity and mortality worldwide. Nanotechnology has provided the possibility of designing nanoparticles that can translocate through tissues and home in to atherosclerotic plaques to achieve desired diagnostic, therapeutic, theranostic or 'theralivery' outcomes. Although nanomedicine approaches have demonstrated exciting possibilities, clinical reality is still distant and challenges are aplenty, such as specificity of targeting and nanotoxicity. Nevertheless, developments in formulations, delivery strategies and experimental models over the coming years will generate new knowledge to define the true potential of this field. This review discusses the most recent developments, current challenges and future possibilities.

Cultivation of human dermal fibroblasts and epidermal keratinocytes on keratin-coated silica bead substrates.

Human hair keratin is promising as a bioactive material platform for various biomedical applications. To explore its versatility further, human hair keratin was coated onto monolayers of silica beads to produce film-like substrates. This combination was hypothesized to provide a synergistic effect in improving the biochemical properties of the resultant composite. Atomic force microscopy analysis showed uniform coatings of keratin on the silica beads with a slight increase in the resulting surface roughness. Keratin-coated silica beads had higher surface energy and relatively lower negative charge than those of bare silica beads. To investigate cell response, human dermal fibroblasts (HDFs), and human epidermal keratinocytes (HEKs) were cultured on the substrates over 4 days. Results showed that keratin coatings significantly enhanced the metabolic activity of HDFs and encouraged cell spreading but did not exert any significant effects on HEKs. HDF expression of collagen I was significantly more intense on the keratin-coated compared to the bare silica substrates. Furthermore, HDF secretion of various cytokines suggested that keratin coatings triggered active cell responses related to wound healing. Collectively, our study demonstrated that human hair keratin-coated silica bead monolayers have the potential to modulate HDF behavior in culture and may be exploited further. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2789-2798, 2017.

Fabrication and characterization of a novel crosslinked human keratin-alginate sponge.

Human hair keratins have been explored for biomedical applications because of their abundance, bioactivity and processability. However, pure keratin templates have poor mechanical properties, which limit their practical relevance. Herein, we described a novel composite sponge, consisting of human hair keratins chemically crosslinked with alginate using 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride, with improved mechanical properties. Fourier transform infrared spectroscopy (FTIR) and free amine group quantification using ninhydrin revealed a maximum crosslinking index of 82.1 ± 1.3%. With increasing alginate proportions, the sponges exhibited increased tensile strength, tensile modulus and compression modulus at maximum values of 10.3 ± 1.92 kPa, 219.07 ± 52.39 kPa and 191.48 ± 32.89 kPa, respectively. The crosslinked sponges also demonstrated water vapour transmission rates comparable to commercial wound dressings. Meanwhile, sponges with higher proportions of keratin showed lower water uptake capacities and higher degradation rates by proteinase K, in comparison with sponges with higher proportions of alginate. Higher proportions of keratin on coated two-dimensional surfaces and in three-dimensional sponges resulted in more attachment and improved proliferation of L929 fibroblasts, verifying the bioactive role of keratin in the composites. In addition, subcutaneous implantation of the keratin-alginate sponges into C57BL/6NTac mice over 4 weeks showed no significant immunological reaction and minimal formation of fibrotic capsules. Furthermore, the sponges supported cellular infiltration, neo-tissue formation and vascularization in vivo. These findings demonstrated the feasibility of producing crosslinked human hair keratin-alginate sponges, with tuneable physical and mechanical properties, which are cell compliant in vitro and biocompatible in vivo, suggesting their potential for clinically relevant exploitations. Copyright © 2016 John Wiley & Sons, Ltd.

Cell viability and angiogenic potential of a bioartificial adipose substitute.

An implantable scaffold pre-seeded with cells needs to remain viable and encourage rapid angiogenesis in order to replace injured tissues, especially for tissue defect repairs. We created a bioartificial adipose graft composed of an electrospun 3D nanofibrous scaffold and fat tissue excised from New Zealand white rabbits. Cell viability and angiogenesis potential of the bioartificial substitute were examined during four weeks of culture in Dulbecco's Modified Eagle Medium by immunohistochemical staining with LIVE/DEAD® cell kit and PECAM-1 antibody, respectively. In addition, a Matrigel® assay was performed to examine the possibility of blood vessels sprouting from the bioartificial graft. Our results showed that cells within the graft were viable and vascular tubes were present at week 4, while cells in a fat tissue block were dead in vitro. In addition, capillaries were observed sprouting from the graft into the Matrigel, demonstrating its angiogenic potential. We expect that improved cell viability and angiogenesis in the bioartificial substitute, compared to intact autologous graft, could potentially contribute to its survival following implantation.