Reciprocal influence of hMSCs/HaCaT cultivated on electrospun scaffolds.

Here, we investigated the synergistic effect of electrospun nanofibrous scaffolds made of gelatin /sulfated hyaluronan (sHA) or native hyaluronan (HA)/chondroitin sulfate (CS) and, keratinocytes (HaCaT)-human mesenchymal stem cells (hMSCs) contact co-culture on epithelial differentiation of hMSCs. The hMSCs were co-cultured in contact with HaCaT cells for 5 days on electrospun scaffold. Results show that electrospun scaffolds containing sulfated glycosaminoglycans (GAGs) stimulate epithelial differentiation in terms of various protein expression markers (keratin 14, ΔNp63α and Pan-cytokeratin) and gene expression of several dermal proteins (keratin 14, ΔNp63α). Electrospun scaffold independent of GAGs alone did not affect the epithelial differentiation of hMSCs but combination of keratinocyte-hMSC contact co-culture and electrospun scaffold promotes the epithelial differentiation of hMSCs.

Fabrication of transparent quaternized PVA/silver nanocomposite hydrogel and its evaluation as an antimicrobial patch for wound care systems.

Grafting of quaternary nitrogen atoms into the backbone of polymer is an efficient way of developing new generation antimicrobial polymeric wound dressing. In this study, an elastic, non-adhesive and antimicrobial transparent hydrogel based dressing has been designed, which might be helpful for routine observation of wound area without removing the dressing material along with maintaining a sterile environment for a longer period of time. Green synthesized silver nanoparticles have been loaded into the quaternized PVA hydrogel matrix to improve its antimicrobial property. Silver nanoparticles loaded quaternized PVA hydrogel showed enhanced mechanical and swelling properties compared to native quaternized PVA hydrogel. Release kinetics evaluated by atomic absorption spectroscopy revealed that the release mechanism of silver nanoparticles from the hydrogel follows Fickian diffusion. Antimicrobial efficacy of the hydrogels was evaluated by disk diffusion test on Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. After 96 h of release in phosphate buffer, the growth inhibition zone created by silver nanoparticless loaded quaternized PVA hydrogel is comparable to that created by ampicillin. These observations assert that the silver nanoparticles loaded quaternized PVA hydrogel acts as a reservoir of silver nanoparticles, which helps in maintaining a sterile environment for longer time duration by releasing Ag nanocrystallite in sustained manner.

Localized temporal co-delivery of interleukin 10 and decorin genes using amediated by collagen-based biphasic scaffold modulates the expression of TGF-ß1/ß2 in a rabbit ear hypertrophic scarring model.

Hypertrophic scarring (HS) is an intractable complication associated with cutaneous wound healing. Although transforming growth factor ß1 (TGF-ß1) has long been documented as a central regulatory cytokine in fibrogenesis and fibroplasia, there is currently no cure. Gene therapy is emerging as a powerful tool to attenuate the overexpression of TGF-ß1 and its signaling activities. An effective approach may require transferring multiple genes to regulate different aspects of TGF-ß1 signaling activities in a Spatio-temporal manner. Herein we report the additive anti-fibrotic effects of two plasmid DNAs encoding interleukin 10 (IL-10) and decorin (DCN) co-delivered via a biphasic 3D collagen scaffold reservoir platform. Combined gene therapy significantly attenuated inflammation and extracellular matrix components' accumulation in a rabbit ear ulcer model; and suppressed the expressions of genes associated with fibrogenesis, including collagen type I, as well as TGF-ß1 and TGF-ß2, while enhancing the genes commonly associated with regenerative healing including collagen type III. These findings may serve to provide a non-viral gene therapy platform that is safe, optimized, and effective to deliver multiple genes onto the diseased tissue in a wider range of tissue fibrosis-related maladies.

Microchip Cytometry for Multiplexed Single-Cell Protein Detection in a Low-Resource Setting toward Point of Care Diagnosis.

Multiplex measurement of protein expression with the single-cell resolution has been challenging. Although a few conventional approaches including flow cytometry and immunofluorescence-based methods have been developed to detect proteins in individual cells, they are either dependent on bulky instrument or not multiplexed and high-throughput enough. Here we present a portable single-cell analysis system that is operable in a resource-limited environment. A stand-sit microchip housed in a clamp enables simple and instrument-free operation of all necessary steps, and the detection based on immunogold enhancement exonerates the reliance on fluorescence optics and electronics. The quantified sensitivity was found comparable to the conventional fluorescence approaches. We used this system to analyze five immune effector proteins and found the system is equally effective to detect those proteins in hundreds of single cells. Significant increase of cytokine protein production by THP1 monocytes was observed upon stimulation by lipopolysaccharide. Further study showed that a low-end imaging setup with low resolution can also detect signals without much loss of sensitivity. Taken together, this portable multiplex single-cell system may find broad biomedical applications in a field setting.

Highly Multiplexed Single-Cell Protein Profiling with Large-Scale Convertible DNA-Antibody Barcoded Arrays.

Highly multiplexed detection of proteins secreted by single cells is always challenging. Herein, a multiplexed in situ tagging technique based on single-stranded DNA encoded microbead arrays and multicolor successive imaging for assaying single-cell secreted proteins with high throughput and high sensitivity is presented. This technology is demonstrated to be capable of increasing the multiplexity exponentially. Upon integration with polydimethylsiloxane microwells, this platform is applied to detect ten immune effector proteins from differentiated single macrophages stimulated with lipopolysaccharide. Significant heterogeneity is observed when the derived human primary macrophages are analyzed. This versatile technology is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.

Nanofibrous artificial skin substitute composed of mPEG-PCL grafted gelatin/hyaluronan/chondroitin sulfate/sericin for 2nd degree burn care: in vitro and in vivo study.

The aim of this study was to investigate the efficacy of a skin substitute composed of mPEG-PCL-grafted-gelatin (Bio-Syn)/hyaluronan/chondroitin sulfate/sericin and to study its in vitro biocompatibility with human fibroblasts, human keratinocytes and hMSCs in terms of cellular adhesion and proliferation (∼5-6 fold). mPEG-PCL was grafted into a gelatin backbone via a Michael addition reaction to prepare Bio-Syn and it was characterized using ATR-FTIR, 1H NMR and TNBS assay. Additionally, keratinocyte-hMSC contact co-culture studies showed that Bio-Syn composite scaffolds loaded with sericin promote hMSCs' epithelial differentiation with regard to qRT-PCR gene expression (ΔNp63α and keratin 14) and expression of various epithelial markers (Pan-cytokeratin, ΔNp63α and keratin 14). In vivo efficacy studies on a 2nd degree burn wound model in Wistar rats showed an improved rate of wound contraction, histology (H&E and Van Gieson's staining) and pro-healing marker (hexosamine, hydroxyproline, etc.) expression in granular tissue compared to using the commercial dressing Neuskin™ and a cotton gauze control.

Biomimetic electrospun scaffolds from main extracellular matrix components for skin tissue engineering application - The role of chondroitin sulfate and sulfated hyaluronan.

Incorporation of bioactive components like glycosaminoglycans (GAGs) into tissue engineering scaffolds, is a promising approach towards developing new generation functional biomaterial. Here, we have designed electrospun nanofibrous scaffolds made of gelatin and different concentrations of chemically sulfated or non-sulfated hyaluronan (sHA or HA) and chondroitin sulfate (CS). Evenly distributed fiber morphology was observed with no differences between varying concentrations and types of GAGs. In vitro release kinetics revealed that GAGs release is driven by diffusion. The effects of these scaffolds were analyzed on human keratinocyte (HaCaT), fibroblast (Hs27) and mesenchymal stem cells (hMSCs) adhesion and proliferation. A significant increase in cell number (~5 fold) was observed when cultivating all three cell types alone on scaffolds containing sHA and CS. These findings suggest that sulfated GAG-containing electrospun nanofibrous scaffolds might be beneficial for the development of effective skin tissue engineered constructs by stimulating cellular performance and therefore accelerate epidermal-dermal regeneration processes.

Assessment of PVA/silver nanocomposite hydrogel patch as antimicrobial dressing scaffold: Synthesis, characterization and biological evaluation.

A novel, elastic, non-adhesive and antimicrobial hydrogel PVA scaffold (loaded with AgNPs) synthesized using freeze-thaw method has been characterized in this study. The direct visualization of the as synthesized (one-pot green synthesis methodology) AgNPs using TEM shows particle size in the range of 7±3nm. The minimum inhibitory concentration (MIC) of AgNPs for Staphylococcus aureus and Escherichia coli was estimated to be 7.81µg/mL, whereas for Pseudomonas aeruginosa (gram negative) it was around 3.90µg/mL. The antimicrobial efficacy of AgNPs was further studied by protein leakage, ROS and LDH activity assay. The quantitative elemental analysis of silver was calculated before and after release in phosphate buffer (pH-7.4) by atomic absorption spectroscopy. The antimicrobial efficacy of the scaffold was retained even after 96h of release of AgNPs which suggests that the scaffold can be used as a reservoir for AgNPs to maintain a moist and sterile environment for a long period of time.

Co-cultivation of keratinocyte-human mesenchymal stem cell (hMSC) on sericin loaded electrospun nanofibrous composite scaffold (cationic gelatin/hyaluronan/chondroitin sulfate) stimulates epithelial differentiation in hMSCs: In vitro study.

Fortifying the scaffold with bioactive molecules and glycosaminoglycans (GAGs), is an efficient way to design new generation tissue engineered biomaterials. In this study, we evaluated the synergistic effect of electrospun nanofibrous composite scaffold (cationic gelatin/hyaluronan/chondroitin sulfate) loaded with sericin and, contact co-culture of human mesenchymal stem cells (hMSCs)-keratinocytes on hMSCs' differentiation towards epithelial lineage. Cationic gelatin is prepared with one step novel synthesis process by grafting quaternary ammonium salts to the backbone of gelatin. Release kinetics studies showed that Fickian diffusion is the major release mechanism for both GAGs and sericin/gelatin. In vitro biocompatibility of the electrospun scaffold was evaluated in terms of LDH and DNA quantification assay on human foreskin fibroblast, human keratinocyte and hMSC. Significant proliferation (∼ 4-6 fold) was detected after culturing all three cell on the electrospun scaffold containing sericin. After 5 days of contact co-culture, results revealed that electrospun scaffold containing sericin promote epithelial differentiation of hMSC in terms of several protein markers (keratin 14, ΔNp63α and Pan-cytokeratin) and gene expression of some dermal proteins (keratin 14, ΔNp63α). Findings of this study will foster the progress of current skin tissue engineering scaffolds by understanding the skin regeneration and wound healing process.

Structural and textural classification of erythrocytes in anaemic cases: a scanning electron microscopic study.

The objective of this study is to address quantitative microscopic approach for automated screening of erythrocytes in anaemic cases using scanning electron microscopic (SEM) images of unstained blood cells. Erythrocytes were separated from blood samples and processed for SEM imaging. Thereafter, erythrocytes were segmented using marker controlled watershed transformation technique. Total 47 structural and textural features of erythrocytes were extracted using various mathematical measures for six types of anaemic cases as compared to the control group. These features were statistically evaluated at 1% level of significance and subsequently ranked using Fisher's F-statistic describing the group discriminating potentiality. Amongst all extracted features, twenty nine features were found to be statistically significant (p<0.001). Finally, Bayesian classifier was applied to classify six types of anaemia based on top seventeen ranked features those of which are of course statistically significant. The present study yielded a predictive accuracy of 88.99%.