Origins and diversity of pan-isotype human bone marrow plasma cells.

Bone marrow plasma cells (BMPCs) produce durable, protective IgM, IgG, and IgA antibodies, and in some cases, pro-allergic IgE antibodies, but their properties and sources are unclear. We charted single BMPC transcriptional and clonal heterogeneity in food-allergic and non-allergic individuals across CD19 protein expression given its inverse correlation to BMPC longevity. Transcriptional and clonal diversity revealed distinct functional profiles. Additionally, distribution of somatic hypermutation and intraclonal antibody sequence variance suggest that CD19low and CD19high BMPCs arise from recalled memory and germinal center B cells, respectively. Most IgE BMPCs were from peanut-allergic individuals; two out of 32 from independent donors bound peanut antigens in vitro and in vivo. These findings shed light on BMPC origins and highlight the bone marrow as a source of pathogenic IgE in peanut allergy.

Propolis as an adjunctive therapy for treatment of uncomplicated cystitis in women: A randomized double-blind placebo-controlled trial.

The current research is designed to investigate the effect of propolis supplementation on the clinical manifestations in women suffering from uncomplicated cystitis. In this randomized double-blind, placebo-controlled trial, 120 women with uncomplicated cystitis were selected and randomly assigned into two groups to receive two 500 mg capsules of propolis or placebo daily for 7 days along with ciprofloxacin (250 mg). Clinical symptoms including hematuria, urinary frequency, dysuria, suprapubic pain, and urgency, as well as bacteriuria, were assessed before and after the intervention. After supplementation, participants in the intervention group had significantly fewer days of urinary frequency (p < 0.001), dysuria (p = 0.005), and urgency (p = 0.03). However, there was no significant difference between the two groups regarding hematuria and suprapubic pain (p > 0.05). Furthermore, the severity of bacteriuria decreased significantly in both groups. In conclusion, it seems that propolis supplementation in women with uncomplicated cystitis could improve urinary frequency, dysuria, and urgency. However, further clinical trials should be conducted to fully understand the effects of propolis in women suffering from uncomplicated cystitis.

Optimization of Parylene C and Parylene N thin films for use in cellular co-culture and tissue barrier models.

Parylene has been used widely used as a coating on medical devices. It has also been used to fabricate thin films and porous membranes upon which to grow cells. Porous membranes are integral components of in vitro tissue barrier and co-culture models, and their interaction with cells and tissues affects the performance and physiological relevance of these model systems. Parylene C and Parylene N are two biocompatible Parylene variants with potential for use in these models, but their effect on cellular behavior is not as well understood as more commonly used cell culture substrates, such as tissue culture treated polystyrene and glass. Here, we use a simple approach for benchtop oxygen plasma treatment and investigate the changes in cell spreading and extracellular matrix deposition as well as the physical and chemical changes in material surface properties. Our results support and build on previous findings of positive effects of plasma treatment on Parylene biocompatibility while showing a more pronounced improvement for Parylene C compared to Parylene N. We measured relatively minor changes in surface roughness following plasma treatments, but significant changes in oxygen concentration at the surface persisted for 7 days and was likely the dominant factor in improving cellular behavior. Overall, this study offers facile and relatively low-cost plasma treatment protocols that provide persistent improvements in cell-substrate interactions on Parylene that match and exceed tissue culture polystyrene.

Engineering cell-substrate interactions on porous membranes for microphysiological systems.

Microphysiological systems are now widely used to recapitulate physiological and pathological microenvironments in order to study and understand a variety of cellular processes as well as drug delivery and stem cell differentiation. Central to many of these systems are porous membranes that enable tissue barrier formation as well as compartmentalization while still facilitating small molecule diffusion, cellular transmigration and cell-cell communication. The role or impact of porous membranes on the cells cultured upon them has not been widely studied or reviewed. Although many chemical and physical substrate characteristics have been shown to be effective in controlling and directing cellular behavior, the influence of pore characteristics and the ability to engineer porous membranes to influence these responses is not fully understood. In this mini-review, we show that many studies point to a multiphasic cell-substrate response, where increasing pore sizes and pore-pore spacing generally leads to improved cell-substrate interactions. However, the smallest pores in the nano-scale sometimes promote the strongest cell-substrate interactions, while the very largest micron-scale pores hinder cell-substrate interactions. This synopsis provides an insight into the importance of membrane pores in controlling cellular responses, and may help with the design and utilization of porous membranes for induction of desired cell processes in the development of biomimetic platforms.

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes.

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis while decreasing osteogenesis.

Microengineered 3D Collagen Gels with Independently Tunable Fiber Anisotropy and Directionality.

Cellular processes, including differentiation, proliferation, and migration, have been linked to the alignment (anisotropy) and orientation (directionality) of collagen fibers in the native extracellular matrix (ECM). Given the critical role that biophysical cell-matrix interactions play in regulating biological functions, several microfluidic-based methods have been used to establish 3D collagen gels with defined fiber properties; these gels have helped to establish quantitative relationships between structural ECM cues and observed cell responses. Although existing microfluidic fabrication methods provide excellent definition over collagen fiber anisotropy, they have not demonstrated the independent control over fiber anisotropy and directionality necessary to replicate in vivo collagen architecture. Therefore, to advance collagen microengineering capabilities, we present a user-friendly technology platform that uses controlled fluid flows within a non-uniform microfluidic channel network to create collagen landscapes that can be tuned as a function of extensional strain rate. Herein, we demonstrate capabilities to i) control the degree of fiber anisotropy, ii) create spatial gradients in fiber anisotropy, iii) independently define fiber directionality, and iv) generate multi-material interfaces within a 3D environment. We then address the practical issue of integrating cells into microfluidic systems by using a peel-off template technique to provide direct access to microengineered collagen gels, and demonstrate that cells respond to the defined properties of the landscape. Finally, the platform's modular capability is highlighted by integrating a sub-micrometer thick porous parylene membrane onto the microengineered collagen as a method to define cell-substrate interactions.

Simple and robust fabrication and characterization of conductive carbonized nanofibers loaded with gold nanoparticles for bone tissue engineering applications.

Bone tissue engineering is a new and applicable emerging approach to repair bone defects. Electrical conductive scaffolds through a physiologically relevant physical signaling, i.e., electrical stimulation, are highly promising candidates for tissue engineering applications. In this paper, we fabricated carbon nanofiber/gold nanoparticle (CNF/AuNP) conductive scaffolds using two distinct methods. These methods are blending electrospinning in which AuNPs were blended with electrospinning solution, and electrospinning/electrospraying in which AuNPs were electrosprayed simultaneously with electrospinning. The obtained electrospun mats underwent a stabilization/carbonization process. The scaffolds were characterized by SEM, XRD, FT-IR, and Raman spectroscopy. SEM characterizations showed improved morphology and a slight decrease in the diameter of the electrospinned and electrosprayed nanofibers (from 178.66 ± 38.40 nm to 157.94 ± 24.14 nm and 120.81 ± 13.77 nm, respectively). Raman spectroscopy showed improvement in the graphitization. Electrical conductivity improved by up to 29.2% and 81% in electrospraying and blending electrospinning modes, respectively. Indirect MTT and LDH toxicity assays directly were performed to assess MG63 cell toxicity, but no significant toxicity was observed, and the scaffolds did not adversely affect cell proliferation. It can be concluded these scaffolds have the potential for bone tissue engineering applications.

Micropatterned Poly(ethylene glycol) Islands Disrupt Endothelial Cell-Substrate Interactions Differently from Microporous Membranes.

Porous membranes are ubiquitous in cell co-culture and tissue-on-a-chip studies. These materials are predominantly chosen for their semi-permeable and size exclusion properties to restrict or permit transmigration and cell-cell communication. However, previous studies have shown pore size, spacing and orientation affect cell behavior including extracellular matrix production and migration. The mechanism behind this behavior is not fully understood. In this study, we fabricated micropatterned non-fouling polyethylene glycol (PEG) islands to mimic pore openings in order to decouple the effect of surface discontinuity from potential grip on the vertical contact area provided by pore wall edges. Similar to previous findings on porous membranes, we found that the PEG islands hindered fibronectin fibrillogenesis with cells on patterned substrates producing shorter fibrils. Additionally, cell migration speed over micropatterned PEG islands was greater than unpatterned controls, suggesting that disruption of cell-substrate interactions by PEG islands promoted a more dynamic and migratory behavior, similarly to enhanced cell migration on microporous membranes. Preferred cellular directionality during migration was nearly indistinguishable between substrates with identically patterned PEG islands and previously reported behavior over micropores of the same geometry, further confirming disruption of cell-substrate interactions as a common mechanism behind the cellular responses on these substrates. Interestingly, compared to respective controls, there were differences in cell spreading and a lower increase in migration speed over PEG islands compared prior results on micropores with identical feature size and spacing. This suggests that membrane pores not only disrupt cell-substrate interactions, but also provide additional physical factors that affect cellular response.

Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: in vitro and in vivo studies.

Functional wound dressing with tailored physicochemical and biological properties is vital for diabetic foot ulcer (DFU) treatment. Our main objective in the current study was to fabricate Cellulose Acetate/Gelatin (CA/Gel) electrospun mat loaded with berberine (Beri) as the DFU-specific wound dressing. The wound healing efficacy of the fabricated dressings was evaluated in streptozotocin-induced diabetic rats. The results demonstrated an average nanofiber diameter of 502 ± 150 nm, and the tensile strength, contact angle, porosity, water vapor permeability and water uptake ratio of CA/Gel nanofibers were around 2.83 ± 0.08 MPa, 58.07 ± 2.35°, 78.17 ± 1.04%, 11.23 ± 1.05 mg/cm2/hr, and 12.78 ± 0.32%, respectively, while these values for CA/Gel/Beri nanofibers were 2.69 ± 0.05 MPa, 56.93 ± 1°, 76.17 ± 0.76%, 10.17 ± 0.21 mg/cm2/hr, and 14.37 ± 0.42%, respectively. The antibacterial evaluations demonstrated that the dressings exhibited potent antibacterial activity. The collagen density of 88.8 ± 6.7% and the angiogenesis score of 19.8 ± 3.8 obtained in the animal studies indicate a proper wound healing. These findings implied that the incorporation of berberine did not compromise the physical properties of dressing, while improving the biological activities. In conclusion, our results indicated that the prepared mat is a proper wound dressing for DFU management and treatment.

Naturally occurring biological macromolecules-based hydrogels: Potential biomaterials for peripheral nerve regeneration.

Despite the recent advances in the treatment strategies of peripheral nerve system defects, peripheral nerve injury (PNI) is still one of the most important health issues with increasing incidence worldwide. The most commonly used treatment approaches are allografts, xenografts, and autologous, which have some drawbacks, including complications, limited source of the donor tissue, tubular collapse, and scar tissue formation. In this context, regenerative medicine has been introduced as a powerful approach to improve the healing process and obtain acceptable functional recovery in the injury site using living cells, scaffold, and bioactive (macro-) molecules. Amongst them, scaffold as a three-dimensional (3D) support biomaterial, structurally bridged the gap or site of injury in order to provide physical and chemical cues to promote correct reinnervation and functional regeneration. Amongst different scaffolding biomaterials, naturally occurring biological macromolecules (more especially proteins and polysaccharides)-based hydrogels exhibited promising results due to their fascinating physicochemical, as well as physiologically relevant properties. This review highlights the recent progress in the development of natural hydrogels-based neural scaffolds. Furthermore, PNI healing process, current status, and challenges are also shortly discussed.

In vitro Studies of Transendothelial Migration for Biological and Drug Discovery.

Inflammatory diseases and cancer metastases lack concrete pharmaceuticals for their effective treatment despite great strides in advancing our understanding of disease progression. One feature of these disease pathogeneses that remains to be fully explored, both biologically and pharmaceutically, is the passage of cancer and immune cells from the blood to the underlying tissue in the process of extravasation. Regardless of migratory cell type, all steps in extravasation involve molecular interactions that serve as a rich landscape of targets for pharmaceutical inhibition or promotion. Transendothelial migration (TEM), or the migration of the cell through the vascular endothelium, is a particularly promising area of interest as it constitutes the final and most involved step in the extravasation cascade. While in vivo models of cancer metastasis and inflammatory diseases have contributed to our current understanding of TEM, the knowledge surrounding this phenomenon would be significantly lacking without the use of in vitro platforms. In addition to the ease of use, low cost, and high controllability, in vitro platforms permit the use of human cell lines to represent certain features of disease pathology better, as seen in the clinic. These benefits over traditional pre-clinical models for efficacy and toxicity testing are especially important in the modern pursuit of novel drug candidates. Here, we review the cellular and molecular events involved in leukocyte and cancer cell extravasation, with a keen focus on TEM, as discovered by seminal and progressive in vitro platforms. In vitro studies of TEM, specifically, showcase the great experimental progress at the lab bench and highlight the historical success of in vitro platforms for biological discovery. This success shows the potential for applying these platforms for pharmaceutical compound screening. In addition to immune and cancer cell TEM, we discuss the promise of hepatocyte transplantation, a process in which systemically delivered hepatocytes must transmigrate across the liver sinusoidal endothelium to successfully engraft and restore liver function. Lastly, we concisely summarize the evolving field of porous membranes for the study of TEM.

Robust and Gradient Thickness Porous Membranes for In Vitro Modeling of Physiological Barriers.

Porous membranes are fundamental elements for tissue-chip barrier and co-culture models. However, the exaggerated thickness of commonly available membranes may represent a stumbling block impeding a more accurate in vitro modeling. Existing techniques to fabricate membranes such as solvent cast, spin-coating, sputtering and PE-CVD result in uniform thickness films. Here, we developed a robust method to generate ultrathin porous parylene C (UPP) membranes not just with precise thicknesses down to 300 nm, but with variable gradients in thicknesses, while at the same time having porosities up to 25%. We also show surface etching and increased roughness lead to improved cell attachment. Next, we examined the mechanical properties of UPP membranes with varying porosity and thickness and fit our data to previously published models, which can help determine practical upper limits of porosity and lower limits of thickness. Lastly, we validate a straightforward approach allowing the successful integration of the UPP membranes into a prototyped 3D-printed scaffold, demonstrating mechanical robustness and allowing cell adhesion under varying flow conditions. Collectively, our results support the integration and the use of UPP membranes to examine cell-cell interaction in vitro.

Preparation and characterization of novel functionalized multiwalled carbon nanotubes/chitosan/ß-Glycerophosphate scaffolds for bone tissue engineering.

A major limitation in current tissue engineering scaffolds is that some of the most important characteristics of the intended tissue are ignored. As piezoelectricity and high mechanical strength are two of the most important characteristics of the bone tissue, carbon nanotubes are getting a lot of attention as a bone tissue scaffold component in recent years. In the present study, composite scaffolds comprised of functionalized Multiwalled Carbon Nanotubes (f-MWCNT), medium molecular weight chitosan and ß-Glycerophosphate were fabricated and characterized. Biodegradability and mechanical tests indicate that while increasing f-MWCNT content can improve electrical conductivity and mechanical properties, there are some limitations for these increases, such as a decrease in mechanical properties and biodegradability in 1w/v% content of f-MWCNTs. Also, MTT cytotoxicity assay was conducted for the scaffolds and no significant cytotoxicity was observed. Increasing f-MWCNT content led to higher alkaline Phosphatase activity. The overall results show that composites with f-MWCNT content between 0.1w/v% and 0.5w/v% are the most suitable for bone tissue engineering application. Additionally, Preliminary cell electrical tests proved the efficiency of the prepared scaffolds for cell electrical applications.