Fabrication and characterization of bioprints with Lactobacillus crispatus for vaginal application.

Bacterial vaginosis (BV) is characterized by low levels of lactobacilli and overgrowth of potential pathogens in the female genital tract. Current antibiotic treatments often fail to treat BV in a sustained manner, and > 50% of women experience recurrence within 6 months post-treatment. Recently, lactobacilli have shown promise for acting as probiotics by offering health benefits in BV. However, as with other active agents, probiotics often require intensive administration schedules incurring difficult user adherence. Three-dimensional (3D)-bioprinting enables fabrication of well-defined architectures with tunable release of active agents, including live mammalian cells, offering the potential for long-acting probiotic delivery. One promising bioink, gelatin alginate has been previously shown to provide structural stability, host compatibility, viable probiotic incorporation, and cellular nutrient diffusion. This study formulates and characterizes 3D-bioprinted Lactobacillus crispatus-containing gelatin alginate scaffolds for gynecologic applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine formulations with highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity via mass loss and swelling measurements. Post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated greatest structural stability with dual genipin and calcium crosslinking, showing minimal mass loss and swelling over 28 days. 3D-bioprinted L. crispatus-containing scaffolds demonstrated sustained release and proliferation of live bacteria over 28 days, without impacting viability of vaginal epithelial cells. This study provides in vitro evidence for 3D-bioprinted scaffolds as a novel strategy to sustain probiotic delivery with the ultimate goal of restoring vaginal lactobacilli following microbiological disturbances.

Polymer Surface Dissection for Correlated Microscopic and Compositional Analysis of Bacterial Aggregates during Membrane Biofouling.

Multispecies biofilms are a common limitation in membrane bioreactors, causing membrane clogging, degradation, and failure. There is a poor understanding of biological fouling mechanisms in these systems due to the limited number of experimental techniques useful for probing microbial interactions at the membrane interface. Here, we develop a new experimental method, termed polymer surface dissection (PSD), to investigate multispecies assembly processes over membrane surfaces. The PSD method uses photodegradable polyethylene glycol hydrogels functionalized with bioaffinity ligands to bind and detach microscale, microbial aggregates from the membrane for microscopic observation. Subsequent exposure of the hydrogel to high resolution, patterned UV light allows for controlled release of any selected aggregate of desired size at high purity for DNA extraction. Follow-up 16S community analysis reveals aggregate composition, correlating microscopic images with the bacterial community structure. The optimized approach can isolate aggregates with microscale spatial precision and yields genomic DNA at sufficient quantity and quality for sequencing from aggregates with areas as low as 2000 µm2, without the need of culturing for sample enrichment. To demonstrate the value of the approach, PSD was used to reveal the composition of microscale aggregates of different sizes during early-stage biofouling of aerobic wastewater communities over PVDF membranes. Larger aggregates exhibited lower diversity of bacterial communities, and a shift in the community structure was found as aggregate size increased to areas between 25,000 and 45,000 µm2, below which aggregates were more enriched in Bacteroidetes and above which aggregates were more enriched with Proteobacteria. The findings demonstrate that community succession can be observed within microscale aggregates and that the PSD method is useful for identification and characterization of early colonizing bacteria that drive biofouling on membrane surfaces.

Perspectives on Existing and Novel Alternative Intravaginal Probiotic Delivery Methods in the Context of Bacterial Vaginosis Infection.

Bacterial vaginosis (BV) is one of the most common vaginal infections that affects hundreds of millions of women of reproductive age, worldwide. Traditional treatment strategies, such as oral and topical antibiotics, have shown efficacy against BV, but frequent recurrence of infection and the development of antibiotic-resistant bacteria remain as significant challenges. Alternatively, recent progress in understanding immune, microbiological, and metabolic interactions in the vaginal microbiota has prompted the consideration of administering probiotic organisms to restore and maintain vaginal health within the context of BV prevention and treatment. Given this, the objective of this review is to discuss existing and potential alternative approaches to deliver, and to potentially sustain the delivery of probiotics, to prevent and/or treat BV infections. First, a brief overview is provided regarding the probiotic species and combinatorial probiotic strategies that have shown promise in the treatment of BV and in restoring female reproductive health. Additionally, the advantages and challenges associated with current oral and intravaginal probiotic delivery platforms are discussed. Lastly, we present emerging and promising alternative dosage forms, such as electrospun fibers and 3D bioprinted scaffolds, that may be adapted as new strategies to intravaginally deliver probiotic organisms. Graphical abstract.

Photodegradable Hydrogels for Rapid Screening, Isolation, and Genetic Characterization of Bacteria with Rare Phenotypes.

Screening mutant libraries (MLs) of bacteria for strains with specific phenotypes is often a slow and laborious process that requires assessment of tens of thousands of individual cell colonies after plating and culturing on solid media. In this report, we develop a three-dimensional, photodegradable hydrogel interface designed to dramatically improve the throughput of ML screening by combining high-density cell culture with precision extraction and the recovery of individual, microscale colonies for follow-up genetic and phenotypic characterization. ML populations are first added to a hydrogel precursor solution consisting of polyethylene glycol (PEG) o-nitrobenzyl diacrylate and PEG-tetrathiol macromers, where they become encapsulated into 13 µm thick hydrogel layers at a density of 90 cells/mm2, enabling parallel monitoring of 2.8 × 104 mutants per hydrogel. Encapsulated cells remain confined within the elastic matrix during culture, allowing one to track individual cells that grow into small, stable microcolonies (45 ± 4 µm in diameter) over the course of 72 h. Colonies with rare growth profiles can then be identified, extracted, and recovered from the hydrogel in a sequential manner and with minimal damage using a high-resolution, 365 nm patterned light source. The light pattern can be varied to release motile cells, cellular aggregates, or microcolonies encapsulated in protective PEG coatings. To access the benefits of this approach for ML screening, an Agrobacterium tumefaciens C58 transposon ML was screened for rare, resistant mutants able to grow in the presence of cell free culture media from Rhizobium rhizogenes K84, a well-known inhibitor of C58 cell growth. Subsequent genomic analysis of rare cells (9/28,000) that developed into microcolonies identified that seven of the resistant strains had mutations in the acc locus of the Ti plasmid. These observations are consistent with past research demonstrating that the disruption of this locus confers resistance to agrocin 84, an inhibitory molecule produced by K84. The high-throughput nature of the screen allows the A. tumefaciens genome (approximately 5.6 Mbps) to be screened to saturation in a single experimental trial, compared to hundreds of platings required by conventional plating approaches. As a miniaturized version of the gold-standard plating assay, this materials-based approach offers a simple, inexpensive, and highly translational screening technique that does not require microfluidic devices or complex liquid handling steps. The approach is readily adaptable to other applications that require isolation and study of rare or phenotypically pure cell populations.

Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution.

Lectin-functional interfaces are useful for isolation of bacteria from solution because they are low-cost and allow nondestructive, reversible capture. This study provides a systematic investigation of physical and chemical surface parameters that influence bacteria capture over lectin-functionalized polymer interfaces and then applies these findings to construct surfaces with significantly enhanced bacteria capture. The designer block copolymer poly(glycidyl methacrylate)- block-poly(vinyldimethyl azlactone) was used as a lectin attachment layer, and lectin coupling into the polymer film through azlactone-lectin coupling reactions was first characterized. Here, experimental parameters including polymer areal chain density, lectin molecular weight, and lectin coupling buffer were systematically varied to identify parameters driving highest azlactone conversions and corresponding lectin surface densities. To introduce physical nanostructures into the attachment layer, nanopillar arrays (NPAs) of varied heights (300 and 2100 nm) were then used to provide an underlying surface template for the functional polymer layer. Capture of Escherichia coli on lectin-polymer surfaces coated over both flat and NPA surfaces was then investigated. For flat polymer interfaces, bacteria were detected on the surface after incubation at a solution concentration of 103 cfu/mL, and a corresponding detection limit of 1.7 × 103 cfu/mL was quantified. This detection limit was 1 order of magnitude lower than control lectin surfaces functionalized with standard, carbodiimide coupling chemistry. NPA surfaces containing 300 nm tall pillars further improved the detection limit to 2.1 × 102 cfu/mL, but also reduced the viability of captured cells. Finally, to investigate the impact of cell surface parameters on capture, we used Agrobacterium tumefaciens cells genetically modified to allow manipulation of exopolysaccharide adhesin production levels. Statistical analysis of surface capture levels revealed that lectin surface density was the primary factor driving capture, as opposed to exopolysaccharide adhesin expression. These findings emphasize the critical importance of the synthetic interface and the development of surfaces that combine high lectin densities with tailored physical features to drive high levels of capture. These insights will aid in design of biofunctional interfaces with physicochemical surface properties favorable for capture and isolation of bacteria cells from solutions.

An investigation into electrochemical properties of poly(ether sulfone)/poly(vinyl pyrrolidone) heterogeneous cation-exchange membranes by using design of experiment method.

Poly(ether sulfone) (PES)/poly(vinyl pyrrolidone) (PVP) blend heterogeneous cation exchange membranes were prepared by solution casting technique using dimethylformamide as solvent and cation exchange resin powder as functional groups agent. In this study, Taguchi experiment design method was employed for investigating the effects of controlling variables including polymer binder (PVP + PES) to total casting solution ratio, blend ratio of polymer binders (PVP to PES), resin to polymer binder ratio, and casting temperature on electrochemical characteristics of PES/PVP heterogeneous membranes. To this aim, each factor was considered at 4 different levels and therefore, 16 experiments were designed. To improve the quality of the membranes ultrasonic was used for appropriate dispersing of resin particles in the matrix of the membranes. According to the results, the averaged maximum values of 1.535 meq/g and 46.6 mV were obtained for IEC and membrane potential, respectively. Also, the highest obtained value of ion permeability tests was equal to 1.33 m/s. Finally, the synthesis conditions was optimized by considering the IEC and membrane potential as the objective functions which gave the values of 1.55 meq/g and 1.39 m/s for IEC and membrane potential, respectively, which proved that the synthesized membrane can be considered as a promising heterogeneous membrane.

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers.

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 µm). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.