Genome-wide identification and characterization of major latex-like protein genes responsible for crop contamination in Cucurbita pepo.

BACKGROUND: Zucchini plants (Cucurbita pepo) accumulate persistent organic pollutants (POPs) at high concentrations in their aerial parts, and major latex-like proteins (MLPs) play crucial roles in their accumulation. MLPs bind to POPs in root cells, MLP-POP complexes are then translocated into xylem vessels, and POPs are transported to the aerial parts. We previously identified three CpMLP genes (MLP-PG1, MLP-GR1, and MLP-GR3) as transporting factors for POPs; however, other studies have shown that the genomes of several plant species contain more than 10 MLP genes, thus, further MLP genes responsible for POP accumulation may have been overlooked. METHODS AND RESULTS: Here, we investigated the number of CpMLP genes by performing a hidden Markov model search against the C. pepo genome database and characterized their effects on POP accumulation by performing the expression analysis in the organs and in silico structural analysis. The C. pepo genome contained 21 CpMLP genes, and several CpMLP genes, including MLP-PG1 and MLP-GR3, were highly expressed in roots. 3D structural prediction showed that all examined CpMLPs contained a cavity with a hydrophobic region, which facilitated binding to POPs. CONCLUSIONS: The present study provides insights regarding CpMLP genes responsible for POP accumulation.

A comparison between ß-tricalcium phosphate and chitosan poly-caprolactone-based 3D melt extruded composite scaffolds.

Melt extrusion 3D printing has become an attractive additive manufacturing technology to construct degradable scaffolds as tissue precursors in order to create clinically relevant medical devices. Towards this end, a commonly used synthetic polyester, poly-caprolactone (PCL), was used to make scaffolds composed of different biomaterial compositions to increase bioactivity using 3D melt pneumatic extrusion technology. Varying ratios of the natural biopolymer, chitosan, or the bioceramic, ß-tricalcium phosphate (TCP) were blended with PCL to fabricate support scaffolds with three-dimensional (3D) architecture for human bone-marrow derived mesenchymal stem cell (hBMSC) growth for potential bone regeneration application. In this study, basic printing requirements as well as biomaterial dynamic mechanical (DMA), elemental, and thermogravimetric (TGA) analysis results demonstrate material homogeneity as well as thermal stability. Scaffold morphology and microarchitecture were assessed using scanning electron microscopy (SEM) alongside in vitro scaffold degradation and biological characterisation. Human BMSC proliferation was assessed using fluorescence imaging, and quantitated via the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. These in vitro cell viability studies revealed that the highest chitosan concentration blend of 20% favoured the most hBMSC growth, exhibited the most swelling, and showed minimal degradation after 28 days. The 20% TCP blend had the second highest hBMSC growth, exhibited moderate swelling, and the fastest degradation rate. Overall, this study demonstrates the first direct comparison of a natural biopolymer-based, that is, chitosan, 3D melt extruded PCL composite with that of a bioceramic-based, that is, ß-TCP, PCL composite and their effects on hBMSC 3D proliferation. 3D melt extruded PCL-based composite scaffolds methodology offers a straightforward way to print scaffolds with good shape fidelity, interconnected porosities and enhanced bioactivity; and demonstrates their potential use for regenerative, bone repair applications.

Intervertebral Disc Tissue Engineering Using Additive Manufacturing.

Intervertebral disc (IVD) degeneration is one of the major causes of lower back pain, a common health condition that greatly affects the quality of life. With an increasing elderly population and changes in lifestyle, there exists a high demand for novel treatment strategies for damaged IVDs. Researchers have investigated IVD tissue engineering (TE) as a way to restore biological and mechanical functions by regenerating or replacing damaged discs using scaffolds with suitable cells. These scaffolds can be constructed using material extrusion additive manufacturing (AM), a technique used to build three-dimensional (3D), custom discs utilising computer-aided design (CAD). Structural geometry can be controlled via the manipulation of printing parameters, material selection, temperature, and various other processing parameters. To date, there are no clinically relevant TE-IVDs available. In this review, advances in AM-based approaches for IVD TE are briefly discussed in order to achieve a better understanding of the requirements needed to obtain more effective, and ultimately clinically relevant, IVD TE constructs.

Three-Dimensional Melt-Electrowritten Polycaprolactone/Chitosan Scaffolds Enhance Mesenchymal Stem Cell Behavior.

Melt electrowriting (MEW) is an emerging technique that precisely fabricates microfibrous scaffolds, ideal for tissue engineering, where biomimetic microarchitectural detail is required. Polycaprolactone (PCL), a synthetic polymer, was selected as the scaffold material due to its biocompatibility, biodegradability, mechanical strength, and melt processability. To increase PCL bioactivity, a natural polymer, chitosan, was added to construct MEW fibrous composite scaffolds. To date, this is the first study of its kind detailing the effects of stem cell behavior on PCL containing chitosan MEW scaffolds. The aim of this study was to melt electrowrite a range of PCL/chitosan tissue-engineered constructs (TECs) and assess their suitability to promote the growth of human bone-marrow-derived mesenchymal stem cells (hBMSCs). In vitro physical and biological characterizations of melt-electrowritten TECs were performed. Physical characterization showed that reproducible, layered micron-range scaffolds could be successfully fabricated. As well, cell migration and proliferation were assessed via an assay to monitor cell infiltration throughout the three-dimensional (3D) melt-electrowritten scaffold structure. A statistically significant increase (∼140%) in hBMSC proliferation in 1 wt % chitosan PCL blends in comparison to PCL-only scaffolds was found when monitored over two weeks. Overall, our study demonstrates the fabrication of melt-electrowritten PCL/chitosan composite scaffolds with controlled microarchitecture and their potential use for regenerative, tissue engineering applications.

Melt Electrowritten Sandwich Scaffold Technique Using Sulforhodamine B to Monitor Stem Cell Behavior.

Background: Three-dimensional (3D) printing using melt electrowriting (MEW) technology is a recently developed technique to produce biocompatible micron-level mesh scaffolds layer-by-layer that can be seeded with cells for tissue engineering. Examining cell behavior, such as growth rate and migration, can be problematic in these opaque 3D scaffolds. A straightforward and quantitative method was developed to examine these cellular parameters on poly-ɛ-caprolactone (PCL) multilayered MEW scaffolds developed as components of the annulus fibrosus region of bioengineered intervertebral discs. Experiment: The anti-adhesion protein, bovine serum albumin (BSA), was used to coat plasticware to improve mesenchymal stem cell (T0523) adhesion to MEW scaffolds. Cells were seeded on circular MEW (cMEW) discs as defined growth starting points sandwiched between two test template scaffolds investigated at varying pore sizes. Cell expansion, growth, and migration were quantitated utilizing the protein-specific dye sulforhodamine B (SRB). Live cell imaging combined with image analysis were used to examine cell motility and expansion on 3D scaffolds. Results: After one coating of BSA, cells remained nonadherent for the duration of the study with cell spheroids formed and enlarging over 21 days and becoming entangled in MEW scaffold pores. Cells grown on the 250 µm pore size scaffolds exhibited a doubling time of 7 days, whereas the 400 µm pore size scaffolds time was 11.5 days. Conclusions: BSA coating of tissue culture dishes prevented surface adhesion of cells to vessel surfaces and promoted spheroid formation that encouraged attachment to the PCL scaffolds. Batch-printed cMEW scaffolds were useful as a defined starting point for quantitative assays that successfully measured cell migration, expansion and proliferation on test scaffolds. The SRB assay was shown to be a useful and straightforward way to quantitate cell numbers in multilayered MEW scaffolds. A pore size of 250 µm exhibited the fastest cell growth, spread, and expansion. Impact statement In this article, a new, useful, and straightforward method to quantitate cell numbers on three-dimensional (3D) melt electrowritten (MEW) scaffolds is presented. By using the sulforhodamine B assay on bovine serum albumin-coated dishes cell migration, expansion and proliferation in 3D printed MEW test scaffolds were quantitatively measured. Printed circular MEW (cMEW) scaffolds sandwiched between two MEW test scaffolds (Fig. 3) were used as defined cellular growth starting points with a particular pore size of 250 µm displaying the fastest cell growth and migration. This MEW sandwich technique could potentially be used to quantitate cell numbers and migration in other 3D multilayered MEW scaffold systems.

Alveolar bone loss in rats infected with a strain of Prevotella intermedia and Fusobacterium nucleatum isolated from a child with prepubertal periodontitis.

Prevotella intermedia and fusobacterium nucleatum are associated with various forms of periodontal disease. The purpose of the present study was to infect the clinical isolates of these periodontopathic bacteria and to induce a significant loss of alveolar bone in specific pathogen-free (SPF) rats in the absence of ligatures. P. intermedia YKD8 and F. nucleatum YKZ5 were isolated from a prepubertal periodontitis patient, while P. gingivalis MWB13 was from a patient with juvenile periodontitis. At first, SPF Sprague-Dawley rats (70 days of age, male) were infected with A. viscosus Ny1R and subsequently superinfected with P. gingivalis MWB13, P. intermedia YKD8, or F. nucleatum YKZ5, respectively. The control group was infected with A. viscosus Ny1R alone. All rats were killed and periodontal bone levels were assessed morphometrically 135 days after the first infection with A. viscosus. P. intermedia YKD8 was recovered frequently from rats, with serum antibody levels remaining highly elevated throughout the experiment. Significant loss of alveolar bone was found in rats infected with P. intermedia YKD8, the virulence of which was equivalent to that of P. gingivalis MWB13. F. nucleatum YKZ5 also induced alveolar bone loss, but not significantly when compared with rats infected with A. viscosus Ny1R alone.

Clinical, microbiological and host defense parameters associated with a case of localized prepubertal periodontitis.

A 4-year-old Japanese boy was referred to Osaka University Dental Hospital because of severe mobility and pain of the right lower primary canine. The canine had severe bone loss and a pocket depth exceeding 5-6 mm. The left lower canine showed slight mobility and moderate alveolar bone loss. The other primary teeth showed no pathogenic findings. The subgingival microflora from the right lower canine was dominated by gram-negative rods, especially capnocytophaga and fusobacterium, while actinomyces sp. were the most common gram-positive bacteria. While neutrophil functions of the patient were within the normal ranges of healthy subjects, some lymphocyte functions such as IL-2 production and IgG and IgM syntheses were lower in the patient. 7 months after the extraction of the right lower primary canine, the patient complained of pain around the right lower primary lateral incisor. In 3-4 weeks, the alveolar bone was lost rapidly and mobility of the lower anterior teeth increased significantly. The primary lateral incisor was extracted and the other primary teeth were treated by sealing and systemic and local administration of antibiotics. After treatment, the lower anterior teeth became less mobile and the gram-positive cocci predominated.