Robotics-Driven Manufacturing of Cartilaginous Microtissues for Skeletal Tissue Engineering Applications.

Automated technologies are attractive for enhancing the robust manufacturing of tissue-engineered products for clinical translation. In this work, we present an automation strategy using a robotics platform for media changes, and imaging of cartilaginous microtissues cultured in static microwell platforms. We use an automated image analysis pipeline to extract microtissue displacements and morphological features as noninvasive quality attributes. As a result, empty microwells were identified with a 96% accuracy, and dice coefficient of 0.84 for segmentation. Design of experiment are used for the optimization of liquid handling parameters to minimize empty microwells during long-term differentiation protocols. We found no significant effect of aspiration or dispension speeds at and beyond manual speed. Instead, repeated media changes and time in culture were the driving force or microtissue displacements. As the ovine model is the preclinical model of choice for large skeletal defects, we used ovine periosteum-derived cells to form cartilage-intermediate microtissues. Increased expression of COL2A1 confirms chondrogenic differentiation and RUNX2 shows no osteogenic specification. Histological analysis shows an increased secretion of cartilaginous extracellular matrix and glycosaminoglycans in larger microtissues. Furthermore, microtissue-based implants are capable of forming mineralized tissues and bone after 4 weeks of ectopic implantation in nude mice. We demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous microtissues and anticipate the progressive substitution of manual operations with automated solutions for the manufacturing of microtissue-based living implants.

Engineering bone-forming biohybrid sheets through the integration of melt electrowritten membranes and cartilaginous microspheroids.

Bone fractures are one of the most common traumatic large-organ injuries and although many fractures can heal on their own, 2-12% of fractures are slow healing or do not heal (non-unions). Autologous grafts are currently used for treatment of non-unions but are associated with limited healthy bone tissue. Tissue engineered cell-based products have promise for an alternative treatment method. It was previously demonstrated that cartilaginous microspheroids of periosteum-derived cells could be assembled into scaffold-free constructs and heal murine critically-sized long bone defects (non-unions). However, the handleability of such scaffold-free implants can be compromised when scaling-up. In this work, cartilaginous spheroids were combined with melt electrowritten (MEW) meshes to create an engineered cell-based implant, able to induce in vivo bone formation. MEW polycaprolactone meshes were tailored to contain pores (116 ± 28 µm) of a size that captured microspheroids (180 ± 15 µm). Periosteum-derived microspheroids pre-cultured for 4 days, were seeded on MEW meshes and gene expression analysis demonstrated up-regulation of chondrogenic (SOX9, COL2) and prehypertrophic (VEGF) gene markers after 14 days, creating a biohybrid sheet. When implanted subcutaneously (4 weeks), the biohybrid sheets mineralized (23 ± 3% MV/TV) and formed bone and bone marrow. Bone formation was also observed when implanted in a murine critically-sized long bone defect, though a high variation between samples was detected. The high versatility of this biofabrication approach lies in the possibility to tailor the scaffolds to shape and dimensions corresponding to the large bone defects and the individual patient using robust bone forming building blocks. These strategies are instrumental in the development of personalized regenerative therapies with predictive clinical outcomes. STATEMENT OF SIGNIFICANCE: Successful treatments for healing of large long bone defects are still limited and 2-12% of fractures do not heal properly. We combined a novel biofabrication technique: melt electrowriting (MEW), with robust biology: bone forming cartilaginous spheroids to create biohybrid sheets able to form bone upon implantation. MEW enabled the fabrication of scaffolds with micrometer-sized fibers in defined patterns which allowed the capturing of and merging with cartilaginous spheroids which had the potency to mature into bone via the developmental process of endochondral ossification. The present study contributes to the rapidly growing field of "Biofabrication with Spheroid and Organoid Materials'' and demonstrates design considerations that are of great importance for biofabrication of functional tissues through the assembly of cellular spheroids.

Vicia faba Hypersensitivity and ASA Intolerance in a Farmer: A Case Report.

The IgE-mediated allergic reactions to food are caused, generally, by ingestion. However, they can be rarely induced by exposure to airborne food particles through the handling or the cooking. Vicia faba is a vegetable which belongs to Legumes or Fabaceae family, Fabales order. Allergic reactions after ingestion of legumes and cases of asthma after exposure to the cooking vapors have been reported in the literature. A paper assessed the volatile substances (insect repellents) released by V. faba. The authors demonstrated that this plant produces several chemical substances, such as small quantities of methyl salicylate. We describe a case of occupational allergy, induced by handling during picking up of fresh broad beans, in a farmer with history of adverse reaction after eating the cooked and raw vegetable.

Biological and genetic evolution of HIV type 1 in two siblings with different patterns of disease progression.

To investigate the immunological and virological factors that may lead to different patterns of disease progression characteristic of HIV-1-infected children, two HIV-1-infected siblings, a slow and a fast progressor, were followed prospectively before the onset of highly active antiretroviral therapy. Viral coreceptor usage, including the use of CCR5/CXCR4 chimeric receptors, macrophage tropism, and sensitivity to the CC-chemokine RANTES, has been studied. An autologous and heterologous neutralizing antibody response has been documented using peripheral blood mononuclear cells- and GHOST(3) cell line-based assays. Viral evolution was investigated by env C2-V3 region sequence analysis. Although both siblings were infected with HIV-1 of the R5 phenotype, their viruses showed important biological differences. In the fast progressor there was a higher RANTES sensitivity of the early virus, an increased trend to change the mode of CCR5 receptor use, and a larger genetic evolution. Both children developed an autologous neutralizing antibody response starting from the second year with evidence of the continuous emergence of resistant variants. A marked viral genetic and phenotypic evolution was documented in the fast progressor sibling, which is accompanied by a high viral RANTES sensitivity and persistent neutralizing antibodies.

Potential role of cholinesterases in the invasive capacity of the freshwater bivalve, Anodonta woodiana (Bivalvia: Unionacea): a comparative study with the indigenous species of the genus, Anodonta sp.

To address the potential role of cholinesterase enzymes in the invasive capacity of species, the present study investigated ChE activity in the invasive freshwater bivalve Anodonta woodiana (Lea, 1834) comparing it with that of the indigenous species, Anodonta sp. (Linnaeus, 1758). The invasive capacity of pests has often been linked to their ecological plasticity and high intrinsic genetic variability; however the role played by molecular and cellular mechanisms, generally known as an organism's response to pollution, is unclear. Different substrates and selective ChE enzyme inhibitors were investigated in digestive gland, foot, gonad, adductor muscle and gill tissues while sensitivity to four organophosphate (OP) insecticides was investigated in vitro only in adductor muscle. The invasive species (A. woodiana) showed significantly greater (at least one order of magnitude) ChE activity than the endemic species (Anodonta sp.) (p<0.05) using acetylthiocholine (ASCh) as substrate and the activity was more widely distributed in tissues involved in movement (adductor muscle and foot), respiration, feeding (gills) and reproduction (gonads). Moreover, only the invasive species, A. woodiana, showed detectable ChE (vs. ASCh) activity in gill tissue. No substrate specificity was observed in any tissue of either species as already described for other bivalve species. ChE activity was not inhibited by Iso-OMPA but showed high sensitivity to BW248c51 and eserine. Both species showed moderate to low sensitivities in vitro to OP insecticides in the range 10(-7)-10(-2) M. Calculated IC(50) for fenitrothion and chlorpyrifos was in the range 10(-6)-10(-3) M in muscle of A. woodiana while a higher inhibition was observed for fenitrothion (10(-7) M) and lower for chlorpyrifos (10(-2) M) in the indigenous species Anodonta sp. Similar IC(50) of 10(-5)-10(-6) M were observed for DFP and azamethiphos in both species. The hypotheses of other authors that acetylcholinesterase (AChE) is involved in the control of many essential functions, such as frontal ciliary activity of gill epithelium, temperature resistance, ciliary activity for transport of suspended particulate, valve opening and embryo development, suggest that the high catalytic efficiency of the invasive species may endow it with a competitive advantage over the endemic species. In view of the peculiar reproductive strategy of these mussels, higher ChE vs. ASCh activity in gonads of the invasive species could also favour glochidium production and embryo development under a wider range of environmental conditions.

Prognostic value of the stromal cell-derived factor 1 3'A mutation in pediatric human immunodeficiency virus type 1 infection.

A mutation of the stromal cell-derived factor 1 gene (SDF-1 3'A) was shown to protect adults exposed to human immunodeficiency virus type 1 (HIV-1) from infection and to affect HIV disease progression in adults. The presence of this mutation in HIV-1-infected Kenyan children did not predict mother-to-child virus transmission. The SDF-1 3'A polymorphism was studied in 256 HIV-1-infected, 118 HIV-1-exposed but uninfected, and 170 unexposed and uninfected children of Italian origin, and the frequency of SDF-1 3'A heterozygosity and homozygosity in each of the 3 groups was similar. Of the 256 HIV-1-infected children, 194 were regularly followed up and were assigned to groups according to disease progression. The frequency of the SDF-1 3'A allele was substantially lower among children with long-term nonprogression than among children with rapid (P =.0329) or delayed (P =.0375) progression. We show that the presence of the SDF-1 3'A gene correlates with accelerated disease progression in HIV-1-infected children born to seropositive mothers but does not protect against mother-to-child HIV-1 transmission.