Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Proteomic Fingerprint of Lung Fibrosis Progression and Response to Therapy in Bleomycin-Induced Mouse Model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.

Modeling pulmonary fibrosis through bleomycin delivered by osmotic minipump: a new histomorphometric method of evaluation.

The systemic delivery of bleomycin (BLM) to mice through subcutaneously implanted osmotic minipumps may be used to experimentally mimic the typical features of systemic sclerosis and related interstitial lung diseases. The published studies on this model principally have focused on induced dermal modifications, probably because lung lesions are typically mild, subpleurally localized, and difficult to analyze. The use of high BLM doses to increase their severity has been proposed but is ethically questionable because of the compromising of animal welfare. We propose a tailored histomorphometric method suitable to detect and quantify this type of mild lung lesions. Using a two-step automated image analysis, a peripheral region of interest with a depth of 250 µm from the pleural edge was defined on whole slide images, and the fibrotic foci were histomorphometrically characterized. The effects of different BLM doses on lung alterations were evaluated in C57BL/6 mice and 60 U/kg resulted in a fair compromise between fibrotic lesions and animal welfare. This dose was also tested in time course experiments. The analysis revealed a peak of histological fibrotic-like alterations, cytokine expression, metalloprotease, and macrophagic activation between the 21st and 28th day after pump implant. The induced dermal fibrosis was characterized by the progressive loss of the white dermal adipose layer, an increase in dermal thickness, dermal hyperplasia, and more compacted collagen fibers. Despite the trend toward spontaneous resolution, our model allowed a double organ readout of the BLM effect and the identification of a therapeutic window for testing pharmacological compounds without using life-threatening doses.

In vitro and in vivo characterization of poractant alfa supplemented with budesonide for safe and effective intratracheal administration.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

The integration of orthodontic miniscrews under mechanical loading: a pre-clinical study in rabbit.

INTRODUCTION: Orthodontic miniscrews are an increasingly popular choice to achieve absolute anchorage. The temporary use of miniscrews and their recent introduction have limited the debate over the biological aspect of the materials to that of the surface that permeates the field of dental implants. The aim of the present study was to investigate the integration of grade 5 titanium mini-implants with machined or sand blasted acid etched surface (SAE) under mechanical load in a rabbit tibia model of implant integration. METHODS: A total of 64 miniscrews (Ti6Al4V) of 1.5 mm diameter and 6.5 mm length were inserted in the proximal medial surface of each tibia in eight male rabbits aged 6 months. Each tibia received four miniscrews. A 100 g nickel-titanium coil spring (Neosentalloy) was applied between two miniscrews along the main axis while two miniscrews were left unloaded. The removal torque was measured for loaded and unloaded miniscrews after 12 weeks. Two miniscrews were harvested for histology. RESULTS: Removal torque was significantly higher for SAE mini-implants than for machined screws, under both loading conditions. Although no difference in bone to implant contact was observed among the groups, cortical area significantly decreased with both surfaces under loading. CONCLUSIONS: Our data indicate that SAE miniscrews have higher bone retention than MA miniscrews, although the effects of mechanical loading of these devices on cortical bone require further investigations.

In vivo monitoring of lung inflammation in CFTR-deficient mice.

BACKGROUND: Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis. METHODS: Wild type (WT) and CF mice were stimulated with P. aeruginosa LPS, TNF-alpha and culture supernatant derived from P. aeruginosa (strain VR1). Lung inflammation was detected by measuring bioluminescence in vivo in mice transiently transgenized with a luciferase reporter gene under the control of a bovine IL-8 gene promoter. RESULTS: Differences in bioluminescence (BLI) signal were revealed by comparing the two types of mice after intratracheal challenge with pro-inflammatory stimuli. BLI increased at 4 h after stimulation with TNF-alpha and at 24 h after administration of LPS and VR1 supernatant in CF mice with respect to untreated animals. The BLI signal was significantly more intense and lasted for longer times in CF animals when compared to WT mice. Analysis of BALF markers: leukocytes, cytokines and histology revealed no significant differences between CF and WT mice. CONCLUSIONS: In vivo gene delivery technology and non-invasive bioluminescent imaging has been successfully adapted to CFTR-deficient mice. Activation of bIL-8 transgene promoter can be monitored by non-invasive BLI imaging in the lung of the same animal and compared longitudinally in both CF or WT mice, after challenge with pro-inflammatory stimuli. The combination of these technologies and the use of CF mice offer the unique opportunity of evaluating the impact of therapies aimed to control inflammation in a CF background.

Morphological analysis of the urethral muscle of the male pig with relevance to urinary continence and micturition.

To investigate whether the pig could be considered a suitable model to study lower urinary tract function and dysfunction, the pelvic urethra of 24 slaughtered male pigs were collected, and the associated muscles were macroscopically, histologically and histochemically analyzed. In cross-sections of the urethra, a muscular complex composed of an inner layer of smooth muscle and an outer layer of striated muscle that are not separated by fascial planes was observed. A tunica muscularis, composed of differently oriented smooth muscle bundles, is only evident in the proximal part of the pelvic urethra while, in the remaining part, it contributes to form the prostatic fibromuscular stroma. The striated urethral muscle surrounds the pelvic urethra in a horseshoe-like configuration with a dorsal longitudinal raphe, extending from the bladder neck to the central tendon of perineum. Proximally to the bladder, it is constituted of slow-twitch and fast-twitch myofibers of very small diameter, and embedded in an abundant collagen and elastic fiber net. Moving caudally it is gradually encircled and then completely substituted by larger and compact myofibers, principally presenting circular orientation and fast-twitch histochemical characteristics. So, like in humans, the cranial tract of the muscular system surrounding the pelvic urethra is principally composed of smooth musculature. The striated component cranially may have a role in blocking retrograde ejaculation, while the middle and caudal tracts may facilitate urine and semen flow, and seem especially concerned with the rapid and forceful urethral closure during active continence. Some differences in the morphology and structure between pigs and humans seem due to the different morphology of the 'secondary' sexual organs that develop from the urethral wall and to the different effect of gravity on the mechanics of the urinary system in quadruped and bipedal mammals.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study.

BACKGROUND: Osteochondral defects significantly affect patients' quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically. RESULTS: The retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage. CONCLUSIONS: In conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.

Short-chain fatty acids modulate the IPEC-J2 cell response to pathogenic E. coli LPS-activated PBMC.

Intestinal disorders can affect pigs of any age, especially when animals are young and more susceptible to infections and environmental stressors. For instance, pathogenic E. coli can alter intestinal functions, thus leading to altered nutrient adsorption by interacting with local cells through lipopolysaccharide (LPS). Among several compounds studied to counteract the negative effects on the intestine, short-chain fatty acids (SCFA) were demonstrated to exert beneficial effects on gut epithelial cells and resident immune cells. In this study, acetate and propionate were tested for their beneficial effects in a co-culture model of IPEC-J2 and porcine PBMC pre-stimulated with LPS from E. coli 0111:B4 aimed at mimicking the interaction between intestinal cells and immune cells in an inflammatory/activated status. IPEC-J2 viability was partially reduced when co-cultured with activated PBMC and nitric oxide concentration increased. IPEC-J2 up-regulated innate and inflammatory markers, namely BD-1, TLR-4, IL-8, TNF-α, NF-κB, and TGF-ß. Acetate and propionate positively modulated the inflammatory condition by sustaining cell viability, reducing the oxidative stress, and down-regulating the expression of inflammatory mediators. TNF-α expression and secretion showed an opposite effect in IPEC-J2 depending on the extent of LPS stimulation of PBMC and TGF-ß modulation. Therefore, SCFA proved to mediate a differential effect depending on the degree and duration of inflammation. The expression of the tight junction proteins (TJp) claudin-4 and zonula occludens-1 was up-regulated by LPS while SCFA influenced TJp with a different kinetics depending on PBMC stimulation. The co-culture model of IPEC-J2 and LPS-activated PBMC proved to be feasible to address the modulation of markers related to anti-bacterial immunity and inflammation, and intestinal epithelial barrier integrity, which are involved in the in vivo responsiveness and plasticity to infections.

A bioartificial and vasculomorphic bone matrix-based organoid mimicking microanatomy of flat and short bones.

We engineered an in vitro model of bioartificial 3D bone organoid consistent with an anatomical and vascular microenvironment common to mammalian flat and short bones. To achieve this, we chose the decellularized-decalcified matrix of the adult male rat scapula, implemented with the reconstruction of its intrinsic vessels, obtained through an original intravascular perfusion with polylevolactic (PLLA), followed by coating of the PLLA-fabricated vascularization with rat tail collagen. As a result, the 3D bone and vascular geometry of the native bone cortical and cancellous compartments was reproduced, and the rat tail collagen-PLLA biomaterial could in vitro act as a surrogate of the perivascular extracellular matrix (ECM) around the wall of the biomaterial-reconstituted cancellous vessels. As a proof-of-concept of cell compatibility and site-dependent osteoinductive properties of this bioartificial 3D construct, we show that it in vitro leads to a time-dependent microtopographic positioning of rat mesenchymal stromal cells (MSCs), initiating an osteogenic fate in relation to the bone compartment. In addition, coating of PLLA-reconstructed vessels with rat tail collagen favored perivascular attachment and survival of MSC-like cells (mouse embryonic fibroblasts), confirming its potentiality as a perivascular stroma for triggering competence of seeded MSCs. Finally, in vivo radiographic topography of bone lesions in the human jaw and foot tarsus of subjects with primary osteoporosis revealed selective bone cortical versus cancellous involvement, suggesting usefulness of a human 3D bone organoid engineered with the same principles of our rat organoid, to in vitro investigate compartment-dependent activities of human MSC in flat and short bones under experimental osteoporotic challenge. We conclude that our 3D bioartificial construct offers a reliable replica of flat and short bones microanatomy, and promises to help in building a compartment-dependent mechanistic perspective of bone remodeling, including the microtopographic dysregulation of osteoporosis.

Preterm rabbit-derived Precision Cut Lung Slices as alternative model of bronchopulmonary dysplasia in preclinical study: a morphological fine-tuning approach.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm delivery, with significant morbidity and mortality in a neonatal intensive care setting. Research in this field aims to identify the mechanisms of late lung development with possible therapeutic targets and the improvement of medical management. Rabbits represent a suitable lab preclinical tool for mimicking the clinical BPD phenotype. Rabbits are born at term in the alveolar phase as occurs in large animals and humans and in addition, they can be delivered prematurely in contrast to mice and rats. Continuous exposure to high oxygen concentration (95% O2) for 7 days induces functional and morphological lung changes in preterm rabbits that resemble those observed in BPD-affected babies. The preclinical research pays great attention to optimize the experimental procedures, reduce the number of animals used in experiments and, where possible, replace animal models with alternative assays, following the principle of the 3 Rs (Replace, Reduce and Refine). The use of in vitro assays based on the ex vivo culture of Precision Cut Lung Slices (PCLS) goes in this direction, representing a good compromise between controlled and flexible in vitro models and the more physiologically relevant in vivo ones. This work aims to set up morphological analyses to be applied in preclinical tests using preterm rabbits derived PCLS, cultured up to 7 days in different oxygen conditions, as a model. After a preliminary optimization of both lung preparation and histological processing methods of the lung slices of 300 µm, the morphological analysis was conducted evaluating a series of histomorphometric parameters derived from those widely used to follow the phases of lung development and its alterations in vivo. Our histomorphometric results demonstrated that the greatest differences from pseudo-normoxia and hyperoxia exposed samples at day 0, used as starting points to compare changes due to treatments and time, are detectable after 4 days of in vitro culture, representing the most suitable time point for analysis in preclinical screening. The combination of parameters suitable for evaluating PCLS morphology in vitro resulted to be Tissue Density and Septal Thickness. Shape Factor and Roughness, evaluated to highlight the increasing complexity of the airspaces, due to the formation of septal crests, gave useful information, however, without significant differences up to day 4. Other parameters like Mean Linear Intercept and Septal Density did not allow to highlight significant differences between different oxygen conditions and time points. Instead, Radial Alveolar Count, could not be applied to PCLS, due to the tissue changes following agar infusion and culture conditions.

Acetate and propionate effects in response to LPS in a porcine intestinal co-culture model.

BACKGROUND: The interest in acetate and propionate as short chain fatty acids (SCFA) derives from research on alternative strategies to the utilization of antibiotics in pig farms. SCFA have a protective role on the intestinal epithelial barrier and improve intestinal immunity by regulating the inflammatory and immune response. This regulation is associated with an increase in intestinal barrier integrity, mediated by the enhancement of tight junction protein (TJp) functions, which prevent the passage of pathogens through the paracellular space. The purpose of this study was to evaluate the effect of in vitro supplementation with SCFA (5 mM acetate and 1 mM propionate) on viability, nitric oxide (NO) release (oxidative stress), NF-κB gene expression, and gene and protein expression of major TJp (occludin [OCLN], zonula occludens-1 [ZO-1], and claudin-4 [CLDN4]) in a porcine intestinal epithelial cell (IPEC-J2) and peripheral blood mononuclear cell (PBMC) co-culture model upon LPS stimulation, through which an acute inflammatory state was simulated. RESULTS: Firstly, the inflammatory stimulus induced by LPS evaluated in the IPEC-J2 monoculture was characterized by a reduction of viability, gene expression of TJp and OCLN protein synthesis, and an increase of NO release. The response evaluated in the co-culture showed that acetate positively stimulated the viability of both untreated and LPS-stimulated IPEC-J2 and reduced the release of NO in LPS-stimulated cells. Acetate also promoted an increase of gene expression of CLDN4, ZO-1, and OCLN, and protein synthesis of CLDN4, OCLN and ZO-1 in untreated and LPS-stimulated cells. Propionate induced a reduction of NO release in both untreated and LPS-stimulated IPEC-J2. In untreated cells, propionate induced an increase of TJp gene expression and of CLDN4 and OCLN protein synthesis. Contrarily, propionate in LPS-stimulated cells induced an increase of CLDN4 and OCLN gene expression and protein synthesis. PBMC were influenced by acetate and propionate supplementation, in that NF-κB expression was strongly downregulated in LPS-stimulated cells. CONCLUSIONS: The present study demonstrates the protective effect of acetate and propionate upon acute inflammation by regulating epithelial tight junction expression and protein synthesis in a co-culture model, which simulates the in vivo interaction between epithelial intestinal cells and local immune cells.

Osmolarity modulates the de-differentiation of horse articular chondrocytes during cell expansion in vitro: implications for tissue engineering in cartilage repair.

Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmolarity (350-450 mOsm/L) compared to normal physiological fluids (~ 300 mOsm/L) and some studies have demonstrated that osmolarity has a chondroprotective effect in vitro and in vivo. Therefore, the response of horse articular chondrocytes to osmolarity changes (280, 380, and 480 mOsm/L) was studied both in proliferating, de-differentiated chondrocytes grown in adhesion, and in differentiated chondrocytes grown in a 3D culture system. To this aim, cell proliferation (cell counting), morphology (optical microscopy), and differentiation (gene expression of specific markers) were monitored along with the expression of osmolyte transporters involved in volume regulation [betaine-GABA transporter (BGT-1), taurine transporter (SLC6A6), and neutral amino acid transporter (SNAT)] real-time qPCR. Proliferating chondrocytes cultured under hyperosmolar conditions showed low proliferation, spheroidal morphology, a significant reduction of de-differentiation markers [collagen type I (Col1) and RUNX2] and an increase of differentiation markers [collagen type II (Col2) and aggrecan]. Notably, a persistently high level of BGT-1 gene expression was maintained in chondrocyte cultures at 380 mOsm/L, and particularly at 480 mOsm/L both in proliferating and differentiated chondrocytes. These preliminary data encourage the study of osmolarity as a microenvironmental co-factor to promote/maintain chondrocyte differentiation in both 2D and 3D in vitro culture systems.

Integration of micro-CT and histology data for vasculature morpho-functional analysis in tissue regeneration.

The demand for artificial or bioartificial engineered tissues is increasing today in regenerative medicine techniques to replace and restore the physiological function of damaged tissues. Such engineered constructs hold different properties depending on the tissue to be replicated. As for vascularized tissues, complex biocompatible structures, namely scaffolds, play a key role in supporting oxygen and nutrient supply, thus sustaining tissue neoformation and integration with the host. Scaffold architecture significantly impacts its regenerative potential, while preclinical trials are essential to define scaffold-host interactions. In compliance with the 3 R principle, there is a clear need to optimize both the procedures to evaluate scaffold performance and the analysis methodology decreasing the number of animals required to gain consistent data. In parallel, current technologies used in preclinical research generate huge amounts of data that need to be elaborated and interpreted correctly. Therefore, we designed this study to evaluate the results of scaffold integration with the host tissue after implantation in a mouse subcutaneous pocket model. We evaluated the angiogenic response developed by the host and the degree of scaffold integration by using a combined morphometric approach based on both histological and micro-CT analyses. Six-layer scaffolds, made of polycaprolactone (PCL) microspheres, with an ordered structure were produced by thermal sintering. Scaffolds were then implanted in BALB/c mice and retrieved 21 days post-implantation when the animals were deeply anesthetized and perfused with Microfil, a contrast agent for micro-CT. Here, we describe a method to extract quantitative data from micro-CT reconstructions such as (i) total vessel volume; (ii)% of vessel penetration; (iii) distribution of vessel diameters. The general principle of this approach is the refinement of the region of interest (ROI), thus producing a volume of interest (VOI) that matches scaffold volume. This VOI serves as a dataset from which to extract volumetric information. Then VOIs are divided into three identical parts, proximal, median, and distal, to follow the vessel progression into the scaffold, thus obtaining their depth of penetration (DoP). By this methodology, we observed in mean, among the analyzed samples, a vessel invasion for 1,38 mm3 corresponding to the 1,53% of the scaffold volume. We then looked at the diameter distribution being this value a key indicator of vessel maturity, highlighting that 55% of vessels fall into the range from 5,99-53.99 µm while the remaining 45% are distributed into intervals from 54 to 136 µm. In parallel, to evaluate tissue integration in detail, histological and immunofluorescent analyses were performed to look at vessel distribution and collagen synthesis. Histological results strongly correlate with the micro-CT data providing, however, an overview of the ingrowth tissues. In addition, by immunofluorescent analysis we demonstrate that newly formed vessels are mature at the considered time point and tissue collagen deposition is widespread within the scaffolds. Collectively, we propose a new method to track vessel formation by using a multi-modal approach posing the basis for: i) the fabrication of novel scaffolds for Tissue Engineering; ii) the integration of detailed information for a wide range of morphological and functional analyses.

Effect of surface treatment on cell responses to grades 4 and 5 titanium for orthodontic mini-implants.

INTRODUCTION: Mini-implants are used to improve orthodontic anchorage, but optimal composition and surface characteristics have yet to be determined. We investigated the behavior of osteoblast-like cells on grade 4 commercially pure titanium and grade 5 titanium alloy with different surface treatments for mini-implants. METHODS: MC3T3 cells were plated on machined, acid-etched, or acid-etched grade 4 titanium enriched with calcium phosphate, or machined, anodized, or anodized and calcium phosphate-enriched grade 5 titanium disks. Surface and cell morphologies were assessed by scanning electron microscopy. Cell viability was measured by chemiluminescence, cytoskeletal organization was investigated by immunofluorescence, and real-time polymerase chain reaction for osteoblast-specific genes was performed to measure cell differentiation. RESULTS: Flattened shapes and strong stress fibers were observed on the machined surfaces; cells on the rough surfaces had a spindle shape, with lower cytoskeletal polarization. Cell proliferation was highest on smooth grade 4 titanium surfaces, whereas cells quickly reached a plateau on rough grade 4 titanium; no difference was observed after 72 hours in the grade 5 titanium groups. Calcium phosphate enrichment on grade 4 titanium significantly increased the messenger RNA levels for alkaline phosphatase and osteocalcin. Osteoblastic markers were higher on the grade 5 titanium machined surfaces than on the rough surfaces, and comparable with acid-etched grade 4 titanium. CONCLUSIONS: Although the grade 4 titanium enriched with calcium phosphate had the highest level of differentiation in vitro, the grade 5 titanium machined surfaces supported cell proliferation and matrix synthesis, and induced high expression of early differentiation markers. Increased mechanical resistance of grade 5 titanium makes it a potential candidate for orthodontic mini-implants.

The normal and fibrotic mouse lung classified by spatial proteomic analysis.

Single cell classification is elucidating homeostasis and pathology in tissues and whole organs. We applied in situ spatial proteomics by multiplex antibody staining to routinely processed mouse lung, healthy and during a fibrosis model. With a limited validated antibody panel (24) we classify the normal constituents (alveolar type I and II, bronchial epithelia, endothelial, muscular, stromal and hematopoietic cells) and by quantitative measurements, we show the progress of lung fibrosis over a 4 weeks course, the changing landscape and the cell-specific quantitative variation of a multidrug transporter. An early decline in AT2 alveolar cells and a progressive increase in stromal cells seems at the core of the fibrotic process.

Effects of different short-chain fatty acids (SCFA) on gene expression of proteins involved in barrier function in IPEC-J2.

BACKGROUND: Gut microbial anaerobic fermentation produces short-chain fatty acids (SCFA), which are important substrates for energy metabolism and anabolic processes in mammals. SCFA can regulate the inflammatory response and increase the intestinal barrier integrity by enhancing the tight junction protein (TJp) functions, which prevent the passage of antigens through the paracellular space. The aim of this study was to evaluate the effect of in vitro supplementation with SCFA (acetate, propionate, butyrate, and lactate) at different concentrations on viability, nitric oxide (NO) release (oxidative stress parameter) in cell culture supernatants, and gene expression of TJp (occludin, zonula occludens-1, and claudin-4) and pro-inflammatory pathway-related mediators (ß-defensin 1, TNF-α, and NF-κB) in intestinal porcine epithelial cell line J2 (IPEC-J2). RESULTS: The SCFA tested showed significant effects on IPEC-J2, which proved to be dependent on the type and specific concentration of the fatty acid. Acetate stimulated cell viability and NO production in a dose-dependent manner (P < 0.05), and specifically, 5 mM acetate activated the barrier response through claudin-4, and immunity through ß-defensin 1 (P < 0.05). The same effect on these parameters was shown by propionate supplementation, especially at 1 mM (P < 0.05). Contrarily, lactate and butyrate showed different effects compared to acetate and propionate, as they did not stimulate an increase of cell viability and regulated barrier integrity through zonula occludens-1 and occludin, especially at 30 mM and 0.5 mM, respectively (P < 0.05). Upon supplementation with SCFA, the increase of NO release at low levels proved not to have detrimental effects on IPEC-J2 proliferation/survival, and in the case of acetate and propionate, such levels were associated with beneficial effects. Furthermore, the results showed that SCFA supplementation induced ß-defensin 1 (P < 0.05) that, in turn, may have been involved in the inhibition of TNF-α and NF-κB gene expression (P < 0.05). CONCLUSIONS: The present study demonstrates that the supplementation with specific SCFA in IPEC-J2 can significantly modulate the process of barrier protection, and that particularly acetate and propionate sustain cell viability, low oxidative stress activity and intestinal barrier function.

Pivotal role of micro-CT technology in setting up an optimized lung fibrosis mouse model for drug screening.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with no curative pharmacological treatment. The most used animal model of IPF for anti-fibrotic drug screening is bleomycin (BLM)-induced lung fibrosis. However, several issues have been reported: the balance among disease resolution, an appropriate time window for therapeutic intervention and animal welfare remain critical aspects yet to be fully elucidated. In this study, C57Bl/6 male mice were treated with BLM via oropharyngeal aspiration (OA) following either double or triple administration. The fibrosis progression was longitudinally assessed by micro-CT every 7 days for 4 weeks after BLM administration. Quantitative micro-CT measurements highlighted that triple BLM administration was the ideal dose regimen to provoke sustained lung fibrosis up to 28 days. These results were corroborated with lung histology and Bronchoalveolar Lavage Fluid cells. We have developed a mouse model with prolonged lung fibrosis enabling three weeks of a curative therapeutic window for the screening of putative anti-fibrotic drugs. Moreover, we have demonstrated the pivotal role of longitudinal micro-CT imaging in reducing the number of animals required per experiment in which each animal can be its own control. This approach permits a valuable decrease in costs and time to develop disease animal models.

Hypoxia and platelet lysate sustain differentiation of primary horse articular chondrocytes in xeno-free supplementation culture.

Currently, the main limitation for the use of adult differentiated chondrocytes in cell-based therapy and tissue engineering for the repair of articular cartilage is the difficulty of maintaining their state of differentiation during cell expansion. The adult articular cartilage has no direct blood supply, and local oxygen concentrations range from 5%-10% at the surface near the synovial fluid to less than 1% in the deep layer. Low oxygen tension is currently considered an important environmental condition for chondrocytes, and hypoxia has been explored as a signal potentially promoting differentiation and matrix deposition. In the present study, hypoxia and PL supplementation were studied to maintain differentiation in adult articular chondrocytes. Freshly isolated equine articular chondrocytes were grown in monolayer culture at a low seeding density (condition favoring proliferation and dedifferentiation) and in alginate beads (3D culture condition maintaining chondrocyte differentiation) both in normoxic and hypoxic conditions and in various conditions of supplementation or deprivation (fetal bovine serum [FBS]- and PL-free; 10% FBS; 5% PL; 10% PL). Results demonstrated that hypoxia is a micro-environmental condition that reduces chondrocyte dedifferentiation or maintains differentiation during in vitro expansion, as shown by the sustained expression of differentiation markers (COL2, ACAN, SOX9, HIF1a) and the reduction of dedifferentiation marker expression (COL1, RUNX2). In association with hypoxia, PL supplementation demonstrated a positive effect on chondrocyte differentiation in association with hypoxia. This promising result should be confirmed in other conditions of chondrocyte differentiation before proposing PL as a complete alternative to xenogenic serum for the expansion of articular chondrocytes.

Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies.

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.