Heterologous matrix metalloproteinase gene promoter activity allows In Vivo real-time imaging of Bleomycin-induced Lung fibrosis in transiently transgenized mice

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure.Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycintreated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on doublebleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.Fil: Stellari, Fabio Franco. Chiese Farmaceutici; ItaliaFil: Ruscitti, Francesca. Chiese Farmaceutici; ItaliaFil: Pompilio, Daniela. Chiese Farmaceutici; ItaliaFil: Ravanetti, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Tebaldi, Giulia. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Macchi, Francesca. Università di Parma. Dipartimento di Scienze Medico Veterinarie; ItaliaFil: Verna, Andrea Elizabeth. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Chiese Farmaceutici; ItaliaFil: Villetti, Gino. Chiese Farmaceutici; ItaliaFil: Donofrio, Gaetano. Università di Parma. Dipartimento di Scienze Medico Veterinarie ; Itali

Influence of genetic selection on the myofibre type composition of porcine biceps femoris muscle: a comparative study of a purebred (Nero di Parma) and commercial hybrid pigs (Large White × Landrace × Duroc)

This study is aimed to evaluate histological features related to the different quality of meat cuts obtained from different pig breeds. We compared animals genetically selected to restore the original local purebred “Nero di Parma” pig, and highly selected fast-growing commercial hybrids. As weight is the main factor determining the time of slaughter, we compared equally weighing animals, sampled from slaughter for edible use, regardless of their age and sex, and immunohistochemically demonstrated the myofibre type composition of their biceps femoris muscle. In both groups we observed type I myofibres, situated as central islets, encircled by type IIA, IIX and IIB myofibres ordered in concentric rings according to the dynamic of their differentiation/maturation. However, the purebred pig muscles contained a smaller quantity of myofibres expressing the MyHC-IIb isoform, related to rapid postmortem glycolytic rate, meat toughness and poorer quality, in comparison to commercial hybrids. This proves that the latter are subjected to a more rapid transition in the expression of the different MyHC, probably as a consequence of genetic selection and breeding conditions, such as different feeding and housing reducing the possibility of continuous physical exercise. Further studies on the postnatal transitions timing of myosin heavy chain isoforms in functionally different muscles of various breeds are necessary to verify if they might be “artificially modulated”, with the aim to design breeding programs allowing a good balance between growth performance, muscularity and meat quality

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

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

BackgroundOsteochondral 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.ResultsThe 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.ConclusionsIn 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

Immunophenotypical characterization of canine mesenchymal stem cells from perivisceral and subcutaneous adipose tissue by a species-specific panel of antibodies

Immunophenotypical characterization of mesenchymal stem cells is fundamental for the design and execution of sound experimental and clinical studies. The scarce availability of species-specific antibodies for canine antigens has hampered the immunophenotypical characterization of canine mesenchymal stem cells (MSC). The aim of this study was to select a panel of species-specific direct antibodies readily useful for canine mesenchymal stem cells characterization. They were isolated from perivisceral and subcutaneous adipose tissue samples collected during regular surgeries from 8 dogs. Single color flow cytometric analysis of mesenchymal stem cells (P3) deriving from subcutaneous and perivisceral adipose tissue with a panel of 7 direct anti-canine antibodies revealed two largely homogenous cell populations with a similar pattern: CD29+, CD44+, CD73+, CD90+, CD34−, CD45− and MHC-II− with no statistically significant differences among them. Antibody reactivity was demonstrated on canine peripheral blood mononuclear cells. The similarities are reinforced by their in vitro cell morphology, trilineage differentiation ability and RT-PCR analysis (CD90+, CD73+, CD105+, CD44+, CD13+, CD29+, Oct-4+ gene and CD31− and CD45− expression). Our results report for the first time a comparison between the immunophenotypic profile of canine MSC deriving from perivisceral and subcutaneous adipose tissue. The substantial equivalence between the two populations has practical implication on clinical applications, giving the opportunity to choose the source depending on the patient needs. The results contribute to routine characterization of MSC populations grown in vitro, a mandatory process for the definition of solid and reproducible laboratory and therapeutic procedures

Quantification of Lung Fibrosis in IPF-Like Mouse Model and Pharmacological Response to Treatment by Micro-Computed Tomography

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive degenerative lung disease leading to respiratory failure and death. Although anti-fibrotic drugs are now available for treating IPF, their clinical efficacy is limited and lung transplantation remains the only modality to prolong survival of IPF patients. Despite its limitations, the bleomycin (BLM) animal model remains the best characterized experimental tool for studying disease pathogenesis and assessing efficacy of novel potential drugs. In the present study, the effects of oropharyngeal (OA) and intratracheal (IT) administration of BLM were compared in C57BL/6 mice. The development of lung fibrosis was followed in vivo for 28 days after BLM administration by micro-computed tomography and ex vivo by histological analyses (bronchoalveolar lavage, histology in the left lung to stage fibrosis severity and hydroxyproline determination in the right lung). In a separate study, the antifibrotic effect of Nintedanib was investigated after oral administration (60 mg/kg for two weeks) in the OA BLM model. Lung fibrosis severity and duration after BLM OA and IT administration was comparable. However, a more homogeneous distribution of fibrotic lesions among lung lobes was apparent after OA administration. Quantification of fibrosis by micro-CT based on % of poorly aerated tissue revealed that this readout correlated significantly with the standard histological methods in the OA model. These findings were further confirmed in a second study in the OA model, evaluating Nintedanib anti-fibrotic effects. Indeed, compared to the BLM group, Nintedanib inhibited significantly the increase in % of poorly aerated areas (26%) and reduced ex vivo histological lesions and hydroxyproline levels by 49 and 41%, respectively. This study indicated that micro-computed tomography is a valuable in vivo technology for lung fibrosis quantification, which will be very helpful in the future to better evaluate new anti-fibrotic drug candidates

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

Cyto-histological study of biomimetic surfaces with high osteogenic function

La ricerca implantologica attuale in materia di impianti osteointegrati è volta al disegno di materiali, definiti bioattivi, che permettano il controllo, guidino e rendano possibile una più rapida guarigione. In questo ambito il materiale di elezione è rappresentato dal titanio e dalle sue leghe. Materiale bioinerete con proprietà intermedie, il titanio, rappresenta un giusto compromesso tra necessità meccaniche e biologiche, grazie alle sue proprietà specifiche di ottima biocompatibilità, elevata resistenza meccanica ed elevata resistenza alla corrosione. È possibile affermare che con gli attuali sviluppi gli impianti in titanio hanno raggiunto una buona osteointegrazione sia in termini qualitativi sia quantitativi; quindi lo scopo principale delle attuali ricerche non è il miglioramento dell’osteointegrazione, ma l’accelerazione nei processi che vi concorrono. Questo consentirebbe non solo un più rapido recupero da parte del paziente ma anche una stabile fissazione tra osso e impianto, che permetterebbe un carico più precoce. La ricerca di metodiche volte a migliorare l’interfaccia osso-impianto che permettano di considerare il titanio non più come materiale bioinerte, ma bioattivo si è orientata secondo tre approcci diversi: metodi fisici, metodi chimici-elettrochimici e biochimici. Con il presente studio si intende indagare, mediante ricerche in vitro ed in vivo, il comportamento delle cellule e l’osteointegrazione primaria del tessuto osseo in risposta a impianti in titanio con superfici ad elevata funzione osteogenica. Nello specifico saranno descritti due differenti studi: il primo volto a valutare due superfici ottenute mediante trattamenti elettrochimici di deposizione anodica, il secondo volto a valutare un trattamento biochimico di funzionalizzazione peptidica con il nonapeptide HVP (351-359) mappato dalla vitronectina umana mediante approccio di mimica peptidica ed immobilizzato covalentemente. Studi effettuati in diversi modelli animali sono concordi nell’affermare che, per quanto concerne la risposta ad impianti endossei, le prime fasi del processo di osteointegrazione con relativa neoformazione ossea all’interfaccia sono già osservabili entro le due settimane dall’impianto. In relazione alle finalità dei trattamenti di superficie del titanio, nei presenti studi abbiamo voluto analizzare la risposta ossea primaria all’impianto considerando brevi tempi sperimentali; a questo scopo abbiamo utilizzato un modello animale a rapido accrescimento e turn over osseo come il coniglio ed effettuato l’analisi istologica e l’analisi istomorfometrica di parametri sia statici sia dinamici