A novel ventilated thermoplastic mesh bandage for post-operative management of large soft tissue defects: a case series of three dogs treated with autologous platelet concentrates

A ventilated thermoplastic mesh bandage was used for the post-operative management of large soft tissue defects in three dogs. Once the granulation tissue appeared, the wounds were treated with liquid or jellified autologous platelet concentrates, Platelet Rich Plasma (PRP) and Platelet Lysate (PL), to improve the wound healing process. After cleaning the wound with sterile physiological solution, a dressing was performed with several layers of cotton. A window through the layers of cotton was opened above the wound. Then, the platelet concentrate was topically applied, and the bandage was completed by placing, over the access window, a ventilated thermoplasticmeshmodeled according to the size and shape of the wound. After 24 h, it was replaced by a low adhesion bandage. The thermoplastic mesh avoids the direct contact between the wound and the external layers of the bandage, preventing the drainage of the topical agent and the removal of the growing healthy granulation tissue. The bandage proposed in this study is easily applied by the veterinarian and well-tolerated by the animal, ensuring high welfare standards in stressed patients presenting compromised clinical conditions

In Vitro Evaluation of Cytotoxicity and Proliferative Effects of Lyophilized Porcine Liver Tissue on HepG2 Hepatoma Cells and Adipose-Tissue-Derived Mesenchymal Stromal Cells

In recent years, nutritional supplements from different sources have been widely considered to support medical treatments in patients affected by chronic hepatopathies. Their potential therapeutic benefit has been recognized, but some evidence of safety issues has been reported. Recently it has been hypothesized that the liver could produce various of bioactive factors to maintain organ homeostasis and promote tissue healing. Thus, liver-specific preparations containing bioactive factors could provide a suitable substrate for in vitro study of liver tissue maintenance/healing, as a prospective regenerative medicine approach. Furthermore, they could represent a dietary supplement or nutraceutical for adjuvant therapies when correctly prepared and formulated. This work aims to provide data about the safety and biological activity of a freeze-dried porcine liver preparation. The lyophilized powder obtained from the whole organ has been tested in term of in vitro cell cytotoxicity (MTT assay) and proliferation assays (bromo-deoxyuridine incorporation and direct cell count) in two different cell types: human hepatoma HepG2 cell line and adipose-tissue-derived canine mesenchymal stromal cells (At-MSCs). At concentration levels between 100 to 500 g/mL, the lyophilized liver powder stimulated mitochondrial metabolism as assessed by MTT assay (p 0.001 for HepG2 and for At-MSCs) and induced an increase in bromo-deoxyuridine incorporation in both cell types (p 0.01 for HepG2 and p < 0.001 for At-MSCs). In addition, direct cell count demonstrated a higher proliferative activity in treated At-MSCs (p < 0.001). Although preliminary, these data suggest that the whole-liver powder is noncytotoxic in vitro and may represent a stimulus to cell metabolism and proliferation. Further studies are needed to detect the bioactive components of the supplement and characterize in deeper detail the cellular pathways that they can modulate

Effects of orexin B on swine granulosa and endothelial cells

In addition to the well-known central modulatory role of orexins, we recently demonstrated a peripheral involvement in swine granulosa cells for orexin A and in adipose tissue for orexin B (OXB). The aim of present research was to verify immunolocalization of OXB and its potential role in modulating the main features of swine granulosa cells. In particular, we explored the effects on granulosa cell proliferation (through the incorporation of bromodeoxyuridine), cell metabolic activity (as indirect evaluation by the assessment of ATP), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test and the non-enzymatic reducing power by FRAP test). Our data point out that OXB does not modify granulosa cell growth, steroidogenesis and superoxide anion generation. On the contrary, the peptide stimulates (p &lt; 0.05) nitric oxide output and non-enzymatic reducing power. Since new vessel growth is crucial for ovarian follicle development, a further aim of this study was to explore the expression of prepro-orexin and the effects of OXB on swine aortic endothelial cells. We found that the peptide is ineffective in modulating cell growth, while it inhibits redox status parameters. In addition, we demonstrated a stimulatory effect on angiogenesis evaluated in fibrin gel angiogenesis assay. Taken together, OXB appears to be potentially involved in the modulation of redox status in granulosa and endothelial cells and we could argue an involvement of the peptide in the follicular angiogenic event

Evaluation of Oxidative Stress Parameters in Healthy Saddle Horses in Relation to Housing Conditions, Presence of Stereotypies, Age, Sex and Breed

Oxidative stress plays an important role in the development of many horse diseases and it has been shown that housing has important implications for the psychophysical well-being of horses. The aim of this study is to determine if there are any dierences between the redox status in horses in relation to housing conditions. The four housing conditions analyzed were: single box, without external access and without contact (Cat A), single box with external access and possibility of partial contact (Cat B), group housing with box and large paddock (Cat C), pasture with more than 7 horses and the possibility of green forage for the whole year (Cat D). A group of 117 healthy horses were selected in several private stables in Northern Italy. All subjects treated with any type of drug were excluded. At the end of the enrollment, the 117 selected horses were divided into the four housing categories. Stereotypies were highest in the group of horses in single box, without external access and without contact (Cat A). Oxidative stress was evaluated by testing plasma or serum samples for the following parameters: superoxide anion (WST), nitric oxide (NO), reactive oxygen species (d-ROMs), ferric reducing ability of plasma (FRAP), and the activity of superoxide dismutase (SOD). Simultaneously with the blood sampling, the owners completed a questionnaire with all the management aspects of the horse (signaling, feeding, equestrian activity, vaccinations, foot management etc.). The statistical evaluation was carried out based on the categories previously described, on the presence and absence of stereotypies and on some signaling data obtained from the questionnaire. There were no significant dierences in the parameters analyzed between the categories. No significant redox status dierences were detected based on the presence or absence of stereotypies. Interestingly, when the age was introduced as selection (&lt;14 and &gt;14 years old) parameter inside the categories, statistical significance was observed for some of the stress markers considered. Finally, independently of the housing conditions, the horses of the most two represented breeds exhibited dierent values of FRAP. All these aspects are commented in the discussion

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products

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

Virally and physically transgenized equine adipose-derived stromal cells as a cargo for paracrine secreted factors

<p>Abstract</p> <p>Background</p> <p>Adipose-Derived Stromal Cells have been shown to have multiple lineage differentiation properties and to be suitable for tissues regeneration in many degenerative processes. Their use has been proposed for the therapy of joint diseases and tendon injuries in the horse. In the present report the genetic manipulation of Equine Adipose-Derived Stromal Cells has been investigated.</p> <p>Results</p> <p>Equine Adipose-Derived Stromal Cells were successfully virally transduced as well as transiently and stably transfected with appropriate parameters, without detrimental effect on their differentiation properties. Moreover, green fluorescent protein alone, fused to <it>neo </it>gene, or co-expressed as bi-cistronic reporter constructs, driven by viral and house-keeping gene promoters, were tested. The better expressed cassette was employed to stably transfect Adipose-Derived Stromal Cells for cell therapy purposes. Stably transfected Equine Adipose-Derived Stromal Cells with a heterologous secreted viral antigen were able to immunize horses upon injection into the lateral wall of the neck.</p> <p>Conclusion</p> <p>This study provides the methods to successfully transgenize Adipose-Derived Stromal Cells both by lentiviral vector and by transfection using optimized constructs with suitable promoters and reporter genes. In conclusion these findings provide a working platform for the delivery of potentially therapeutic proteins to the site of cells injection via transgenized Equine Adipose-Derived Stromal Cells.</p