Mesenchymal Stromal Cells Derived from Canine Adipose Tissue: Evaluation of the Effect of Different Shipping Vehicles Used for Clinical Administration.

Mesenchymal Stromal Cells (MSCs)-based therapies are rapidly gaining interest in veterinary medicine. Cellular therapy represents a new challenge for practitioners and requires precise coordination between the cell processing laboratory and the veterinary clinic. Cryopreservation is the best method to provide fast, in-time, and long-distance delivery of cells for therapeutic applications. However, potentially toxic cryoprotectants and xenobiotic products make the direct administration of cells impracticable for patients. Alternatively, the cells may be resuspended in a ready-to-use vehicle and shipped to the veterinary clinic. In this study, two nutrient-poor vehicles (physiologic saline and ringer lactate solutions) and two nutrient-rich vehicles (the releasate derived from autologous Platelet Poor Plasma and Platelet Rich Plasma) were tested on adipose tissue-derived canine MSCs (AD-MSCs). AD-MSCs stored for 2, 4, or 24 h in the different media were compared regarding mortality, metabolic activity, and replicative capacity. Furthermore, antioxidant activity and the pattern of expression of genes related to AD-MSCs function were performed following 24 h of storage. The results showed that all the different vehicles preserve cell vitality and replication following short-term storage. In long-term storage, the vehicle and cell density affect cell vitality, proliferation, and gene expression (CCL-2, CXCR-4, and TSG-6). Nutrient-rich vehicles seem better suited to preserve cell functionalities in this contest.

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.

Canine Mesenchymal Cell Lyosecretome Production and Safety Evaluation after Allogenic Intraarticular Injection in Osteoarthritic Dogs.

In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 106 cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.

Case Report: 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 thermoplastic mesh modeled 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.

Cultured Horse Articular Chondrocytes in 3D-Printed Chitosan Scaffold With Hyaluronic Acid and Platelet Lysate.

Three-dimensional (3D) printing has gained popularity in tissue engineering and in the field of cartilage regeneration. This is due to its potential to generate scaffolds with spatial variation of cell distribution or mechanical properties, built with a variety of materials that can mimic complex tissue architecture. In the present study, horse articular chondrocytes were cultured for 2 and 4 weeks in 3D-printed chitosan (CH)-based scaffolds prepared with or without hyaluronic acid and in the presence of fetal bovine serum (FBS) or platelet lysate (PL). These 3D culture systems were analyzed in terms of their capability to maintain chondrocyte differentiation in vitro. This was achieved by evaluating cell morphology, immunohistochemistry (IHC), gene expression of relevant cartilage markers (collagen type II, aggrecan, and Sox9), and specific markers of dedifferentiated phenotype (collagen type I, Runx2). The morphological, histochemical, immunohistochemical, and molecular results demonstrated that the 3D CH scaffold is sufficiently porous to be colonized by primary chondrocytes. Thereby, it provides an optimal environment for the colonization and synthetic activity of chondrocytes during a long culture period where a higher rate of dedifferentiation can be generally observed. Enrichment with hyaluronic acid provides an optimal microenvironment for a more stable maintenance of the chondrocyte phenotype. The use of 3D CH scaffolds causes a further increase in the gene expression of most relevant ECM components when PL is added as a substitute for FBS in the medium. This indicates that the latter system enables a better maintenance of the chondrocyte phenotype, thereby highlighting a fair balance between proliferation and differentiation.

Equine Mesenchymal Stem/Stromal Cells Freeze-Dried Secretome (Lyosecretome) for the Treatment of Musculoskeletal Diseases: Production Process Validation and Batch Release Test for Clinical Use.

In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases.

OBP-functionalized/hybrid superparamagnetic nanoparticles for Candida albicans treatment.

Infections caused by the opportunistic yeast Candida albicans are one of the major life threats for hospitalized and immunocompromised patients, as a result of antibiotic and long-term antifungal treatment abuse. Odorant binding proteins can be considered interesting candidates to develop systems able to reduce the proliferation and virulence of this yeast, because of their intrinsic antimicrobial properties and complexation capabilities toward farnesol, the major quorum sensing molecule of Candida albicans. In the present study, a hybrid system characterized by a superparamagnetic iron oxide core functionalized with bovine odorant binding protein (bOBP) was successfully developed. The nanoparticles were designed to be suitable for magnetic protein delivery to inflamed areas of the body. The inorganic superparamagnetic core was characterized by an average diameter of 6.5 ± 1.1 nm and a spherical shape. Nanoparticles were functionalized by using 11-phosphonoundecanoic acid as spacer and linked to bOBP via amide bonds, resulting in a concentration level of 26.0 ± 1.2 mg bOBP/g SPIONs. Finally, both the biocompatibility of the developed hybrid system and the fungistatic activity against Candida albicans by submicromolar OBP levels were demonstrated by in vitro experiments.

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.

Platelet lysate reduces the chondrocyte dedifferentiation during in vitro expansion: Implications for cartilage tissue engineering.

In vitro studies have demonstrated that platelet lysate (PL) can serve as an alternative to platelet-rich plasma (PRP) to sustain chondrocyte proliferation and production of extracellular matrix components in chondrocytes. The present study aimed to evaluate the direct effects of PL on equine articular chondrocytes in vitro in order to provide a rationale for in vivo use of PL. An in vitro cell proliferation and de-differentiation model was used: primary articular chondrocytes isolated from horse articular cartilage were cultured at low density under adherent conditions to promote cell proliferation. Chondrocytes were cultured in serum-free medium, 10% foetal bovine serum (FBS) supplemented medium, or in the presence of alginate beads containing 5%, 10% and 20% PL. Cell proliferation and gene expression of relevant chondrocyte differentiation markers were investigated. The proliferative capacity of cultured chondrocytes, was sustained more effectively at certain concentrations of PL as compared to that with FBS. In addition, as opposed to FBS, PL, particularly at percentages of 5% and 10%, could maintain the gene expression pattern of relevant chondrocyte differentiation markers. In particular, 5% PL supplementation showed the best compromise between chondrocyte proliferation capacity and maintenance of differentiation. The results of the present study provide a rationale for using PL as an alternative to FBS for in vitro expansion of chondrocytes for matrix-assisted chondrocyte implantation, construction of 3D scaffolds for tissue engineering, and treatment of damaged articular cartilage.

Platelet-rich Plasma Combined With a Sterile 3D Polylactic Acid Scaffold for Postoperative Management of Complete Hoof Wall Resection for Keratoma in Four Horses.

Keratoma is a nonmalignant horse tumor that grows in the space between the horn of the hoof and the distal phalanx. Keratoma causes lameness in the horse, and surgical excision is the treatment of choice. Four horses underwent removal of a keratoma by complete hoof wall resection. The remaining wound was treated with platelet-rich plasma (PRP) combined with a sterile three-dimensional polylactic acid scaffold. The PRP was applied at 3, 6, 9, 12, 15, and 18 days postoperatively. The surgical site was cleaned with gauzes and swabs soaked in Ringer's lactate solution before applying PRP and the foot bandage. Healthy granulation tissue developed at 6-21 days postoperatively. The hoof wall defect was completely filled with new hoof wall within 6-8 months after surgery. All horses returned to their previous exercise level, and no recurrence of lameness was reported by the owner.

Vertebrate odorant binding proteins as antimicrobial humoral components of innate immunity for pathogenic microorganisms.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

Use of platelet-rich plasma for the treatment of prostatic cysts in dogs.

Abnormalities of the canine prostate gland, in particular, benign hyperplasia and cysts, are frequent in dogs over 5 years of age. Treatment strategies for dogs with prostatic cysts include ultrasound-guided percutaneous drainage with alcohol sclerotherapy, reduction surgery, and omentalization. The aim of this study was to evaluate the use of platelet-rich plasma (PRP) for the treatment of prostatic cysts in dogs. Ten mixed breed adult male dogs were included in the study. Percutaneous drainage was performed prior to ultrasound-guided PRP injection with a 21G spinal needle. The PRP dose contained half of the solution drained from the cyst. Follow-ups were performed 6, 12, 24, and 60 d after treatment. Ten prostatic cysts were successfully treated with PRP after the ultrasound-guided drainage of the cystic cavities. No side effects were observed during the procedures. After the introduction of PRP, cysts detected by ultrasound appeared hypoechoic. On the sixth day, the hypoechoic appearance was maintained and margins were still identifiable. At 24-day and 60-day follow-ups, cysts were no longer detectable by ultrasound. One of the ten dogs had to be euthanized because of the presence of a malignant mesenchymal neoplasm of the oral cavity (fibroblastic sarcoma) and histological examination of serial sections of the prostate gland was performed. Histologic evaluation 1 year after treatment showed a regression of the cystic lesion injected with PRP. The therapy, which uses PRP injection in combination with ultrasound-guided drainage, can be considered a safe and effective strategy for the treatment of prostatic cysts in dogs.

Les anomalies de la prostate chez le chien, plus particulièrement l'hyperplasie bénigne et les kystes, sont fréquentes chez les chiens âgés de 5 ans ou plus. Les stratégies de traitement pour les chiens avec des kystes prostatiques incluent le drainage transcutané par échographie guidée avec sclérothérapie à l'alcool, chirurgie de réduction, et omentalisation. Le but de la présente étude était d'évaluer l'utilisation de plasma enrichi de plaquettes (PEP) pour le traitement de kystes prostatiques chez des chiens. Dix chiens mâles adultes de race mélangée ont été inclus dans l'étude. Le drainage transcutané a été effectué avant l'injection par échographie guidée de PEP avec une aiguille spinale de 21G. La dose de PEP contenait la moitié de la solution drainée du kyste. Des suivis ont été faits à 6, 12, 24, et 60 j suivant le traitement. Dix kystes prostatiques ont été traités avec succès avec le PEP suite au drainage des cavités kystiques par échographie guidée. Aucun effet secondaire ne fut observé durant les procédures. Après l'introduction du PEP, les kystes détestés par échographie apparaissaient hypoéchogène. Au sixième jour, l'apparence hypoéchogène était maintenue et les bordures étaient toujours identifiables. Lors des suivis aux jours 24 et 60, les kystes n'étaient plus détectables par échographie. Un des dix chiens dû être euthanasié à cause de la présence d'un néoplasme mésenchymateux malin de la cavité orale (sarcome fibroblastique) et l'examen histologique de sections sériées de la prostate a été effectué. L'évaluation histologique 1 an après le traitement a montré une régression de la lésion kystique injectée avec du PEP. La thérapie, qui utilise l'injection de PEP en combinaison avec un drainage par échographie guidée, peut être considérée comme une stratégie sécuritaire et efficace pour le traitement de kystes prostatiques chez le chien(Traduit par Docteur Serge Messier).

Platelets are involved in in vitro swine granulosa cell luteinization and angiogenesis.

During corpus luteum formation, impressive biological events take place to guarantee the transition from original follicular to luteal cells and to support required massive angiogenesis. It has been demonstrated that these phenomena resemble those essential for wound healing. After ovulation, blood vessels release their content in the antral cavity and coagulation takes place. Involvement of platelets in corpus luteum growth has been hypothesized both in human and in rat. On this basis, using platelet lysate (PL), a blood derivative with a higher platelet concentration, we aimed to assess a potential involvement of platelets in swine granulosa cell luteinization and on new blood vessel growth. Our results demonstrate, for the first time in the swine, that platelets could be directly involved in granulosa cell physiological luteinization, since the treatment with PL shifted steroid production from estradiol 17ß to progesterone. Moreover, PL stimulated angiogenesis. Nitric oxide could be involved in these effects. These results are important to clarify complex intrafollicular molecular machinery. A better understanding of these mechanisms can be useful to develop more focused therapeutic strategies to contrast sow infertility. In addition, since the pig represents a model for translational studies, collected data could be of interest for human medicine because reproductive pathologies such as Polycystic Ovary Syndrome (PCOS) and endometriosis are often accompanied by platelet dysfunctions.

The Contribution of Adipose Tissue-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma to the Treatment of Chronic Equine Laminitis: A Proof of Concept.

Laminitis, a highly debilitating disease of the foot in ungulates, is characterized by pathological changes of the complex lamellar structures that maintain the appendicular skeleton within the hoof. Laminitis is a multifactorial disease that involves perturbation of the vascular, hematological, and inflammatory homeostasis of the foot. Interestingly, the pathogenesis of the disease resembles what is observed in metabolic syndromes and sepsis-induced organ failure in humans and animals. We hypothesized that local administration of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) might contribute to establishing an anti-inflammatory and pro-angiogenic environment, and could stimulate the injured tissue in order to restore its functional integrity. According to this assumption, an experimental protocol based on the local intravenous administration of adipose tissue-derived MSCs (aMSCs) in combination with PRP was developed for the treatment of horses affected by chronic laminitis. Nine horses with severely compromised venograms (showing grade III and IV laminitis) that had been unsuccessfully treated with conventional therapies were enrolled. aMSCs and PRP (15 × 106 cells resuspended in 15 mL of PRP) were injected into the lateral or medial digital vein three times, at one-month intervals. The first administration was performed with allogeneic aMSCs, while for the following administrations, autologous aMSCs were used. There was no adverse short-term reaction to the intravenous injection of aMSCs. In the long term, venograms outlined, in all subjects, a progressive amelioration of the vascularization of the foot. An improvement in the structure and function of the hoof was also observed. No adverse events were reported during the follow-up, and the horses returned to a comfortable quality of life. Although the number of animals enrolled in the study is limited, both clinical observations and venography demonstrated an enhancement in the condition of all horses, suggesting that the regenerative therapies in chronic laminitis could be useful, and are worthy of further investigation.

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.

Regenerative therapy for the management of a large skin wound in a dog.

Extensive full-thickness skin wounds are quite common in domestic animals. In these report, following the failure of reconstructive surgery, adipose tissue-derived mesenchymal stem cells, and platelet-rich plasma were successfully used in a dog to improve speed and quality of skin tissue healing, avoiding suffering, and debilitating effects.

Preparation and application of an innovative thrombocyte/leukocyte-enriched plasma to promote tissue repair in chelonians.

Platelet concentrates are widely used in mammalian regenerative medicine to improve tissue healing. Chelonians (Testudines) would benefit from the application of thrombocyte preparations to regenerate damaged tissues, since traumatic injuries are leading causes of morbidity and mortality for both wild-living and domesticated animals. The aim of this study was to establish a protocol that optimized the recovery of the thrombocytes from blood samples and to show the efficacy of thrombocyte-enriched plasma in chelonians. Peripheral blood samples were obtained from Testudo spp. (n = 12) and Trachemys scripta elegans (n = 10). Blood cells were fractionated by sodium diatrizoate-sodium polysucrose density gradient using a two-step centrifugation protocol. Thrombocytes and leukocytes were isolated and resuspended to obtain thrombocyte-leucocyte rich plasma (TLRP). The mean recovery of leukocytes and thrombocytes was 48.9% (±4.0 SEM, n = 22) of the whole blood cell content. No statistically significant difference was observed between blood samples collected from different turtle species. The ability of TLRP to form a gel was evaluated by adding variable concentrations of calcium gluconate at room temperature and at 37°C. A reliable and consistent clotting of the TLRP was obtained in glass tubes and dishes by adding 5-20% v/v of a 100 mg/ml solution of calcium gluconate. Furthermore, in order to test the clinical efficacy of TLRP, a preliminary evaluation was performed on four turtles (Testudo spp.) with traumatic injuries. In all the four animals, a successful clinical outcome was observed. The results demonstrated that a thrombocyte-enriched plasma, comparable to mammalian platelet rich plasma, can be prepared from chelonian blood samples. Furthermore, although the low number of cases presented does not allow definitive conclusions from a clinical point of view, their outcome suggests that TLRP application could be further investigated to improve the healing process of both soft and hard tissue injuries in chelonians.

An innovative bovine odorant binding protein-based filtering cartridge for the removal of triazine herbicides from water.

Odorant binding protein (OBP) is a multi-functional scavenger for small hydrophobic molecules dissolved in the mucus lining the nasal epithelia of mammals, characterized by broad ligand binding specificity towards a large number of structurally unrelated natural and synthetic molecules of different chemical classes. Here, we demonstrate for the first time the application of OBP as the active element of an innovative filtering matrix for the removal of environmental pollutants such as triazine herbicides from water samples. The filtering device, obtained by coupling histidine-tagged bovine OBP to a nickel nitrilotriacetic acid (Ni-NTA) agarose resin, was characterized in terms of retention capacity for the herbicides atrazine, simazine, and propazine. Analysis of these herbicides at trace levels with solid-phase microextraction followed by gas chromatography-mass spectrometry using the selected ion monitoring mode proved the capabilities of the proposed device for the decontamination of surface and groundwater samples in the 0.2-2,300 µg/L concentration range, obtaining a reduction in the triazine content greater than 97 %, thus suggesting its possible use for the potabilization of water.

Atrazine disrupts steroidogenesis, VEGF and NO production in swine granulosa cells.

Atrazine is one of the most widely employed herbicides. Due to its environmental persistence, it can be detected in ground and water thus becoming the subject of a serious concern because of its potential endocrine disrupting activity. In particular, several in vitro and in vivo studies point out adverse effects on reproduction. However, these data were mainly collected in the male, while studies on females are lacking. Present work was therefore set up on swine ovarian granulosa cells to investigate the effect of atrazine on steroidogenesis and proliferation. Moreover, since vessel growth is fundamental for reproductive function, we evaluated the herbicide's effect on two of the main angiogenesis signaling molecules, VEGF and NO. Our data show that atrazine markedly interferes with steroidogenesis while it does not modify cell proliferation; in addition, the herbicide has also been found to affect the production of the examined angiogenesis molecules. Collectively, these results indicate for the first time a potential negative effect of atrazine on ovarian functions in the swine species.

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

BACKGROUND: 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. RESULTS: 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 neo 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. CONCLUSION: 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.