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

ABSTRACT: 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.

SUBMITTER: Di Ianni F

PROVIDER: S-EPMC4406685 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Di Ianni Francesco F Merli Elisa E Burtini Francesca F Conti Virna V Pelizzone Igor I Di Lecce Rosanna R Parmigiani Enrico E Squassino Gian Paolo GP Del Bue Maurizio M Lucarelli Enrico E Ramoni Roberto R Grolli Stefano S

PloS one 20150422 4

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-enriche ...[more]

PMID: 25901960

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Project description:BackgroundMesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been found to have a great potential for soft tissue repair due to various biological functions, including pro-angiogenesis and low immunogenicity. However, the low yield and heterogeneity of MSC-EVs limited their clinical transformation. This study was designed to develop a novel adipose-derived stem cell engineered nanovesicles (ADSC-NVs) with high production and explore its pro-angiogenetic effect and application in adipose tissue regeneration.MethodsAdipose-derived stem cell-derived extracellular vesicles (ADSC-EVs) were isolated from an EVs-free culture medium for human ADSCs (hADSCs). ADSC-NVs were prepared by sequentially extruding ADSCs followed by iodixanol density gradient ultracentrifugation and were compared with ADSC-EVs in morphology, size distribution, protein contents and yield. The pro-angiogenetic effect of ADSC-NVs in different doses (0, 5, 20 and 80 μg/mL) in vitro was determined using transwell assay, tube formation assay, western blot and qRT-PCR. In vivo, BALB/c nude mice were administered injection of a mixture of fat granules and different dose of ADSC-NVs and grafts were harvested at 12 weeks post-transplantation for further analysis. By analyzing the weight and volume of grafts and histological evaluation, we investigated the effect of ADSC-NVs in vessel formation and adipose tissue regeneration.ResultsOur results showed yield of purified ADSC-NVs was approximately 20 times more than that of ADSC-EVs secreted by the same number of ADSCs. In vitro, both ADSC-NVs and ADSC-EVs exhibited a dose-dependent pro-angiogenetic effect, despite their distinct miRNA profiles. These effects of ADSC-NVs may be mediated by enriched miR-21-5p via PTEN inhibition and PI3K/p-Akt signaling activation. Furthermore, after a mixed injection of ADSC-NVs, vessel formation and adipose regeneration were observed in vivo in fat implants.ConclusionsOur study developed a potent alternative of ADSC-EVs. ADSC-NVs have a high pro-angiogenesis potential and can be used as cell-free therapeutic biomaterials in soft tissue regeneration.

Dataset Information

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

Publications

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

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