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.

Effects of topical insulin on second-intention wound healing in the red-eared slider turtle (Trachemys scripta elegans) – a controlled study

“…Skin wounds are a common reason for veterinary consultation in reptiles kept in captivity. The list of potential causes includes thermal burns, trauma, surgery, improper husbandry, unsanitary conditions, and stress among others [ 1 – 4 ]. Compared with mammals, skin wounds of reptiles heal more slowly and their rate of healing is influenced by ambient temperature [ 5 , 6 ].…”

“…Unfortunately, the high cost of producing purified growth factors has prevented their integration into standard therapies in veterinary practice. Recently, a convenient protocol to concentrate platelets from chelonian whole blood has been described and the thrombocyte enriched plasma obtained was used successfully to treat several clinical cases of traumatic injuries [ 4 ]. As another alternative, insulin has been reported to promote wound healing in mammals and, in contrast to other growth factors, is affordable and readily available worldwide [ 15 , 16 ].…”

Use of Thrombocyte–Leukocyte-Rich Plasma in the Treatment of Chronic Oral Cavity Disorders in Reptiles: Two Case Reports

“…13,[33][34][35] The aim of this work was to assess the potential role of plateletconcentrates, on the basis of their regenerative and antimicrobial properties, in treating reptilian oral disorders, and halting chronic bone lysis. TLRP was obtained following a previous protocol 20 with thrombocyte-leukocyte counts paralleling those previously documented. 20 TLRP was preferred to simple PRP since, platelets and leukocytes release growth factors and chemotactic molecules including interleukin-6 and -4 (IL-6, IL-4), tumor necrosis factor-a (TNF-a), transforming growth factor-b (TGF-b), insulin-like growth factor 1 (IGF-1), basic-fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), or platelet-derived growth factor (PDGF) 9,12,36 able to stimulate proliferation of mesenchymal cells including fibroblasts, osteoblasts, and endothelial cells.…”

A novel method to in vitro evaluate biocompatibility of nanoscaled scaffolds

“…From a physiological point of view, blood leukocytes are necessary for controlled tissue integration because they are one kind of the first cells to get into contact with the implanted biomaterials. Additionally, they provide growth factors necessary to initiate and accelerate wound healing, tissue repair, and regeneration . On the other hand, leukocytes accumulation has also been considered as a major component in the pathogenesis of inflammatory tissue injuries .…”

“…Additionally, they provide growth factors necessary to initiate and accelerate wound healing, tissue repair, and regeneration. 35,36 On the other hand, leukocytes accumulation has also been considered as a major component in the pathogenesis of inflammatory tissue injuries. 37 The function of neutrophils, which account for most part of blood leukocytes, is to initially react nonspecially to foreign objects.…”