Di Ianni Francesco, Merli Elisa, Burtini Francesca, Conti Virna, Pelizzone Igor, Di Lecce Rosanna, Parmigiani Enrico, Squassino Gian Paolo, Del Bue Maurizio, Lucarelli Enrico, Ramoni Roberto, Grolli Stefano
Department of Veterinary Science, Università degli Studi di Parma, Via Del Taglio 10, 43126, Parma, Italy.
Clinica Veterinaria Ambulatorio Squassino, Asti, Italy.
PLoS One. 2015 Apr 22;10(4):e0122595. doi: 10.1371/journal.pone.0122595. eCollection 2015.
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.
血小板浓缩物在哺乳动物再生医学中被广泛用于促进组织愈合。龟类（龟鳖目）可从应用血小板制剂来再生受损组织中获益，因为创伤性损伤是野生和家养龟类发病和死亡的主要原因。本研究的目的是建立一种优化从血液样本中回收血小板的方案，并展示富含血小板血浆在龟类中的疗效。从陆龟属（n = 12）和红耳龟（n = 10）获取外周血样本。使用两步离心方案通过泛影酸钠-聚蔗糖钠密度梯度对血细胞进行分离。分离并重悬血小板和白细胞以获得富含血小板-白细胞血浆（TLRP）。白细胞和血小板的平均回收率为全血细胞含量的48.9%（±4.0 SEM，n = 22）。在从不同龟种采集的血液样本之间未观察到统计学上的显著差异。通过在室温及37°C下添加不同浓度的葡萄糖酸钙来评估TLRP形成凝胶的能力。通过添加5-20% v/v的100 mg/ml葡萄糖酸钙溶液，在玻璃管和培养皿中获得了TLRP可靠且一致的凝血。此外，为了测试TLRP的临床疗效，对四只患有创伤性损伤的龟（陆龟属）进行了初步评估。在所有四只动物中，均观察到了成功的临床结果。结果表明，可以从龟类血液样本中制备出与哺乳动物富含血小板血浆相当的富含血小板血浆。此外，尽管所呈现的病例数量较少，从临床角度无法得出确定性结论，但其结果表明，可以进一步研究应用TLRP来改善龟类软硬组织损伤的愈合过程。
