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.

血小板浓缩物（platelet concentrates）已广泛应用于哺乳动物再生医学领域，以促进组织修复。龟类（龟鳖目，Testudines）的受损组织再生可受益于凝血细胞制剂的应用，因为创伤是野生及圈养龟类发病与死亡的主要诱因。本研究旨在建立一套优化血液样本中凝血细胞（thrombocyte）回收率的实验方案，并验证富凝血细胞血浆（thrombocyte-enriched plasma）制剂在龟类中的应用效果。本研究采集了12份陆龟属（Testudo spp.）个体及10份红耳滑龟（Trachemys scripta elegans）的外周血样本。采用两步离心法，通过泛影酸钠-聚蔗糖密度梯度离心分离血液细胞组分。分离得到凝血细胞与白细胞，并重悬制备得到富凝血细胞-白细胞血浆（thrombocyte-leucocyte rich plasma，TLRP）。白细胞与凝血细胞的平均回收率为全血细胞总量的48.9%（±4.0 SEM，n=22）。不同龟类物种的血液样本之间未检测到统计学显著性差异。通过在室温及37℃条件下添加不同浓度的葡萄糖酸钙，评估TLRP的凝胶形成能力。当添加体积分数5%~20%的100 mg/ml葡萄糖酸钙溶液时，可在玻璃试管与培养皿中实现TLRP稳定且一致的凝固效果。此外，为验证TLRP的临床应用效果，本研究对4只患有创伤性损伤的陆龟属个体开展了初步评估。4只受试个体均取得了良好的临床转归。研究结果证实，可从龟类血液样本中制备出与哺乳动物富血小板血浆效果相当的富凝血细胞血浆。尽管本研究纳入的病例数量较少，无法从临床层面得出确定性结论，但受试个体的转归结果提示，可进一步探索TLRP的应用，以促进龟类软组织与硬组织损伤的修复过程。