Influence of cryopreservation and mechanical stimulation on equine Autologous Conditioned Plasma (ACP®).

OBJECTIVE: To determine the influence of cryopreservation at two different temperatures on platelet concentration, growth factor (GF) levels and platelet activation parameters in equine ACP®; moreover, to determine if adding mechanical ACP® stimulation to freeze-thaw activation amplifies GF release from platelets. MATERIAL AND METHODS: Firstly, blood from five horses was used to prepare ACP®. Platelet, platelet derived growth factor BB (PDGF-BB) and transforming growth factor ß1 (TGF-ß1) concentrations as well as mean platelet volume (MPV) and mean platelet component (MPC) were determined in fresh and corresponding ACP® samples after 2 months cryopreservation at -20 °C and -80 °C, respectively. Secondly, ACP® was prepared from blood of nine horses. Half of ACP® was activated using one freeze-thaw-cycle at -20 °C, whereas the rest was first vortexed. Their PDGF-BB and TGF-ß1 concentrations were subsequently determined. RESULTS: Platelet concentration significantly decreased after -80 °C cryopreservation. PDGF-BB level augmented significantly after both storage methods, whereas TGF-ß1 concentration was not significantly altered. MPV significantly increased after -20 °C cryopreservation. Both storage regimens induced a significant MPC decrease. No significant differences in GF concentrations between the vortexed and non-vortexed samples were detected. DISCUSSION: Both cryopreservation methods induced platelet activation, but storage at -80 °C apparently harmed the platelets without generating higher GF release than -20 °C. The mechanical stimulation process could not enhance GF release in subsequently frozen-thawed ACP®. CONCLUSION AND CLINICAL RELEVANCE: Storage of ACP® at -20 °C could be useful in equine practice, but, before this procedure can be recommended, further qualitative tests are needed. The mechanical stimulation technique should be adjusted in order to increase platelet activation.

Equine autologous platelet concentrates: A comparative study between different available systems.

REASONS FOR PERFORMING STUDY: Autologous platelet concentrates (APCs) are being used increasingly in horses to enhance regeneration in tissues that have poor natural healing capabilities. Numerous APC systems, which are based on different preparation techniques and were originally developed for human patients, are now routinely used in equine cases. However, preliminary process validation and adequate in vitro biochemical characterisation of most of these systems do not exist for horses. OBJECTIVES: To compare haematological findings and growth factor concentrations of equine APCs obtained with 4 commercially available systems and a noncommercial double-centrifugation technique. STUDY DESIGN: Nonrandomised in vitro experiment. METHODS: Blood samples from 6 horses were processed to produce APCs using one equine-specific filtration-based and 4 different centrifugation-based techniques. Platelet, leucocyte, platelet-derived growth factor-BB and transforming growth factor-ß1 concentrations were measured in all APCs, and their respective enrichment factors were compared. RESULTS: Mean platelet concentration increased in all systems in comparison to baseline; however, the mean enrichment factor, which ranged from 130% to 527% depending on the APC, was statistically significant in only 2 products. One method reduced total leucocyte counts to 9% of the baseline value, while the others had a mean fold increase varying from 116 to 663% of the baseline. Differential leucocyte count also differed between the products. Moreover, the various systems had significantly different mean growth factor enrichments (184-1255% for platelet-derived growth factor-BB and 93-560% for transforming growth factor-ß1 ). CONCLUSIONS: Haematological and biochemical characteristics varied markedly among 5 techniques used in the field to produce APCs in horses. These discrepancies could have an impact on clinical outcomes, and further studies are needed to determine their influence on the quality of tissue regeneration. Clinicians should not rely on the manufacturers' data relating to human patients to select the most appropriate method for horses.

Comparison of healing in forelimb and hindlimb surgically induced core lesions of the equine superficial digital flexor tendon.

OBJECTIVE: Even though equine multi-limb tendinopathy models have been reported, it is unknown if fore- and hindlimb tendon healing behave similarly. The aim of this study was to compare the healing process of surgically induced superficial digital flexor tendon (SDFT) core lesions of fore- and hindlimbs in horses. METHODS: Tendon core lesions were surgically induced in the SDFT of both fore- and hindlimbs in eight horses. One randomly assigned forelimb and one randomly assigned hindlimb were injected with saline one and two weeks post-surgery. The healing process was monitored clinically and ultrasonographically. After 24 weeks, the tendons were harvested and biochemical, biomechanical and histological parameters were evaluated. RESULTS: Twenty-four weeks post-surgery, the forelimb SDFT lesions had a significantly higher colour Doppler ultrasound vascularization score (p = 0.02) and glycosaminoglycan concentration (p = 0.04) and a significantly lower hydroxylysylpyridinoline content (p = 0.03). CLINICAL RELEVANCE: Our results indicate that fore- and hindlimb SDFT surgically induced lesions exhibit significant differences in several important parameters of tendon healing 24 weeks post-surgery. These differences create significant challenges in using all four limbs and accurately interpreting the results that one might generate. Therefore these findings do not support the use of four-limb models for study of tendon injury until the reasons for these differences are much better understood.

Morphometrical analysis of the thoracolumbar dural sac in sheep using computed assisted myelography.

OBJECTIVES: Sheep are frequently used as animal models in experimental spinal injury studies. Therefore, extensive knowledge of ovine spinal dimensions is essential for experimental design and interpretation of results obtained in these trials. This study aimed to obtain quantitative morphometrical data of the thoracolumbar dural sac in sheep and determine the anatomical relationship between the dural sac and the vertebral canal. METHODS: Computed assisted myelography imaging was carried out in five adult German Black-Headed Mutton sheep under general anaesthesia. Transverse images were acquired with 2 mm slice thickness from the first thoracic to the sixth lumbar vertebrae. Sagittal and transverse diameters and the cross-sectional area of the dural sac and vertebral canal were measured. To determine the anatomical relationship between the dural sac and vertebral canal, the pedicle-dural sac distance (PPSD) and the epidural space as well as the SAC (available space for the dural sac) were calculated. RESULTS: Sagittal diameters of the dural sac ranged from 5.1 to 12.0 mm. Transverse diameters ranged from 5.6 to 12.2 mm. The dural sac area covered 45.9% and 49.0% of the thoracic and lumbar vertebral canal area. The PDSD in the lumbar vertebrae was up to 15.8% larger than in the thoracic ones. The dural sac area was significantly positively correlated with the transverse diameter and area of the vertebral canal. CLINICAL SIGNIFICANCE: The lumbar vertebral canal contained more space for the dural sac, which seems to be safer for testing spinal implants.

Concentration of platelets and growth factors in canine autologous conditioned plasma.

OBJECTIVES: To report the concentration of blood cells and selected growth factors in canine autologous conditioned plasma (ACP). METHODS: The density of blood cells in whole blood (WB), ACP and standard plasma preparation (SP) of 10 healthy mature dogs was determined. In both ACP and SP, the concentration of insulin-like growth factor-1 (IGF-1), epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor-AA, platelet-derived growth factor-AB, platelet-derived growth factor-BB, transforming growth factor-ß1 (TGF-ß1), and transforming growth factor-ß2 was measured using the ELISA technique. In another ten dogs, ACP was prepared using an ultra-soft spinning protocol, and again blood cell density was compared to that obtained in WB. RESULTS: The density of platelets in ACP was significantly higher than that in SP (p = 0.0002), but there was not any significant difference between ACP and WB, nor between WB and ACP prepared using softer centrifugations. Interestingly, only for IGF-1, PDGF-BB, and TGF-ß1 could reliable measurements be obtained, showing a significant increase in PDGF-BB and TGF-ß1 concentrations in ACP compared to SP (p = 0.001, p = 0.0028). Regarding IGF-1 content, there was not any significant difference between ACP and SP. CLINICAL SIGNIFICANCE: Canine ACP prepared according to the manufacturer's recommendations, or by using a softer spin does not show the same specifications as human ACP, which shows a doubling in platelet count compared to WB. Even though canine ACP has a similar number of platelets per injected volume and consequently, probably the same amount of injected growth factors than WB, application of canine ACP would not be associated with the proinflammatory potential reported for WB, as it is almost free of erythrocytes and nucleated cells.

[The influence of the socio-economic structure of the breeding farms of Franches-Montagnes horses on the conditions of husbandry]. / L'influence de la structure socio-economique des exploitations d'elevage de chevaux de race franches-montagnes sur les conditions de détention.

The socio-economic structure of the breeding farms of Franches-Montagnes horses (FM) in Switzerland is evaluated on the basis of an investigation carried out in 2002 by the Swiss FM breeding federation. Questionnaires were sent to 3500 of its members and the results include data from 968 breeding enterprises, housing a total of 3965 FM. The quality of the husbandry of FM varies according to factors such as the altitude and the geographical situation of the farms and studs. Socio-economic parameters, such as the role of FM in the business, their use (breeding, driving, riding) and the age and level of professional education of the owners may also have an effect on standards of husbandry. The results show that the owners for whom FM represent a source of income more frequently keep their horses in standing stalls, but give them more time to exercise at liberty than the horses belonging to amateur breeders. Younger and better educated breeders are more likely to house their animals in groups.

[Evaluation of the quality of husbandry of Franches-Montagnes horses in their breeding farms]. / Les conditions de détention des chevaux de race franches-montagnes dans leurs exploitations d'elevage.

The quality of husbandry of Franches-Montagnes horses (FM) in Switzerland is evaluated on the basis of an investigation carried out in 2002 by the Swiss FM breeding federation. Questionnaires were sent to 3500 of its members and the results include data from 968 breeding enterprises, housing a total of 3965 FM: 46.1% were breeding mares (61.0% with foal at foot), 26.5% young stock, 1.3% stallions and 26.0% non breeding stock (74.6% of which were pleasure horses and 25.4% working horses). 57.6% of the FM were housed in individual boxes with or without permanent outdoor access, 25.4% were hold in groups with or without permanent outdoor access, the remaining 17.0% were kept in standing stalls. 95.0% of the FM had at least visual contact with other equines and 99.2% had sufficient light in their stable. 88.1% were stabled on long stalk straw, while only 4.3% were bedded on other materials other than straw. The average time spent at pasture per horse and per week ranged from 96.5 +/- 51.6 hours in summer to 27.2 +/- 26.7 hours in winter. On average, a FM is used for 8.3 +/- 6.5 hours per week. Horses with an paddock at their disposal spend an average of 39.8 +/- 45.9 hours there per week.