Effect of allogeneic pure platelet-rich plasma, sodium cloxacillin, and their combination for the treatment of subclinical mastitis in crossbred cows.

Introduction: Bovine subclinical mastitis (SCM) caused by Gram-positive bacteria is a major cause of economic loss in the dairy industry, exacerbated in situations where antimicrobial resistance is present. Pure platelet-rich plasma (P-PRP) may be a therapeutic alternative for SCM, when used alone or with antibiotics, such as sodium cloxacillin (SC). This study aimed 1) to evaluate the therapeutic efficacy of allogeneic P-PRP, SC, and their combination (P-PRP+SC) in cows with SCM caused by Staphylococcus aureus and by streptococci (Staphylococcus aureus and S. dysgalactiae); 2) to determine the concentrations of somatic cells (SCC), interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α) and TGF-ß1 in milk samples of the cows. Methods: 130 cows from 4 dairy herds completed the study, of which 40 cows were treated with P-PRP (10 mL), 28 cows with SC (5g), 36 with P-PRP+SC (10mL/5g), and 26 did not receive no treatment (negative control group, NCG). Results: The overall bacteriological cure was observed in 10/40 (25%) cows in the P-PRP group, 9/28 (32.14%) animals in the SC group, 26/36 (72.22%) cows in the P-PRP+SC group, and 10/26 (38.46%) animals in the NCG. SCM caused by S. aureus (82/130, 63.08%), was cured in 6/24 (25%) cows treated with P-PRP, 7/24 (29.2%) cows treated with SC, 8/16 (50%) animals treated with P-PRP+SC, and in 8/18 (44.4%) cows in NCG. For SCM caused by the streptococci (48/130, 36.91%), the cure was achieved in 4/12 (33.3%) cows treated with P-PRP, 2/4 (50%) cows treated with SC, 18/20 (90%) cows treated with P-PRP+SC, and in 2/8 (25%) cows of the NCG. SCC was significantly (p < 0.001) affected by the treatment, herd, cure, bacteria group, and number of calvings factors. IL-1ß milk concentrations were significantly (p < 0.001) influenced by treatment and farm factors, and the interaction between these factors. TNF-α milk concentrations were significantly (p < 0.001) influenced by time factor. TGF-ß1 milk concentrations were significantly affected by the time and cure factors. Conclusion: The combination of P-PRP and SC showed the best therapeutic response (90%) against bovine SCM caused by streptococci. However, none of the treatments showed an effective therapeutic response against S. aureus.

Effects of Temperature and Time on the Denaturation of Transforming Growth Factor Beta-1 and Cytokines from Bovine Platelet-Rich Gel Supernatants.

There is a lack of information about transforming growth factor beta-1 (TGF-ß1) and cytokines contained in pure platelet-rich plasma (P-PRP) and release from pure-platelet-rich gel supernatants (P-PRGS) might be affected by the temperature and time factors; P-PRP from 6 heifers was activated with calcium gluconate. Thereafter, P-PRG and their supernatants (P-PRGS) were maintained at -80, -20, 4, 21, and 37 °C and collected at 3, 6, 12, 24, 48, 96, 144, 192, 240, and 280 h for subsequent determination of TGF-ß1, tumor necrosis factor alfa (TNF-α), interleukin (IL)-2, and IL-6; TGF-ß1 concentrations were significantly (p < 0.05) higher in PRGS maintained at 21 and 37 °C when compared to PRGS maintained at 4, -20, and -80 °C; PRGS TNF-α concentrations were not influenced by temperature and time factors. However, PRGS maintained at 4 °C showed significantly (p < 0.05) higher concentrations when compared to PRGS maintained at -20, and -80 °C at 144, and 192 h. IL-6 concentrations were significantly (p < 0.05) higher in PRGS stored at -20, and -80 over the first 48 h and at 10 days when compared to PRGS stored at 4, 21, and 37 °C. These results could suggest that P-PRP/P-PRGS could be maintained and well preserved for at least 12 days at room temperature for clinical use in bovine therapeutic massive protocols.

Platelet-Rich Plasma and Related Orthobiologics for the Treatment of Equine Musculoskeletal Disorders-A Bibliometric Analysis from 2000 to 2024.

(1) Background: There is increasing interest in the use of platelet-rich plasma and related orthobiologics for the treatment of chronic musculoskeletal disorders in horses; however, there is no information on the bibliometric impact of the literature published in this area. (2) Methods: A bibliometric analysis was performed using the bibliometrix R package by analyzing the documents registered in the WOS and Scopus databases from 2000 to 2024. The included registers were evaluated according to the menu of results from the biblioshiny web app (overview, sources, authors, documents, words, trending topics, clustering, conceptual structure, and social structure). (3) Conclusions: The documents produced were mainly published in Frontiers in Veterinary Science, Journal of Equine Veterinary Science, BMC Veterinary Research, and the American Journal of Veterinary Research). The most productive institutions were Universidad de Caldas, Colorado State University, University of California-Davis, and University of Leipzig, and the most productive countries were the USA, Brazil, and Colombia. Horse, platelet-rich plasma, equine, osteoarthritis, and autologous conditioned serum were the most frequently used keywords. The trending topics in this area are platelet lysates and orthobiologics. The collaboration network of authors, institutions, and countries shows an isolated development of individual author networks with modest collaboration between institutions and countries.

Short-Term Effects of Two COX-2 Selective Non-Steroidal Anti-Inflammatory Drugs on the Release of Growth Factors and Cytokines from Canine Platelet-Rich Gel Supernatants.

(1) Background: There is a lack of knowledge about how a single dose of COX-2 selective non-steroidal anti-inflammatory drugs (NSAIDs) might affect the release of growth factors (GFs) and cytokines from canine platelet-rich gels (PRGs) and other hemocomponents. (2) Methods: A crossover study was conducted in six adult mongrel dogs. Animals were randomized to receive a single dose of either carprofen or firocoxib. PRG, temperature-induced platelet lysate (TIPL), chemically induced PL (CIPL), and plasma hemocomponents were obtained from each dog before (1 h) and after (6 h) the treatments. Platelet and leukocyte counts and determination of the concentrations of platelet-derived growth factor-BB, (PDGF-BB), transforming growth factor beta-1 (TGF-ß1), interleukin 1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α) and IL-10 concentrations were assayed by ELISA in all hemocomponents. (3) Results: Both platelet and leukocyte counts and PDGF-BB concentrations were not affected by NSAIDs and time. Total TGF-ß1 concentrations were not affected by NSAIDs; however, the release of this GF was increased in PRG supernatants (PRGS) at 6 h. IL-1ß and TNF-α concentrations were significantly (p < 0.001) lower in both firocoxib PRGS and plasma at 6 h, respectively. IL-10 concentrations were significantly (p < 0.001) lower at 6 h in all hemocomponents treated with both NSAIDs. (4) Conclusions: The clinical implications of our findings could indicate that these drugs should be withdrawn from patients to allow their clearance before the clinical use of PRP/PRG. On the other hand, the prophylactic use of NSAIDs to avoid the inflammatory reactions that some patients might have after PRP/PRG treatment should be performed only in those animals with severe reactive inflammation to the treatment.

Comparison of Two Methods for the Measurement of Blood Plasma and Capillary Blood Glucose in Tropical Highland Grassing Dairy Cows.

(1) Background: There is lack of published studies validating specific cow-side glucometers such as Centrivet GK (CVGK). (2) Methods: The aims were (1) to measure and compare the blood glucose concentrations in 52 tropic highland grassing cows by using CVGK and the traditional enzymatic/photometric assay (EPA) in plasma and serum (reference method) and (2) to establish if glucose concentrations obtained via these methods could be affected by several demographic and zootechnical parameters of the dairy herd evaluated. (3) Results: Glucose concentrations were significantly (p = 0.00) affected by the method used for their measurement. The intra-assay coefficient of variation (CV) for glucose concentrations in plasma EPA and for CVGK was 14% for both methods with serum EPA, whereas the inter-assay CV for plasma EPA and CVGK was 8% and 13.7%, respectively, with serum EPA. Pearson correlation coefficient calculations between the reference method in serum and plasma presented a slightly positive significant (p = <0.000) correlation (r = 0.56), whereas there was not a significant (p = 0.413) correlation between serum EPA and CVGK (r = 0.135). The Passing and Bablok regressions were out of the ideal expected values for the slope (ß = 1) and the intercept (α = 0) (11), whereas the Bland-Altman plots showed a bias of 5.29 ± 11.73 (mg/dL) for serum and plasma and 11.01 ± 15.74 (mg/dL) for serum and CVGK. The ROC curve showed no sensitivity in detecting normoglycemic cows (area = 53.7 %, e.d = 12.5 %, p = 0.759) for CVGK when compared to plasma EPA (area = 36.1 %, e.d = 14.2 %, p = 0.256). Plasma EPA exhibited a better but not significant effect in detecting hyperglycemic cows (area = 63.9%, e.d = 14.2%, p = 0.256) when compared to HHD (area = 46.3 %, e.d = 12.5 %, p = 0.759). General glucose concentrations, independently of the method used, were significantly (p = <0.001) greater in young cows when compared to adult and old cows. (4) Conclusions: Glucose concentration measurement in plasma by using EPA or in capillary blood via CVGK were not reliable methods when compared with the reference method.

Temporal Release and Denature of Several Mediators in Pure Platelet-Rich Plasma and Temperature-Induced Platelet Lysates Derived from a Similar Bovine Platelet Concentrate.

There is scarce information about bovine platelet-rich plasma/platelet-rich gel (PRP/PRG) and related hemocomponents (HCs), such as platelet lysates (PLs), including growth factor (GF) and cytokine concentrations, and how the stability of these biomolecules could be affected by time and temperature. This study aimed to evaluate the release and stability of transforming growth factor beta 1 (TGF-ß 1), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNF-α) contained in bovine pure PRP (P-PRP) and temperature-induced PL (TIPL) coming from a similar platelet concentrate (PC) at 4 and 37°C at 3 and 96 h. Platelet concentrates (PCs) presented a 1.7-fold concentration of platelets (PLTs) with negligible counts of white blood cells (WBCs) when compared to the counts for these cells in whole blood. TGF-ß 1 concentrations were significantly lowest in plasma followed by TIPL, chemical-induced PL (CIPL), and P-PRP. IL-4 and TNF-α concentrations did not differ between HCs. TGF-ß 1 concentrations were negatively affected in P-PRPs stored at 4°C at 3 and 96 h, whereas those from P-PRP maintained at 37°C presented similar concentrations to TIPL stored at both temperatures over time. IL-4 and TNF-α concentrations were not affected by time or temperature in any of the HCs evaluated. Pure PRGs released additional quantities of GF and cytokines over time when compared with HCs stored over 96 h at 4 and 37°C. The method, either chemical or physical, used for platelet activation or damage produces a different GF and cytokine release pattern, which makes to each evaluated HCs different despite they come from a similar bovine PC. P-PRP activated with calcium gluconate and maintained at 37°C, which polymerizes in P-PRG, showed the best GF and cytokine release/denature profile compared with the rest of the HCs evaluated.

Evaluation of the Pro-, Anti-Inflammatory, and Anabolic Effects of Autologous Platelet-Rich Gel Supernatants in an in vitro Coculture System of Canine Osteoarthritis.

There are scarce in vitro studies indicating the basic mechanisms of why platelet-rich plasma (PRP) is useful in the clinical management of dogs with naturally occurring OA. Methods. Cartilage and synovial membrane explants from six dogs were challenged with lipopolysaccharide (LPS) and cultured for 48 h with platelet-poor gel supernatant (PPGS) and platelet-rich gel supernatant (PRGS) at concentrations of 25 and 50%, respectively. The tissue explants challenged with LPS were cocultured over 48 h and culture media were sampled at 1 and 48 h for the determination of IL-1ß, IL-10, hyaluronan, TGF-ß1, and PDGF-BB by ELISA. Results. IL-1ß concentrations were significantly higher in tissue explant groups cultured for 48 h with PRGS at 50% and with PPGS at 25% when compared to the remaining experimental groups at any time. IL-10 and HA presented similar concentrations in all evaluated groups at any time. TGF-ß1 and PDGF-BB presented higher concentrations in the culture media of tissue explants cultured with PPGS and PRGS at 50%, which diminished with time. Conclusions. Both PPGS and PRGS at both concentrations showed a limited biological effect on cartilage and synovial membrane explants in coculture with LPS. Even PPGS at 25% and PRGS at 50% exhibited proinflammatory effects on these tissues at 48 h.

Intramammary treatment using allogeneic pure platelet-rich plasma in cows with subclinical mastitis caused by Gram-positive bacteria.

The aims of the study were (1) to compare the cure risk of intramammary treatment of pure platelet rich plasma (P-PRP) or cefquinome sulfate (CS) in cows with subclinical mastitis (SCM) caused by Gram-positive bacteria, evaluated via somatic cell count (SCC) and the microbiological analysis of milk; (2) to compare the inflammatory/anti-inflammatory response of mammary gland to both treatments through the analyses of interleukins (IL), interferon gamma (IFN-γ), and tumour necrosis factor alpha (TNF-α) in milk. A non-inferiority randomized clinical trial was conducted. The null hypothesis was that cure risk in the experimental group (P-PRP) was inferior to the reference group (CS). A total of 103 cows were selected according to SCC and presence of Gram-positive bacteria, 49 cows were treated with CS and 54 cows were treated with P-PRP after determination of its cellular and molecular quality control. Cure was assessed by milk analyses at day 21 and 22 after treatment. Cows that remained with SCM were retreated at day 26, and cure assessed at day 47 and 48. Overall, bacteriological cure was observed in 16 cows (30%) of the P-PRP group, and 35 cows (71%) in CS group. Staphylococcus aureus cure risk was higher in CS group, but inconclusive for Streptococcus spp. The mean SCC increased in relation to time only in the P-PRP group. A direct relation between time and treatment for IL-1, IL-2, and IL-6 was observed, while no differences were observed for IL-4. Furthermore, IL-1 and IL-2 increased in cows treated twice in both groups. IL-8, IFN-γ, and TNF-α showed a significant interaction between time and treatment. IFN-γ concentration was lower in the P-PRP group compared to the CS on days 0 and 22. Leukocyte counts were lower in P-PRP when compared to whole blood. TGF-ß1 and PF4 concentrations were higher in platelet lysates in comparison to P-PRGS and plasma. Moreover, PDGF-BB concentration was significantly higher in platelet lysates in comparison to plasma. Results obtained in this study demonstrate that SCM treated with PRP showed a lower rate of bacteriologic cure when compared to animals treated with CS.

Application of udder surface temperature by infrared thermography for diagnosis of subclinical mastitis in Holstein cows located in tropical highlands.

Several reports have indicated that udder surface temperature (UST) can be a useful indicator of subclinical mastitis (SCM). The objective was to evaluate UST by infrared thermography (IRT) as a diagnostic tool for SCM and intramammary infection (IMI), and to assess the influence of environmental conditions in the potential diagnosis of this disease in dairy cows located at high-altitude tropical regions. A total of 105 cows (397 quarters) from 3 dairy farms with mechanical and manual milking methods were enrolled in the study. Subclinical mastitis was diagnosed when quarter samples had a somatic cell count (SCC) ≥200 × 103 cells/mL, microbial growth (MG) was defined when a major pathogen (≥1 cfu/plate) or Corynebacterium spp. (≥10 cfu/plate) was isolated, and IMI was defined as the presence of MG and SCC ≥100 × 103 cells/mL. Infrared images were taken with a thermal camera placed 1 m away from the udder, and shots of the rear and left and right lateral view were made during the morning milking, before any manipulation of the udder and employing dark cardboard on the contralateral side to avoid artifacts in the background. A multilevel mixed effects linear regression model clustered within cows and herd was performed to evaluate the associations with UST. Clinical performance was evaluated using the Youden index to establish the optimum UST thresholds, which were set at 32.6°C for any case definition when milking was by hand, at 33.7°C for MG, and at 34°C for SCM and IMI in machine-milked quarters. Sensitivity (Se), specificity (Sp), area under curve (AUC), and positive likelihood ratio (+LR) were also assessed. Test agreement was assessed by kappa coefficient (κ). The UST of healthy quarters ranged between (95% CI) 32.4 and 32.6°C, lower than SCM quarters (n = 88) at 32.9°C (95% CI: 32.7-33.1 °C), MG quarters (n = 56) at 33.5°C (95% CI: 33.3-33.7°C), and IMI quarters (n = 50) at 33.5°C (95% CI: 33.2-33.7 °C). The UST was also related to the milking method: higher temperatures were observed for hand milking (n = 90) compared with machine milking (n = 185). No relation between environmental conditions such as wind speed, atmospheric temperature, relative humidity, and temperature-humidity index and UST were observed during this study. For hand milking, the optimal UST threshold was 32.6°C; for SCM, Se = 0.53, Sp = 0.89, AUC = 0.71, κ = 0.4; for MG, Se = 0.83, Sp = 0.93, AUC = 0.88, κ = 0.77; and for IMI, Se = 0.82, Sp = 0.92, AUC = 0.87, κ = 0.74. The machine milking threshold for SCM resulted in Se = 0.42, Sp = 0.97, AUC = 0.70, κ = 0.47; for MG, Se = 0.82, Sp = 0.89, AUC = 0.85, κ = 0.60; and for IMI, Se = 0.82, Sp = 0.98, AUC = 0.90, κ = 0.79. These findings suggest that UST determined by IRT is higher in machine-milked cows and in quarters with MG and IMI than in healthy quarters; therefore, UST by IRT is a reliable, clinically useful method for MG and IMI diagnosis.

A Critical Overview of the Use of Platelet-Rich Plasma in Equine Medicine Over the Last Decade.

In the 1990s, the role of platelets in inflammation and tissue healing was finally recognized. Since then, the clinical use of platelet-derived products (hemocomponents), such as, platelet-rich plasma (PRP), markedly increased. The promise of a more economical option of a disease-modifying treatment led to the intensive and continuous research of PRP products and to its widespread clinical use. A number of protocols and commercial kits have been developed with the intention of creating a more practical and reliable option for clinical use in equine patients. Still, the direct comparison between studies is particularly challenging due to the lack of standardization on the preparation methods and product composition. The incomplete reports on PRP cellular concentration and the poorly designed in vivo studies are additional matters that contest the clinical efficiency of this biomaterial. To overcome such challenges, several in vitro and in vivo studies have been proposed. Specifically, experiments have greatly focused in protocol optimization and its effect in different tissues. Additionally, in vivo studies have proposed different biological products envisioning the upgrade of the anti-inflammatory cytokines trusting to increase its anti-inflammatory effect. The individual variability and health status of the animal, type of tissue and condition treated, and protocol implemented are known to influence on the product's cell and cytokine composition. Such variability is a main clinical concern once it can potentially influence on PRP's therapeutic effects. Thus, lack of qualitative and quantitative evidence-based data supporting PRP's clinical use persists, despite of the numerous studies intended to accomplish this purpose. This narrative review aims to critically evaluate the main research published in the past decade and how it can potentially impact the clinical use of PRP.

Changes on the Structural Architecture and Growth Factor Release, and Degradation in Equine Platelet-Rich Fibrin Clots Cultured Over Time.

The aims of the present study were (1) to describe the microscopic and ultrastructural appearance of equine platelet-rich fibrin (PRF) clots and (2) to determine the release and degradation of transforming growth factor beta 1 (TGF-ß1) and insulin-like growth factor type I (IGF-I) from PRF clots incubated over 14 days. Whole blood from six horses was collected into plain tubes and centrifuged at 240 g for 8 minutes. Clots were evaluated by histology and by both transmission and scanning electronic microscopy (TEM and SEM). Growth factor concentrations were measured by ELISA at 48-hour intervals over 14 days and analyzed by one-way repeated-measures ANOVA. Histology showed a clot composed by a fibrin layer and a cellular layer with platelets and leukocytes. Scanning electron microscopy showed the cells trapped by an incipient fibrin network at 1 hour. At day 8, these cells were embedded by an incipient fibrin network. At day 14, the leukocytes and platelet aggregates from the clot were imbibed in an organized web of fibrin fibrils. TEM exhibited platelets with preserved cytoplasm and alpha granules randomly scattered at day 8, and damaged platelets with interrupted cytoplasm and organelle emigration to the periphery at day 14. TGF-ß1 and IGF-I concentrations showed a progressive increase until day 14. TGF-ß1 was released from PRF clots in a gradual and controlled manner, and increasing its concentration for two weeks, which supports TEM findings indicating that platelets began disintegrating by day 14. Furthermore, IGF-I production and release from PRF clots is sustained over time.

Equine suspensory ligament and tendon explants cultured with platelet-rich gel supernatants release different anti-inflammatory and anabolic mediators.

The aim of this study was to evaluate the release of pro- and anti-inflammatory as well as anabolic mediators stimulated by a leukocyte-reduced platelet-rich gel supernatant (Lr-PRGS) and a leukocyte-reduced plasma supernatant (Lr-PL) at two concentrations (25 and 50%) on normal equine suspensory ligament explants (SLEs) and tendon explants (TEs). SLEs and TEs from six horses were independently incubated for 48 h with Lr-PRGS and Lr-PL at concentrations of 25 and 50%, respectively. Samples were collected from the incubated tissues at 1 h and 48 h, which were employed for ELISA determination of interleukin 1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), IL-4, IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor isoform BB (PDGF-BB), transforming growth factor beta-1 (TGF-ß1, and hyaluronic acid (HA). Overall, 50% Lr-PRGS induced significantly less IL-1ß release than the other hemoderivatives in both tissues. At 48 h, both Lr-PRGS and 25% Lr-PL induced significantly higher TNF-α concentrations in SLEs when compared to TEs, whereas both Lr-PRGS concentrations induced significantly higher IL-4 concentrations in SLEs in comparison to TEs. IL-1ra release was not different between tissues. However, this cytokine was significantly higher in tissue explants cultured with both Lr-PRGS concentrations. HA concentration was lower in tissue explants cultured with all hemoderivatives at two concentrations when compared to the control group. The positive effects observed for ligaments and tendons treated with Lr-PRGS may be mediated by the inhibition of IL-1ß release of and increased release of IL-4 and IL-1ra. Furthermore, PDGF-BB could be a polypeptide responsible for mediating the release of anti-inflammatory cytokines in SLEs and TEs incubated with Lr-PRGS.

Study of a Two-Step Centrifugation Protocol for Concentrating Cells and Growth Factors in Bovine Platelet-Rich Plasma.

There is a lack of information about the methods used for bovine platelet-rich plasma (PRP)/platelet-rich gel (PRG) procurement, including information on platelet (PLT), white blood cell (WBC) in PRP, and growth factor release from PRG supernatants. The aims of this study were to compare and to correlate the PLT, WBC, transforming growth factor beta-1 (TGF-ß1), and platelet-derived growth factor BB (PDGF-BB) concentrations in bovine whole blood, plasma, and four PRP layers and their respective PRG supernatants: A and B (obtained by a single centrifugation tube method at 720g/5 min) and C and D (obtained by a double centrifugation tube method, by using two centrifugation episodes at 720g/5 min). PLT and WBC counts were significantly higher in PRP-C, followed by whole blood, PRP-A, PRP-B, and PRP-D. TGF-ß1 concentrations were significantly higher in PRG-B supernatants and its correspondent PRP-B lysate when compared to the other PRG supernatants and plasma. Supernatants from PRG-A, PRG-B, and PRG-D had equivalent TGF-ß1 concentrations. PDGF-BB concentrations were not statistically different between the hemoderivatives. Significant Pearson correlations were noted between PLT counts and WBC counts (0.8) and between PLT counts and PLT distribution width (0.6). Further studies should be performed to assess the potential clinical applications of these PRPs.

Proinflammatory and Anabolic Gene Expression Effects of Platelet-Rich Gel Supernatants on Equine Synovial Membrane Explants Challenged with Lipopolysaccharide.

Platelet-rich plasma (PRP) preparations are used in horses with osteoarthritis (OA). However, some controversies remain regarding the ideal concentration of platelets and leukocytes to produce an adequate anti-inflammatory and anabolic response in the synovial membrane. The aims of this study were to study the influence of leukoconcentrated platelet-rich gel (Lc-PRG) and leukoreduced platelet-rich gel (Lr-PRG) supernatants on the quantitative expression of some proinflammatory and anabolic genes in equine synovial membrane explants (SMEs) challenged with lipopolysaccharide (LPS). SMEs from six horses were cultured over 96 h. Then, SMEs were harvested for RNA extraction and quantitative gene expression analysis by RT-qPCR for nuclear factor kappa B (NFκB), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1), and cartilage oligomeric matrix protein (COMP). The 25% and 50% Lc-PRG supernatants led to downregulation of NFκB, MMP-13, ADAMTS-4, COL1A1, COL2A1, and COMP in SMEs. Lr-PRG supernatants (particularly at the 50% concentration) induced downregulation of NFκB, MMP-13, ADAMTS-4, and COL1A1 and upregulation of COL2A1 and COMP. Lr-PRG supernatants should be used for the treatment of inflammatory arthropathies in horses because they have anti-inflammatory and anabolic effects in the synovial membrane.

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

OBJECTIVES: To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-ß1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. METHODS: Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. RESULTS: There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. CONCLUSIONS: Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-ß1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals.

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants.

OBJECTIVE: To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-ß1) concentrations in platelet-rich gel (PRG) supernatants. METHODS: Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. RESULTS: Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-ß1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (rs: 0.76), platelet counts in PRP with TGF-ß1 concentrations in PRG supernatants (rs: 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (rs: 0.78), leukocyte counts in PRP with TGF-ß1 concentrations in PRG supernatants (rs: 0.76), and PDGF-BB concentrations with activating substances (rs: 0.72). CLINICAL SIGNIFICANCE: Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.

In vitro effects of platelet-rich gel supernatants on histology and chondrocyte apoptosis scores, hyaluronan release and gene expression of equine cartilage explants challenged with lipopolysaccharide.

BACKGROUND: Platelet-rich plasma (PRP) preparations are a common treatment in equine osteoarthritis (OA). However, there are controversies regarding the ideal concentration of platelets and leukocytes in these biological substances necessary to induce an adequate anti-inflammatory and anabolic response in articular cartilage. The aims were to study the influence of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the histological changes of cartilage, the degree of chondrocyte apoptosis, the production of hyaluronan (HA) and the gene expression of nuclear factor kappa beta (NFkß), matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), collagen type I alpha 1 (COL1A1), collagen type II alpha 1 (COL2A1) and cartilage oligomeric matrix protein (COMP) in normal cartilage explants (CEs) challenged with lipopolysaccharide (LPS). RESULTS: Overall, 25 % L-PRG supernatant (followed in order of importance by, 50 % P-PRG, 25 % P-PRG and 50 % L-PRG) represented the substance with the most important anti-inflammatory and anabolic effect. 25 % P-PRG supernatant presented important anabolic effects, but it induced a more severe chondrocyte apoptosis than the other evaluated substances. CONCLUSIONS: 25 % L-PRG supernatant presented the best therapeutic profile. Our results demonstrate that the biological variability of PRP preparations makes their application rather challenging. Additional in vivo research is necessary to know the effect of PRP preparations at different concentrations.

Implications of anticoagulants and gender on cell counts and growth factor concentration in platelet-rich plasma and platelet-rich gel supernatants from rabbits.

OBJECTIVES: Our objectives were as follows: 1) to validate a protocol for producing rabbit platelet-rich plasma (PRP); 2) to determine the influence of two anticoagulants, sodium citrate and acid-citrate-dextrose solution A, and gender on cell count in PRP and growth factor concentration in pure platelet-rich gel supernatants; 3) to correlate the variables evaluated. METHODS: Whole blood from 18 New Zealand rabbits (9 males and 9 females) was obtained with sodium citrate and acid-citrate-dextrose solution A for processing PRP fractions (A and B), which were evaluated for haematology. The PRP fractions were either activated with calcium gluconate or lysated with a detergent. The concentrations of transforming growth factor beta 1 and platelet-derived growth factor BB were assayed by ELISA. RESULTS: The sodium citrate PRP-B had significantly higher counts of platelets in comparison to PRP-A and whole blood obtained with the same anticoagulant and the homologous acid-citrate-dextrose solution A PRP fraction. The sodium citrate PRP-A had a significantly higher count of leukocytes compared to the homologous acid-citrate-dextrose solution A fraction. All the PRP fractions had a significant leuko-reduction when compared to whole blood. The sodium citrate PRP-A fraction from female rabbits had significantly lower platelet counts and significantly higher leukocyte counts than the same acid-citrate-dextrose solution A fraction. Growth factor concentration was not affected by the type of anticoagulant or gender. CLINICAL SIGNIFICANCE: The type of anticoagulant and gender affected the cell counts in PRP, but they did not influence the growth factor concentration. More complete rabbit PRP studies should be performed before evaluating this type of substance in models of disease.

Autologous leukocyte-reduced platelet-rich plasma therapy for Achilles tendinopathy induced by collagenase in a rabbit model.

Leukocyte-reduced platelet-rich plasma (LR-PRP) is a therapy for tendinopathy of the Achilles tendon (TAT); however, there is scarce information regarding LR-PRP effects in rabbit models of TAT. We compared, at 4 and 12 weeks (w), the LR-PRP and placebo (PBS) effects on ultrasonography, histology and relative gene expression of collagen types I (COL1A1) and III (COL3A1) and vascular endothelial growth factor (VEGF) in 24 rabbits with TAT induced by collagenase. The rabbits (treated with both treatments) were euthanatised after either 4 or 12 w. A healthy group (HG (n = 6)) was included. At 4 and 12 w, the LR-PRP group had a no statistically different histology score to the HG. At w 4, the COL1A1 expression was significantly higher in the LR-PRP group when compared to HG, and the expression of COL3A1 from both LR-PRP and PBS-treated tendons was significantly higher when compared to the HG. At w 12, the expression of COL3A1 remained significantly higher in the PBS group in comparison to the LR-PRP group and the HG. At w 4, the LR-PRP group presented a significantly higher expression of VEGF when compared to the PBS group and the HG. In conclusion, LR-PRP treatment showed regenerative properties in rabbits with TAT.