Extracorporeal Shockwave Therapy Increases Growth Factor Release from Equine Platelet-Rich Plasma In Vitro

IntroductionExtracorporeal shockwave therapy (ESWT) and platelet-rich plasma (PRP) are common treatments for soft tissue injuries in horses. Shockwave triggers cell specific responses to promote healing. Growth factors released from PRP also promote healing. It has been hypothesized that greater growth factor release would amplify the healing process. The combination of ESWT and PRP could promote healing in injured tendons and ligaments in the horse. The objective of this study was to determine if application of shockwaves to PRP samples increases the concentration of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor ββ (PDGF-ββ) released from the platelets in vitro.Materials and methodsPRP was produced from blood drawn from six horses. The PRP from each horse was exposed to the following treatments: (1) positive control (freeze-thaw cycle), (2) untreated negative control, or shockwaves with either (3) a “standard probe” (ESWT-S) with a 2 cm focal width and medium energy density or (4) a “power probe” (ESWT-P) with a 1 cm focal width and high energy density. After each treatment, the samples were centrifuged, and the supernatant was harvested. The supernatant was then used for growth factor quantification via commercially available ELISA kits for TGF-β1 and PDGF-ββ.ResultsConcentrations of TGF-β1 and PDGF-ββ in PRP that underwent a freeze-thaw cycle were significantly increased compared with all other treatments. Both ESWT-S and ESWT-P resulted in significantly increased TGF-β1 concentrations, 46 and 33%, respectively, when compared with the negative control. Both ESWT-S and ESWT-P resulted in significantly increased PDGF-ββ concentrations, 219 and 190%, respectively, when compared with the negative control.DiscussionThese data indicate that the application of ESWT to PRP increases the expression of growth factors in vitro. This suggests that the combination therapy of local PRP injection followed by ESWT may stimulate release of growth factors from platelets after they have been injected into the area of injury.ConclusionThe combination of PRP and ESWT might result in synergism of two modalities previously utilized individually for tendon and ligament injuries in horses

MRI-Based Assessment of Intralesional Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Equine Tendonitis

Ultrasound-guided intralesional injection of mesenchymal stem cells (MSCs) is held as the benchmark for cell delivery in tendonitis. The primary objective of this study was to investigate the immediate cell distribution following intralesional injection of MSCs. Unilateral superficial digital flexor tendon (SDFT) lesions were created in the forelimb of six horses and injected with 10 × 106 MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIOs) under ultrasound guidance. Assays were performed to confirm that there were no significant changes in cell viability, proliferation, migration, or trilineage differentiation due to the presence of SPIOs. Limbs were imaged on a 1.5-tesla clinical MRI scanner postmortem before and after injection to determine the extent of tendonitis and detect SPIO MSCs. Clusters of labeled cells were visible as signal voids in 6/6 subjects. Coalescing regions of signal void were diffusely present in the peritendinous tissues. Although previous reports have determined that local injury retains cells within a small radius of the site of injection, our study shows greater than expected delocalization and relatively few cells retained within collagenous tendon compared to surrounding fascia. Further work is needed if this is a reality in vivo and to determine if directed intralesional delivery of MSCs is as critical as presently thought

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle

Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA)

Abstract Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA–LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells

Abstract Background Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Methods Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Results Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS. Conclusion ePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS

Local and Systemic Antibody Responses in Beef Calves Vaccinated with a Modified-Live Virus Bovine Respiratory Syncytial Virus (BRSV) Vaccine at Birth following BRSV Infection

Maternal antibodies interfere with BRSV vaccine responses and efficacy in young calves. The objective of this study was to determine if vaccination before the complete absorption of colostral antibodies results in adequate immune priming and clinical protection of beef calves. Within 6 h of life, calves were randomly assigned to 2 different treatment groups. Group Vacc (n = 25) received a single dose of a modified-live virus (MLV) BRSV vaccine intranasally (IN) and group Control (n = 25) received 2 mL of 0.9% saline IN. At approximately 3 months of age, all calves were experimentally challenged with BRSV. Serum and nasal secretion samples were collected before and after challenge for BRSV real-time RT-PCR and antibody testing. Respiratory signs were not observed before challenge. After challenge, respiratory scores were similar between groups. On the challenge day, >40% of calves in each group were febrile. The mean serum and nasal BRSV-specific antibody titers indicated natural BRSV exposure before the experimental challenge in both groups. All calves tested positive for BRSV and had a similar duration of shedding after challenge. Based on these results, vaccination at birth does not offer advantages for immune priming or clinical protection for beef calves in BRSV-endemic cow-calf herds