A safety evaluation of allogeneic freeze-dried platelet-rich plasma or conditioned serum compared to autologous frozen products equivalents in equine healthy joints.

BACKGROUND: Hemoderivatives such as autologous conditioned serum (ACS) and platelet-rich plasma (PRP) have been used as potential disease-modifying therapies in musculoskeletal disorders such as osteoarthritis (OA). These therapies are based on the delivery of multiple growth factors and anti-inflammatory cytokines that are known to participate in inflammatory processes. The variability of cytokine content due to the autologous nature of the product, the non-availability for immediate use and need for storage at low temperatures are limitations for its use in the field. An allogeneic freeze-dried conditioned serum (CS) and PRP would provide field clinicians with a more practical approach to use such products in daily practice. Based on in vitro preliminary data, this experimental study aimed to test the in vivo safety of allogeneic freeze-dried CS and PRP in healthy joints, using the horse as a model. RESULTS: Eight horses were randomly assigned and treated with PRP or CS. Horses had three joints injected with ALLO-FD PRP or CS, and three contralateral joints injected with the AUTO version of the same product, by a blinded clinician. Horses were evaluated clinically, and had synovial fluid collected at different time points and evaluated for cell content, PGE2 and protein. Both CS and PRP products triggered a self-limiting and mild inflammatory response in equine healthy joints. This was indicated by the transient increase in nucleated cell count, PGE2 and total protein in synovial fluid. This mild inflammatory response did not result in significant lameness and was not different among the groups. CONCLUSIONS: The allogeneic freeze-dried PRP and CS showed to be overall safe and not dissimilar compared to their autologous frozen version in equine healthy joints. Further studies are necessary to evaluate the modulatory effects of these therapies in a clinical setting.

Use of allogeneic freeze-dried conditioned serum for the prevention of degradation in cartilage exposed to IL-1ß.

BACKGROUND: Autologous conditioned serum (ACS) has been extensively used in the field of veterinary orthopaedics and sports medicine. Due to the autologous and blood-derived nature of this product, issues such as individual variability, need for storage at low temperatures and non-availability for immediate are frequently encountered for ACS use in the field. To address those issues, we proposed the evaluation of an off-the-shelf allogeneic freeze-dried version of conditioned serum in an in vitro model of osteoarthritis. In this study, we evaluated if origin (autologous and allogeneic) and preparation (frozen and freeze-dried) of conditioned serum could influence in its effect in an in vitro model. RESULTS: IL-1ß stimulation in cartilage led to a significant increase in media GAG and decreased levels of GAG in cartilage explants at the termination of the experiment. No significant differences were noted in outcomes measured in the cartilage explants with respect to the main effects of treatment (frozen versus freeze-dried serum), autologous versus allogeneic preparations or based on serum concentration. CONCLUSIONS: The study did not observe any substantial differences in the response of cartilage to allogeneic freeze-dried CS when compared to other independent parameters (autologous and frozen preparations). Further investigation using in vivo systems appears warranted.

Platelet-Rich Products and Their Application to Osteoarthritis.

Autologous platelet-rich plasma (PRP) is a biological preparation made from the patient's own plasma that contains a platelet concentration above the whole blood baseline. Owing to the release of growth factors and other cytokines after degranulation, platelets have a central role in inflammation and in different stages of the healing process. For this reason, PRP-derived products have been used to enhance healing of musculoskeletal injuries and modulate progression of inflammatory processes, including osteoarthritis (OA). Osteoarthritis is one of the main causes of musculoskeletal disabilities in horses, and currently, there is no effective treatment for this disease. Treatments that focus on the modulation of inflammation and disease progression offer new hope for OA. Platelet-rich plasma provides a more practical and accessible option of therapy compared to other forms of biological treatment (i.e., stem cell therapies) and is believed to induce the production of functional matrix. However, several factors related to PRP production, including methods of preparation and application, and intraindividual variability, lead to an inconsistent product, precluding reliable conclusions about its efficacy for clinical use. The aim of this study was to review the benefits related to the clinical use of PRP in OA as well as factors that influence its use, the limitations of this treatment, and future directions of PRP research and therapy.

Avaliação histológica e imunoistoquímica de dois protocolos de terapia celular no reparo do ligamento suspensório equino

O objetivo foi avaliar histologicamente a qualidade do reparo tecidual em amostras de ligamento suspensório equino tratadas com dois protocolos de terapia celular. Os quatro membros de seis animais foram operados simultaneamente para remoção de um fragmento com punch de biópsia. Dois dias após, cada um dos ligamentos recebeu aplicação local de fração mononuclear de medula óssea (MO), células mononucleares cultivadas derivadas de tecido adiposo (TA), solução fisiológica 0,9% (CP) ou nenhum tratamento (CN) como controles. Dois meses após, foram realizadas biópsias dos ligamentos para análise histológica (HE, tricrômio de Masson e pricosírius red) e imunoistoquímica (colágeno tipo III). Verificaram-se alinhamento de fibras, presença de células inflamatórias e presença de vasos (HE e tricrômio de Masson), intensidade de birrefringência e organização tecidual (pricosírius) e deposição de colágeno tipo III (imunoistoquímica). Os dados foram comparados pelo teste de Kruskal-Wallis. Apesar de não ter alcançado significância estatística (P = 0,08), verificou-se melhor alinhamento nos grupos tratados. Na intensidade de birrefringência, verificado pela coloração Pricosírius, não houve diferença com relação aos grupos tratados (MO e TA), contudo os valores se mostraram superiores aos grupos controles, indicando presença de fibras colágenas de melhor qualidade nos grupos submetidos à terapia celular. A presença de células inflamatórias e a intensa marcação para colágeno tipo III demonstram processo cicatricial em evolução em todos os grupos. Concluiu-se que os protocolos utilizados demonstraram potencial para o tratamento de lesões do ligamento suspensório equino, produzindo melhor organização tecidual no tempo estudado

This study aimed to histologically evaluate the quality of the repair tissue in experimentally injured equine suspensory ligament samples treated with two different protocols of cell therapy. The four members of six animals were operated simultaneously to remove a fragment with punch biopsy. Two days later, each ligament received local application of a mononuclear cell fraction of bone marrow (BM), cultured mononuclear cells cultivated from adipose tissue (TA), saline (CP) or no treatment (CN) as controls. Two months later, biopsies were performed for histological analysis of the ligaments (HE, Masson trichrome and pricosirius red) and immunohistochemistry (collagen type III). Collagen fiber alignment, presence of inflammatory cells and presence of vessels (HE and Masson thichrome), birefringence intensity and tissue organization (pricosirius) and type III deposition (immunohistochemistry) were analysed. Data were compared using the Kruskal-Wallis test. Although no statistical significance (P = 0.08) was observed for fiber alignment, there was better alignment in the treated groups. Even though for the birefringence intensity (verified by pricosirius staining), there was no difference between the treated groups (MO and TA) the values were higher than control groups, indicating better deposition of collagen fiber in ligaments subjected to cell therapy. The presence of inflammatory cells and intense staining for collagen type III demonstrate the healing process in all groups. In conclusion, the results demonstrate a potential for the treatment of equine suspensory ligament injuries, producing better tissue organization during the study period