Lower centrifugation speed and time are positively associated with platelet concentration in a canine autologous conditioned plasma system.

OBJECTIVE: To evaluate the effect of variable centrifugation protocols on the cellular composition of the final product of a canine autologous conditioned plasma double-syringe system. ANIMALS: 30 client-owned healthy adult medium- to large-breed (17- to 45-kg) dogs. METHODS: 35 mL of anticoagulated whole blood from each subject was aliquoted into 3 samples: a baseline and 2 double syringes. The syringes were processed for platelet-rich plasma (PRP). Each double syringe was randomly assigned to 1 of 5 groups, which varied in centrifugation settings between 580 and 1,304 X g and 5 and 10 minutes. CBC analysis was performed on each of the samples to determine cellular composition. A mixed-effect linear model was fit to the data. RESULTS: 60 PRP samples and 30 whole blood samples were analyzed. Manufacturer settings generated a platelet fold change > 1 but did not increase concentration to the extent expected. When comparing speed alone, increased centrifugation force was associated with lower platelet fold change. When comparing time alone, increased centrifugation time was also associated with lower platelet fold change and lower leukocyte concentration. CLINICAL RELEVANCE: Autologous conditioned plasma double syringes require a low volume of initial whole blood, making them preferable for canine PRP in clinical settings. This study aimed to evaluate the effect of the centrifugation protocol on the final product cellular composition in dogs and add to the available data on protocols to maximize platelet yield in PRP. Due to inherent individual variability, this study emphasized the importance of evaluating biological samples prior to administration to predict and improve patient outcomes.

Dose-Dependent Increase in Whole Blood Omega-3 Fatty Acid Concentration in Horses Receiving a Marine-Based Fatty-Acid Supplement.

The objective of this study was to determine the effects of an oral, commercially available, marine based omega-3 fatty acid (n3-FA) supplement on fatty acid characteristics in horse whole blood. Fifty healthy, adult horses of various light breeds were assigned to one of two treatment groups: Group 1 receiving 7.5g/day of the test supplement, and Group 2 receiving 15g/day of the test supplement. The supplement contained 0.092g docosahexaenoic acid (DHA) and 0.148g eicosapentaenoic acid (EPA) per gram. Therefore, Group 1 received 1.11g of EPA and 0.69g of DHA daily, while Group 2 received 2.22g of EPA and 1.38g DHA daily. Blood was taken at time of enrollment and after 6 and 12 weeks of supplementation. Blood was subjected to gas chromatography to quantify the fatty acid characteristics of whole blood. At both 6 and 12 weeks following supplementation, there was a significant increase in all n3-FAs evaluated, including DHA and EPA, compared to baseline values, with Group 2 significantly increased compared to Group 1 at both time points. There was also a significant decrease in omega-6 fatty acids (n6-FAs) between baseline and 6 weeks of supplementation in both groups, with a larger decrease seen in Group 2. The dose-dependent increases in concentration of all n3-FAs evaluated at all-time points validates the use of this product as a n3-FA targeted supplement in horses. These findings also suggest that dose of supplement has a greater effect on increasing whole blood n3-FAs compared to duration of treatment.

The Effect of Single vs Serial Platelet-Rich Plasma Injections in Osteochondral Lesions Treated With Microfracture: An In Vivo Rabbit Model.

BACKGROUND: Biological adjuvants are used after a musculoskeletal injury to improve healing, decrease inflammation, and restore joint homeostasis. Work on 1 such adjuvant, platelet-rich plasma (PRP), has suggested a positive effect when introduced during cartilage repair. However, it remains unknown whether healing osteochondral injuries benefit from serial PRP injections. PURPOSE: To evaluate the effects of serial PRP injections versus a single PRP injection on reparative cartilaginous tissue, subchondral bone remodeling, and the expression of inflammatory cytokines in joint synovium. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 New Zealand White rabbits were randomly assigned to receive 1 (1P), 2 (2P), or 3 (3P) PRP injections. Cylindrical full-thickness cartilage defects (2.9 × 2.9 mm) with microdrillings (0.6-mm diameter) were created on the medial condyles of both knees. PRP was injected into the right knee after closure (groups 1P, 2P, and 3P), at 2 weeks after surgery (groups 2P and 3P), and at 4 weeks after surgery (group 3P). The left knees did not receive any PRP injections. A total of 6 rabbits in each group were euthanized at 3, 6, and 12 weeks postoperatively. Cartilage repair tissue was assessed using the Goebel macroscopic and modified International Cartilage Regeneration & Joint Preservation Society (ICRS) histological scoring systems. Subchondral bone remodeling was evaluated by micro-computed tomography analysis (micro-CT). Inflammatory cytokine levels were assessed by quantitative polymerase chain reaction. RESULTS: No significant differences were found for the mean macroscopic score between the PRP groups at 12 weeks (control, 6.1 ± 3.3; group 1P, 3.4 ± 2.7; group 2P, 4.2 ± 2.9; group 3P, 0.7 ± 1.5). All PRP groups had a significantly higher mean modified ICRS histological score compared with the control group, but no significant difference was found among the PRP groups. No significant differences were seen in outcomes for the tested micro-CT parameters or cytokine expression levels. CONCLUSION: Serial PRP injections conferred no apparent advantage over single injections according to evaluations of the macroscopic and histological appearance of the cartilaginous tissue, subchondral bone healing, and inflammatory cytokine expression levels in the synovium. CLINICAL RELEVANCE: The use of PRP as a biological adjuvant to bone marrow stimulation for osteochondral lesions has the potential to enhance the quality of regenerative cartilaginous tissue. We recommend only a single PRP injection if the use of PRP is indicated by the operating surgeon as an adjuvant therapy for osteochondral lesions.

Biological Mechanisms for Cartilage Repair Using a BioCartilage Scaffold: Cellular Adhesion/Migration and Bioactive Proteins.

Objective. BioCartilage is a desiccated, particulated cartilage allograft used for repair of focal cartilage defects. It is mixed with a biologic such as bone marrow concentrate (BMC), pressed into a contained defect, and sealed with fibrin glue. The objective of this study was to assess if BioCartilage could serve as a bioactive scaffold by affecting cellular adhesion, cellular migration, or the release interleukin-1 receptor antagonist protein (IL-1RA), and to identify its full proteomic makeup. Design. Cartilage explants were used to model confined defects. BioCartilage was mixed with BMC, grafted into defects, and sealed with 1 of 5 fibrin glues. Constructs were cultured for 24 or 48 hours and then processed for live/dead microscopy. Chondrocyte and mesenchymal stem cell (MSC) adhesion on BioCartilage was assessed using scanning electron microscopy. Conditioned medium from cultures and the biologics used in the study were assayed for IL-1RA. The protein footprint of BioCartilage was determined using bottom-up proteomics. Results. BioCartilage supported chondrocyte and MSC attachment within 24 hours, and cell viability was retained in all constructs at 24 and 48 hours. Fibrin glue did not inhibit cell attachment. BMC had the highest concentration of IL-1RA. Proteomics yielded 254 proteins, including collagens, proteoglycans, and several bioactive proteins with known anabolic roles including cartilage oligomeric matrix protein. Conclusions. This study suggests that BioCartilage has the chemical composition and architecture to support cell adherence and migration and to provide bioactive proteins, which together should have biologics advantages in cartilage repair beyond its role as a scaffold.

Platelet and Leukocyte Concentration in Equine Autologous Conditioned Plasma Are Inversely Distributed by Layer and Are Not Affected by Centrifugation Rate.

Background: Platelet rich plasma (PRP) is used extensively in equine regenerative medicine. Differences in preparation protocols give rise to significant variability in the cellular composition of PRP making it very difficult to establish a standard of care in the field. This study aimed to optimize the preparation protocol for leukocyte-reduced PRP (P-PRP). Methods: Blood (100 mL) was collected from horses (n = 5) and divided into 2 purple top EDTA tubes and 6 (15 mL) double syringesa with a final concentration of 10% acid citrate dextrose anticoagulant. Six double syringesa were collected from each horse; PRP samples were prepared in duplicate and centrifuged at 1,100 rpm (188 × g), 1,300 rpm (263 × g), or 1,500 rpm (350 × g). Duplicates were subjected to +/- braking at the end of centrifugation. The total volume of PRP generated was measured and divided into thirds. Each third (top, middle, and bottom) were drawn off separately using the inner (6 mL syringe) and placed in purple top EDTA tubes. Automated complete blood counts were performed on all peripheral whole blood and PRP samples. Results: The concentration of leukocytes was higher in the bottom layer of PRP compared to the top and middle layers (p < 0.0001). The concentration of platelets was slightly lower in the bottom layer of PRP than the middle layer (p = 0.02). Centrifugation braking increased the leukocyte concentration in the top (p = 0.03) and middle layers of PRP (p = 0.001). Centrifugation rate had no effect on the cellular composition of PRP (p = 0.1-0.6). Conclusions: Because layer of plasma affected both platelet and leukocyte concentrations in PRP, the most important modification for the current single spin, double syringe, plasma based PRP preparation protocols is to exclude the bottom 1/3 layer of PRP.

Integrin α10ß1-Selected Mesenchymal Stem Cells Mitigate the Progression of Osteoarthritis in an Equine Talar Impact Model.

BACKGROUND: Early intervention with mesenchymal stem cells (MSCs) after articular trauma has the potential to limit progression of focal lesions and prevent ongoing cartilage degeneration by modulating the joint environment and/or contributing to repair. Integrin α10ß1 is the main collagen type II binding receptor on chondrocytes, and MSCs that are selected for high expression of the α10 subunit have improved chondrogenic potential. The ability of α10ß1-selected (integrin α10high) MSCs to protect cartilage after injury has not been investigated. PURPOSE: To investigate integrin α10high MSCs to prevent posttraumatic osteoarthritis in an equine model of impact-induced talar injury. STUDY DESIGN: Controlled laboratory study. METHODS: Focal cartilage injuries were created on the tali of horses (2-5 years, n = 8) by using an impacting device equipped to measure impact stress. Joints were treated with 20 × 106 allogenic adipose-derived α10high MSCs or saline vehicle (control) 4 days after injury. Synovial fluid was collected serially and analyzed for protein content, cell counts, markers of inflammation (prostaglandin E2, tumor necrosis factor α) and collagen homeostasis (procollagen II C-propeptide, collagen type II cleavage product), and glycosaminoglycan content. Second-look arthroscopy was performed at 6 weeks, and horses were euthanized at 6 months. Joints were imaged with radiographs and quantitative 3-T magnetic resonance imaging. Postmortem examinations were performed, and India ink was applied to the talar articular surface to identify areas of cartilage fibrillation. Synovial membrane and osteochondral histology was performed, and immunohistochemistry was used to assess type I and II collagen and lubricin. A mixed effect model with Tukey post hoc and linear contrasts or paired t tests were used, as appropriate. RESULTS: Integrin α10high MSC-treated joints had less subchondral bone sclerosis on radiographs (P = .04) and histology (P = .006) and less cartilage fibrillation (P = .04) as compared with control joints. On gross pathology, less India ink adhered to impact sites in treated joints than in controls, which may be explained by the finding of more prominent lubricin immunostaining in treated joints. Prostaglandin E2 concentration in synovial fluid and mononuclear cell synovial infiltrate were increased in treated joints, suggesting possible immunomodulation by integrin α10high MSCs. CONCLUSION: Intra-articular administration of integrin α10high MSCs is safe, and evidence suggests that the cells mitigate the effects of joint trauma. CLINICAL RELEVANCE: This preclinical study indicates that intra-articular therapy with integrin α10high MSCs after joint trauma may be protective against posttraumatic osteoarthritis.

Long-term Evaluation of Meniscal Tissue Formation in 3-dimensional-Printed Scaffolds With Sequential Release of Connective Tissue Growth Factor and TGF-ß3 in an Ovine Model.

BACKGROUND: Artificial meniscal scaffolds are being developed to prevent development of osteoarthritis after meniscectomy. Previously, it was reported that 3-dimensional (3D) anatomic scaffolds loaded with connective tissue growth factor (CTGF) and transforming growth factor ß3 (TGF-ß3) achieved meniscal regeneration in an ovine model. This was a relatively short-term study (3 months postoperative), and outcome analyses did not include magnetic resonance imaging (MRI). PURPOSE: To evaluate long-term outcome of meniscal replacement with growth factor-laden poly-ε-caprolactone (PCL) scaffolds. STUDY DESIGN: Controlled laboratory study. METHODS: Anatomically shaped ovine meniscal scaffolds were fabricated from PCL with a 3D printer based on MRI data. Skeletally mature sheep (N = 34) were randomly allocated to 3 groups: scaffold without growth factor (0-µg group), scaffold with CTGF microspheres (µS) (5 µg) + TGF-ß3 µS (5 µg) (5-µg group), and scaffold with CTGF µS (10 µg) + TGF-ß3 µS (10 µg) (10-µg group). Unilateral medial meniscal replacement was performed. Animals were euthanized at 6 or 12 months. Regenerated meniscus, articular cartilage status, and synovial reaction were evaluated quantitatively with gross inspection, histology, and MRI. Kruskal-Wallis and Dunn tests were used to compare the 3 groups. RESULTS: Remnants of the PCL scaffold were evident in the 6-month specimens and were decreased but still present at 12 months in most animals. There were no significant differences among groups in gross inspection, histology, or MRI for either meniscal regeneration or articular cartilage protection. All experimental groups exhibited articular cartilage degeneration as compared with control (nonoperated). In terms of synovitis, there were no clear differences among groups, suggesting that growth factors did not increase inflammation and fibrosis. MRI revealed that meniscal extrusion was observed in most animals (82.7%). CONCLUSION: Previously, the combination of CTGF and TGF-ß3 was shown to stimulate mesenchymal stem cells into a fibrochondrocyte lineage. CTGF and TGF-ß3 did not aggravate synovitis, suggesting no adverse response to the combination of 3D-printed PCL scaffold combined with CTGF and TGF-ß3. Further work will be required to improve scaffold fixation to avoid meniscal extrusion. CLINICAL RELEVANCE: A significant advantage of this technique is the ability to print custom-fit scaffolds from MRI-generated templates. In addition, average-size menisci could be printed and available for off-the-shelf applications. Based on the 1-year duration of the study, the approach appears to be promising for meniscal regeneration in humans.

Short-Term Storage of Platelet-Rich Plasma at Room Temperature Does Not Affect Growth Factor or Catabolic Cytokine Concentration.

The aim of this study was to provide clinical recommendations about the use of platelet-rich plasma (PRP) that was subjected to short-term storage at room temperature. We determined bioactive growth factor and cytokine concentrations as indicators of platelet and white blood cell degranulation in blood and PRP. Additionally, this study sought to validate the use of manual, direct smear analysis as an alternative to automated methods for platelet quantification in PRP. Blood was used to generate low-leukocyte PRP (Llo PRP) or high-leukocyte PRP (Lhi PRP). Blood was either processed immediately or kept at room temperature for 2 or 4 hours prior to generation of PRP, which was then held at room temperature for 0, 1, 2, or 4 hours. Subsequently, bioactive transforming growth factor beta-1 and matrix metalloproteinase-9 were measured by ELISA (enzyme-linked immunosorbent assay). Manual and automated platelet counts were performed on all blood and PRP samples. There were no differences in growth factor or cytokine concentration when blood or Llo PRP or Lhi PRP was retained at room temperature for up to 4 hours. Manual, direct smear analysis for platelet quantification was not different from the use of automated machine counting for PRP samples, but in the starting blood samples, manual platelet counts were significantly higher than those generated using automated technology. When there is a delay of up to 4 hours in the generation of PRP from blood or in the application of PRP to the patient, bioactive growth factor and cytokine concentrations remain stable in both blood and PRP. A manual direct counting method is a simple, cost-effective, and valid method to measure the contents of the PRP product being delivered to the patient.

Inflammatory licensed equine MSCs are chondroprotective and exhibit enhanced immunomodulation in an inflammatory environment.

BACKGROUND: Inflammatory licensed mesenchymal stem cells (MSCs) have the ability to promote functional tissue repair. This study specifically sought to understand how the recipient tissue environment reciprocally affects MSC function. Inflammatory polarized macrophages, modeling an injured tissue environment, were exposed to licensed MSCs, and the resultant effects of MSC immunomodulation and functionality of the MSC secretome on chondrocyte homeostasis were studied. METHODS: Inflammatory licensed MSCs were generated through priming with either IFN-γ or polyinosinic:polycytidylic acid (poly I:C). Macrophages were polarized to an inflammatory phenotype using IFN-γ. Licensed MSCs were co-cultured with inflammatory macrophages and immunomodulation of MSCs was assessed in a T-cell proliferation assay. MSC gene expression was analyzed for changes in immunogenicity (MHC-I, MHC-II), immunomodulation (IDO, PTGS2, NOS2, TGF-ß1), cytokine (IL-6, IL-8), and chemokine (CCL2, CXCL10) expression. Macrophages were assessed for changes in cytokine (IL-6, IL-10, TNF-α, IFN-γ) and chemokine (CCL2, CXCL10) expression. Conditioned medium representing the secretome from IFN-γ or poly I:C-primed MSCs was applied to IL-1ß-stimulated chondrocytes, which were analyzed for catabolic (IL-6, TNF-α, CCL2, CXCL10, MMP-13, PTGS2) and matrix synthesis (ACAN, COL2A1) genes. RESULTS: IFN-γ-primed MSCs had a superior ability to suppress T-cell proliferation compared to naïve MSCs, and this ability was maintained following exposure to proinflammatory macrophages. In naïve and licensed MSCs exposed to inflammatory macrophages, MHC-I and MHC-II gene expression was upregulated. The secretome from licensed MSCs was chondroprotective and downregulated inflammatory gene expression in IL-1ß-stimulated chondrocytes. CONCLUSIONS: In-vitro inflammatory licensing agents enhanced the immunomodulatory ability of MSCs exposed to inflammatory macrophages, and the resultant secretome was biologically active, protecting chondrocytes from catabolic stimulation. Use of licensing agents produced a more consistent immunomodulatory MSC population compared to exposure to inflammatory macrophages. The clinical implications of this study are that in-vitro licensing prior to therapeutic application could result in a more predictable immunomodulatory and reparative response to MSC therapy compared to in-vivo inflammatory licensing by the recipient environment.

Bone marrow concentrate and platelet-rich plasma differ in cell distribution and interleukin 1 receptor antagonist protein concentration.

PURPOSE: Bone marrow concentrate (BMC) and platelet-rich plasma (PRP) are used extensively in regenerative medicine. The aim of this study was to determine differences in the cellular composition and cytokine concentrations of BMC and PRP and to compare two commercial BMC systems in the same patient cohort. METHODS: Patients (29) undergoing orthopaedic surgery were enrolled. Bone marrow aspirate (BMA) was processed to generate BMC from two commercial systems (BMC-A and BMC-B). Blood was obtained to make PRP utilizing the same system as BMC-A. Bone marrow-derived samples were cultured to measure colony-forming units, and flow cytometry was performed to assess mesenchymal stem cell (MSC) markers. Cellular concentrations were assessed for all samples. Catabolic cytokines and growth factors important for cartilage repair were measured using multiplex ELISA. RESULTS: Colony-forming units were increased in both BMCs compared to BMA (p < 0.0001). Surface markers were consistent with MSCs. Platelet counts were not significantly different between BMC-A and PRP, but there were differences in leucocyte concentrations. TGF-ß1 and PDGF were not different between BMC-A and PRP. IL-1ra concentrations were greater (p = 0.0018) in BMC-A samples (13,432 pg/mL) than in PRP (588 pg/mL). The IL-1ra/IL-1ß ratio in all BMC samples was above the value reported to inhibit IL-1ß. CONCLUSIONS: The bioactive factors examined in this study have differing clinical effects on musculoskeletal tissue. Differences in the cellular and cytokine composition between PRP and BMC and between BMC systems should be taken into consideration by the clinician when choosing a biologic for therapeutic application. LEVEL OF EVIDENCE: Clinical, Level II.

The immunomodulatory function of equine MSCs is enhanced by priming through an inflammatory microenvironment or TLR3 ligand.

Mesenchymal stem cells (MSCs) have the therapeutic potential to treat a variety of inflammatory and degenerative disease processes, however the effects of the tissue environment on MSCs have been overlooked. Our hypothesis was that the immunomodulatory function of MSCs would be impaired by TLR4 stimulation or exposure to inflammatory macrophages, whereas their immunosuppressive properties would be enhanced by TLR3 stimulation. MSCs were exposed to polyinosinic:polycytidylic acid (poly I:C) to stimulate TLR3 receptors or lipopolysaccharide (LPS) to stimulate TLR4 receptors. MSC1 proinflammatory phenotype in human MSCs was associated with increased IL-6 and IL-8 and MSC2 regenerative phenotype was associated with increased CCL2 and CXCL10. MSC immunomodulatory function was assessed by measuring the ability of primed MSCs to suppress mitogen-stimulated T cell proliferation. Peripheral blood monocytes were isolated using CD14 MACs positive selection, differentiated into macrophages, and polarized using interferon-gamma (IFN-γ). Polarization was confirmed by increased gene expression of TNFα, CCL2, and CXCL10. Inflammatory macrophages were co-cultured with MSCs for 6h, and the resultant MSC phenotype was analyzed as described above. Both TLR3 and TLR4 priming and co-culture of MSCs with inflammatory macrophages resulted in increased expression of IL-6, CCL2, and CXCL10 in MSCs. Both TLR3 and TLR4 priming or exposure of MSCs to inflammatory macrophages significantly (p<0.05) enhanced their immunomodulatory function, demonstrated by a decrease in T cell proliferation in the presence of poly I:C primed MSCs (11%), LPS primed MSCs (7%), or MSCs exposed to inflammatory macrophages (12%), compared to unstimulated MSCs. Additionally, MHC class II positive MSCs tended to have a greater magnitude of response to priming compared to MHC class II negative MSCs. These results suggest that MSCs can be activated by a variety of inflammatory stimuli, but the recipient injured tissue bed in chronic injuries may not contain sufficient inflammatory signals to activate MSC immunomodulatory function. Enhancement of MSCs immunomodulatory function through inflammatory priming prior to clinical application might improve the therapeutic effect of MSC treatments.

Antigenicity of mesenchymal stem cells in an inflamed joint environment.

OBJECTIVE To determine whether major histocompatability complex (MHC) class II expression in equine mesenchymal stem cells (MSCs) changes with exposure to a proinflammatory environment reflective of an inflamed joint. SAMPLE Cryopreserved bone marrow-derived MSCs from 12 horses and cartilage and synovium samples from 1 horse euthanized for reasons other than lameness. PROCEDURES In part 1 of a 3-part study, the suitability of a quantitative reverse transcriptase PCR (qRT-PCR) assay for measurement of MHC class II expression in MSCs following stimulation with interferon (IFN)-γ was assessed. In part 2, synoviocyte-cartilage cocultures were or were not stimulated with interleukin (IL)-1ß (10 ng/mL) to generate conditioned media that did and did not (control) mimic an inflamed joint environment. In part 3, a qRT-PCR assay was used to measure MSC MHC class II expression after 96 hours of incubation with 1 of 6 treatments (control-conditioned medium, IL-1ß-conditioned medium, and MSC medium alone [untreated control] or with IL-1ß [10 ng/mL], tumor necrosis factor-α [10 ng/mL], or IFN-γ [100 ng/mL]). RESULTS The qRT-PCR assay accurately measured MHC class II expression. Compared with MHC class II expression for MSCs exposed to the untreated control medium, that for MSCs exposed to IL-1ß was decreased, whereas that for MSCs exposed to IFN-γ was increased. Neither the control-conditioned nor tumor necrosis factor-α medium altered MHC class II expression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that MSC exposure to proinflammatory cytokine IL-1ß decreased MHC class II expression and antigenicity. Treatment of inflamed joints with allogeneic MSCs might not be contraindicated, but further investigation is warranted.