Leukocyte-rich Plasma Research Articles

Release kinetic of pro- and anti-inflammatory biomolecules from platelet-rich plasma and functional study on osteoarthritis synovial fibroblasts

Background aimsThis study evaluated the release kinetics of numerous representative and less studied platelet-rich plasma (PRP) cytokines/chemokines with regard to the effects of various cellular compositions and incubation times. In addition, the biological effects of different PRPs on osteoarthritis synovial fibroblasts in vitro were tested. MethodsPeripheral whole blood was collected from healthy donors, and pure platelet-rich plasma (P-PRP), leukocyte-rich platelet-rich plasma (L-PRP) and platelet-poor plasma (PPP) were prepared for the analysis of the following biomolecules: IL-1β, IL-4, IL-6, IL-10, IL-17a, IL-22, MIP-1α/CCL-3, RANTES/CCL-5, MCP-3/CCL-7, Gro-α/CXCL-1, PF-4/CXCL-4, ENA-78/CXCL-5, NAP-2/CXCL-7, IL-8/CXCL-8, Fractalkine/CX3CL-1, s-CD40L P-PRP, L-PRP and PPP. Their effect on osteoarthritis synovial fibroblasts in vitro was tested by analyzing changes induced in both gene expression on a panel of representative molecules involved in physiopathology of joint environment and synthesis of IL-1β, IL-8 and hyaluronic acid. ResultsThis study demonstrated that among the 16 analyzed biomolecules, four were undetectable, whereas most of the detected biomolecules were more concentrated in L-PRP even when concentrations were normalized to platelet number. Despite the pro-inflammatory boost, the various PRP preparations did not alter synovial fibroblast gene expression of specific factors that play a pivotal role in joint tissue homeostasis and are able to induce anti-inflammatory (TIMP-1) biomolecules. DiscussionThis study provides a set of reference data on the concentration and release kinetics of some less explored biomolecules that could represent potential specific effectors in the modulation of inflammatory processes and in tissue repair after treatment with PRP.

Comparison of Leukocyte-Rich Platelet-Rich Plasma and Leukocyte-Poor Platelet-Rich Plasma on Achilles Tendinopathy at an Early Stage in a Rabbit Model

Background: Tendinopathy is still a great challenge in clinical practice, and the role of platelet-rich plasma (PRP) is controversial. The influence of leukocytes on tendinopathy at an early stage has not been defined so far. Purpose: To compare the effects of leukocyte-rich PRP (Lr-PRP) and leukocyte-poor PRP (Lp-PRP) on Achilles tendinopathy when applied at an early stage. Study Design: Controlled laboratory study. Methods: A rabbit Achilles tendinopathy model was induced by a collagenase injection. A week later, treatments were applied randomly on local Achilles tendon lesions: (1) 200 μL of Lr-PRP (16 legs), (2) 200 μL of Lp-PRP (16 legs), and (3) 200 μL of saline (16 legs). At 3 and 6 weeks after the collagenase injection, outcomes were evaluated by histology, magnetic resonance imaging (MRI), real-time polymerase chain reaction analysis, immunohistochemistry, and transmission electron microscopy (TEM). Results: The Lr-PRP group had a lower T2 signal intensity (P = .0377) and smaller diameter (P = .0193) and cross-sectional area (P = .0194) than the Lp-PRP group on MRI. Histologically, the Lr-PRP group had better scores than the Lp-PRP group (P = .0284 and P = .0188, respectively). Compared with the Lp-PRP group, higher gene expression and more protein synthesis of collagen I (P = .0160 and P = .0309, respectively) and CD163 (P < .0001 and P = .0411, respectively) were found in the Lr-PRP group. Considering TEM and biomechanical testing, the Lr-PRP group demonstrated more mature collagen fibers (P < .0001), a larger fiber diameter (P = .0005), a higher failure load (P = .00417), and higher tensile stress (P < .0001) than the Lp-PRP group. Conclusion: Lr-PRP had more beneficial effects than Lp-PRP when delivered at an early stage during tendon repair. Clinical Relevance: Here, we showed that tendinopathy influenced the curative effects of PRP in vivo. An early-stage application of Lr-PRP had more benefits for the repair of tendinopathy than Lp-PRP in a rabbit model, which will supplement guidelines of PRP treatment on tendinopathy clinically.

A dextran-based warming method for preparing leukocyte-rich plasma and its clinical application for endotoxin assay.

We devised a method using dextran for obtaining leukocyte-rich plasma (LRP) to measure endotoxin in blood. In order to find the optimal temperature for obtaining LRP, the measurement results were examined using samples prepared at 37 and 0°C. Sample separation time of LRP was significantly shorter at 37°C than at 0°C. Endotoxin measurement values showed a strong correlation between the two groups but many of the LRPs made at 37°C had measurements above those of the LRPs prepared at 0°C. The diagnostic accuracy for gram-negative bacterial infection was superior for LRP prepared at 37°C, with sensitivity and specificity of 96.8 and 100%, respectively.

Intratendon delivery of leukocyte-rich platelet-rich plasma at early stage promotes tendon repair in a rabbit Achilles tendinopathy model.

Tendinopathy is a great obstacle in clinical practice due to its poor regenerative capacity. The influence of different stages of tendinopathy on effects of leukocyte-rich platelet-rich plasma (Lr-PRP) has not been elucidated. The aim of this study is to investigate the optimal time point for delivery of Lr-PRP on tendinopathy. A tendinopathy model was established by local collagenase injection on the rabbit Achilles tendon. Then after collagenase induction, following treatments were applied randomly on the lesion: (a) 200 μl of Lr-PRP at 1 week (PRP-1 group), (b) 200 μl of saline at 1 week (Saline-1 group), (c) 200 μl of Lr-PRP at 4 weeks (PRP-2 group), and (d) 200 μl of saline at 4 weeks (Saline-2 group). Six weeks after collagenase induction, outcomes were assessed by magnetic resonance imaging, cytokine quantification, gene expression, histology, and transmission electron microscopy. Our results demonstrated that PRP-1 group had the least cross-sectional area and lesion percent of the involved tendon, as well as the lowest signal intensity in magnetic resonance imaging among all groups. However, the PRP-2 group showed larger cross-sectional area than saline groups. Enzyme-linked immunosorbent assay indicated that PRP-1 group had a higher level of interleukin-10 but lower level of interleukin-6 when compared with PRP-2 and saline groups. Meanwhile, the highest expression of collagen (Col) 1 in PRP-1 and Col 3, matrix metalloproteinase (MMP)-1, and MMP-3 in PRP-2 was found. Histologically, the PRP-1 showed better general scores than PRP-2, and no significant difference was found between the PRP-2 and saline groups. For transmission electron microscopy, PRP-1 had the largest mean collagen fibril diameter, and the PRP-2 group showed even smaller mean collagen fibril diameter than saline groups. In conclusion, intratendon delivery of Lr-PRP at early stage showed beneficial effect for repair of tendinopathy but not at late stage. For translation of our results to clinical circumstances, further studies are still needed.

The Effect of Delayed Injection of Leukocyte-Rich Platelet-Rich Plasma Following Rotator Cuff Repair on Patient Function: A Randomized Double-Blind Controlled Trial

To investigate the effect of delayed application of leukocyte-rich platelet-rich plasma (PRP) on rotator cuff repair results as assessed by outcome scores and imaging at one year. Patients with a symptomatic rotator cuff tear awaiting arthroscopic repair were approached to take part in the study. Final eligibility for the study was confirmed at the time of surgery. A total of 97 patients were randomized to an ultrasound guided injection of leukocyte-rich PRP or normal saline between 10 and 14 days postsurgery. A total of 87 patients completed clinical evaluation and underwent magnetic resonance imaging imaging at 1 year. Outcome scores included the American Shoulder and Elbow Score, Constant score, Western Ontario Rotator Cuff Index, and the Disabilities of the Arm, Shoulder and Hand Score. Structural integrity of the repair was assessed according to the Sugaya grading. Muscle fatty infiltration was assessed on magnetic resonance imaging using the Goutallier classification. At 1 year postsurgery, there was no significant difference between the treatment groups on any of the patient-reported outcome measures or Constant score. On postoperative imaging analysis, there was no difference in the retear rates (Sugaya 4 and 5) between the groups (21% in control group vs 15.3% in PRP group). Fatty infiltration on postoperative imaging was found to be significantly higher in the normal saline group compared with the PRP group (Kendall's tau-b P= .032). The delayed application of PRP postrotator cuff repair did not improve function as measured by patient-reported outcome measures and Constant score at 1 year postoperatively. Level II, prospective randomized therapeutic trial.

Clinical outcomes following injections of leukocyte-rich platelet-rich plasma in osteoarthritis patients

BackgroundScientists are trying to discover how to repair cartilage defects in knee osteoarthritis (KOA). In our previous study, we found a fibrocartilage-rich cover over the defective portions of cartilage after administering leukocyte-rich platelet-rich plasma (LR-PRP). This study aimed to investigate the efficacy of multiple injections of LR-PRP for the treatment of KOA and determine an LR-PRP treatment protocol for KOA in actual clinical practice. HypothesisWe hypothesized that using abundant LR-PRP would improve outcomes in patients with KOA. Study designProspective, cross-sectional, interventional, randomized trial. MethodsIntra-articular LR-PRP injections were administered to 50 knees. Patients received six injections of LR-PRP in total, which were administered at 4-week intervals. Patients were evaluated based on clinical outcomes, including visual analog scale (VAS) scores, Knee injury and Osteoarthritis Outcome Scores (KOOS), and magnetic resonance images (MRI) and radiographic findings before treatment and at 3 and 6 months after treatment. We investigated the recurrence of pain and presence/absence of MRI changes. Furthermore, we examined the Outcome Measures In Rheumatology-Osteoarthritis Research Society International (OMERACT-OARSI) responder criteria. ResultsThe mean improvement rate, as assessed by VAS, was 61.6% (P < .0001). Concerning OMERACT-OARSI, 37 of 50 knees (74%) were considered responders. There was a significant difference in the follow-up MRI findings, as assessed by the MRI Osteoarthritis Knee Score for bone marrow lesions (P < .007). No significant difference in osteoarthritis grade was observed. ConclusionOur LR-PRP procedure resulted in 74% of knees being classified as responders, regardless of the degree of knee deformation. Multiple injections of LR-PRP was effective for advanced grades of KOA. Thus, based on the results of our study, we believe that LR-PRP should be implemented as an additional conservative treatment option for non-operative management of OA. Trial registrationJapan Medical Association Center for Clinical Trials (JMA-XXX).

The Behavior of Tendon Progenitor Cells from Tendinopathic Tendons: Implications for Treatment.

Tendinopathy remains a significant clinical challenge. Although there is some evidence that leukocyte-rich platelet-rich plasma can improve the symptoms of tendinopathy, more efficacious treatments will be required in the future to improve probability of successfully resolving this condition in athletes. Because optimal treatments are not currently available, there is a need to better understand the pathology of tendinopathy from the perspective of tendon progenitor cells (TPCs). TPCs isolated from normal and tendinopathy donors were characterized by their stem cell properties and proliferation capacities, along with their ability to become tenocytes under mechanical loading. The results showed a significant 2.6-fold increase in the viable cell population in tendinopathy versus normal donors. Although the percentage of self-renewing cells was similar, the total number of TPCs in tendinopathy was significantly higher (1.6-fold) than normal TPCs based on the colony formation assays. In contrast, TPCs from tendinopathy tissue showed significantly lower cellular proliferation rate by cumulative population doublings. Next, the expanded TPCs from both tissues successfully demonstrated the trilineage differentiation capabilities with specific gene markers, staining, and biochemical assays. To induce tenogenic differentiation, stretchable silicone wells were designed and fabricated, plus the creation of an adaptor platform used on a syringe pump for mechanical stretch. This economic design provided the adequate cyclic loading to drive tenogenic differentiation. With these devices, the stretch duration was optimized and showed the significant increase in scleraxis (SCX) and tenomodulin (TNMD) expression at 2.60 (fold change) and 3.86 (fold change in logarithm), respectively, by reverse transcription-quantitative polymerase chain reaction in normal TPCs after stretch. This assay also demonstrated the widespread cell reorientation following stretch in normal TPCs. In contrast, the mechanical loading did not increase the SCX gene expression; TNMD expression remained undetectable, and cell realignment was significantly less in tendinopathy TPCs. In addition, western blot analysis confirmed the elevated TNMD protein expression in normal TPCs following stretch and the lack of expression in tendinopathy TPCs. In summary, tendinopathy TPCs were unable to differentiate into tenocytes following mechanical stretch. Future studies may aim to reprogram tendinopathy TPCs to allow tenogenic induction. Impact Statement This article presents a model to distinguish between normal and tendinopathy progenitor cell behavior, which reveals insight into the pathophysiology of tendinopathy. With the design of a platform adaptor, mechanical stretch was applied to tendon progenitor cells (TPCs) that promoted tenogenic differentiation. This design provided programmable features for more flexible application with low cost. These devices successfully stimulated tenogenic differentiation of TPCs from normal, but not tendinopathic tendons under cyclic stretch. The scientific method provided in this article will allow testing of biologics, exosomes, and other treatment strategies to derive new, more efficient treatment of tendinopathy in the future.

Effects of Aspirin on Growth Factor Release From Freshly Isolated Leukocyte-Rich Platelet-Rich Plasma in Healthy Men: A Prospective Fixed-Sequence Controlled Laboratory Study

Background: The benefits of platelet-rich plasma (PRP) are believed to be in part dependent on growth factor release after platelet activation. Platelet activation is complex and involves multiple mechanisms. One important mechanism is driven by cyclooxygenase 1 (COX-1)–mediated conversion of arachidonic acid (AA) to precursor prostaglandins that then mediate proinflammatory responses that trigger growth factor release. Acetylsalicylic acid (ASA; also known as aspirin) is known to irreversibly inhibit COX-1, thereby blocking AA-mediated signaling; however, it is unclear whether ASA use alters growth factor release from freshly isolated PRP. Purpose: To assess the effects of low-dose ASA use on activation of growth factor release from freshly isolated human PRP via AA and thrombin (TBN). Study Design: Controlled laboratory study. Methods: Twelve healthy men underwent blood collection and leukocyte-rich PRP (LR-PRP) preparation through a double-spin protocol to obtain baseline whole blood and PRP counts the same day. PRP was aliquoted into 3 groups: nonactivated, AA activated, and TBN activated. Immediately after activation, the concentrations of transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), and platelet-derived growth factor AB (PDGF-AB) were measured using enzyme-linked immunosorbent assays (ELISAs). The same 12 participants were then placed on an 81-mg daily dose of oral ASA for 14 days. Repeat characterization of whole blood and PRP analyses was done on day 14, followed by repeat ELISAs of growth factors under the same nonactivated and activated settings as previously stated. Results: Fourteen days of daily ASA had no effect on the number of platelets and leukocytes measured in whole blood and LR-PRP. Compared with nonactivated LR-PRP, AA- and TBN-mediated activation led to significant release of VEGF and PDGF-AB. In contrast, release of TGF-β1 from LR-PRP was observed only with activation by AA, not with TBN. Consistent with its inhibitory role in AA signaling, ASA significantly inhibited AA-mediated release of all 3 growth factors measured in this study. Although ASA had no effect on TBN-mediated release of VEGF and TGF-β1 from LR-PRP, ASA did partially block TBN-mediated release of PDGF-AB, although the mechanism remains unclear. Conclusion: Daily use of low-dose ASA reduces VEGF, PDGF-AB, and TGF-β1 expression in freshly isolated human LR-PRP when activated with AA. Clinical Relevance: Reduction in growth factor release attributed to daily use of low-dose ASA or other COX inhibitors can be mitigated when PRP samples are activated with TBN. Clinical studies are needed to determine whether activation before PRP injection is needed in all applications where ASA is in use and to what extent ASA may inhibit growth factor release in vivo at the site of injury.

The Influence of Naproxen on Biological Factors in Leukocyte-Rich Platelet-Rich Plasma: A Prospective Comparative Study

To quantify and compare normative catabolic and anabolic factor concentrations in leukocyte-rich platelet-rich plasma (LR-PRP) at various time points, including baseline, 1week after initiating naproxen use, and after a 1-week washout period. Asymptomatic healthy donors aged between 18 and 70years were recruited (average age, 36.6years; range, 25-64years). Subjects were excluded from the study if they were actively taking any prescribed medications or nonsteroidal anti-inflammatory drugs (NSAIDs) or if they had any of the following at present or previously: blood or immunosuppression disorders, cancer, osteonecrosis, rheumatoid arthritis, avascular necrosis, NSAID intolerance, gastrointestinal or peptic ulcer disease, or kidney dysfunction. The anabolic factors vascular endothelial growth factor, fibroblast growth factor 2, platelet-derived growth factor AB (PDGF-AB), and platelet-derived growth factor AA (PDGF-AA) and the catabolic factors interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor α in LR-PRP were measured. Peripheral blood was drawn at 3 time points: baseline, after 1week of naproxen use, and after a 1-week washout period. The angiogenic factors PDGF-AA (44% decrease in median) and PDGF-AB (47% decrease) significantly declined from baseline (P < .05) after 1week of naproxen use. There was a significant recovery (P < .05) of PDGF-AA (94% increase) and PDGF-AB (153% increase) levels after the 1-week washout period, with a return to baseline levels. The catabolic factor IL-6 also had a significant decline from baseline (77% decrease in median, P < .05) after 1week of naproxen use. After a 1-week washout period, the IL-6 level was similar to the baseline level (130% increase, P < .05). Naproxen use diminished several biological factors in LR-PRP; however, a 1-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, and IL-6 to return to baseline levels. Tumor necrosis factor α, IL-1β, IL-8, vascular endothelial growth factor, and fibroblast growth factor 2 did not show differences between the 3 time points of data collection. Discontinuing NSAIDs for a minimum of 1week before LR-PRP treatment may improve certain biological factor levels. Level II, prospective comparative study.

Comparing leukocyte-rich platelet-rich plasma injection with surgical intervention for the management of refractory tennis elbow. A prospective randomised trial.

Patients with ongoing symptoms after non-operative treatment of lateral epicondylosis are usually treated with surgical release. Platelet-rich plasma injection is an alternative treatment option. This study aims to determine whether there is a difference in outcome from platelet-rich plasma injection or surgical release for refractory tennis elbow. Eighty-one patients with a diagnosis of tennis elbow for a minimum of six months, treated with previous steroid injection and a minimum visual analogue scale pain score of 50/100 were randomised to open surgery release (41 patients) or leucocyte rich platelet-rich plasma (L-PRP) (40 patients). Patients completed the Patient-Rated Tennis Elbow Evaluation and Disability of the Arm Shoulder and Hand at baseline, 1.5, 3, 6 and 12 months post-intervention. The primary endpoint was change in Patient-Rated Tennis Elbow Evaluation pain score at 12 months. Fifty-two patients completed final follow-up. Functional and pain scores improved in both groups. No differences in functional improvements were found but greater improvements in Patient-Rated Tennis Elbow Evaluation pain scores were seen after surgery. Thirteen patients crossed over from platelet-rich plasma to surgery within 12 months, and one surgical patient underwent a platelet-rich plasma injection. L-PRP and surgery produce equivalent functional outcome but surgery may result in lower pain scores at 12 months. Seventy per cent of patients treated with platelet-rich plasma avoided surgical intervention.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

Comparison of Effects of Leukocyte-Rich and Leukocyte-Poor Platelet-Rich Plasma on Pain and Functionality in Patients With Lateral Epicondylitis.

This study aims to compare the effect of leukocyte concentration in platelet-rich plasma (PRP) on pain, functionality and post-injection local inflammatory reactions in patients with lateral epicondylitis. The study included 90 patients (26 males, 64 females; mean age 38.6 years; range 18 to 75 years) with lateral epicondylitis- related pain visual analog scale (VAS) score of ≥5 for more than three months. Patients were randomly assigned into three groups. Normal saline (1.5 mL) was injected in group 1 (control group) while a single dose of leukocyte-poor-PRP (1.5 mL) and leukocyte-rich-PRP (1.5 mL) were injected in groups 2 and 3, respectively. An exercise program was recommended to patients in all three groups. Patients were assessed according to VAS, Patient-Rated Tennis Elbow Evaluation, grip dynamometer and pinchmeter, extensor tendon thickness and cortical derangement at baseline and at fourth and eighth weeks after therapy. All patients were questioned regarding paracetamol use and adverse effects after therapy. No significant differences were detected between groups regarding VAS, Patient-Rated Tennis Elbow Evaluation, grip and pinch measurements, extensor tendon thickness and cortical derangement (p>0.05). In intra-group comparisons, VAS and Patient-Rated Tennis Elbow Evaluation scores obtained at fourth and eighth weeks were significantly decreased in all groups when compared to baseline values (p>0.05). Again, there was no significant difference in the control visit at eighth week when compared to baseline. Assessment of grip and pinch measurements revealed that values obtained at fourth and eighth weeks were significantly increased compared to baseline in all three groups (p<0.05). In leukocyte-rich-PRP group, a significant increase was detected in the values obtained at eighth week compared to those obtained at fourth week, but no significant change was detected in other groups. No significant difference was detected in extensor tendon thickness in any group. No significant difference was detected between groups in terms of paracetamol use and post-injection reactions. According to our study findings, lateral epicondylitis does not seem to affected either leukocyte-rich-PRP or leukocyte-poor-PRP on pain and function in the short term. Leukocyte concentration had no association with post-injection local inflammatory reactions.

Inhibition of microsomal prostaglandin E-synthase-1 (mPGES-1) selectively suppresses PGE2 in an in vitro equine inflammation model.

Inhibition of prostaglandin E2 (PGE2) production effectively limits inflammation in horses, however nonspecific prostaglandin blockade via cyclooxygenase (COX) inhibition elicits deleterious gastrointestinal side effects in equine patients. Thus, more selective PGE2 targeting therapeutics are needed to treat inflammatory disease in horses. One potential target is microsomal prostaglandin E-synthase-1 (mPGES-1), which is the terminal enzyme downstream of COX-2 in the inducible PGE2 synthesis cascade. This enzyme has yet to be studied in equine leukocytes, which play a pivotal role in equine inflammatory disease. The objective of this study was to determine if mPGES-1 is a PGE2-selective anti-inflammatory target in equine leukocytes. To evaluate this objective, leukocyte-rich plasma (LRP) was isolated from equine whole blood collected via jugular venipuncture of six healthy adult horses of mixed breeds and genders. LRP was primed with granulocyte-monocyte colony-stimulating factor (GM-CSF) and stimulated with lipopolysaccharide (LPS) in the presence or absence of an mPGES-1 inhibitor (MF63), a COX-2 inhibitor (NS-398), or a nonselective COX inhibitor (indomethacin). Following treatment, mPGES-1 and COX-2 mRNA and protein levels were measured via qPCR and western blot, respectively, and PGE2, thromboxane (TXA2) and prostacyclin (PGI2) levels were measured in cellular supernatants via ELISA. This study revealed that LPS significantly increased mPGES-1 mRNA, but not protein levels in equine LRP as measured by qPCR and western blot, respectively. In contrast, COX-2 mRNA and protein were coordinately induced by LPS. Importantly, treatment of LPS-stimulated leukocytes with indomethacin and NS-398 significantly reduced extracellular concentrations of multiple prostanoids (PGE2, TXA2 and PGI2), while the mPGES-1 inhibitor MF63 selectively inhibited PGE2 production only. mPGES-1 inhibition also preserved higher basal levels of PGE2 production when compared to either COX inhibitor, which might be beneficial in a clinical setting. In conclusion, this work identifies mPGES-1 as a key regulator of PGE2 production and a PGE2-selective target in equine leukocytes. This study demonstrates that mPGES-1 is a potentially safer and effective therapeutic target for treatment of equine inflammatory disease when compared to traditional non-steroidal anti-inflammatory drugs.

Misoprostol Inhibits Lipopolysaccharide-Induced Pro-inflammatory Cytokine Production by Equine Leukocytes.

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm–Sidak post hoc testing) or Friedman’s RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3–6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1β protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1β protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1β, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo.

Effect of oral magnesium supplementation on the transcription of TRPM6, TRPM7, and SLC41A1 in individuals newly diagnosed of pre-hypertension. A randomized, double-blind, placebo-controlled trial.

A stringent regulation of influx and efflux of magnesium by cation transporters seems to play an important role in the regulation of blood pressure (BP). With this regard, we evaluate the effect of oral magnesium supplementation on the transcription of TRPM6, TRPM7, and SLC41A1, in individuals with incident pre-hypertension (preHTN). For such purpose, we conducted a randomized, double-blind, placebo-controlled trial that compared 18 individuals who received oral magnesium lactate (360 mg elemental magnesium) versus 18 individuals who received placebo, during 4 months. Diagnosis of hypertension or normal BP, diabetes, alcohol intake, chronic diarrhea, use of diuretics, intake of magnesium supplementation, and reduced renal function were exclusion criteria. Regarding the transcription analysis of TRPM6, TRPM7, and SLC41A1 using RT-qPCR, leukocyte-rich plasma was obtained and total RNA was isolated with the kit Direct-zol™ RNA MiniPrep (Zymo). The leukocyte TRPM6 mRNA relative expression showed a significant increase (2.1±1.37 and 0.8±0.4, P<0.05), whereas the mRNA relative expression of both leukocyte TRPM7 (0.8±1.1 and 0.9±0.6, pNS) and SLC41A1 (0.9±1.0 and 0.7±0.6, pNS) showed no significant differences, between the magnesium and placebo groups, respectively. Oral magnesium supplementation increases the leukocyte TRPM6 mRNA relative expression, in subjects with new diagnosis of preHTN.

Does leukocyte-poor or leukocyte-rich platelet-rich plasma applied with biopolymers have superiority to conventional platelet-rich plasma applications on chondrocyte proliferation?

This study aims to investigate the possible effects of leukocyte concentration in the content of platelet-rich plasma (PRP) and the administration of PRP using a drug delivery system on chondrocyte proliferation in vitro conditions. Blood from nine male patients (mean age 65 years; range 49 to 81 years) with advanced stage osteoarthritis who had not responded to medical or conservative treatments and underwent total knee arthroplasty was used to prepare two formulations: PRP with low concentration leukocytes (2000-4000 leukocytes/µL) was designated as pure PRP (P-PRP), whereas PRP with high concentration leukocytes (9000-11000 leukocytes/µL) as leukocyte-rich PRP (L-PRP). Samples were divided into five groups as control group (group 1), chondrocyte cultures with P-PRP applied directly (group 2), chondrocyte cultures with L-PRP applied directly (group 3), chondrocytes co-cultured with P-PRP applied hydrogel (group 4), and chondrocytes co-cultured with L-PRP applied hydrogel (group 5). In all groups; cell morphology, viability and proliferation were compared with the expression of stage-specific embryonic antigen-1 (SSEA-1), a precondrocyte marker. Maximum cell proliferation and SSEA-1 expression occurred in group 4, with a statistically significant correlation between SSEA-1 expression and cell proliferation. Our study showed the importance of leukocyte concentration of PRP and efficiency of delivery systems such as hydrogel and that L-PRP administered with a delivery system is more efficient than conventional applications of PRP in the treatment of cartilage damage.

Leukocyte-Rich Platelet-Rich Plasma Injections Do Not Up-Modulate Intra-Articular Pro-Inflammatory Cytokines in the Osteoarthritic Knee.

IntroductionThe presence of leukocytes in platelet concentrates is deemed to cause deleterious effects when injected intra articularly. The aim of this study is to analyse both local and systemic effects induced by leukocyte-rich Platelet-rich Plasma (PRP) injections through a proteomic characterization of serial synovial fluid and blood samples obtained from subjects treated for knee OA. Secondary aim was to compare the effects on knee homeostasis and systemic response with those obtained with visco-supplementation.MethodsThirty-six OA patients treated either by autologous L-PRP or HA intra-articular knee injections, administered in series of three at one-week intervals, were analyzed. Just before the injection, 1 ml of synovial fluid was collected through the same needle way. In the same time, a peripheral blood sample was obtained and plasma separated. A further peripheral blood sample was collected at 2, 6, and 12 months. L-PRP, plasma and synovial fluid were tested by multiplex bead-based sandwich immunoassay by means of the Bio-Plex suspension array system (Bio-Rad Laboratories) for the presence of pro- and anti-inflammatory cytokines (IL-1beta, IL-6, IL-8, IL-17 and IL-4, IL-10, IL-13) and growth factors (FGF-b, HGF, PDGF-AB/BB).ResultsIn general, pro-inflammatory cytokine levels were similar at basal condition and after treatment whereas anti-inflammatory ones were nearly undetectable. L-PRP administration did not modulate significant changes of cytokine concentrations either in synovial fluid or plasma, whatever the time points analyzed. No different trend was observed between L-PRP and HA administration in terms of pro- and anti-inflammatory cytokines, as well as growth factors.ConclusionsIn contrast with the evidence reported by “in vitro” studies, where a cellular pro-inflammatory response appears to be induced by the presence of leukocytes, these results suggest that the presence leukocyte-rich PRP doesn’t induce a relevant in vivo up regulation of pro-inflammatory mediators.

Effect of autologous platelet leukocyte rich plasma injections on atrophied lumbar multifidus muscle in low back pain patients with monosegmental degenerative disc disease

Background: Lumbar multifidus muscle dysfunction and chronic low back pain are strongly correlated. There is no consensus regarding treatment of chronic LBP. The effect of platelet leukocyte rich plasma (PLRP) injections on atrophied lumbar multifidus (LMF) muscle and chronic low back pain has never been studied before. Patients and methods: One hundred fifteen patients with chronic non-specific LBP fulfilled the inclusion criteria. Patients were treated with weekly PLRP injections for six weeks and followed up for 24 months. Primary outcome measures included Numerical Rating Scale (NRS) and Oswestry Disability Index (ODI). Secondary outcome measures included Patient Satisfaction Index (PSI), modified MacNab criteria, and lumbar MRI at 12 months follow-up. Results: One hundred and four patients completed the trial. There were no serious complications. NRS significantly improved gradually from a mean of 8.8 ± 8 pre-injection to 3.45 ± 2.9 by 12 months and ODI significantly improved gradually from a mean of 36.7 ± 3.9 to 14.6 ± 12.8 by 12 months (P < 0.005). After reaching maximum improvement between 12 and 18 months, all outcome measures remained stable till the end of the 24 months follow-up period with statistically insignificant changes (P > 0.05). 87.8% (65/74) of the satisfied patients showed increased cross-sectional area and decreased fatty degeneration of LMF muscle on MRI at 12 months follow-up. Conclusion: PLRP injections into atrophied lumbar multifidus muscle represent a safe, effective method for relieving chronic low back pain and disability with long-term patient satisfaction and success rate of 71.2%. We recommend the use of the lumbar PLRP injections of LMF muscle to refine the inclusion criteria of lumbar fusion to avoid failed back syndrome.

A manual method to obtain platelet rich plasma.

OBJECTIVE: This study is to report a manual method to obtain platelet rich plasma (PRP). METHODS: For this study 61 ml of peripheral blood was obtained and submitted to centrifugation at 541g for 5 min. The centrifugation separates the blood into three components: red blood cells, buffy coat and platelet rich plasma. Blood and platelet rich plasma samples were sent to the Hospital's Laboratory and platelets and leukocytes were measured. RESULTS: A sample of 637 blood donors was evaluated. The platelet yield efficiency was 86.77% and the increase in platelet concentration factor was 2.89 times. The increase in leukocyte concentration factor was 1.97 times. CONCLUSION: The method described here produces leukocyte-rich and platelet-rich plasma with a high platelet and leukocyte increased factor. Level of Evidence IV, Controlled Laboratory Study.

Injection of platelet- and leukocyte-rich plasma at the junction of the proximal sesamoid bone and the suspensory ligament branch for treatment of yearling Thoroughbreds with proximal sesamoid bone inflammation and associated suspensory ligament branch desmitis

To determine the effects of treatment with platelet- and leukocyte-rich plasma (PRP) on future 2-, 3-, and 4-year-old racing performance of yearling Thoroughbreds with proximal sesamoid bone inflammation and associated suspensory ligament branch (SLB) desmitis. Randomized clinical trial. 39 yearling Thoroughbreds. Yearling Thoroughbreds with radiographic evidence of performance-limiting proximal sesamoid bone inflammation and ultrasonographic evidence of associated SLB desmitis were identified and randomly assigned to undergo PRP (treatment group) or saline (0.9% NaCl) solution (control group) injection at the affected SLB-proximal sesamoid bone junction. Race records of horses for the 2-, 3-, and 4-year-old racing years were obtained. Data regarding amount of money earned and number of races started were used as outcome measures, and results for groups were compared. Horses treated with PRP were significantly more likely to start at least 1 race during the 2-year-old racing year than were horses treated with saline solution; no significant differences were detected between groups regarding that variable for the 3- and 4-year-old racing years. No significant differences between groups were detected regarding earnings for any racing year. Although PRP-treated horses were more likely to start a race during the 2-year-old racing year versus control group horses, results for horses in each group were not significantly different for the 3- and 4-year-old racing years. Therefore, the PRP treatment protocol evaluated in this study did not seem to improve future racing performance of yearling Thoroughbreds with proximal sesamoid bone inflammation and associated SLB desmitis, compared with injection of saline solution.