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Effects over time of two platelet gel supernatants on growth factor, cytokine and hyaluronan concentrations in normal synovial membrane explants challenged with lipopolysaccharide.

ABSTRACT: Platelet-rich plasma (PRP) preparations are a common treatment in osteoarthritis (OA) and inflammatory synovitis. However, there is ambiguity regarding the ideal concentration of leukocytes and platelets in these preparations necessary to induce an adequate anti-inflammatory and anabolic response in joint tissues, such as the synovial membrane. This research aimed to study, in normal synovial membrane explants (SME) challenged with lipopolysaccharide (LPS), the temporal effects (at 48 and 96h) of leukocyte- and platelet-rich gel (L-PRG) and pure platelet-rich gel (P-PRG) supernatants on the production and degradation of platelet associated growth factors (GF) (platelet derived GF isoform BB (PDGF-BB) and transforming growth factor beta-1 (TGF-?1)), pro-inflammatory (tumour necrosis factor alpha (TNF-?)) and anti-inflammatory cytokines (interleukin 4 (IL-4) and IL-1 receptor antagonists (IL-1ra)) and hyaluronan (HA).Synovial membrane explants (SMEs) from 6 horses were challenged with LPS and cultured for 96h with L-PRG and P-PRG supernatants at concentrations of 25 and 50 %, respectively. The SME culture medium was changed every 48h and used for determination by ELISA of PDGF-BB, TGF-?1, TNF-?, IL-4, IL-1ra and HA. These molecules were also determined in synovial fluid from the horses.Both the 25 and 50 % PRG supernatants produced a molecular profile in the culture media unlike that of the SME challenged with LPS only. They presented GF, cytokine and HA concentrations very near to the concentrations of these molecules in normal synovial fluid when compared with the SME control groups (either with LPS or without LPS). However, in comparison with the rest of the SME treated groups, the 25 % L-PRG produced the most IL-1ra, and the 50 % P-PRG induced the sustained production of IL-4 and HA.These in vitro findings suggest that anabolic and anti-inflammatory joint responses depend on the leukocyte and platelet concentration of the PRP preparation and on the volume of this substance injected. Moreover, it is possible, that leukoreduced PRP preparations are more effective for the medical treatment of patients with OA and inflammatory synovitis.

SUBMITTER: Rios DL

PROVIDER: S-EPMC4475292 | biostudies-other | 2015 Jun

REPOSITORIES: biostudies-other

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Effects over time of two platelet gel supernatants on growth factor, cytokine and hyaluronan concentrations in normal synovial membrane explants challenged with lipopolysaccharide.

Ríos Diana L DL López Catalina C Álvarez María E ME Samudio Ismael J IJ Carmona Jorge U JU

BMC musculoskeletal disorders 20150620

<h4>Background</h4>Platelet-rich plasma (PRP) preparations are a common treatment in osteoarthritis (OA) and inflammatory synovitis. However, there is ambiguity regarding the ideal concentration of leukocytes and platelets in these preparations necessary to induce an adequate anti-inflammatory and anabolic response in joint tissues, such as the synovial membrane. This research aimed to study, in normal synovial membrane explants (SME) challenged with lipopolysaccharide (LPS), the temporal effect ...[more]

PMID: 26092588

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Project description:Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E2 (PGE2) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE2 production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE2, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE2, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.

Project description:BackgroundThe preimplantation embryo is sensitive to culture conditions in vitro and poor maternal diet in vivo. Such environmental perturbations can have long-lasting detrimental consequences for offspring health and physiology. However, early embryo susceptibility to other aspects of maternal health and their potential long-term influence into adulthood is relatively unexplored. In this study, we established an in vivo mouse model of maternal periconceptional systemic inflammation by intraperitoneal lipopolysaccharide (LPS) administration on the day of zygote formation and investigated the consequences into adulthood.ResultsIn the short term, maternal LPS challenge induced a transient and typical maternal sickness response (elevated serum proinflammatory cytokines and hypoactive behaviour). Maternal LPS challenge altered preimplantation embryo morphogenesis and cell lineage allocation, resulting in reduced blastocyst inner cell mass (ICM) cell number and a reduced ICM:trophectoderm cell ratio. In the long term, diverse aspects of offspring physiology were affected by maternal LPS treatment. Whilst birthweight, growth and adult blood pressure were unaltered, reduced activity in an open-field behaviour test, increased fat pad:body weight ratio and increased body mass index were observed in male, but not female, offspring. Most importantly, the maternal LPS challenge caused corticosterone-independent blunting of the serum proinflammatory cytokine response to innate immune challenge in both male and female offspring. The suppressed state of innate immunity in challenged offspring was dose-dependent with respect to the maternal LPS concentration administered.ConclusionsThese results demonstrate for the first time that the preimplantation embryo in vivo is sensitive to maternal systemic inflammation, with effects on blastocyst cell lineage allocation and consequences for behaviour, adiposity and innate immune response in adult offspring. Critically, we identify a novel mechanism mediated through maternal-embryonic interactions that confers plasticity in the development of the innate immune system, which is potentially important in setting postnatal tolerance to environmental pathogens. Our study extends the concept of developmental programming of health and disease to include maternal health at the time of conception.

Project description:Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender.The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet releasates from apheresis platelets have not been hitherto addressed by means of extensive proteomics technologies. Indeed, apheresis platelets are resuspended in donors' plasma, which hampers detection of less abundant proteins, owing to the overwhelming abundance of albumin (and a handful of other proteins), and the dynamic range of protein concentrations of plasma proteins. In order to cope with these issues, we hereby performed an immuno-affinity column-based depletion of the 14 most abundant plasma proteins. Samples were thus assayed via GeLC-MS, a workflow that allowed us to cover an unprecedented portion of the platelet supernatant proteome, in comparison to previous transfusion medicine-oriented studies in the literature. Finally, we hereby address the issue of biological variability, by considering the donor gender as a key factor influencing the composition of apheresis platelet supernatants. As a result, we could conclude that platelet supernatants from male and female donors are not only different in the first place, but they also store differently. This conclusion has been so far only suggested by classic transfusion medicine studies, but has been hitherto unsupported by actual biochemistry/proteomics investigations. In our opinion, the main strengths of this study are related to the analytical workflow (immunodepletion and GeLC-MS) and proteome coverage, the translational validity of the results (from a transfusion medicine standpoint) and the biological conclusion about the intrinsic (and storage-dependent) gender-related differences of platelet supernatants.

Project description:The purpose of the current study was to establish an in vitro model for osteoarthritis (OA) by co-culture of osteochondral and synovial membrane explants. Osteochondral explants were cultured alone (control-1) or in co-culture with synovial membrane explants (control-2) in standard culture medium or with interleukin-1β (IL1β) and tumor necrosis factor (TNFα) added to the culture medium (OA-model-1 = osteochondral explant; OA-model-2 = osteochondroal-synovial explant). In addition, in OA-model groups a 2-mm partial-thickness defect was created in the centre of the cartilage explant. Changes in the expression of extracellular matrix (ECM) genes (collagen type-1 (Col1), Col2, Col10 and aggrecan) as well as presence and quantity of inflammatory marker genes (IL6, matrix metalloproteinase-1 (MMP1), MMP3, MMP13, a disintegrin and metalloproteinase with-thrombospondin-motif-5 (ADAMTS5) were analysed by immunohistochemistry, qPCR and ELISA. To monitor the activity of classically-activated pro-inflammatory (M1) versus alternatively-activated anti-inflammatory/repair (M2) synovial macrophages, the nitric oxide/urea ratio in the supernatant of osteochondral-synovial explant co-cultures was determined. In both OA-model groups immunohistochemistry and qPCR showed a significantly increased expression of MMPs and IL6 compared to their respective control group. ELISA results confirmed a statistically significant increase in MMP1and MMP3 production over the culturing period. In the osteochondral-synovial explant co-culture OA-model the nitric oxide/urea ratio was increased compared to the control group, indicating a shift toward M1 synovial macrophages. In summary, chemical damage (TNFα, IL1β) in combination with a partial-thickness cartilage defect elicits an inflammatory response similar to naturally occurring OA in osteochondral explants with and without osteochondral-synovial explant co-cultures and OA-model-2 showing a closer approximation of OA due to the additional shift of synovial macrophages toward the pro-inflammatory M1 phenotype.

Dataset Information

Effects over time of two platelet gel supernatants on growth factor, cytokine and hyaluronan concentrations in normal synovial membrane explants challenged with lipopolysaccharide.

Publications

Effects over time of two platelet gel supernatants on growth factor, cytokine and hyaluronan concentrations in normal synovial membrane explants challenged with lipopolysaccharide.

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