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Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells.

ABSTRACT: BACKGROUND:The mechanisms by which macrophage phenotype contributes to mesenchymal stem cells (MSC)-mediated bone repair remain unclear. In this work, we investigated the influence of factors released by human macrophages polarized to a pro-inflammatory or an anti-inflammatory phenotype on the ability of human MSC to attach, migrate, and differentiate toward the osteoblastic lineage. We focused on the role of TNF-? and IL-10, key pro-inflammatory and anti-inflammatory cytokines, respectively, in regulating MSC functions. METHODS:MSC were treated with media conditioned by pro-inflammatory or anti-inflammatory macrophages to study their influence in cell attachment, migration, and osteogenic differentiation. The involvement of TNF-? and IL-10 in the regulation of MSC functions was investigated using neutralizing antibodies and recombinant cytokines. RESULTS:Treatment of MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages promoted cell elongation and enhanced MSC ability to attach and migrate. These effects were more noticeable when MSC were treated with media from pro-inflammatory macrophages. Interestingly, MSC osteogenic activity was enhanced by factors released by anti-inflammatory macrophages, but not by pro-inflammatory macrophages. Significant IL-10 levels originated from anti-inflammatory macrophages enhanced MSC osteogenesis by increasing ALP activity and mineralization in MSC layers cultured under osteogenic conditions. Moreover, macrophage-derived IL-10 regulated the expression of the osteogenic markers RUNX2, COL1A1, and ALPL. Notably, low TNF-? levels secreted by anti-inflammatory macrophages increased ALP activity in differentiating MSC whereas high TNF-? levels produced by pro-inflammatory macrophages had no effects on osteogenesis. Experiments in which MSC were treated with cytokines revealed that IL-10 was more effective in promoting matrix maturation and mineralization than TNF-?. CONCLUSIONS:Factors secreted by pro-inflammatory macrophages substantially increased MSC attachment and migration whereas those released by anti-inflammatory macrophages enhanced MSC osteogenic activity as well as cell migration. IL-10 was identified as an important cytokine secreted by anti-inflammatory macrophages that potentiates MSC osteogenesis. Our findings provide novel insights into how environments provided by macrophages regulate MSC osteogenesis, which may be helpful to develop strategies to enhance bone regeneration.

SUBMITTER: Valles G

PROVIDER: S-EPMC7020593 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells.

Vallés Gema G Bensiamar Fátima F Maestro-Paramio Leila L García-Rey Eduardo E Vilaboa Nuria N Saldaña Laura L

Stem cell research & therapy 20200213 1

<h4>Background</h4>The mechanisms by which macrophage phenotype contributes to mesenchymal stem cells (MSC)-mediated bone repair remain unclear. In this work, we investigated the influence of factors released by human macrophages polarized to a pro-inflammatory or an anti-inflammatory phenotype on the ability of human MSC to attach, migrate, and differentiate toward the osteoblastic lineage. We focused on the role of TNF-α and IL-10, key pro-inflammatory and anti-inflammatory cytokines, respecti ...[more]

PMID: 32054534

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Project description:Total hip arthroplasty (THA) represents one of the commonest surgical procedures in the orthopedic field. Osteointegration of the implant with native bone is essential for an optimal result; thus, the quality of the patient's bone surrounding the implant (i.e., the bone stock) is crucial. However, in some cases, the bone stock is insufficient and needs to be improved with autologous grafts rich in multipotent cells (i.e., from the iliac crest, from the head of the femur, or from the subchondral bone harvested from the acetabulum) or allogenic frozen bone. It is not known if the harvesting site may influence the osteogenic potential of these cells. Thus, our aim was to characterize and compare multipotent cells collected from the bone marrow, acetabular subchondral bone, and trabecular bone on the femoral head with a focus on osteogenic differentiation. The cells from three sources had a fibroblast-like phenotype and expressed surface antigens CD73, CD90, and CD105 and are negative to CD11b, CD34, and CD45. Although all these cells could be induced to differentiate into osteoblasts, chondrocytes, and adipocytes, they displayed different differentiation potentials. In osteogenic differentiation condition, the cells from the acetabulum had the lowest accumulation of calcium deposit while the cells originated from the bone marrow and femur created a considerably increased amount of the deposit. These findings were confirmed by quantitative polymerase chain reaction (qPCR). In chondrogenic and adipogenic conditions, bone marrow cells possessed a predominant differential capacity compared with the others, illustrated by high collagen type II expression together with a cartilage-like lacuna structure and the presence of fat-specific markers, respectively. To our knowledge, this is the first study comparing and demonstrating that the progenitor cells obtained from diverse surgical sites in hip replacement procedure share common characteristics of MSC but differ about plasticity and may provide rational for clinical application in cell therapy and bone grafting. The project number L1033 is registered with ClinicalTrials.gov NCT03369457.

Project description:BackgroundPeriodontal tissue regeneration is the ultimate goal of periodontitis treatment. Exosomes are nanoscale vesicles secreted by cells that participate in and regulate the physiological activities between cells. However, the relationship between inflammatory macrophage-derived exosomes and osteoblast differentiation in periodontitis has not been thoroughly reported. Here, we attempt to explore the role of inflammatory macrophage-derived exosomes in crosstalk with osteoblasts.MethodsPorphyromonas gingivalis lipopolysaccharide was used to stimulate macrophages and inflate their inflammatory cellular state. Exosomes were extracted from inflammatory macrophages using supercentrifugation, and their characteristics were detected by transmission electron microscopy, particle size analysis, and Western blotting. Exosome uptake bybone marrow mesenchymal stem cells (BMSCs) was observed by fluorescence microscopy. The effects of exosomes on the BMSC inflammatory response and on osteogenic differentiation were detected by quantitative polymerase chain reaction and Western blot analysis. Alkaline phosphatase activity was tested for verification.ResultsWe successfully extracted and identified inflammatory macrophage-derived exosomes and observed that BMSCs successfully took up exosomes. Inflammatory macrophage-derived exosomes upregulated the expression levels of the inflammatory factors interleukin-6 and tumour necrosis factor-alpha in BMSCs and mediated inflammatory stimulation. Additionally, they inhibited the transcription levels of the osteogenic genes alkaline phosphatase, type I collagen, and Runt-related transcription factor 2 as well as the alkaline phosphatase activity, while the use of the exosome inhibitor GW4869 attenuated this effect.ConclusionOur study shows that macrophages in periodontitis can mediate inflammatory stimulation and inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through the exosome pathway. Interference with exosome secretion is likely to be a promising method for bone tissue regeneration in inflammatory states.

Project description:Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism. PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways. After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs. Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration.

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Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells.

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Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells.

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