Facultad de Ciencias de la Salud

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes progressive joint destruction. Despite the advances in the treatment of this condition there remains a clinical need for safe therapies leading to clinical remission. Mesenchymal stem/stromal cells (MSCs) play immunomodulatory and regenerative roles which can be partly mediated by their secretome. In recent years, the important contribution of extracellular vesicles (EVs) to MSC actions has received an increasing interest as a new therapeutic approach. We provide an extensive overview of the immunomodulatory properties of MSC EVs and their effects on articular cells such as fibroblast-like synoviocytes that play a central role in joint destruction. This review discusses the anti-arthritic effects of MSC EVs in vitro and in animal models of RA as well as their potential mechanisms. Recent preclinical data suggest that transfer of non-coding RNAs by MSC EVs regulates key signaling pathways involved in the pathogenesis of RA. We also examine a number of EV modifications for improving their anti-arthritic efficacy and carrier ability for drug delivery.

The use of mesenchymal stem cells constitutes a promising therapeutic approach, as it has shown beneficial effects in different pathologies. Numerous in vitro, pre-clinical, and, to a lesser extent, clinical trials have been published for osteoarthritis. Osteoarthritis is a type of arthritis that affects diarthritic joints in which the most common and studied effect is cartilage degradation. Nowadays, it is known that osteoarthritis is a disease with a very powerful inflammatory component that affects the subchondral bone and the rest of the tissues that make up the joint. This inflammatory component may induce the differentiation of osteoclasts, the bone-resorbing cells. Subchondral bone degradation has been suggested as a key process in the pathogenesis of osteoarthritis. However, very few published studies directly focus on the activity of mesenchymal stem cells on osteoclasts, contrary to what happens with other cell types of the joint, such as chondrocytes, synoviocytes, and osteoblasts. In this review, we try to gather the published bibliography in relation to the effects of mesenchymal stem cells on osteoclastogenesis. Although we find promising results, we point out the need for further studies that can support mesenchymal stem cells as a therapeutic tool for osteoclasts and their consequences on the osteoarthritic joint.

Background: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert antiinflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). Methods: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. Results: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1β. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. Conclusion: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV. The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Adipose tissue represents an abundant source of mesenchymal stem cells (MSC) for therapeutic purposes. Previous studies have demonstrated the anti-inflammatory potential of adipose tissue-derived MSC (ASC). Extracellular vesicles (EV) present in the conditioned medium (CM) have been shown to mediate the cytoprotective effects of human ASC secretome. Nevertheless, the role of EV in the anti-inflammatory effects of mouse-derived ASC is not known. The current study has investigated the influence of mouse-derived ASC CM and its fractions on the response of mouse-derived peritoneal macrophages against lipopolysaccharide (LPS). CM and its soluble fraction reduced the release of pro-inflammatory cytokines, adenosine triphosphate and nitric oxide in stimulated cells. They also enhanced the migration of neutrophils or monocytes, in the absence or presence of LPS, respectively, which is likely related to the presence of chemokines, and reduced the phagocytic response. The anti-inflammatory effect of CM may be dependent on the regulation of toll-like receptor 4 expression and nuclear factor- B activation. Our results demonstrate the antiinflammatory effects of mouse-derived ASC secretome in mouse-derived peritoneal macrophages stimulated with LPS and show that they are not mediated by EV.

Mesenchymal stem/stromal cells (MSCs) represent a promising therapy for musculoskeletal diseases. There is compelling evidence indicating that MSC e ects are mainly mediated by paracrine mechanisms and in particular by the secretion of extracellular vesicles (EVs). Many studies have thus suggested that EVs may be an alternative to cell therapy with MSCs in tissue repair. In this review, we summarize the current understanding of MSC EVs actions in preclinical studies of (1) immune regulation and rheumatoid arthritis, (2) bone repair and bone diseases, (3) cartilage repair and osteoarthritis, (4) intervertebral disk degeneration and (5) skeletal muscle and tendon repair. We also discuss the mechanisms underlying these actions and the perspectives of MSC EVs-based strategies for future treatments of musculoskeletal disorders.

Dentistry-applied bioceramic materials are ceramic materials that are categorized asbioinert, bioactive and biodegradable. They share a common characteristic of being specificallydesigned to fulfil their function; they are able to act as root canal sealers, cements, root repair or fillingmaterials. Bioactivity is only attributed to those materials which are capable of inducing a desiredtissue response from the host. The aim of this study is to present a systematic review of availableliterature investigating bioactivity of dentistry-applied bioceramic materials towards dental pulp stemcells, including a bibliometric analysis of such a group of studies and a presentation of the parametersused to assess bioactivity, materials studied and a summary of results. The research question, based onthe PICO model, aimed to assess the current knowledge on dentistry-based bioceramic materials byexploring to what extent they express bioactive properties inin vitroassays and animal studies whenexposed to dental pulp stem cells, as opposed to a control or compared to different bioceramic materialcompositions, for their use in the dentin-pulp complex therapy. A systematic search of the literaturewas performed in six databases, followed by article selection, data extraction, and quality assessment.Studies assessing bioactivity of one or more bioceramic materials (both commercially available ornovel/experimental) towards dental pulp stem cells (DPSCs) were included in our review. A total of37 articles were included in our qualitative review. Quantification of osteogenic, odontogenic andangiogenic markers using reverse transcriptase polymerase chain reaction (RT-PCR) is the prevailingmethod used to evaluate bioceramic material bioactivity towards DPSCs in the current investigativestate, followed by alkaline phosphatase (ALP) enzyme activity assays and Alizarin Red Staining(ARS) to assess mineralization potential. Mineral trioxide aggregate and Biodentine are the prevalentreference materials used to compare with newly introduced bioceramic materials. Available literaturecompares a wide range of bioceramic materials for bioactivity, consisting mostly ofin vitroassays.The desirability of this property added to the rapid introduction of new material compositions makesthis subject a clear candidate for future research.Keywords:bioactivity; bioceramic materials; dental pulp stem cells; systematic reviewMaterials2019,12, 1015; doi:10.3390/ma12071015www.mdpi.com/journal/materials Materials2019,12, 10152 of 301. IntroductionWithin the field of biomedical therapeutics, we can highlight the concept of tissue engineeringto refer to the development of procedures and biomaterials that aim to devise new tissues to replacethose damaged, following the principles of cellular and molecular biology and taking as a premise thesearch for “biological solutions for biological problems” [1].In 2007, the American Association of Endodontists adopted the term “regenerative endodontics”to refer to the concept of tissue engineering applied to the restoration of root canal health, in a waythat continuous development of the root and tissues surrounding it is promoted [2].The introduction of the so-called bioceramic materials meant a great advance for this newparadigm in endodontic therapy [3], given their biocompatible nature and excellent physicochemicalproperties [4]. Categorized as bioinert, bioactive and biodegradable [5], dentistry-applied bioceramicmaterials are ceramic materials which share a common characteristic of being specifically designed tofulfil their function; they are able to act as root canal sealers, cements, root repair or filling materials [4].Applied to vital pulp therapy, bioceramic materials can be used in cases of pulp exposition fromtrauma, caries or other mechanical causes, as direct pulp cappers [6].Properties like biocompatibility and bioactivity are to be expected in dentistry-applied bioceramicmaterials for their use in vital pulp therapy [7]. The first one refers to the “ability to perform as asubstrate that will support the appropriate cellular activity, including the facilitation of molecularand mechanical signaling systems, in order to optimize tissue regeneration, without eliciting anyundesirable local or systemic responses in the eventual host” [8], while bioactivity goes even further,and is only attributed to those materials which are capable of inducing a desired tissue responsefrom the host [9] by the use of biomimetic approaches [10]. The term differs depending on the fieldin which it is implemented, being related to the cellular effects induced by biologically active ionsand substances released from biomaterials in the field of tissue engineering, but referred to as thebiomaterial’s capability of forming hydroxyl apatite mineral on its surface bothin vitroandin vivointhe field of biomaterial science [11].Considering these desirable characteristics of bioceramic materials, it seems convenient to analyzethe interaction between human dental pulp stem cells (hDPSCs), which are post-natal stem cellswith mesenchymal stem cell (MSCs)-like characteristics, like auto-renewal ability and multilineagedifferentiation potential [12], and them; as their combined use could mean and advancement in thefield of regenerative endodontics.Cytotoxicity and biocompatibility of a wide range of bioceramic materials towards dental stemcells (DSCs) have been investigated in numerous studies [13–17]; among others. The well-knownPro-Root MTA (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) has been shown to increaseosteoblast, fibroblast, cementoblast, odontoblast and pulp cell differentiation, but its handling difficultyamong other limitations encourages for a search for alternative materials [13]. Materials like Biodentine(Septodont, Saint Maurdes-Fosses, France) and TheraCal LC (Bisco Inc., Schaumburg, IL, USA) areexamples of bioceramic materials introduced posteriorly in dentistry for their use in vital pulp therapyas blood clot protectors in pulpal revascularization procedures, standing out for their consistency,easier manipulation and tricalcium silicate composition [16].However, to the best of the authors’ knowledge, there has been no effort to sort and summarizestudies analyzing bioactivity of such materials into more homogenous subgroups that would allow foran easier analysis of the evidence.

The inflammatory process is an essential phenomenon in the induction of immune responses. Monocytes are key effector cells during the inflammatory process. A wide range of evidence indicates that mesenchymal stem cells from adipose tissue (ASC) are endowed with immunomodulatory capacity. However, the interaction between ASC and monocytes in the innate immune response is not well understood. The aim of this work was to investigate the possible paracrine anti-inflammatory effects of ASC in human monocytes. Monocytes were isolated from buffy coats and ASC from fat of non-obese patients. Conditioned medium (CM) from ASC in primary culture was used. We have assessed the effects of CM on the production of inflammatory mediators, degranulation, migration, phagocytic activity, senescence, oxidative stress, mitochondrial membrane potential and macrophage polarization. We have shown that ASC exert paracrine antiinflammatory actions on human monocytes. CM significantly reduced the production of TNFa, NO and PGE2 and the activation of NF-kB. In addition, we observed a significant reduction of degranulation, phagocytic activity and their migratory ability in the presence of the chemokine CCL2. The senescence process and the production of oxidative stress and mitochondrial dysfunction were inhibited by CM which also reduced the production of TNFa by M1 macrophages while enhanced TGFb1 and IL-10 release by M2 macrophages. This study have demonstrated relevant interactions of ASC with human monocytes and macrophages which are key players of the innate immune response. Our results indicate that ASC secretome mediates the anti-inflammatory actions of these cells. This paracrine mechanism would limit the duration and amplitude of the inflammatory response.