A microphysiological system for handling graphene related materials under flow conditions.

The field of nanotechnology has developed rapidly in recent decades due to its broad applications in many industrial and biomedical fields. Notably, 2D materials such as graphene-related materials (GRMs) have been extensively explored and, as such, their safety needs to be assessed. However, GRMs tend to deposit quickly, present low stability in aqueous solutions, and adsorb to plastic materials. Consequently, traditional approaches based on static assays facilitate their deposition and adsorption and fail to recreate human physiological conditions. Organ-on-a-chip (OOC) technology could, however, solve these drawbacks and lead to the development of microphysiological systems (MPSs) that mimic the microenvironment present in human tissues. In light of the above, in the present study a microfluidic system under flow conditions has been optimised to minimise graphene oxide (GO) and few-layer graphene (FLG) adsorption and deposition. For that purpose, a kidney-on-a-chip was developed and optimised to evaluate the effects of exposure to GO and FLG flakes at a sublethal dose under fluid flow conditions. In summary, MPSs are an innovative and precise tool for evaluating the effects of exposure to GRMs and other type of nanomaterials.

Human Dermal Decellularized ECM Hydrogels as Scaffolds for 3D In Vitro Skin Aging Models.

Biomaterials play an important role in the development of advancing three dimensional (3D) in vitro skin models, providing valuable insights for drug testing and tissue-specific modeling. Commercial materials, such as collagen, fibrin or alginate, have been widely used in skin modeling. However, they do not adequately represent the molecular complexity of skin components. On this regard, the development of novel biomaterials that represent the complexity of tissues is becoming more important in the design of advanced models. In this study, we have obtained aged human decellularized dermal extracellular matrix (dECM) hydrogels extracted from cadaveric human skin and demonstrated their potential as scaffold for advanced skin models. These dECM hydrogels effectively reproduce the complex fibrillar structure of other common scaffolds, exhibiting similar mechanical properties, while preserving the molecular composition of the native dermis. It is worth noting that fibroblasts embedded within human dECM hydrogels exhibit a behavior more representative of natural skin compared to commercial collagen hydrogels, where uncontrolled cell proliferation leads to material shrinkage. The described human dECM hydrogel is able to be used as scaffold for dermal fibroblasts in a skin aging-on-a-chip model. These results demonstrate that dECM hydrogels preserve essential components of the native human dermis making them a suitable option for the development of 3D skin aging models that accurately represent the cellular microenvironment, improving existing in vitro skin models and allowing for more reliable results in dermatopathological studies.

Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/µL) or basal PL (BPL; 177 × 103 platelets/µL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P < 0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (P < 0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.

Assessment of effectiveness and safety of repeat administration of proinflammatory primed allogeneic mesenchymal stem cells in an equine model of chemically induced osteoarthritis.

BACKGROUND: This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. RESULTS: Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-ß1 and downregulated cyclooxygenase-2 and interleukin (IL)-1ß. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-ß1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1ß (p < 0.01). CONCLUSIONS: Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.

Differentiation of equine bone marrow derived mesenchymal stem cells increases the expression of immunogenic genes.

Mesenchymal stem cells (MSCs) are a promising treatment for equine musculoskeletal injuries because of their ability to regulate the inflammation and to differentiate into other cell types. Since interest in allogeneic therapy is rising, concerns about MSC immunogenicity need to be addressed. Differentiated MSCs from several species increase their expression of immunogenic molecules and induce alloresponses, but equine MSC immunogenic profile after differentiation has not been reported. Therefore, the aim of this study was to assess the gene expression of immunogenic markers in tri-lineage differentiated equine bone marrow derived MSCs (eBM-MSCs). For this purpose, eBM-MSCs (n = 4) were differentiated into osteoblasts, adipocytes and chondrocytes. Differentiation was confirmed by specific staining and gene expression of lineage-related markers. Subsequently, gene expression of MHC-I, MHC-II, CD40 and CD80 was analyzed in undifferentiated (control) and tri-lineage differentiated eBM-MSCs. Osteogenesis and adipogenesis, but not chondrogenesis, significantly upregulated MHC-I; MHC-II expression significantly increased in the three lineages, while CD40 and CD80 expression did not change. Despite this, MHC-I and MHC-II upregulation after differentiation might lead to increased immunogenicity and risk of allorecognition, either eBM-MSCs differentiate in vivo after administration or they are differentiated prior to administration, with potential negative consequences for effectiveness and safety of allogeneic therapy.

Inflammation affects the viability and plasticity of equine mesenchymal stem cells: possible implications in intra-articular treatments.

Mesenchymal stem cells (MSCs) are gaining relevance for treating equine joint injuries because of their ability to limit inflammation and stimulate regeneration. Because inflammation activates MSC immunoregulatory function, proinflammatory priming could improve MSC efficacy. However, inflammatory molecules present in synovial fluid or added to the culture medium might have deleterious effects on MSCs. Therefore, this study was conducted to investigate the effects of inflammatory synovial fluid and proinflammatory cytokines priming on viability and plasticity of equine MSCs. Equine bone marrow derived MSCs (eBM-MSCs) from three animals were cultured for 72 h in media supplemented with: 20% inflammatory synovial fluid (SF); 50 ng/mL IFN-γ and TNF-α (CK50); and 20 ng/mL IFN-γ and TNF-α (CK20). Proliferation assay and expression of proliferation and apoptosis-related genes showed that SF exposed-eBM-MSCs maintained their viability, whereas the viability of CK primed-eBM-MSCs was significantly impaired. Tri-lineage differentiation assay revealed that exposure to inflammatory synovial fluid did not alter eBM-MSCs differentiation potential; however, eBM-MSCs primed with cytokines did not display osteogenic, adipogenic or chondrogenic phenotype. The inflammatory synovial environment is well tolerated by eBM-MSCs, whereas cytokine priming negatively affects the viability and differentiation abilities of eBM-MSCs, which might limit their in vivo efficacy.

Priming Equine Bone Marrow-Derived Mesenchymal Stem Cells with Proinflammatory Cytokines: Implications in Immunomodulation-Immunogenicity Balance, Cell Viability, and Differentiation Potential.

Mesenchymal stem cells (MSCs) have a great potential for treating equine musculoskeletal injuries. Although their mechanisms of action are not completely known, their immunomodulatory properties appear to be key in their functions. The expression of immunoregulatory molecules by MSCs is regulated by proinflammatory cytokines; so inflammatory priming of MSCs might improve their therapeutic potential. However, inflammatory environment could also increase MSC immunogenicity and decrease MSC viability and differentiation capacity. The aim of this study was to assess the effect of cytokine priming on equine bone marrow-derived MSC (eBM-MSC) immunoregulation, immunogenicity, viability, and differentiation potential, to enhance MSC immunoregulatory properties, without impairing their immune-evasive status, viability, and plasticity. Equine BM-MSCs (n = 4) were exposed to 5 ng/mL of TNFα and IFNγ for 12 h (CK5-priming). Subsequently, expression of genes coding for immunomodulatory, immunogenic, and apoptosis-related molecules was analyzed by real-time quantitative polymerase chain reaction. Chromatin integrity and proliferation assays were assessed to evaluate cell viability. Trilineage differentiation was evaluated by specific staining and gene expression. Cells were reseeded in a basal medium for additional 7 days post-CK5 to elucidate if priming-induced changes were maintained along the time. CK5-priming led to an upregulation of immunoregulatory genes IDO, iNOS, IL-6, COX-2, and VCAM-1. MHC-II and CD40 were also upregulated, but no change in other costimulatory molecules was observed. These changes were not maintained 7 days after CK5-priming. Viability and differentiation potential were maintained after CK5-priming. These findings suggest that CK5-priming of eBM-MSCs could improve their in vivo effectiveness without affecting other eBM-MSC properties.

Acute phase protein haptoglobin as inflammatory marker in serum and synovial fluid in an equine model of arthritis.

Acute phase proteins are useful inflammatory markers in horses. Haptoglobin (Hp) serum level is increased in horses undergoing different inflammatory processes, including arthritis. However, Hp concentration has not been assessed in inflammatory synovial fluid (SF). The aim of the present study was to investigate the Hp response in serum and SF in horses undergoing experimentally induced arthritis. For this purpose, serum and SF samples were collected from 12 animals before amphotericin B-induced arthritis was created (T0, healthy) and 15days after the lesion induction (T1, joint inflammation) and Hp was determined by single radial immunodiffusion. The Hp increase between T0 and T1 was significant in both serum and SF, and serum Hp concentration at T0 was significantly higher than in SF, but significant differences were not found at T1, indicating a higher Hp increase in SF. A significant positive correlation for Hp concentration between serum and SF samples was found. These results highlight the potential usefulness of Hp as inflammatory marker in horses, showing for the first time the increase of Hp in SF from joint inflammation in the horse.

Expression of genes involved in immune response and in vitro immunosuppressive effect of equine MSCs.

The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-ß1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-ß1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.

Expansion under hypoxic conditions enhances the chondrogenic potential of equine bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are widely used in regenerative medicine in horses. Most of the molecular characterisations of BM-MSCs have been made at 20% O(2), a higher oxygen level than the one surrounding the cells inside the bone marrow. The present work compares the lifespan and the tri-lineage potential of equine BM-MSCs expanded in normoxia (20% O(2)) and hypoxia (5% O(2)). No significant differences were found in long-term cultures for osteogenesis and adipogenesis between normoxic and hypoxic expanded BM-MSCs. An up-regulation of the chondrogenesis-related genes (COL2A1, ACAN, LUM, BGL, and COMP) and an increase of the extracellular sulphated glycosaminoglycan content were found in cells that were expanded under hypoxia. These results suggest that the expansion of BM-MSCs in hypoxic conditions enhances chondrogenesis in equine BM-MSCs.

Isolation and characterization of ovine mesenchymal stem cells derived from peripheral blood.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells with capacity to differentiate into several mesenchymal lineages. This quality makes MSCs good candidates for use in cell therapy. MSCs can be isolated from a variety of tissues including bone marrow and adipose tissue, which are the most common sources of these cells. However, MSCs can also be isolated from peripheral blood. Sheep has been proposed as an ideal model for biomedical studies including those of orthopaedics and transmissible spongiform encephalopathies (TSEs). The aim of this work was to advance these studies by investigating the possibility of MSC isolation from ovine peripheral blood (oPB-MSCs) and by subsequently characterizing there in vitro properties. RESULTS: Plastic-adherent fibroblast-like cells were obtained from the mononuclear fraction of blood samples. These cells were analysed for their proliferative and differentiation potential into adipocytes, osteoblasts and chondrocytes, as well as for the gene expression of cell surface markers. The isolated cells expressed transcripts for markers CD29, CD73 and CD90, but failed to express the haematopoietic marker CD45 and expressed only low levels of CD105. The expression of CD34 was variable. The differentiation potential of this cell population was evaluated using specific differentiation media. Although the ability of the cultures derived from different animals to differentiate into adipocytes, osteoblasts and chondrocytes was heterogeneous, we confirmed this feature using specific staining and analysing the gene expression of differentiation markers. Finally, we tested the ability of oPB-MSCs to transdifferentiate into neuronal-like cells. Morphological changes were observed after 24-hour culture in neurogenic media, and the transcript levels of the neurogenic markers increased during the prolonged induction period. Moreover, oPB-MSCs expressed the cellular prion protein gene (PRNP), which was up-regulated during neurogenesis. CONCLUSIONS: This study describes for the first time the isolation and characterization of oPB-MSCs. Albeit some variability was observed between animals, these cells retained their capacity to differentiate into mesenchymal lineages and to transdifferentiate into neuron-like cells in vitro. Therefore, oPB-MSCs could serve as a valuable tool for biomedical research in fields including orthopaedics or prion diseases.

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. RESULTS: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. CONCLUSIONS: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.

Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue.

Bone marrow and adipose tissue are the two main sources of mesenchymal stem cell (MSC). The aim of this work was to analyse the immunophenotype of 7 surface markers and the expression of a panel of 13 genes coding for cell surface markers in equine bone marrow and adipose tissue-derived MSCs obtained from 9 horses at third passage. The tri-lineage differentiation was confirmed by specific staining. Equine MSCs from both sources were positive for the MSC markers CD29 and CD90, while were negative for CD44, CD73, CD105, CD45 and CD34. The gene expression of these molecules was also evaluated by reverse transcriptase real-time quantitative PCR along with the expression of 5 other MSC markers. Both populations of cells expressed CD13, CD29, CD44, CD49d, CD73, CD90, CD105, CD106, CD146 and CD166 transcripts. Significant differences in gene expression levels between BM- and AT-MSCs were observed for CD44, CD90, CD29 and CD34. Both cell types were negative for CD45 and CD31. The surface antigens tested revealed a similar phenotypic profile between horse and human MSCs, although specific differences in some surface antigens were noticed.