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Background Progression of senile osteoporosis is associated with deteriorated regenerative potential of bone marrow-derived mesenchymal stem/stromal cells (BMSCs). According to the recent results, the senescent phenotype of osteoporotic cells strongly correlates with impaired regulation of mitochondria dynamics. Moreover, due to the ageing of population and growing osteoporosis incidence, more efficient methods concerning BMSCs rejuvenation are intensely investigated. Recently, miR-21-5p was reported to play a vital role in bone turnover, but its therapeutic mechanisms in progenitor cells delivered from senile osteoporotic patients remain unclear. Therefore, the goal of this paper was to investigate for the first time the regenerative potential of miR-21-5p in the process of mitochondrial network regulation and stemness restoration using the unique model of BMSCs isolated from senile osteoporotic SAM/P6 mice model. Methods BMSCs were isolated from healthy BALB/c and osteoporotic SAM/P6 mice. We analysed the impact of miR-21-5p on the expression of crucial markers related to cells’ viability, mitochondria reconstruction and autophagy progression. Further, we established the expression of markers vital for bone homeostasis, as well as defined the composition of extracellular matrix in osteogenic cultures. The regenerative potential of miR-21 in vivo was also investigated using a critical-size cranial defect model by computed microtomography and SEM–EDX imaging. Results MiR-21 upregulation improved cells’ viability and drove mitochondria dynamics in osteoporotic BMSCs evidenced by the intensification of fission processes. Simultaneously, miR-21 enhanced the osteogenic differentiation of BMSCs evidenced by increased expression of Runx-2 but downregulated Trap, as well as improved calcification of extracellular matrix. Importantly, the analyses using the critical-size cranial defect model indicated on a greater ratio of newly formed tissue after miR-21 application, as well as upregulated content of calcium and phosphorus within the defect site. Conclusions Our results demonstrate that miR-21-5p regulates the fission and fusion processes of mitochondria and facilitates the stemness restoration of senile osteoporotic BMSCs. At the same time, it enhances the expression of RUNX-2, while reduces TRAP accumulation in the cells with deteriorated phenotype. Therefore, miR-21-5p may bring a novel molecular strategy for senile osteoporosis diagnostics and treatment.

Mesenchymal stem cells (MSCs), known from their key role in the regeneration process of tissues, and their abilities to release bioactive factors like extracellular vesicles (EVs) could be considered as a potential, modern tool in the treatment of AKI (acute kidney injury) in both human and veterinary patients. The complex pathophysiology of a renal function disorder (AKI) makes difficult to find a universal therapy, but the treatment strategy is based on MSCs and derived from them, EVs seem to solve this problem. Due to their small size, the ability of the cargo transport, the ease of crossing the barriers and the lack of the ability to proliferate and differentiate, EVs seem to have a significant impact on the development such therapy. Their additional impact associated with their ability to modulate immune response and inflammation process, their strong anti-fibrotic and anti-apoptotic effects and the relation with the releasing of the reactive oxygen species (ROS), that pivotal role in the AKI development is undoubtedly, limits the progress of AKI. Moreover, the availability of EVs from different sources encourages to extend research with using EVs from MSCs in AKI treatment in felines; in that, the possibilities of kidney injuries treatment are still limited to the classical therapies burdened with dangerous side effects. In this review, we underline the significance of the processes, in whose EVs are included during the AKI in order to show the potential benefits of EVs-MSCs-based therapies against AKI in felines.

Available therapies aimed at treating age-related osteoporosis are still insufficient. Therefore, designing reliable in vitro model for the analysis of molecular mechanisms underlying senile osteoporosis is highly required. We have isolated and characterized progenitor cells isolated from bone marrow (BMSCs) of osteoporotic mice strain SAM/P6 (BMSCSAM/P6 ). The cytophysiology of BMSCSAM/P6 was for the first time compared with BMSCs isolated from healthy BALB/c mice (BMSCBALB/c ). Characterization of the cells included evaluation of their multipotency, morphology and determination of specific phenotype. Viability of BMSCs cultures was determined in reference to apoptosis profile, metabolic activity, oxidative stress, mitochondrial membrane potential and caspase activation. Additionally, expression of relevant biomarkers was determined with RT-qPCR. Obtained results indicated that BMSCSAM/P6 and BMSCBALB/c show the typical phenotype of mesenchymal stromal cells (CD44+, CD73+, CD90+) and do not express CD45. Further, BMSCSAM/P6 were characterized by deteriorated multipotency, decreased metabolic activity and increased apoptosis occurrence, accompanied by elevated oxidative stress and mitochondria depolarisation. The transcriptome analyses showed that BMSCSAM/P6 are distinguished by lowered expression of molecules crucial for proper osteogenesis, including Coll-1, Opg and Opn. However, the expression of Trap, DANCR1 and miR-124-3p was significantly up-regulated. Obtained results show that BMSCSAM/P6 present features of progenitor cells with disturbed metabolism and could serve as appropriate model for in vitro investigation of age-dependent osteoporosis.

Laminitis is a life threating, extremely painful and frequently recurrent disease of horses which affects hoof structure. It results from the disruption of blood flow to the laminae, contributing to laminitis and in severe separation of bone from the hoof capsule. Still, the pathophysiology of the disease remains unclear, mainly due to its complexity. In the light of the presented data, in the extremally difficult process of tissue structure restoration after disruption, a novel type of progenitor cells may be involved. Herein, we isolated and performed the initial characterization of stem progenitor cells isolated from the coronary corium of the equine feet (HPC). Phenotype of the cells was investigated with flow cytometry and RT-qPCR revealing the presence of nestin, CD29, and expression of progenitor cell markers including SOX2, OCT4, NANOG and K14. Morphology of HPC was investigated with light, confocal and SEM microscopes. Cultured cells were characterised by spindle shaped morphology, eccentric nuclei, elongated mitochondria, and high proliferation rate. Plasticity and multilineage differentiation potential was confirmed by specific staining and gene expression analysis. We conclude that HPC exhibit in vitro expansion and plasticity similar to mesenchymal stem cells, which can be isolated from the equine foot, and may be directly involved in the pathogenesis and recovery of laminitis. Obtained results are of importance to the field of laminitis treatment as determining the repairing cell populations could contribute to the discovery of novel therapeutic targets and agents including and cell‐based therapies for affected animals. Supplementary Information The online version contains supplementary material available at 10.1007/s12015-021-10187-x.

Krzysztof Marycz,1,2 Agnieszka Smieszek,1 Klaudia Marcinkowska,1 Mateusz Sikora,1 Eliza Turlej,1 Paulina Sobierajska,3 Adrian Patej,3 Alina Bienko,4 Rafal J Wiglusz3 1The Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences, Wroclaw, Poland; 2International Institute of Translational Medicine, Malin, Poland; 3Institute of Low Temperature and Structure Research, PAS, Wroclaw, Poland; 4Faculty of Chemistry, University of Wroclaw, Wroclaw, PolandCorrespondence: Krzysztof MaryczThe Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences Wroclaw, Norwida 27B St, Wroclaw, 50– 375, PolandTel +48 71 320 5201Email krzysztof.marycz@upwr.edu.plPurpose: Osteoporosis results in a severe decrease in the life quality of many people worldwide. The latest data shows that the number of osteoporotic fractures is becoming an increasing international health service problem. Therefore, a new kind of controllable treatment methods for osteoporotic fractures is extensively desired. For that reason, we have manufactured and evaluated nanohydroxyapatite (nHAp)-based composite co-doped with iron oxide (IO) nanoparticles. The biomaterial was used as a matrix for the controlled delivery of miR-21-5p and miR-124-3p, which have a proven impact on bone cell metabolism.Methods: The nanocomposite Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) was obtained by the wet chemistry method and functionalised with microRNAs (nHAp/IO@miR-21/124). Its physicochemical characterization was performed using XRPD, FT-IR, SEM-EDS and HRTEM and SAED methods. The modulatory effect of the composite was tested in vitro using murine pre-osteoblasts MC3T3-E1 and pre-osteoclasts 4B12. Moreover, the anti-inflammatory effects of biomaterial were analysed using a model of LPS-treated murine macrophages RAW 264.7. We have analysed the cells’ viability, mitochondria membrane potential and oxidative stress under magnetic field (MF+) and without (MF-). Moreover, the results were supplemented with RT-qPCR and Western blot assays to evaluate the expression profile for master regulators of bone metabolism.Results: The results indicated pro-osteogenic effects of nHAp/IO@miR-21/124 composite enhanced by exposure to MF. The enhanced osteogenesis guided by nHAp/IO@miR-21/124 presence was associated with increased metabolism of progenitor cells and activation of osteogenic markers (Runx-2, Opn, Coll-1). Simultaneously, nanocomposite decreased metabolism and differentiation of pre-osteoclastic 4B12 cells accompanied by reduced expression of CaII and Ctsk. Obtained composite regulated viability of bone progenitor cells and showed immunomodulatory properties inhibiting the expression of inflammatory markers, ie, TNF-α, iNOs or IL-1β, in LPS-stimulated RAW 264.7 cells.Conclusion: We have described for the first time a new concept of osteoporosis treatment based on nHAp/IO@miR-21/124 application. Obtained results indicated that fabricated nanocomposite might impact proper regeneration of osteoporotic bone, restoring the balance between osteoblasts and osteoclast.Keywords: nanocomposites, hydroxyapatite, osteoblasts, osteoclasts, osteoporosis

Background The senescence of dermal fibroblasts (DFLs) leads to an imbalance in the synthesis and degradation of extracellular matrix (ECM) proteins, presenting so-called senescence-associated secretory phenotype (SASP), which ultimately leads to skin aging. Recently, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been recognized as a promising cell-free therapy for degenerative diseases, which opens a new avenue for skin aging treatment. Methods In this study, we utilized chitosan (CS) hydrogel for effective loading and sustained release of EVs. In vitro, we explored the rejuvenation effects of CS hydrogel-incorporated EVs (CS-EVs) on replicative senescence DFLs through a series of experiments such as senescence-associated β-galactosidase (SA-β-gal) staining, RT-PCR, and Western blot analysis. Besides, we employed local multi-site subcutaneous injection to treat skin aging of naturally aged mice with CS-EVs and DiI fluorescent dye was used to label EVs to achieve in vivo real-time tracking. Results CS-EVs can significantly improve the biological functions of senescent fibroblasts, including promoting their proliferation, enhancing the synthesis of ECM proteins, and inhibiting the overexpression of matrix metalloproteinases (MMPs). Moreover, CS hydrogel could prolong the release of EVs and significantly increase the retention of EVs in vivo. After CS-EVs subcutaneous injection treatment, the aging skin tissues showed a rejuvenation state, manifested explicitly as the enhanced expression of collagen, the decreased expression of SASP-related factors, and the restoration of tissue structures. Conclusions CS hydrogel-encapsulated EVs could delay the skin aging processes by ameliorating the function of aging DFLs. Our results also highlight the potential of CS hydrogel-encapsulated EVs as a novel therapeutic strategy for improving aging skin to rejuvenation.

Extracellular vesicles (EVs), a spherical membrane fragments including exosomes, are released from several cell types, including mesenchymal stromal cells (MSCs), constitutively or under stimulation. As MVs cargo include DNA, RNA, miRNA, lipids and proteins their have gain special attention in the field of regenerative medicine. Depending on the type of transferred molecules, MVs may exert wide range of biological effects in recipient cells including pro-inflammatory and anti-apoptotic action. In presented paper, we isolated MVs form adipose derived mesenchymal stem cells (ASC) which underwent stimulation with 5-azacytydine and resveratrol (AZA/RES) in order to improve their therapeutic potential. Then, isolated MVs were applied to ASC with impaired cytophysiological properties, isolated from equine metabolic syndrome diagnosed animals. Using RT-PCR, immunofluorescence, ELISA, confocal microscopy and western blot, we have evaluated the effects of MVs on recipient cells. We have found, that MVs derived from AZA/RES treated ASC ameliorates apoptosis, senescence and endoplasmic reticulum (ER) stress in deteriorated cells, restoring their proper functions. The work indicates, that cells treated with AZA/RES through their paracrine action can rejuvenate recipient cells. However, further research needs to be performed in order to fully understand the molecular mechanisms of these bioactive factors action. Graphical Abstract Graphical abstract of presented study Electronic supplementary material The online version of this article (10.1007/s12015-020-10035-4) contains supplementary material, which is available to authorized users.

Chronic osteomyelitis, a bone infectious disease, is characterized by dysregulation of bone homeostasis, which results in excessive bone resorption. Lipopolysaccharide (LPS) which is a gram‐negative endotoxin was shown to inhibit osteoblast differentiation and to induce apoptosis and osteoclasts formation in vitro. While effective therapy against bacteria‐induced bone destruction is quite limited, the investigation of potential drugs that restore down‐regulated osteoblast function remains a major goal in the prevention of bone destruction in infective bone diseases. This investigation aimed to rescue LPS‐induced MC3T3‐E1 pre‐osteoblastic cell line using the methanolic extract of Cladophora glomerata enriched with Mn(II) ions by biosorption. LPS‐induced MC3T3‐E1 cultures supplemented with C. glomerata methanolic extract were tested for expression of the main genes and microRNAs involved in the osteogenesis pathway using RT‐PCR. Moreover, osteoclastogenesis of 4B12 cells was also investigated by tartrate‐resistant acid phosphatase (TRAP) assay. Treatment with algal extract significantly restored LPS‐suppressed bone mineralization and the mRNA expression levels of osteoblast‐specific genes such as runt‐related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin (OCN), osteopontin (OPN), miR‐27a and miR‐29b. The extract also inhibited osteoblast apoptosis, significantly restored the down‐regulated expression of Bcl‐2, and decreased the loss of MMP and reactive oxygen spices (ROS) production in MC3T3‐E1 cells induced by LPS. Furthermore, pre‐treatment with algal extract strongly decreased the activation of osteoclast in MC3T3‐E1‐4B12 coculture system stimulated by LPS. Our findings suggest that C. glomerata enriched with Mn(II) ions may be a potential raw material for the development of drug for preventing abnormal bone loss induced by LPS in bacteria‐induced bone osteomyelitis.

Aleksandra Seweryn,1 Ariadna Pielok,2 Krystyna Lawniczak-Jablonska,1 Rafal Pietruszka,1 Klaudia Marcinkowska,2 Mateusz Sikora,2 Bartlomiej S Witkowski,1 Marek Godlewski,1 Krzysztof Marycz,2,3 Agnieszka Smieszek2 1Institute of Physics, Polish Academy of Sciences, Warsaw PL-02668, Poland; 2Wroclaw University of Environmental and Life Sciences, Department of Experimental Biology, Wroclaw PL-50375, Poland; 3Cardinal Stefan Wyszynski University, Collegium Medicum, Warsaw PL-01938, PolandCorrespondence: Aleksandra SewerynPolish Academy of Sciences, Aleja Lotnikow 32/46, Warsaw PL-02668, PolandTel +48 22 116 33 99Fax +48 22 843 09 26Email aseweryn@ifpan.edu.plAgnieszka SmieszekWroclaw University of Environmental and Life Sciences, Norwida St. 27 B, Wroclaw PL-50-375, PolandTel +48 71 320 5229Fax +48 22 843 09 26Email agnieszka.smieszek@upwr.edu.plIntroduction: The development of the field of biomaterials engineering is rapid. Various bioactive coatings are created to improve the biocompatibility of substrates used for bone regeneration, which includes formulation of thin zirconia coatings with pro-osteogenic properties. The aim of this study was to assess the biological properties of ZrO2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO2ALD).Methodology: The cytocompatibility of the obtained layers was analysed using the mice pre-osteoblastic cell line (MC3T3) characterized by decreased expression of microRNA 21-5p (miR-21-5p) in order to evaluate the potential pro-osteogenic properties of the coatings. The in vitro experiments were designed to determine the effect of ZrO2ALD coatings on cell morphology (confocal microscope), proliferative activity (cell cycle analysis) and metabolism, reflected by mitochondrial membrane potential (cytometric-based measurement). Additionally, the influence of layers on the expression of genes associated with cell survival and osteogenesis was studied using RT-qPCR. The following genes were investigated: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), p53 and p21, as well as osteogenic markers, i.e. collagen type 1 (Coll-1), osteopontin (Opn), osteocalcin (Ocl) and runt-related transcription factor 2 (Runx-2). The levels of microRNA (miRNA/miR) involved in the regulation of osteogenic genes were determined, including miR-7, miR-21, miR-124 and miR-223.Results: The analysis revealed that the obtained coatings are cytocompatible and may increase the metabolism of pre-osteoblast, which was correlated with increased mitochondrial membrane potential and extensive development of the mitochondrial network. The obtained coatings affected the viability and proliferative status of cells, reducing the population of actively dividing cells. However, in cultures propagated on ZrO2ALD coatings, the up-regulation of genes essential for bone metabolism was noted.Discussion: The data obtained indicate that ZrO2 coatings created using the ALD method may have pro-osteogenic properties and may improve the metabolism of bone precursor cells. Given the above, further development of ZrO2ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.Keywords: ALD, zirconia-based coatings, pro-osteogenic properties, cytocompatibility, osteoblasts precursors

BackgroundPrevalence of osteoporosis is rapidly growing and so searching for novel therapeutics. Yet, there is no drug on the market available to modulate osteoclasts and osteoblasts activity simultaneously. Thus in presented research we decided to fabricate nanocomposite able to: (i) enhance osteogenic differentiation of osteoblast, (i) reduce osteoclasts activity and (iii) reduce pro-inflammatory microenvironment. As a consequence we expect that fabricated material will be able to inhibit bone loss during osteoporosis.ResultsThe α-Fe2O3/γ-Fe2O3nanocomposite (IOs) was prepared using the modified sol–gel method. The structural properties, size, morphology and Zeta-potential of the particles were studied by means of XRPD (X-ray powder diffraction), SEM (Scanning Electron Microscopy), PALS and DLS techniques. The identification of both phases was checked by the use of Raman spectroscopy and Mössbauer measurement. Moreover, the magnetic properties of the obtained IOs nanoparticles were determined. Then biological properties of material were investigated with osteoblast (MC3T3), osteoclasts (4B12) and macrophages (RAW 264.7) in the presence or absence of magnetic field, using confocal microscope, RT-qPCR, western blot and cell analyser. Here we have found that fabricated IOs: (i) do not elicit immune response; (ii) reduce inflammation; (iii) enhance osteogenic differentiation of osteoblasts; (iv) modulates integrin expression and (v) triggers apoptosis of osteoclasts.ConclusionFabricated by our group α-Fe2O3/γ-Fe2O3nanocomposite may become an justified and effective therapeutic intervention during osteoporosis treatment.

Background Protein tyrosine phosphatase 1B (PTP1B) and low molecular weight protein tyrosine phosphatase (LMPTP) are implicated in the development of metabolic disorders. Yet, their role in progenitor stem cell adipogenic differentiation and modulation of mitochondrial dynamics remains elusive. Methods In this study, we decided to investigate whether inhibition of PTP1B and LMPTP enhance adipogenic differentiation of metabolically impaired progenitor stem cells via modulation of mitochondrial bioenergetics and dynamics. Cells were cultured under adipogenic conditions in the presence of PTP1B and LMPTP inhibitors, and were subjected to the analysis of the main adipogenic-related and mitochondrial-related genes using RT-qPCR. Protein levels were established with western blot while mitochondrial morphology with MicroP software. Results Selective inhibitors of both PTP1B and MPTP enhanced adipogenic differentiation of metabolically impaired progenitor stem cells. We have observed enhanced expression of PPARy and adiponectin in treated cells. What is more, increased antioxidative defence and alternations in mitochondrial bioenergetics were observed. We have found that inhibition of PTP1B as well as C23 activates oxidative phosphorylation and enhances mitochondrial fusion contributing to enhanced adipogenesis. Conclusions The presented data provides evidence that the application of PTP1B and LMPTP inhibitors enhances adipogenesis through the modulation of mitochondrial dynamics.

Recently, metabolic syndrome (MS) has gained attention in human and animal metabolic medicine. Insulin resistance, inflammation, hyperleptinemia, and hyperinsulinemia are critical to its definition. MS is a complex cluster of metabolic risk factors that together exert a wide range of effects on multiple organs, tissues, and cells in the body. Adipose stem cells (ASCs) are multipotent stem cell population residing within the adipose tissue that is inflamed during MS. Studies have indicated that these cells lose their stemness and multipotency during MS, which strongly reduces their therapeutic potential. They suffer from oxidative stress, apoptosis, and mitochondrial deterioration. Thus, the aim of this study was to rejuvenate these cells in vitro in order to improve their chondrogenic differentiation effectiveness. Pharmacotherapy of ASCs was based on resveratrol and 5-azacytidine pretreatment. We evaluated whether those substances are able to reverse aged phenotype of metabolic syndrome-derived ASCs and improve their chondrogenic differentiation at its early stage using immunofluorescence, transmission and scanning electron microscopy, real-time PCR, and flow cytometry. Obtained results indicated that 5-azacytidine and resveratrol modulated mitochondrial dynamics, autophagy, and ER stress, leading to the enhancement of chondrogenesis in metabolically impaired ASCs. Therefore, pretreatment of these cells with 5-azacytidine and resveratrol may become a necessary intervention before clinical application of these cells in order to strengthen their multipotency and therapeutic potential.

Background Chronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard. Methods In this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques. Results Treatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment. Conclusion Overall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways.

Fibrosis is characterized by an abnormal accumulation of extracellular matrix (ECM) constituents in the liver parenchyma that lead to hepatic cirrhosis. After liver injury, the hepatic stellate cells (HSCs) undergo a response called "activation", transforming the cells into proliferative, fibrogenic and contractile myofibroblasts, representing the main collagen-producing cells in the injured tissue. Activated HSCs are considered as pro-inflammatory cells producing cytokines and several hepatomatogens; they are additionally involved in the recruitment of Kupffer cells, circulating monocytes and macrophages through the production of chemokines. Moreover, HSC have been proposed as being involved in the development of insulin resistance mainly mediated by their inflammatory properties, which undeniably links their activation to the development of diabetes and Non-alcoholic fatty liver disease. In addition, when the liver is injured, a complex interaction between hepatocytes and HSCs occurs, inducing mitochondrial dysfunction, which contributes to the accumulation of fats in hepatocytes that trigger to liver lipotoxicity. These mechanisms underlying the activation of HSC suggest their major role in the development of metabolic disorders. It turns out that several molecules including MicroRNAs and proteins have the ability to inhibit the activation and the proliferation of HSCs, which makes them interesting therapeutic targets for the subsequent management of metabolic conditions. This review focuses on the mechanisms and molecular pathways underlying the initiation and onset of metabolic disorders following HSCs activation, as well as on molecular therapeutic targets, which could limit their fibrogenic transdifferentiation and therefore improve the liver condition in the course of metabolic imbalance.

The aim of this study was to establish the potential effect of Laurus nobilis ethanolic extract on improving insulin sensitivity and protecting liver cells from apoptosis, mitochondrial dysfunction, oxidative stress (OS), and inflammation; all of which considered as major alterations occurring during insulin resistance (IR) as well as diabetes onset, in hyperinsulinemic and hyperglycemic-induced HepG2 cell line. Thereby, L. nobilis ethanolic extract has been first chemically characterized using LC-MS/MS technique. Subsequently, HepG2 cells were pre-treated with an optimal concentration of L. nobilis ethanolic extract for 24 h, and then, subjected to 30 mM D-glucose and 500 nM insulin mixture for another 24 h in order to induce hyperinsulinemia and hyperglycaemia (HI/HG) status. Several parameters such as biocompatibility, hepatotoxicity, reactive oxygen species (ROS), mitochondrial transmembrane potential, dynamics, and metabolism, multicaspase activity, glucose uptake, in addition to genes and proteins expression levels were investigated. The obtained results showed that the bioactive extract of Laurus nobilis increased the number of living cells and their proliferation rate, significantly attenuated apoptosis by modulating pro-apoptotic pathways (p21, p53 and Bax genes), allowed a relative normalization of caspases-activity, and decreased the expression of inflammatory markers including c-Jun, NF-κB and Tlr4 transcripts. L. Nobilis ethanolic extract reduced considerably total intracellular ROS levels in challenged HepG2 cells, and regulated the mitochondrial OXPHOS pathway, demonstrating the potential antioxidant effect of the plant. Ethanolic plant extract increased insulin sensitivity, since an elevated expression of master transcripts responsible for insulin sensitivity including IRS1, IRS2, INSR was found. Taken together, obtained data suggest that L. nobilis ethanolic extract offers new insights in the development of potential antioxidant, insulin sensitizing as well as hepatoprotective drugs.

Background Protein tyrosine phosphatase 1B (PTP1B) is one of the major negative regulators of leptin and insulin signaling, and has been strongly implicated in insulin resistance development in the course of obesity and metabolic syndrome conditions; however, its exact role in controlling adipose tissue biogenesis is still poorly understood. Objectives This investigation aimed to elucidate whether selective inhibition of PTP1B using MSI-1436 compound may improve and restore the defective adipogenicity of ASCs isolated from EMS-affected horses. Methods Equine ASC EMS cells were cultured under adipogenic conditions in the presence of PTP1B inhibitor and were subsequently tested for expression of the main adipogenic-related genes using RT-qPCR, changes in free fatty acid profiles by means of GC-MS technique, and for mitochondrial dynamics improvement through the analysis of mitochondrial transmembrane potential and oxidative stress. Results Selective inhibition of PTP1B in equine ASC EMS cells improved substantially adipogenic differentiation by promoting cellular proliferation and normalizing expression of C/EBPalpha, PPARγ, and Adipoq markers that are critical for proper adipogenesis. Levels of secreted adiponectin and PPARγ were also shown to be increased in MSI-1436-conditioned cells, while total leptin levels markedly dropped under the same conditions. Moreover, MSI-1436 treatment enabled the regulation of metabolic-related transcripts that are crosslink to adipogenesis, namely Akt1, Akt2, and SHBG. The obtained results demonstrated also an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the downregulation of Chop, Perk, Atf6, Ire1, and Xbp1 transcripts upon PTP1B inhibition. Furthermore, general fluctuations in FFA composition of all differentiated groups have been highlighted, where palmitic acid, palmitoleic acid, stearic acid, and linolelaidic acid that are known to be associated with the development of metabolic disorders were found to be normalized upon PTP1B inhibition during adipogenic differentiation. Conclusion The presented data provides the evidence that the use of PTP1B inhibitor may be successful in controlling and enhancing adipogenic differentiation of impaired equine ASCs affected by metabolic syndrome, and thus offers new insights for the management of obesity through the regulation of adipose tissue dynamics. Graphical abstract

Krzysztof Marycz,1,2 Agnieszka Śmieszek,1 Katarzyna Kornicka-Garbowska,1,2 Ariadna Pielok,1 Maciej Janeczek,3 Anna Lipińska,3 Anna Nikodem,4 Jarosław Filipiak,4 Paulina Sobierajska,5 Jean-Marie Nedelec,6 Rafał J Wiglusz2,5 1Department of Experimental Biology, Wroclaw University of Environmental and Life Sciences, Wroclaw, 50-375, Poland; 2International Institute of Translational Medicine, Malin, 55-124, Poland; 3Department of Biostructure and Animal Physiology, Wroclaw University of and Life Sciences, Wroclaw, 51-631, Poland; 4Department of Mechanics, Materials and Biomedical Engineering, Wroclaw University of Science and Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland; 5Institute of Low Temperature and Structure Research, Polish Academy of Sciences, Okolna 2, 50-422 Wroclaw, Poland; 6Universite Clermont Auvergne, Clermont Auvergne INP, CNRS, ICCF, Clermont-Ferrand, FranceCorrespondence: Krzysztof MaryczThe Department of Experimental Biology, The Faculty of Biology and Animal Science, University of Environmental and Life Sciences Wroclaw, Norwida 27B St, Wroclaw, 50– 375, PolandTel +48 71 320 5201Email krzysztof.marycz@upwr.edu.plPurpose: Healing of osteoporotic defects is challenging and requires innovative approaches to elicit molecular mechanisms promoting osteoblasts-osteoclasts coupling and bone homeostasis.Methods: Cytocompatibility and biocompatibility of previously characterised nanocomposites, i.e Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) functionalised with microRNAs (nHAp/IO@miR-21/124) was tested. In vitro studies were performed using a direct co-culture system of MC3T3-E1 pre-osteoblast and 4B12 pre-osteoclasts. The analysis included determination of nanocomposite influence on cultures morphology (confocal imaging), viability and metabolic activity (Alamar Blue assay). Pro-osteogenic signals were identified at mRNA, miRNA and protein level with RT-qPCR, Western blotting and immunocytochemistry. Biocompatibility of biomaterials was tested using bilateral cranial defect performed on a senescence-accelerated mouse model, ie SAM/P6 and Balb/c. The effect of biomaterial on the process of bone healing was monitored using microcomputed tomography.Results: The nanocomposites promoted survival and metabolism of bone cells, as well as enhanced functional differentiation of pre-osteoblasts MC3T3-E1 in co-cultures with pre-osteoclasts. Differentiation of MC3T3-E1 driven by nHAp/IO@miR-21/124 nanocomposite was manifested by improved extracellular matrix differentiation and up-regulation of pro-osteogenic transcripts, ie late osteogenesis markers. The nanocomposite triggered bone healing in a cranial defect model in SAM/P6 mice and was replaced by functional bone in Balb/c mice.Conclusion: This study demonstrates that the novel nanocomposite nHAp/IO can serve as a platform for therapeutic miRNA delivery. Obtained nanocomposite elicit pro-osteogenic signals, decreasing osteoclasts differentiation, simultaneously improving osteoblasts metabolism and their transition toward pre-osteocytes and bone mineralisation. The proposed scaffold can be an effective interface for in situ regeneration of osteoporotic bone, especially in elderly patients.Keywords: senile osteoporosis, osteoblasts, osteoclasts, nanohydroxyapatite, iron oxide nanoparticles, small non-coding RNA

Obesity and endocrine disorders have become prevalent issues in the field of both human and veterinary medicine. Equine metabolic syndrome is a complex disorder involving alternation in metabolism and chronic systemic inflammation. It has been shown that unfavourable microenvironment of inflamed adipose tissue negatively affects adipose stem cell population (ASC) residing within, markedly limiting their therapeutic potential. ASCsEMS are characterized by increased senescence apoptosis, excessive accumulation of reactive oxygen species (ROS), mitochondria deterioration and “autophagic flux.” The aim of the present study was to evaluate whether treatment of ASCsEMS with a combination of 5‐azacytydine (AZA) and resveratrol (RES) would reverse aged phenotype of these cells. For this reason, we performed the following analyzes: molecular biology (RT‐PCR), microscopic (immunofluorescence, TEM) and flow cytometry (JC‐1, ROS, Ki67). We evaluated the mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells by measuring the expression of TET genes and analysis of DNA methylation status. We have demonstrated that AZA/RES treatment of ASCsEMS is able to rejuvenate these cells by modulating mitochondrial dynamics, in particular by promoting mitochondrial fusion over fission. After AZA/RES treatment, ASCsEMS were characterized by increased proliferation rate, decreased apoptosis and senescence and lower ROS accumulation. Our findings offer a novel approach and potential targets for the beneficial effects of AZA/RES in ameliorating stem cell dysfunctions.

Tissue engineering and stem cell-based therapies are one of the most rapidly developing fields in medical sciences. Therefore, much attention has been paid to the development of new drug-delivery systems characterized by low cytotoxicity, high efficiency and controlled release. One of the possible strategies to achieve these goals is the application of magnetic field and/or magnetic nanoparticles, which have been shown to exert a wide range of effects on cellular metabolism. Static magnetic field (SMF) has been commonly used in medicine as a tool to increase wound healing, bone regeneration and as a component of magnetic resonance technique. However, recent data shed light on deeper mechanism of SMF action on physiological properties of different cell populations, including stem cells. In the present review, we focused on SMF effects on stem cell biology and its possible application as a tool for controlled drug delivery. We also highlighted the perspectives, in which SMF can be used in future therapies in tissue engineering due to its easy application and a wide range of possible effects on cells and organisms.

Endocrine disorders have become more and more frequently diagnosed in humans and animals. In horses, equine metabolic syndrome (EMS) is characterized by insulin resistance, hyperleptinemia, hyperinsulinemia, inflammation and usually by pathological obesity. Due to an increased inflammatory response in the adipose tissue, cytophysiological properties of adipose derived stem cells (ASC) have been impaired, which strongly limits their therapeutic potential. Excessive accumulation of reactive oxygen species, mitochondria deterioration and accelerated ageing of those cells affect their multipotency and restrict the effectiveness of the differentiation process. In the present study, we have treated ASC isolated from EMS individuals with a combination of 5‐azacytydine (AZA) and resveratrol (RES) in order to reverse their aged phenotype and enhance osteogenic differentiation. Using SEM and confocal microscope, cell morphology, matrix mineralization and mitochondrial dynamics were assessed. Furthermore, we investigated the expression of osteogenic‐related genes with RT‐PCR. We also investigated the role of autophagy during differentiation and silenced PARKIN expression with siRNA. Obtained results indicated that AZA/RES significantly enhanced early osteogenesis of ASC derived from EMS animals. Increased matrix mineralization, RUNX‐2, collagen type I and osteopontin levels were noted. Furthermore, we proved that AZA/RES exerts its beneficial effects by modulating autophagy and mitochondrial dynamics through PARKIN and RUNX‐2 activity.

The rapid aging of the population results in increased number of metabolic and degenerative disorders, especially in the elderly.Thus, a novel approach in the fields of orthopedic and reconstructive surgery for bone regeneration is strongly desirable. A new perspective in the therapy of bone fractures is tissue engineering which combines living cells with biomaterials to develop modern substitutes that can restore tissue functions. Metallic biomaterials, including stainless steel and pure titanium, have been extensively used for the fabrication of surgical implants over decades. Chemical modification of material surface for example incorporation of chemotactic factors may significantly improve the therapeutic effect. In this paper we describe titanium substrate modifications with ZrO2/SiO2coating functionalized with resveratrol using a sol – gel, dip-coating technique. Moreover, we established the effects of fabricated scaffolds on adipose stem cells isolated from elderly patients. Using fluorescence imaging, polymerase chain reaction (PCR)and cytotoxicity tests, we established that 0.5 Res_ZrO2/SiO2significantly reduced apoptosis and accumulation of oxidative stress factors in adipose derived stem cells (ASC). Thus exploitation of fabricated biomaterials in regenerative medicine as a strategy for rejuvenate ASC from elderly patientsin vivo, seems fully justified.

Mesenchymal stem cells (MSC) have become a promising tool for therapeutic intervention. Their unique features, including self-renewal, multipotency and immunomodulatory properties draw the worldwide attention of researchers and physicians with respect to their application in disease treatment. However, the environment (so-called niche) from which MSCs are isolated may determine their usefulness. Many studies indicated the involvement of MSCs in ageing and disease. In this review, we have focused on how type 2 diabetes (T2D) and metabolic syndrome (MS) affect MSC properties, and thus limit their therapeutic potential. Herein, we mainly focus on apoptosis, autophagy and mitochondria deterioration processes that indirectly affect MSC fate. Based on the data presented, special attention should be paid when considering autologous MSC therapy in T2D or MS treatments, as their therapeutic potential may be restricted.

The aim of the study was to describe the morphology of the upper, lower and third eyelid and characterize the organized lymphoid follicles and diffuse lymphocytes from ornamental and wild birds. The goal of these examinations was also to identify avian conjunctiva-associated lymphoid tissue (CALT) and lymphoid tissue that contained specialized high endothelial venules. The upper, lower and third eyelid from 30 species of ornamental and wild birds representing 21 families were examined under light microscopy and using scanning electron microscopy. The third eyelid in all of the examined birds was composed of a free margin, which was divided into two parts. The largest tarsal plate of the third eyelid was observed in the greater rhea (Rheimorphae), the white-tailed eagle and steppe eagle (Accipitrimorphae) and was approximately 13–15 mm wide and 9–11 mm long, respectively. In all of the examined birds, the CALT was associated with a rich network of small vessels. In addition, the presence of characteristic high endothelial venules and roundish bright endothelial cells was confirmed in the upper and lower eyelids or only in the lower eyelid (Phoenicopterimorphae, Procellariimorphae and Strigimorphae).

Spinal cord injuries (SCI) often require simultaneous regeneration of nerve tissue and bone. Hydroxyapatites are described as bioresorbable materials with proper biocompatibility and osteoconductivity, therefore its application for spinal surgery is considered. In this paper, we present repeatable method for developing nanocrystalline calcium hydroxyapatites structurally modified with Li+ ions (nHAP:Li+). Obtained biomaterials were profoundly characterized in terms of their physicochemical properties. Moreover, we have shown that nHAP:Li+ doped with europium (Eu3+) may serve as a theranostic agent, what additionally extend its potential usage for SCI treatment. The biocompatibility of nHAP:Li+ was determined using human olfactory ensheathing cells (hOECs) and adipose tissue-derived multipotent stromal cells (hASCs). Both population of cells are eagerly applied for cell-based therapies in SCI, mainly due to their paracrine activity. The extensive in vitro studies showed that nHAP:Li+ promotes the cells proliferation, viability and cell-cell interactions. Obtained results provides encouraging approach that may have potential application in regenerative medicine and that could fulfil the promise of personalized medicine – important in SCI treatment.

Visual impairment is a common ailment of the current world population, with more exposure to CCD screens and fluorescent lighting, approximately 285 billion people suffer from this deficiency and 13% of those are considered clinically blind. More common causes for visual impairment include age-related macular degeneration (AMD), glaucoma and diabetic retinopathy (Zhu et al. Molecular Medicine Reports, 2015; Kolb et al. 2007; Machalińska et al. Current Eye Research, 34(9),748–760, 2009) among a few. As cases of retinal and optic nerve diseases rise, it is vital to find a treatment, which has led to investigation of the therapeutic potential of various stem cells types (Bull et al. Investigative Opthalmology & Visual Science, 50(9), 4244, 2009; Bull et al. Investigative Opthalmology & Visual Science, 49(8), 3449, 2008; Yu et al. Biochemical and Biophysical Research Communications, 344(4), 1071-1079, 2006; Na et al. Graefe's Archive for Clinical and Experimental Ophthalmology, 247(4), 503-514, 2008). In previous studies, some of the stem cell variants used include human Muller SCs and bone marrow derived SCs. Some of the regenerative potential characteristics of mesenchymal progenitor stem cells (MSCs) include their multilineage differentiation potential, their immunomodulatory effects, their high proliferative activity, they can be easily cultured in vitro, and finally their potential to synthesize and secrete membrane derived vesicles rich in growth factors, mRNA and miRNA which possibly aid in regulation of tissue damage regeneration. These facts alone, explain why MSCs are so widely used in clinical trials, 350 up to date (Switonski, Reproductive Biology, 14(1), 44–50, 2014). Animal studies have demonstrated that sub-retinal transplantation of MSCs delays retinal degeneration and preserves retinal function through trophic response (Inoue et al. Experimental Eye Research, 85(2), 234–241, 2007). Umbilical cord derived MSCs (UC/MSCs) have also been shown to contain neuroprotective features of ganglion cells in rat studies (Zwart et al. Experimental Neurology, 216(2), 439–448, 2009). This review aims to present current MSC therapies in practice, as well as their retinal regeneration potential in animal models, and their innovative prospects for treatment of human retinal diseases.

Mesenchymal stem cells (MSC) stand as a promising tool in regenerative medicine because of their high therapeutic potential in treatment of degenerative, metabolic and other types of diseases. The cellular therapies involving MSCs include their isolation mainly from the bone marrow, adipose tissue or umbilical cord and in vitro expansion for further autologous or allogeneic transplantation. Recent studies revealed, that bioactive compounds, naturally occurring in seaweeds, herbs, fruits and vegetables, possess the ability to modulate self-renewal and differentiation potential of adult stem cells, targeting a broad range of intracellular signal transduction pathways. Number of ongoing trials aim to find a herbal extract that may become less toxic and affordable natural therapeutic. Mesenchymal stem cells are treated with crude extracts or individual compounds to investigate its effects and mechanism on stem cells proliferation and differentiation. Deeply investigated, herbal extract which increases tissue regeneration and promotes stem cell growth may be successfully applied in the field of biomaterials. Promoting the endogenous stem cell multipotency and their differentiation potential may additionally support the regenerative processes after MSCs transplantation. The review focuses on the beneficial effects of chosen plant derived substances on MSCs proliferative activity and their osteogenic differentiation potential. Copyright © 2017 John Wiley & Sons, Ltd.

Background Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young ( 15 years old). Methods ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. Results Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. Conclusions Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential. Graphical abstract

Background In athlete horses, suspensory ligament (SL) injuries are the most common cause of lameness. Healing of SL injury is still problematic, and even proper rehabilitation and pharmacological therapy do not guarantee returning to the initial performance level. In our previous studies, we have shown that a combination of 5-azacytidine (AZA) and resveratrol (RES) exerts beneficial, rejuvenating effects on metabolic syndrome derived adipose-derived stem cells (ASCs). Thus, in the presented research, we investigate whether not only rejuvenated ASC but also microvesicles (MVsAZA/RES) secreted by them possess enhanced regenerative properties in SL injury. Methods In the presented study, a 6-year-old Dutch Warmblood gelding, working in jumping, was diagnosed with SL injury using ultrasonography, Doppler, real-time elastography and thermography. As a therapeutic strategy, the affected animal was treated with extracellular microvesicles derived from ASC treated with the combination of 5-azacytydine (AZA) and resveratrol (RES) (MVsAZA/RES). Results First, anti-apoptotic effects of MVsAZA/RES were tested in co-culture with metabolic syndrome derived ASC. The proliferation of cells and expression of pro-apoptotic genes were investigated. Then, MVsAZA/RES were injected directly into the injured SL of the Dutch Warmblood gelding. In vitro assays revealed that MVsAZA/RES enhance the proliferation of ASC and exert an anti-apoptotic effect. In the affected horse, the application of MVsAZA/RES resulted in increased lesion filling and improvement of angiogenesis and elasticity in injured tissue. Conclusions As MVsAZA/RES mimic several of the biological actions exerted by ASC, they have become an alternative for stem cell-based therapies and can be effectively applied for the treatment of SL injury in horses.

Equine metabolic syndrome (EMS) is a cluster of metabolic disorders, such as obesity, hyperinsulinemia, and hyperleptinemia, as well as insulin resistance (IR). In accordance with the theory linking obesity and IR, excessive accumulation of lipids in insulin-sensitive tissues (lipotoxicity), like liver, alters several cellular functions, including insulin signaling. Therefore, the purpose of the study was to isolate equine hepatic progenitor-like cells (HPCs) and assess whether inhibition of low molecular weight protein tyrosine phosphatase (LMPTP) affects the expression of genes involved in macroautophagy, chaperone-mediated autophagy (CMA), endoplasmic reticulum stress, and mitochondrial dynamics in a palmitate-induced IR model. We demonstrated that LMPTP inhibition significantly enhanced expression of heat shock cognate 70 kDa protein (HSC70), lysosome-associated membrane protein 2 (LAMP2), and parkin (PRKN), all master regulators of selective autophagy. We also observed downregulation of C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and binding immunoglobulin protein encoded by the HSPA gene. Moreover, LMPTP inhibition increased alternative splicing of X-box binding protein 1 (XBP1), suggesting high endonuclease activity of inositol-requiring enzyme 1 alpha (IRE1α). Taken together, our data provide convincing evidence that LMPTP inhibition reverses palmitate-induced insulin resistance and lipotoxicity. In conclusion, this study highlights the role of LMPTP in the regulation of CMA, mitophagy, and ER stress, and provides a new in vitro model for studying HPC lipotoxicity in pre-clinical research.

Background Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. Objectives In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. Methods Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. Results The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. Conclusion Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

Objectives Equine metabolic syndrome (EMS) refers to a cluster of associated abnormalities and metabolic disorders, including insulin resistance and adiposity. The numerous biological properties of mesenchymal stem cells (MSCs), including self-renewal and multipotency, have been the subject of many in-depth studies, for the management of EMS; however, it has been shown that this cell type may be affected by the condition, impairing thus seriously their therapeutic potential. Therefore, an attempt to rescue EMS adipose-derived stem cells (ASCs) with calystegines (polyhydroxylated alkaloids) that are endowed with strong antioxidant and antidiabetic abilities was performed. Methods ASCs isolated from EMS horses were subsequently treated with various concentrations of total calystegines. Different parameters were then assessed using flow cytometry, confocal as well as SE microscopy, and RT-qPCR. Results Our results clearly demonstrated that calystegines could improve EqASC viability and proliferation and significantly reduce apoptosis, via improvement of mitochondrial potentiation and functionality, regulation of pro- and anti-apoptotic pathways, and suppression of ER stress. Furthermore, nortropanes positively upregulated GLUT4 and IRS transcripts, indicating a possible sensitizing or mimetic effect to insulin. Most interesting finding in this investigation lies in the modulatory effect of autophagy, a process that allows the maintenance of cellular homeostasis; calystegines acted as pharmacological chaperones to promote cell survival. Conclusion Obtained data open new perspectives in the development of new drugs, which may improve the metabolic dynamics of cells challenged by MS.

The widespread epidemic of obesity and type 2 diabetes (T2D), suggests that both disorders are closely linked. Several pre-clinical and clinical studies have showed that adipose-derived mesenchymal stem cells (ASC) transplantation is efficient and safe. Moreover, scientists have already highlighted the therapeutic capacity of their secretomes. In this study, we used quantitative PCR, a flow cytometry-based system, the ELISA method, spectrophotometry, and confocal and scanning electron microscopy, to compare the differences in proliferation activity, viability, morphology, mitochondrial dynamics, mRNA and miRNA expression, as well as the secretory activity of ASCs derived from two donor groups—non-diabetic and T2D patients. We demonstrated that ASCs from T2D patients showed a reduced viability and a proliferative potential. Moreover, they exhibited mitochondrial dysfunction and senescence phenotype, due to excessive oxidative stress. Significant differences were observed in the expressions of miRNA involved in cell proliferations (miR-16-5p, miR-146a-5p, and miR-145-5p), as well as miRNA and genes responsible for glucose homeostasis and insulin sensitivity (miR-24-3p, 140-3p, miR-17-5p, SIRT1, HIF-1α, LIN28, FOXO1, and TGFβ). We have observed a similar correlation of miR-16-5p, miR-146a-5p, miR-24-3p, 140-3p, miR-17-5p, and miR-145-5p expression in extracellular vesicles fraction. Furthermore, we have shown that ASCT2D exhibited a lower VEGF, adiponectin, and CXCL-12 secretion, but showed an overproduction of leptin. We have shown that type 2 diabetes attenuated crucial functions of ASC, like proliferation, viability, and secretory activity, which highly reduced their therapeutic efficiency.

Pseudomonas aeruginosa is an opportunistic pathogen infecting human population. The pathogen is becoming a serious health problem due to its ability to evade normal immune response of the host and multiple drug resistance to many antibiotics. The pathogen has 2 major virulence systems of which the type III secretion system (T3SS) is of major concern to humans. A third system, type 2 secretion system (T2SS), is common to bacteria and used to secrete exotoxin A (ExoA) responsible for human cell destruction. To help bypass the drug resistance, a strategy to block the T2SS based on a low similarity between human ATPases and the essential ATPases of the T3SS and T2SS of P. aeruginosa, was used. An in silico-optimized inhibitor of T3SS, made directly from the computer-optimized of previously published compounds and their combinatorial libraries, showed IC50 = 1.3 ± 0.2 μM in the T2SS ExoA secretion blocking test. The compound was non-toxic to human lung epithelial cell line A549 and could block cellular destruction of those cells in a cell infection model at 200 μM for at least 24 h. The compound could be a lead candidate for the development of T2SS virulence blockers of Pseudomonas aeruginosa.

Osteochondrosis (osteochondrosis dissecans; OCD) is a disease syndrome of growing cartilage related to different clinical entities such as epiphysitis, subchondral cysts and angular carpal deformities, which occurs in growing animals of all species, including horses. Nowadays, these disorders are affecting increasing numbers of young horses worldwide. As a complex multifactorial disease, OCD is initiated when failure in cartilage canals because of existing ischemia, chondrocyte biogenesis impairment as well as biochemical and genetic disruptions occur. Recently, particular attention have been accorded to the definition of possible relations between OCD and some metabolic disorders; in this way, implication of mitochondrial dysfunctions, endoplasmic reticulum disruptions, oxidative stress or endocrinological affections are among the most considered axes for future researches. As one of the most frequent cause of impaired orthopaedic potential, which may result in a sharp decrease in athletic performances of the affected animals, and lead to the occurrence of complications such as joint fragility and laminitis, OCD remains as one of the primary causes of considerable economic losses in all sections of the equine industry. It would therefore be important to provide more information on the exact pathophysiological mechanism(s) underlying early OC(D) lesions, in order to implement innovative strategies involving the use of progenitor stem cells, which are considered nowadays as a promising approach to regenerative medicine, with the potential to treat numerous orthopaedic disorders, including osteo-degenerative diseases, for prevention and reduction of incidence of the disease, not only in horses, but also in human medicine, as the equine model is already widely accepted by the scientific community and approved by the FDA, for the research and application of cellular therapies in the treatment of human conditions.

The aim of this study was to evaluate the influence of resveratrol (RES)-doped polyurethane (TPU)–polylactide (PLA) biomaterials on the senescence and oxidative stress factor of adipose-derived stem cells (ASCs) for tissue engineering. Using a four-step method (solution preparation, solvent casting, freezing/lyophilizing, washing and drying), we fabricated TPU/PLA scaffolds doped with different RES concentrations. Then, we established the mass loss and RES release from the obtained films over time using differential scanning calorimetry and UV-Vis spectrophotometry, respectively. ASC was seeded onto the fabricated scaffolds to establish their biocompatibility and biological influence. Using fluorescence microscopy, real-time PCR, and spectrophotometric assays, the proliferation rate, oxidative stress factors, pro-apoptotic, autophagy-related and osteogenic genes expression were evaluated. The obtained data clearly indicated that the TPU/PLA biomaterial doped with 0.5 mmol RES had the most beneficial effect on ASC, whereby it increased proliferation, reduced oxidative stress and apoptosis, and induced the expression of osteogenic genes. Fabricated by our group, innovative biomaterials were characterized by their RES release in a sustained manner. A prolonged release of RES is especially important when considering clinical application. The release of RES was most intense during the first 20 days, which may be beneficial in vivo as inflammation within damaged tissue is most intense during the first days after injury. The fabricated material directed ASC toward an osteoblast-like molecular phenotype, which suggests it could be a promising tool in regenerative medicine. The combination of the scaffold with ASC may improve the effectiveness and shorten the time for bone regeneration. These characteristics presented here make the biomaterial a promising tool in the emerging field of personalized regenerative medicine, especially in geriatrics.

Oxidative stress is the primary cause of lowering of the body's immune status. It arises as a result of intense metabolic processes in humans and animals that involve a decrease in antioxidant enzymes activity at the expense of increased production of free radicals and reactive oxygen species (ROS). Consequently, this leads to oxidation of lipids, proteins, DNA, and RNA, causing cell proliferation reduction, faster cell aging, and subsequent degradation. Currently, there is a search for methods to maintain an oxidation-antioxidant balance. The results of many authors' research point to the effectiveness of the use of mesenchymal stem cells (MSCs) in the treatment of various clinical disorders. Numerous studies indicate that, compared to other cells, MSCs have ability to regulate cell adhesion molecules. They also have specific immunogenic and immunosuppressive properties that determine their application in clinical research and regenerative medicine.

Adipose‐derived mesenchymal stem cells (ASC) hold great promise in the treatment of many disorders including musculoskeletal system, cardiovascular and/or endocrine diseases. However, the cytophysiological condition of cells, used for engraftment seems to be fundamental factor that might determine the effectiveness of clinical therapy. In this study we investigated growth kinetics, senescence, accumulation of oxidative stress factors, mitochondrial biogenesis, autophagy and osteogenic differentiation potential of ASC isolated from horses suffered from equine metabolic syndrome (EMS). We demonstrated that EMS condition impairs multipotency/pluripotency in ASCs causes accumulation of reactive oxygen species and mitochondria deterioration. We found that, cytochrome c is released from mitochondria to the cytoplasm suggesting activation of intrinsic apoptotic pathway in those cells. Moreover, we observed up‐regulation of p21 and decreased ratio of Bcl‐2/BAX. Deteriorations in mitochondria structure caused alternations in osteogenic differentiation of ASCEMS resulting in their decreased proliferation rate and reduced expression of osteogenic markers BMP‐2 and collagen type I. During osteogenic differentiation of ASCEMS, we observed autophagic turnover as probably, an alternative way to generate adenosine triphosphate and amino acids required to increased protein synthesis during differentiation. Downregulation of PGC1α, PARKIN and PDK4 in differentiated ASCEMS confirmed impairments in mitochondrial biogenesis and function. Hence, application of ASCEMS into endocrinological or ortophedical practice requires further investigation and analysis in the context of safeness of their application.

Articular cartilage dysfunctions are major cause of pain and disability and lead to serious health complications. Cell-based therapies are proposed as treatment methods for cartilage regeneration. In this study, we proposed polyurethane/poly(L-lactide-co-D, L-lactide)-based electrospun nonwovens as carriers for the delivery of human adipose-derived stromal stem cells. We found that 6:4 and 8:2 polyurethane/poly(L-lactide-co-D, L-lactide) initially enhance proliferative rate of human adipose-derived stromal stem cells, shorten their population doubling time, promote creation of functional chondrogenic nodules during chondrogenic differentiation, improve the collagen-2-to-collagen-1 protein ratio, and upregulate the expression of collagen-2 and aggrecan genes.

MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor &kappa, B ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts&rsquo, differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-&kappa, B ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.

Endocrine disorders are becoming an increasing problem in both human and veterinary medicine. In recent years, more and more horses worldwide have been suffering from equine metabolic syndrome (EMS). This metabolic disorder is characterized by pathological obesity, hyperinsulinaemia, hyperglycaemia and insulin resistance. Although metabolic disorders, including diabetes, have been extensively studied, there are still no data on the molecular effects of EMS in horses. Thus, the aim of this study was to evaluate apoptosis, oxidative stress, autophagy and microRNA (miR) expression in multipotent intestinal epithelial stem cells (IECs) and pancreatic islets (PIs) isolated post mortem form healthy and EMS diagnosed horses. Our group was the first to describe how EMS affects IEC and PI aging and senescence. First, we evaluated isolation and culture protocol for these cells and subsequently established their metabolic status in vitro. Both IECs and PIs isolated from EMS horses were characterized by increased apoptosis and senescence. Moreover, they accumulated elevated levels of reactive oxygen species (ROS). Here we have observed that autophagy/mitophagy may be a protective mechanism which allows those cells to maintain their physiological function, clear protein aggregates and remove damaged organelles. Furthermore, it may play a crucial role in reducing endoplasmic reticulum (ER) stress. This protective mechanism may help to overcome the harmful effects of ROS and provide building blocks for protein and ATP synthesis.

In the present paper, the potential of canine articular cartilage-derived cells (cACCs) for chondrogenic differentiation was evaluated. The effectiveness of cACCs’ lineage commitment was analyzed after 14 days of culture in chondorgenic and non-chondrogenic conditions. Formation of proteoglycan-rich extracellular matrix was assessed using histochemical staining – Alcian Blue and Safranin-O, while elemental composition was determined by means of SEM-EDX. Additionally, ultrastructure of cACCs was evaluated using TEM. The expression of genes involved in chondrogenesis was monitored with quantitative Real Time PCR. Results obtained indicate that the potential of cACCs for cartilagous extracellular matrix formation may be maintained only in chondrogenic cultures. The formation of specific chondro-nodules was not observed in a non-chondrogenic culture environment. The analysis of cACCs’ ultrastructure, both in non-chondrogenic and chondrogenic cultures, revealed well-developed rough endoplasmatic reticulum and presence of mitochondria. The cACCs in chondrogenic medium shed an increased number of microvesicles. Furthermore, it was shown that the extracellular matrix of cACCs in chondrogenic cultures is rich in potassium and molybdenum. Additionally, it was determined that gene expression of collagen type II, aggrecan and SOX-9 was significantly increased during chondrogenic differentiation of cACCs. Results obtained indicate that the culture environment may significantly influence the cartilage phenotype of cACCs during long term culture.

The aim of this work was to investigate the effects of static magnetic field (SMF) on the osteogenic properties of human adipose derived mesenchymal stem cells (hASCs). In this study in seven days viability assay we examined the impact of SMF on cells proliferation rate, population doubling time, and ability to form single-cell derived colonies. We have also examined cells' morphology, ultrastructure and osteogenic properties on the protein as well as mRNA level. We established a complex approach, which enabled us to obtain information about SMF and hASCs potential in the context of differentiation into osteogenic and adipogenic lineages. We demonstrated that SMF enhances both viability and osteogenic properties of hASCs through higher proliferation factor and shorter population doubling time. We have also observed asymmetrically positioned nuclei and organelles after SMF exposition. With regards to osteogenic properties we observed increased levels of osteogenic markers i.e. osteopontin, osteocalcin and increased ability to form osteonodules with positive reaction to Alizarin Red dye. We have also shown that SMF besides enhancing osteogenic properties of hASCs, simultaneously decreases their ability to differentiate into adipogenic lineage. Our results clearly show a direct influence of SMF on the osteogenic potential of hASCs. These results provide key insights into the role of SMF on their cellular fate and properties.

Nowadays, endocrine disorders have become more frequent in both human and veterinary medicine. In horses, reduced physical activity combined with carbohydrate and sugar overload may result in the development of the so-called equine metabolic syndrome (EMS). EMS is characterized by insulin resistance, hyperinsulinemia, elevated blood triglyceride concentrations and usually obesity. Although the phenotypic features of EMS individuals are well known, the molecular mechanism underlying disease development remains elusive. Therefore, in the present study, we analyzed insulin-sensitive tissues, i.e., muscles, liver and adipose tissue in order to evaluate insulin resistance and apoptosis. Furthermore, we assessed mitochondrial dynamics and mitophagy in those tissues, because mitochondrial dysfunction is linked to the development of metabolic syndrome. We established the expression of genes related to insulin resistance, endoplasmic reticulum (ER) stress and mitochondria clearance by mitophagy using RT-PCR and Western blot. Cell ultrastructure was visualized using electron transmission microscopy. The results indicated that adipose tissue and liver of EMS horses were characterized by increased mitochondrial damage and mitophagy followed by triggering of apoptosis as mitophagy fails to restore cellular homeostasis. However, in muscles, apoptosis was reduced, suggesting the existence of a protective mechanism allowing that tissue to maintain homeostasis.

Aging and sedentary lifestyle are common nowadays and are associated with the increasing number of chronic diseases. Thus, physical activity is recommended as one of three healthy behavior factors that play a crucial role in health prophylaxis. In the present study, we were interested whether physical activity influences the number and potential of bone-marrow-derived mesenchymal stem cells BMMSCs. In this study, four-week-old male C57Bl/6 mice were trained on a treadmill at progressive speeds over a 5-week period. Comparisons made between exercised (EX) and sedentary animal groups revealed (i) significantly higher number of MSCs in EX animals, (ii) elevated alkaline phosphatase (ALP) activity, (iii) increased level of osteopontin (OPN) and osteocalcin (OCL), and (iv) reduced marrow cavity fat. The results obtained support the thesis that EX may play a substantial role in the regeneration of mesenchymal tissues. Therefore, EX may represent a novel, nonpharmacological strategy of slowing down age-related decline of the musculoskeletal functions.

Mesenchymal stem cells (MSCs) are frequently used in both human and veterinary medicine because their unique properties, such as modulating the immune response and differentiating into multiple lineages, make them a valuable tool in cell-based therapies. However, many studies have indicated the age-, lifestyle-, and disease-related deterioration of MSC regenerative characteristics. However, it still needs to be elucidated how the patient’s health status affects the effectiveness of MSC differentiation. In the present study, we isolated mesenchymal stem cells from adipose tissue (adipose-derived mesenchymal stem cells (ASCs)) from horses diagnosed with equine metabolic syndrome (EMS), a common metabolic disorder characterized by pathological obesity and insulin resistance. We investigated the metabolic status of isolated cells during adipogenic differentiation using multiple research methods, such as flow cytometry, PCR, immunofluorescence, or transmission and confocal microscopy. The results indicated the impaired differentiation potential of ASCEMS. Excessive ROS accumulation and ER stress are most likely the major factors limiting the multipotency of these cells. However, we observed autophagic flux during differentiation as a protective mechanism that allows cells to maintain homeostasis and remove dysfunctional mitochondria.