Mitochondria transfer restores fibroblasts-like synoviocytes (FLS) plasticity in LPS-induced, in vitro synovitis model.

BACKGROUND: Synovitis (SI) is one of the most common and serious orthopedic diseases in horses of different age, breed and sex, which contributes to the development of osteoarthritis. The burden of SI includes economic loss and represents a real challenge for current veterinary health care. At the molecular level, fibroblasts-like synoviocytes (FLS) are recognized as major cell populations involved in SI pathogenesis. In the course of SI, FLSs are losing their protective and pro-regenerative cytological features, become highly proliferative and initiate various stress signaling pathways. METHODS: Fibroblast-like synoviocytes were treated with LPS in order to generate SI in vitro model. Mitochondria were isolated from peripheral blood derived mononuclear cells and co-cultured with FLS. After 24 h of culture, cells were subjected to RT-qPCR, western blot, cytometric and confocal microscopy analysis. RESULTS: Mitochondrial transfer (MT) was observed in vitro studies using confocal microscopy. Further studies revealed, that MT to LPS-treated FLS reduced cell proliferation, modulated apoptosis and decreased inflammatory response. Overall, MT Resulted in the considerable recovery of recipient cells cytophysiological properties. CONCLUSIONS: Presented data provides evidence that mitochondria transfersignificantly modulate FLS proliferative and metabolic activity through improved mitochondrial biogenesis and dynamics in activated FLS. Obtained results for the first time demonstrate that horizontal MT might be considered as a therapeutic tool for synovitis treatment; however, further clinical studies are strongly required. Video abstract.

Extracellular Microvesicles (MV's) Isolated from 5-Azacytidine-and-Resveratrol-Treated Cells Improve Viability and Ameliorate Endoplasmic Reticulum Stress in Metabolic Syndrome Derived Mesenchymal Stem Cells.

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.

Hafnium (IV) oxide obtained by atomic layer deposition (ALD) technology promotes early osteogenesis via activation of Runx2-OPN-mir21A axis while inhibits osteoclasts activity.

BACKGROUND: Due to increasing aging of population prevalence of age-related disorders including osteoporosis is rapidly growing. Due to health and economic impact of the disease, there is an urgent need to develop techniques supporting bone metabolism and bone regeneration after fracture. Due to imbalance between bone forming and bone resorbing cells, the healing process of osteoporotic bone is problematic and prolonged. Thus searching for agents able to restore the homeostasis between these cells is strongly desirable. RESULTS: In the present study, using ALD technology, we obtained homogeneous, amorphous layer of hafnium (IV) oxide (HfO2). Considering the specific growth rate (1.9Å/cycle) for the selected process at the temperature of 90 °C, we performed the 100 nm deposition process, which was confirmed by measuring film thickness using reflectometry. Then biological properties of the layer were investigated with pre-osteoblast (MC3T3), pre-osteoclasts (4B12) and macrophages (RAW 264.7) using immunofluorescence and RT-qPCR. We have shown, that HfO2 (i) enhance osteogenesis, (ii) reduce osteoclastogenesis (iii) do not elicit immune response and (iv) exert anti-inflammatory effects. CONCLUSION: HfO2 layer can be applied to cover the surface of metallic biomaterials in order to enhance the healing process of osteoporotic bone fracture.

Correction to: Iron oxides nanoparticles (IOs) exposed to magnetic field promote expression of osteogenic markers in osteoblasts through integrin alpha-3 (INTa-3) activation, inhibits osteoclasts activity and exerts anti-inflammatory action.

An amendment to this paper has been published and can be accessed via the original article.

Iron oxides nanoparticles (IOs) exposed to magnetic field promote expression of osteogenic markers in osteoblasts through integrin alpha-3 (INTa-3) activation, inhibits osteoclasts activity and exerts anti-inflammatory action.

BACKGROUND: Prevalence 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. RESULTS: The α-Fe2O3/γ-Fe2O3 nanocomposite (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. CONCLUSION: Fabricated by our group α-Fe2O3/γ-Fe2O3 nanocomposite may become an justified and effective therapeutic intervention during osteoporosis treatment.

5-Azacytidine and Resveratrol Enhance Chondrogenic Differentiation of Metabolic Syndrome-Derived Mesenchymal Stem Cells by Modulating Autophagy.

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.

Lithium ions (Li+) and nanohydroxyapatite (nHAp) doped with Li+ enhance expression of late osteogenic markers in adipose-derived stem cells. Potential theranostic application of nHAp doped with Li+ and co-doped with europium (III) and samarium (III) ions.

Lithium (Li+) ion due to its excellent bioactivity is one of the most well-studied element in bone-tissue engineering. In this study, we fabricated nanohydroxyapatite (nHAp) doped with Li+ ions (5 mol% Li+:nHAp) and co-doped with lanthanide ions. We investigated the effects of nHAp, 5 mol% Li+:nHAp or Li+ alone, on osteogenic differentiation of human Adipose Tissue-derived Stem Cells (hASCs), their proliferation, mitochondrial dynamics and apoptosis. Moreover, we monitored cell proliferation after treatment with samarium (III) (Sm3+) and europium (III) (Eu3+) ions co-doped 5 mol% Li+:nHAp as well as their luminescent property. The hASCs treated with 5 mol% Li+:nHAp and Li+ ions proliferated more rapidly and differentiated effectively than control cells without undergoing apoptosis. Both, 5 mol% Li+:nHAp and Li+ ions improved osteogenic differentiation of hASCs. Moreover they decreased expression of glycogen synthase kinase 3ß (GSK3ß) while increased ß-catenin mRNA level. In addition, Li+, nHAp and 5 mol% Li+:nHAp improved mitochondrial dynamics and enhanced expression of neural differentiation marker genes. Collectively, the study indicates on pro-osteogenic and anti-apoptotic properties of nHAp doped with Li+ and Li+ alone. Moreover, unique properties of 5 mol% Li+:nHAp and 5 mol% Li+:nHAp co-doped with rare earth ions, such as Sm3+ and Eu3+ have shed a promising light on their potential application in theranostics.

Subchondral bone cyst surgical treatment using the application of stem progenitor cells combined with alginate hydrogel in small joints in horses.

One of the most common reasons for horse lameness is subchondral bone cysts (SBCs), which are especially evident in young horse athletes. It is believed that SBC development is strongly associated with an individual's bone growth and/or bone microstructure impairment. Current methods of SBC treatment include pharmacological treatment or surgical procedures which may allow the bone within the cyst to rebuild and be restored to properly developed bone tissue. Thus, we propose filling the SBCs with a 3D complex of alginate hydrogel and autologous adipose derived mesenchymal stem cells (ASCs). We have observed at the in vitro level, that this hydrogel complex induces osteogenic and chondrogenic differentiation potential through the upregulation of bone morphogenetic protein, osteopontin, collagen type I and aggrecan mRNA levels. Moreover, we detected the creation of a 3D extracellular matrix (EM). To investigate the complex in vivo, we chose 8 horses of varying age suffering from SBC, which resulted in lameness, to undergo experimental surgery. We documented the horses' clinical appearance, lameness and radiographic appearance, to determine that there was clinical improvement in 87.75% of the patients (n=7, out of 8 horses) 6 months postoperatively and 100% (n=8, out of 8 horses) a year after surgery. These results are promising for the potential of this procedure to become the standard in SBC treatment.

Efficient synthesis of PMMA@Co0.5Ni0.5Fe2O4 organic-inorganic hybrids containing hyamine 1622 - Physicochemical properties, cytotoxic assessment and antimicrobial activity.

The PMMA@Co0.5Ni0.5Fe2O4 ferrite containing hybrid nanomaterials with hyamine were prepared using emulsion polymerization method. Structural and morphological properties were evaluated using XRD, FT-IR, SEM techniques. The TGA and DTA analysis were performed in order to study the thermal properties of hybrid materials in contrast to reference material. Magnetic properties were studied using Quantum Design PPMS (VSM option) in a constant external magnetic field equal (100 Oe and 1000 Oe) in the temperature range from 2 to 380 K. Both the pure Co0.5Ni0.5Fe2O4and the sample with 85% of PMMA exhibit superparamagnetic behavior whereas blocking temperatureTB decreases with increase of PMMA content. The cytotoxicity assessment of PMMA@Co0.5Ni0.5Fe2O4 with hyamine in J774.E murine macrophages and U2OS human osteosarcoma cell lines was performed. Additionally, sensitivity of bacteria Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 25923 to hybrid materials (with/without hyamine) was investigated using a of Kirby-Bauer disc method.

The Effect of Co0.2Mn0.8Fe2O4 Ferrite Nanoparticles on the C2 Canine Mastocytoma Cell Line and Adipose-Derived Mesenchymal Stromal Stem Cells (ASCs) Cultured Under a Static Magnetic Field: Possible Implications in the Treatment of Dog Mastocytoma.

Cobalt manganese ferrite nanoparticles have application potential in the biomedical field, however there is limited information concerning the biological response. The aim of this work was to investigate the cytotoxic potential of cobalt-manganese ferrite nanoparticles in canine mastocytoma tumor cells (C2) and adipose-derived mesenchymal stromal stem cells (ASCs) cultured under a static magnetic field (MF). In this study, we investigated the viability and proliferation rate of ASC and C2 cells cultured with Co0.2Mn0.8Fe2O4 nanoparticles under 0.5T MF. We observed cells morphology and measured intracellular ROS generation. Thermal observations were used to characterize the thermotrophic cell behavior in different condition and RNA level of heat shock proteins and apoptotic genes was measured. Nanoparticles reduced cell viability, caused cell damage, i.e., through the formation of reactive oxygen species (ROS) and increased transcriptional level of apoptotic genes (Bcl-2, Bax, p53, p21). In addition, we have found that C2 mastocytoma cells cultured with metal oxide nanoparticles under MF exhibited unexpected biological responses, including thermotolerance and apoptotic response induced by the expression of heat shock proteins and ROS produced under a MF. Our results suggest that stimulation using MF and Co0.2Mn0.8Fe2O4 nanoparticles is involved in mechanisms associated with controlling cell proliferative potential signaling events. We can state that significant differences between normal and cancer cells in response to nanoparticles and MF are apparent. Our results show that nanoparticles and MF elevate the temperature in vitro in tumor cells, thereby increasing the expression of ROS as well as heat shock proteins.

Multifunctional nanocrystalline calcium phosphates loaded with Tetracycline antibiotic combined with human adipose derived mesenchymal stromal stem cells (hASCs).

Osteoconductive drug delivery system composed of nanocrystalline calcium phosphates (Ca10(PO4)6(OH)2/ß-Ca3(PO4)2) co-doped with Yb(3+)/Er(3+) ions loaded with Tetracycline antibiotic (TC) was developed. Their effect on human adipose derived mesenchymal stromal stem cells (hASCs) as a potential reconstructive biomaterial for bone tissue regeneration was studied. The XRD and TEM measurements were used in order to determine the crystal structure and morphology of the final products. The characteristics of nanocomposites with the TC and hASCs as potential regenerative materials as well as the antimicrobial activity of the nanoparticles against: Staphylococcus aureus ATCC 25923 as a model of the Gram-positive bacteria, Escherichia coli ATCC 8739 of the Gram-negative bacteria, were shown. These combinations can be a promising material for theranostic due to its regenerative, antimicrobial and fluorescent properties.

Therapeutic effect of adipose-derived mesenchymal stem cell injection in horses suffering from bone spavin.

In this article we demonstrate the efficiency of autologous transplantations of adipose-derived mesenchymal stem cells for equine bone spavin treatment. Horses qualified to the study were divided into three groups: (i) research - treated with intra-articular injections of autologous stem cells, (ii) comparison treated with steroid drugs and (iii) control - untreated. All animals underwent comprehensive clinical examination before and after treatment. Our research confirms the long-term beneficial influence resulting from stem cell therapy in horse bone spavin treatment, in contrast to routine steroid usage.

Gastrointestinal development of dairy calves fed low- or high-starch concentrate at two milk allowances.

The objective was to study the effect of type of concentrate with varying starch and fibre content on growth and gastrointestinal development in preweaned dairy calves. Thirty-two newborn Danish Holstein male calves were allocated to four treatment groups in eight blocks of four calves. An experimental low-starch, high-molasses, high-fibre (EXP) concentrate or a traditional high-starch (TRA) concentrate were fed either at a high (HIGH; 2 × 3.2 kg/day) or a low (LOW; 2 × 1.6 kg/day) whole milk allowance in a 2 × 2 factorial design. TRA contained 350 and EXP 107 g starch/kg dry matter (DM), whereas the NDF content was 136 and 296 g/kg DM, respectively. Metabolizable energy (ME) was 11.2 and 12.2 MJ ME/kg DM in EXP and TRA, respectively. All calves had free access to artificially dried grass hay (9.8 MJ ME/kg DM). Four calves were culled during the experiment. The calves were euthanized either at 38 (12 calves) or 56 days (16 calves) of age. Evaluated across both slaughter ages, there was no difference between TRA and EXP in concentrate and hay intake, rumen weight and papillation. EXP resulted in increased villi number in duodenum and jejunum compared with TRA. Concentrate intake and reticulo-rumen weight was higher for LOW compared with HIGH milk allowance, whereas live weight gain was 20% lower. The results show that a low-starch, high-molasses, high-fibre concentrate with 8% lower ME content tended to reduce daily gain compared with a traditional calf starter concentrate, but resulted in similar ruminal development in preweaned calves both on a high and a low milk allowance fed along with grass hay. Furthermore, the results suggest that the experimental concentrate stimulated intestinal villi growth over that of the traditional concentrate.