Effect of storage conditions on the quality of equine and canine mesenchymal stem cell derived nanoparticles including extracellular vesicles for research and therapy.

Nanoparticles including extracellular vesicles derived from mesenchymal stem cells are of increasing interest for research and clinical use in regenerative medicine. Extracellular vesicles (EVs), including also previously named exosomes, provide a promising cell-free tool for therapeutic applications, which is probably a safer approach to achieve sufficient healing. Storage of EVs may be necessary for clinical applications as well as for further experiments, as the preparation is sometimes laborious and larger quantities tend to be gained. For this purpose, nanoparticles were obtained from mesenchymal stem cells from adipose tissue (AdMSC) of horses and dogs. The EVs were then stored for 7 days under different conditions (- 20 °C, 4 °C, 37 °C) and with the addition of various additives (5 mM EDTA, 25-250 µM trehalose). Afterwards, the size and number of EVs was determined using the nano tracking analyzing method. With our investigations, we were able to show that storage of EVs for up to 7 days at 4 °C does not require the addition of supplements. For the other storage conditions, in particular freezing and storage at room temperature, the addition of EDTA was found to be suitable for preventing aggregation of the particles. Contrary to previous publications, trehalose seems not to be a suitable cryoprotectant for AdMSC-derived EVs. The data are useful for processing and storage of isolated EVs for further experiments or clinical approaches in veterinary medicine.

Optimizing the Adipogenic Induction Protocol Using Rosiglitazone Improves the Physiological Parameters and Differentiation Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells for Horses, Sheep, Dogs, Murines, and Humans.

The investigation of adipose tissue-derived mesenchymal stem cells (ASCs) has received considerable interest in regenerative medicine. A nontoxic adipogenic induction protocol valid for cells of different mammalian species has not been described. This study aims to establish an adipogenic differentiation protocol suitable for horses, sheep, dogs, murines, and human cells. An optimized rosiglitazone protocol, consisting of 5% fetal calf serum in Dulbecco's Modified Eagle's Medium, 10 µg/mL insulin, 0.55 µg/mL transferrin, 6.8 ng sodium selenite, 1 µM dexamethasone, and 1-5 µM of rosiglitazone, is compared to the 3-isobutyl-1-methylxantine (IBMX) protocol, where rosiglitazone was replaced with 0.5 mM IBMX and 0.2 mM indomethacin. Cell viability, cytotoxicity, a morphometric analysis of the lipid, and the expression of adipogenic markers for 14 days were assessed. The data revealed that using 5 µM of rosiglitazone promotes the adipogenic differentiation capacity in horse, sheep, and dog cells compared to IBMX induction. Meanwhile, marked reductions in the cell viability and cell number with the IBMX protocol were detected, and rosiglitazone increased the cell number and lipid droplet size, prevented apoptosis, and upregulated FABP-4 and Leptin expression in the cells of most of the species. Our data revealed that the rosiglitazone protocol improves the adipogenesis of ASCs, together with having less toxicity, and should be considered for cell reproducibility and clinical applications targeting obesity.

Frequency and Characterisation of Anomalies and Fractures of the Caudal Spine in Sheep with Undocked Tails.

As tails are often docked within the first days of life, studies investigating tail malformations and injuries in sheep do not exist thus far. To address this gap in the literature, this research aimed to analyse the occurrence of vertebral anomalies and fractures in the tail within an undocked Merinoland sheep population. At 14 weeks of age, the caudal spines of 216 undocked Merinoland lambs was radiographically examined, and tail length and circumference were measured. Anomalies were documented and statistical correlation and model calculations were performed. The occurrence of block vertebrae was observed in 12.96% and wedged vertebrae in 8.33% of the sample. Of the animals, 59 (27.31%) exhibited at least one vertebral fracture, which were observed in the middle and caudal third of the tail. A significant correlation was found between the occurrence of fractures and tail length (r = 0.168) and number of vertebrae (r = 0.155). Conversely, the presence of block and wedged vertebrae was not significantly correlated with tail length, circumference, or number of vertebrae. Only the sex showed significant differences in the probability of axis deviation. These results emphasize the importance of breeding for short tails to avoid fractures.

[Phenotyping the sheep tail - Presentation of characterization methods in the context of breeding for short-tailedness]. / Phänotypisierung des Schafschwanzes ­ Darstellung von Methoden zur Charakterisierung im Rahmen der Zucht auf Kurzschwänzigkeit.

OBJECTIVE: In order to establish targeted breeding for short-tailedness, a suitable method must initially be found that allows phenotyping of the sheep tail beyond tail length. In this study, in addition to assessing body measurements, more advanced studies such as ultrasonography and radiology were performed on the caudal spine of sheep for the first time. The objective of this work was to analyze the physiological variation of tail lengths and vertebrae within a merino sheep population. It also aimed to validate the use of sonographic gray scale analysis and perfusion measurement on the sheep tail. MATERIAL AND METHOD: Tail length and circumference in centimeters were measured in 256 Merino lambs on the first or second day of life. At 14 weeks of age the caudal spine of these animals was examined radiographically. Sonographic gray scale analysis and measurement of the perfusion velocity of the caudal artery mediana were also performed in a portion of the animals. RESULTS: The tested method of measurement showed a standard error of 0,08 cm and a coefficient of variation of 0,23% for tail length and 0,78% for tail circumference. The animals had a mean tail length of 22,5±2,32 cm and a mean tail circumference of 6,53±0,49 cm. The mean caudal vertebrae count for this population was 20,4±1,6. The use of a mobile radiographic unit is well suited for imaging the caudal spine in sheep. It was demonstrated that the caudal median artery could be imaged for measurement of perfusion velocity (cm/s), and sonographic gray-scale analysis also showed good feasibility. The mean gray scale value is 19,74±4,5 and the modal value for the most commonly found gray scale pixels is 191,53±120,2. The mean perfusion velocity for the caudal artery mediana is 5,83±3,04 cm/s. CONCLUSION: The results show that the methods presented are well suited for further characterization of the ovine tail. For the first time, gray values for the tail tissue and the perfusion velocity of the caudal artery mediana were determined.

The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice.

BACKGROUND: Skeletal muscle-derived stem cells (SC) have become a promising approach for investigating myogenic differentiation and optimizing tissue regeneration. Muscle regeneration is performed by SC, a self-renewal cell population underlying the basal lamina of muscle fibers. Here, we examined the impact of hypoxia condition on the regenerative capacity of SC either in their native microenvironment or via isolation in a monolayer culture using ectopic differentiation inductions. Furthermore, the effect of low oxygen tension on myogenic differentiation protocols of the myoblasts cell line C2C12 was examined. METHODS: Hind limb muscles of wild type mice were processed for both SC/fiber isolation and myoblast extraction using magnetic beads. SC were induced for myogenic, adipogenic and osteogenic commitments under normoxic (21% O2) and hypoxic (3% O2) conditions. SC proliferation and differentiation were evaluated using histological staining, immunohistochemistry, morphometric analysis and RT-qPCR. The data were statistically analyzed using ANOVA. RESULTS: The data revealed enhanced SC proliferation and motility following differentiation induction after 48 h under hypoxia. Following myogenic induction, the number of undifferentiated cells positive for Pax7 were increased at 72 h under hypoxia. Hypoxia upregulated MyoD and downregulated Myogenin expression at day-7 post-myogenic induction. Hypoxia promoted both SC adipogenesis and osteogenesis under respective induction as shown by using Oil Red O and Alizarin Red S staining. The expression of adipogenic markers; peroxisome proliferator activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4) were upregulated under hypoxia up to day 14 compared to normoxic condition. Enhanced osteogenic differentiation was detected under hypoxic condition via upregulation of osteocalcin and osteopontin expression up to day 14 as well as, increased calcium deposition at day 21. Hypoxia exposure increases the number of adipocytes and the size of fat vacuoles per adipocyte compared to normoxic culture. Combining the differentiation medium with dexamethasone under hypoxia improves the efficiency of the myogenic differentiation protocol of C2C12 by increasing the length of the myotubes. CONCLUSIONS: Hypoxia exposure increases cell resources for clinical applications and promotes SC multipotency and thus beneficial for tissue regeneration.

Distribution pattern of local immune cells within the lower urinary tract of male sheep lambs.

The local immunity of the lower urinary tract (LUT) is often presumed to influence the development of ascending infections and local inflammation. Due to small ruminants being at a higher risk of developing obstructive urolithiasis after early castration, a relationship is expected to exist between disturbed local immunity, castration and disease. However, the underlying pathophysiology and histological correlation of this assumption are unknown. This study examines the local cellular immunity of the LUT in male lambs with respect to castration status or a recent history of obstructive urolithiasis. Various tissue samples were taken and examined. The sample consisted of 34 male lambs, aged six months (n = 11 early and n = 11 late castration; n = 12 intact) and eight rams that had undergone necropsy due to fatal outcome after obstructive urolithiasis. Immunohistochemical stainings for CD3-T-cells, CD79α-B-cells and MAC 387-macrophages were performed and compared among the groups. Whereas no global group differences were evident, significant differences were found for the localizations (P = 0.002) with a significant interaction between group and localization (P = 0.004). The immunohistochemical results suggest that castration did not affect the cell number, but did have an effect on the distribution pattern of local T-cells within the urethra. In the urolithiasis cases, a reduction of CD3-positive cells along the middle part of the urethra was noticeable.

Podosome-Driven Defect Development in Lamellar Bone under the Conditions of Senile Osteoporosis Observed at the Nanometer Scale.

The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 µm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.

Characterisation of stemness and multipotency of ovine muscle-derived stem cells from various muscle sources.

Muscle stem cells (MSCs) are a promising tool for cell-based therapy and tissue regeneration in veterinary medicine. Evaluation of MSCs from muscles of different origins improves our understanding of their regenerative potential. The present study compared the stemness, cell proliferation, migration potential, myogenic differentiation (MD), and multipotency of MSCs for four developmentally different muscles of ovine origin. MSCs were isolated from the hind limb (HL), diaphragm (DI), extraocular (EO), and masseter (MS) muscles. Cell proliferation, migration, and stemness were examined using sulforhodamine B, and colony formation assays. Evaluation of multipotency was examined using histological and morphometric analyses, alkaline phosphatase (ALP) activity, and the expression of myogenic, adipogenic, and osteogenic markers using RT-qPCR. Data were statistically analysed using analysis of variance. The results revealed that all experimental groups expressed stem cell markers paired box transcription factor Pax7, α7-integrin, CD90, and platelet-derived growth factor receptor alpha. DI and HL muscle cells displayed higher proliferation, migration, and colony formation capacities compared to the EO and MS muscle cells. HL and DI muscle cells showed increased MD, as indicated by myotube formation and relative expression of MyoD at day 7 and Myogenin at day 14. Although MS and EO muscle cells displayed impaired MD, these cells were more prone to adipogenic differentiation, as indicated by Oil Red O staining and upregulated fatty acid-binding protein 4 and peroxisome proliferator-activated receptor gamma expression. DI muscle cells demonstrated a higher osteogenic differentiation capability, as shown by the upregulation of osteopontin expression and an elevated ALP activity. Our data indicate that ovine HL and DI MSCs have a higher regenerative and multipotent potential than the EO and MS muscle cells. These results could be valuable for regional muscle biopsies and cell-based therapies.

Combined macromolecule biomaterials together with fluid shear stress promote the osteogenic differentiation capacity of equine adipose-derived mesenchymal stem cells.

BACKGROUND: Combination of mesenchymal stem cells (MSCs) and biomaterials is a rapidly growing approach in regenerative medicine particularly for chronic degenerative disorders including osteoarthritis and osteoporosis. The present study examined the effect of biomaterial scaffolds on equine adipose-derived MSC morphology, viability, adherence, migration, and osteogenic differentiation. METHODS: MSCs were cultivated in conjunction with collagen CultiSpher-S Microcarrier (MC), nanocomposite xerogels B30 and combined B30 with strontium (B30Str) biomaterials in osteogenic differentiation medium either under static or mechanical fluid shear stress (FSS) culture conditions. The data were generated by histological means, live cell imaging, cell viability, adherence and migration assays, semi-quantification of alkaline phosphatase (ALP) activity, and quantification of the osteogenic markers runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) expression. RESULTS: The data revealed that combined mechanical FSS with MC but not B30 enhanced MSC viability and promoted their migration. Combined osteogenic medium with MC, B30, and B30Str increased ALP activity compared to cultivation in basal medium. Osteogenic induction with MC, B30, and B30Str resulted in diffused matrix mineralization. The combined osteogenic induction with biomaterials under mechanical FSS increased Runx2 protein expression either in comparison to those cells cultivated in BM or those cells induced under static culture. Runx2 and ALP expression was upregulated following combined osteogenic differentiation together with B30 and B30Str regardless of static or FSS culture. CONCLUSIONS: Taken together, the data revealed that FSS in conjunction with biomaterials promoted osteogenic differentiation of MSCs. This combination may be considered as a marked improvement for clinical applications to cure bone defects.

Neuropeptides in the urinary tract of male sheep lambs.

Small ruminants are often presumed to be at a higher risk of developing obstructive urolithiasis after early castration. However, the underlying pathophysiology and histological correlation of this assumption is unknown. This study examines the neuropeptide distribution of the lower urinary tract in male lambs in respect to castration status or a recent history of obstructive urolithiasis. Various tissue samples were taken and examined. The sample consisted of 34 male lambs, aged six months (n = 11 early and n = 11 late castration; n = 12 intact), and 8 rams that had undergone necropsy due to fatal outcome after obstructive urolithiasis. Immunohistochemical stainings for substance P (SP), vasoactive intestinal polypeptide (VIP) and neurofilaments (NF) were performed and compared between the groups. A significant reduction in immunoreactive signals of SP, VIP and NF was evident in the urolithiasis group (SP and NF: P < 0.0001; VIP: P = 0.02). The results of immunohistochemistry suggest that castration had no effect on the content of neuropeptides, as well as the innervation density of the urethra in the male lambs. In the case of. obstructive urolithiasis, the pattern of neuropeptide distribution was severely disturbed and cell damage lead to a reduction in detectable periurethral bundles of nerve fibers. The severe tissue damage was assumed to have a negative impact on the outcome of treatment, leading to complications such as urethral strictures. These, in turn, often result in relapses.

Effect of long term palmitate treatment on osteogenic differentiation of human mesenchymal stromal cells - Impact of albumin.

The long-term effects of palmitate (PA) on osteogenic differentiation capacity of human mesenchymal stromal cells (hMSCs) were investigated by cultivating the cells in osteogenic differentiation medium (O-w/o) and osteogenic medium containing PA (O-BSA-PA) for 21 days. Osteogenic medium containing BSA (O-BSA) was used as a control. By means of rt-qPCR, successful osteogenic differentiation was observed in the O-w/o regarding the levels of osteogenic and cell-communication related genes (OCN, Col1, BMP2, ITGA1, ITGB1, Cx43, sp1) in contrast to expression levels observed in cells incubated within basal medium. However, in the O-BSA, these genes were found to have decreased significantly. In cases of Cx43 and sp1, PA significantly reinforced the reductive effect of BSA alone. O-BSA notably decreased glucose and pyruvate consumption, whereas glutamine consumption significantly increased. In comparison to O-BSA addition of PA significantly reduced glycolysis and glutaminolysis. ToF-SIMS analysis confirmed increased incorporation of supplemented deuterated PA into the cell membranes, while the overall PA-concentration remained unchanged compared to cells incubated in the O-BSA and O-w/o. Therefore, the effects on gene expression and the metabolism did not result from the membrane alterations, but may have risen from inter- and intracellular effects brought on by BSA and PA.

In Vitro and In Vivo Biocompatibility Studies of a Cast and Coated Titanium Alloy.

The biocompatibility of a cast porous and with a calcium titanate reaction layer functionalized titanium alloy (Ti-6Al-7Nb) was tested by means of cell culture, and a small (rat) and large animal (sheep) model. The uncoated titanium material served as a control. In-vitro tests included the validation of osteoblast-like cells attached to the surface of the material with scanning electron microscopy and immunofluorescence of cytoskeletal actin as well as their osteogenic development, the ability to mineralize, and their vitality. Following the in-vitro tests a small animal (rat) and big animal (sheep) model were accomplished by inserting a cylindrical titanium implant into a drill hole defect in the femoral condyle. After 7, 14, and 30 days (rat) and 6 months (sheep) the condyles were studied regarding histological and histomorphometrical characteristics. Uncoated and coated material showed a good biocompatibility both in cell culture and animal models. While the defect area in the rat is well consolidated after 30 days, the sheep show only little bone inside the implant after 6 months, possibly due to stress shielding. None of the executed methods indicated a statistically significant difference between coated and uncoated material.

Effects of testosterone and 17ß-estradiol on osteogenic and adipogenic differentiation capacity of human bone-derived mesenchymal stromal cells of postmenopausal women.

Progressive bone loss is a predominant symptom of aging and osteoporosis. Therefore, the effects of sex steroids (i.e. testosterone and 17ß-estradiol) on the differentiation capacity of human bone-derived mesenchymal stromal cells (hMSCs), as progenitors of osteoblasts and adipocytes, are of particular interest. The objectives of the present study were, thus, to elucidate whether bone-derived hMSCs of postmenopausal women produce aromatase (CYP19A1) and, whether they modulate their differentiation behaviour in response to testosterone and 17ß-estradiol (E2), in relation to their steroid receptor expression. Supplementation of testosterone resulted in a considerable formation of E2 under osteogenic and adipogenic culture conditions, whereas E2 synthesis remained minimal in the cells cultured in basal medium. Concomitant with high aromatase expression and 17ß-estradiol formation of the cells cultured in osteogenic medium supplemented with testosterone, a distinct promotion of late-stage osteogenesis was found, as shown by significant matrix mineralization and a notable increase in osteogenic markers. These effects were abrogated by the aromatase inhibitor anastrozole. Under adipogenic conditions, testosterone reduced the occurrence of lipid droplets and led to a decrease in PPARγ and AR expression, independent of anastrozole. Regardless of the culture conditions, ERα was detectable whilst ERß was not. In conclusion, aromatase activity is limited to differentiated hMSCs and the resulting 17ß-estradiol enhances late osteogenic differentiation stages via ERα. Adipogenic differentiation, on the other hand, is reduced by both sex steroids: testosterone via AR and 17ß-estradiol.

Biomaterial based treatment of osteoclastic/osteoblastic cell imbalance - Gelatin-modified calcium/strontium phosphates.

Osteoporotic bone represents - particularly in case of fractures - difficult conditions for its regeneration. In the present study, the focus was put on a degradable bone substitute material of gelatin-modified calcium and strontium phosphates facing the special demands of osteoporotic bone. The release of strontium ions from the material ought to stimulate osteoblastogenesis either direct by ion release or indirect after material resorption by increased presence and activity of osteoclasts, which subsequently stimulate osteoblasts. A new porous material was produced from calcium phosphate, strontium phosphate and a mixed phase of calcium/strontium phosphate precipitated in presence of gelatin. Initially, ion release was analyzed in standard­calcium containing (2.0 mM) and low-calcium (0.4 mM) minimum essential medium. The cultivation of human peripheral blood mononuclear cells next to the material led to formation of osteoclast-like cells, able to migrate, fuse, and differentiate. Especially, the mixed gelatin-modified calcium/strontium phosphate allowed osteoclastogenesis as proven morphologically and by real-time quantitative polymerase chain reaction (RT-qPCR). It was precisely this material that led to the best osteoblastic reaction of human bone marrow stromal cells cultured on the material. The investigations of the bone substitute material indicate active involvement in the balance of cells of the bone morphogenetic unit.

The effect of high-fat diet on the morphological properties of the forelimb musculature in hypertrophic myostatin null mice.

Obesity is a worldwide nutritional disorder affecting body performance, including skeletal muscle. Inhibition of myostatin not only increases the muscle mass but also it reduces body fat accumulation. We examined the effect of high-fat diet on the phenotypic properties of forelimb muscles from myostatin null mice. Male wild-type and myostatin null mice were fed on either a normal diet or a high-fat diet (45% fat) for 10 weeks. Musculus triceps brachii Caput longum; M. triceps brachii Caput laterale; M. triceps brachii Caput mediale; M. extensor carpi ulnaris and M. flexor carpi ulnaris were processed for fiber type composition using immunohistochemistry and morphometric analysis. Although the muscle mass revealed no change under a high-fat diet, there were morphometric alterations in the absence of myostatin. We show that high-fat diet reduces the cross-sectional area of the fast (IIB and IIX) fibers in M. triceps brachii Caput longum and M. triceps brachii Caput laterale of both genotypes. In contrast, increases of fast fiber areas were observed in both M. extensor carpi ulnaris of wild-type and M. flexor carpi ulnaris of myostatin null mice. Meanwhile, a high-fat diet increased the area of the fast IIA fibers in wild-type mice; myostatin null mice display a muscle-dependent alteration in the area of the same fiber type. The combined high-fat diet and myostatin deletion shows no effect on the area of slow type I fibers. Although a high-fat diet causes a reduction in the area of the peripheral IIB fibers in both genotypes, only myostatin null mice show an increase in the area of the central IIB fibers. We provide evidence that a high-fat diet induces a muscle-dependent fast to slow myofiber shift in the absence of myostatin. The data suggest that the morphological alterations of muscle fibers under a combined high-fat diet and myostatin deletion reflect a functional adaptation of the muscle to utilize the high energy intake.

The Addition of High Doses of Hyaluronic Acid to a Biphasic Bone Substitute Decreases the Proinflammatory Tissue Response.

Biphasic bone substitutes (BBS) are currently well-established biomaterials. Through their constant development, even natural components like hyaluronic acid (HY) have been added to improve both their handling and also their regenerative properties. However, little knowledge exists regarding the consequences of the addition of HY to their biocompatibility and the inflammatory tissue reactions. Thus, the present study was conducted, aiming to analyze the influence of two different amounts of high molecular weight HY (HMWHY), combined with a BBS, on in vitro biocompatibility and in vivo tissue reaction. Established in vitro procedures, using L929 cells, were used for cytocompatibility analyses under the test conditions of DIN EN:ISO 10993-5. For the in vivo part of the study, calvarial defects were created in 20 Wistar rats and subsequently filled with BBS, and BBS combined with two different HMWHY amounts, i.e., BBS + HY(L) and BBS + HY(H). As controls, empty defects were used. Established histological, immunohistochemical, and histomorphometrical methods were applied to analyze the tissue reactions to the three different materials, including the induction of pro- and anti-inflammatory macrophages and multinucleated giant cells (BMGCs). The in vitro results showed that none of the materials or compositions caused biological damage to the L929 cells and can be considered to be non-toxic. The in vivo results showed that only the addition of high doses of HY to a biphasic bone substitute significantly decreases the occurrence of pro-inflammatory macrophages (* p < 0.05), comparable to the numbers found in the control group, while no significant differences within the three study groups for M2-macrophages nor BMGCs were detected. In conclusion, the addition of different amounts of HMWHY does not seem to affect the inflammation response to BBS, while improving the material handling properties.

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells.

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5 mM CaCl2 and 7.5 mM CaCl2 protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5 mM CaCl2 and 7.5 mM CaCl2 promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.

Exosomes isolation and identification from equine mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays. RESULTS: It was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 108 (ultracentrifugation), 2.02 × 109 (precipitation) and 52.5 × 109 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens. CONCLUSION: Exosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation.

Incorporation of silicon into strontium modified calcium phosphate bone cements promotes osteoclastogenesis of human peripheral mononuclear blood cells.

Given the important effects of strontium and silicon on cells of the bone as well as the increasing incidence of osteoporotic fractures, calcium phosphate-based bone cements containing silicon and strontium might represent a promising tool for bone replacement therapies of systemically altered bone. However, information about combined effects of strontium and silicon on osteoclastogenesis is still not available. Therefore, differentiation capacity of human peripheral blood mononuclear cells into osteoclast-like cells was investigated by culturing the cells in combination with a strontium- (pS100) and a strontium/silicon-modified calcium phosphate bone cement (pS100-G). Following culturing expression patterns of the cells in respect of their differentiation- and fusion-capacity were determined by real-time quantitative polymerase chain reaction, while cell morphology was visualized by phalloidin staining of the actin cytoskeleton. Additionally, strontium and silicon release from the bone cements into the cultivation media was determined using inductively coupled plasma mass spectrometry while surface topography of the cements was investigated by scanning electron microscopy. The results show that simultaneous incorporation of strontium and silicon into calcium phosphate cements changes properties of the cement such as solubility, and nearly abrogates the inhibitory effects of strontium on osteoclastogenesis.