MicroRNA-200c promotes osteogenic differentiation of human bone mesenchymal stem cells through activating the AKT/ß-Catenin signaling pathway via downregulating Myd88.

During the human bone formation, the event of osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) is vital, and recent evidence has emphasized the important role of microRNAs (miRNAs) in osteogenic differentiation of hBMSCs. This study aims to examine the potential effects of miR-200c in osteogenic differentiation of hBMSCs and understand their underlying mechanisms. HBMSCs were obtained via human bone marrow. During osteogenic induction and differentiation, cells were transfected with different plasmids with the intention of investigating the roles of miR-200c on osteogenic differentiation, calcium salt deposition, alkaline-phosphatase (ALP) activity, mineralized nodule formation, osteocalcin (OCN) content, and proliferation of osteoblasts. Following transfection, dual luciferase reporter gene assay was conducted so as to explore the correlation between miR-200c and Myd88. Moreover, the AKT/ß-Catenin signaling pathway was blocked with an AKT/ß-Catenin inhibitor, AKTi, to investigate its involvement. The hBMSCs were successfully isolated from human bone marrow. Myd88 was determined as a target gene of miR-200c. Gain and loss-of-function assays confirmed that overexpression of miR-200c, or silencing of Myd88 promoted osteogenic differentiation, increased calcium salt deposition, ALP activity, mineralized nodule formation, and enhanced the proliferation of osteoblasts following osteogenic differentiation of hBMSCs. Meanwhile, the downregulation of miR-200c has been shown to have the opposite effect. Furthermore, these findings showed that the miR-200c overexpression activated the AKT/ß-Catenin signaling pathway by targeting Myd88. To sum up, the miR-200c upregulation induces osteogenic differentiation of hBMSCs by activating the AKT/ß-Catenin signaling pathway via the inhibition of Myd88, providing a target for treatment of bone repair.

Identifying the optimum source of mesenchymal stem cells for use in knee surgery.

Single sitting procedures where the mononuclear cell fraction is extracted from bone marrow and implanted directly into cartilage and bone defects are becoming more popular as novel treatments for cartilage defects which have, until now had few treatment options. This is on the basis that the mesenchymal stem cells (MSCs) contained within will repair the damaged tissue. This study sought to determine if the femur and tibia could provide equivalent amounts of mesenchymal stem cells, with equivalent viability and proliferative capacity, to that obtained from the gold standard of the pelvis in order to potentially reduce the morbidity associated with these procedures. Bone marrow was extracted from the pelvis, femur, and tibia of human subjects. The mononuclear cell fraction was extracted and cultured in the laboratory. Mesenchymal stem cell populations were assessed using a colony forming unit count. Viability was assessed using a PrestoBlue viability assay. Population doubling number was calculated between the end of passage 0 and passage three to determine the proliferative abilities of the different populations. Finally, the cell surface phenotype of the cells was determined by flow cytometry. The results showed that the pelvis was superior to the femur and tibia in terms of the number of stem cells isolated. There was no statistically significant difference in the phenotype of the cells isolated from different locations. This work shows that when undertaking single sitting procedures, the pelvis remains the optimum source for obtaining MSCs, despite the morbidity associated with bone marrow collection from the pelvis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1868-1875, 2017.

Characterization of bursa subacromialis-derived mesenchymal stem cells.

INTRODUCTION: The bursa subacromialis (BS) provides the gliding mechanism of the shoulder and regenerates itself after surgical removal. Therefore, we explored the presence of mesenchymal stem cells (MSCs) within the human adult BS tissue and characterized the BS cells compared to MSCs from bone marrow (BMSCs) on a molecular level. METHODS: BS cells were isolated by collagenase digest from BS tissues derived from patients with degenerative rotator cuff tears, and BMSCs were recovered by adherent culture from bone-marrow of patients with osteoarthritis of the hip. BS cells and BMSCs were compared upon their potential to proliferate and differentiate along chondrogenic, osteogenic and adipogenic lineages under specific culture conditions. Expression profiles of markers associated with mesenchymal phenotypes were comparatively evaluated by flow cytometry, immunohistochemistry, and whole genome array analyses. RESULTS: BS cells and BMSCs appeared mainly fibroblastic and revealed almost similar surface antigen expression profiles, which was CD44(+), CD73(+), CD90(+), CD105(+), CD106(+), STRO-1(+), CD14(-), CD31(-), CD34(-), CD45(-), CD144(-). Array analyses revealed 1969 genes upregulated and 1184 genes downregulated in BS cells vs. BMSCs, indicating a high level of transcriptome similarity. After 3 weeks of differentiation culture, BS cells and BMSCs showed a similar strong chondrogenic, adipogenic and osteogenic potential, as shown by histological, immunohistochemical and RT-PCR analyses in contrast to the respective negative controls. CONCLUSIONS: Our in vitro characterizations show that BS cells fulfill all characteristics of mesenchymal stem cells, and therefore merit further attention for the development of improved therapies for various shoulder pathologies.

Caracterización histoquímica y morfométrica de la musculatura esquelética del miembro pelviano del caprino (Capra hircus) / Histochemical and morphometric characterization of the skeletal musculature of the hind limb of the caprine (Capra hircus)

Se estudiaron los músculos de la región de la cadera, del muslo y del pie del miembro pelviano del caprino, en condiciones de explotación semi-intensiva con técnicas histoquímicas, con la finalidad de conocer la distribución porcentual de los distintos tipos de fibras. Además, contribuye a la caracterización del músculo normal, condición indispensable para el estudio posterior de la patología, la genética y eventualmente, para determinar algunas características organolépticas de la carne. Se tomaron muestras in vivo de los músculos bíceps femoral, glúteo medio, glúteo profundo, vasto intermedio, vasto lateral, gastrocnemios, soleo, semimembranoso, semitendinoso y tensor de la fascia lata, en cinco (5) cabras de 21-30 Kg. de peso. Las muestras se congelaron en isopentano enfriado en nitrógeno líquido. Los cortes se realizaron en un críostato a -20°C, con un espesor de 10 µm. Se realizaron las reacciones de laAdenosina Trifosfatasa (ATP-asa) con un pH de preincubación 4,33 y 4,37 para determinar la distribución porcentual de tipos de fibras. El músculo vasto intermedio presentó 100 por ciento de fibras tipo I. Los tres músculos glúteos mostraron alrededor de 45 por ciento fibras tipo I, 22 por ciento de tipo IIa y 33 por ciento tipo IIb. El resto de los músculos tuvo 29 por ciento de las fibras tipo I, 31por ciento del tipo IIa y 40 por ciento del tipo IIb. El promedio de área de fibra más alto correspondió al músculo vasto intermedio con 4.070 µm² y el promedio de área por tipo de fibra reveló que la fibra más grande fue la fibra tipo I con 2.682 µm² y la más pequeña fue la tipo IIb con 2.046 µm². En conclusión, se caracterizó la composición miofibrilar de diez (10) músculos del miembro pelviano de caprino de la raza Alpino Francés.

The muscles of the hip, high and leg region of the goats were studied under conditions of explotation semi intensive with technical histochemical, with the purpose of knowing the percentage distribution of the different types of fibers, that which contributes to the characterization of the normal muscle, indispensable condition for the later study of the pathology, the genetic one and possibly to determine some organoleptics characteristic of the meat. They took samples in alive of the muscles Biceps femoris, Middle gluteal, Deep gluteal, Vastus intermedius, Vastus lateralis, Gastrocnemius, Soleus, Semimembronosus, Semitendinosus and Tensor fasciae latae, in five (5) goats of 21-30 Kg. of weight. The samples froze in isopentano cooled in liquid nitrogen. The cuts were carried out in a cryostat to -20°C, with a thickness of 10 µm. They were carried out the reactions of the Adenosine Trifosfatase (ATP-handle) with a pH of preincubation 4.33 and 4.37 to determine the percentage distribution of types of fibers. The muscle Vastus intermedius presents 100% of fibers type I, almost all of high capacity oxidative. The three muscles Gluteal showed around 45% fibers type I, 22% typeIIa and 33% type IIb. The rest of the muscles had 29% of the fibers type I, 31% of the type IIa and 40% of the type IIb. The highest average of fiber area was that of the m. Vastus intermedius with 4,070 µm² and the area average for fiber type revealed that the biggest fiber was the fiber type I with 2,682 µm² and the smallest was the type IIb with 2,046 µm². In conclusion, the composition myofibril of ten was characterized (10) muscles of the later member of goats of the French Alpine beed.

Spaceflight inhibits bone formation independent of corticosteroid status in growing rats.

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (-12%; p < 0.05) associated with approximately 50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (-42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.

Enhanced osteoblast development after continuous infusion of hPTH(1-84) in the rat.

Rats and humans respond to intermittent treatment with parathyroid hormone (PTH) with increased bone density and cancellous bone volume. In the rat, osteoblast expression of insulin-like growth factor-I (IGF-I) is elevated by intermittent PTH. We examined the effect of continuous infusion of rhPTH(1-84), a bone catabolic regime, on the IGF system in rat pelvis. Female Sprague-Dawley rats (12 weeks, 250 g) were randomly assigned to receive 0, 0.1, 1, or 5 microg/100 g body weight (b.w.) rhPTH(1-84) (0, 0.106, 1.06, or 5.305 nmol/kg) in vehicle (1% normal rat serum in saline) delivered by subcutaneous Alzet minipump. After 7 days, blood was taken for serum chemistry and pelvises were processed for immunocytochemistry. Sections of pelvis from rats continuously infused with 0.1 or 1 microg/100 g b.w. rhPTH(1-84) for 7 days did not differ significantly from those of the vehicle-treated controls. However, continuous infusion of 5 microg/100 g b.w. rhPTH(1-84) resulted in a dramatic increase in cellular development, with trabeculae surrounded by many layers of large, plump osteoblasts. All pelvis osteoblasts expressed osteocalcin, but only those from rats that received 0, 0.1, or 1 microg/100 g b.w. rhPTH(1-84) showed positive staining for IGF-I. The extra-abundant osteoblasts from rats that received 5 microg/100 g b.w. rhPTH(1-84) did not stain for IGF-I. However, although all osteoblasts stained positively for IGF binding proteins (IGFBPs)-3, -4, and -5, staining for these IGFBPs increased as the dose of rhPTH(1-84) (and osteoblast number) increased. These results suggest that continuous infusion of PTH has a direct effect on osteoblast development (either recruitment or proliferation), decreases the expression of IGF-I, and enhances the expression of IGFBPs in pelvis, factors which may interact to bring about negative bone balance.