Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

Background： The use of equine bone marrow mesenchymal stem cells （BMSC） is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine： 1） if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2） the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum （FBS） or autologous horse serum （HS）, and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 （BMP2）, dexamethasone （DEX）, or combination of the two. Alkaline phosphatase （ALP） activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-reloted tronscrJption foctor2 （Runx2）, osterix （Osx）, and T-box3 （Tbx3）. Data were analyzed by ANOVA. Results： Relative to control, FBS and HS increased cell number （133 ± 5 and 116 ± 5%, respectively; P 〈 0.001） and 5-bromo- 2＇-deoxyuridine （BrdU） incorporation （167 ± 6 and 120 ± 6%, respectively; P 〈 0.001）. Treatment with DEX increased ALP activity compared with control （1,638 ± 38%; P 〈 0.001）. In the absence and presence of Dex, BMP-2 did not alter ALP activity （P 〉 0.8）. Runt-reloted transcription foctor2 expression increased 3-fold （P 〈 0.001） by d 6 of culture. Osterix expression increased 94old （P 〈 0.05） by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 （P 〈 0.01）; however expression was reduced 4-fold at d 18 （P 〈 0.01）. Conclusions： Dexamethasone, but not BMP-2, is required for differentiation of equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation

RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells(BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting β-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs.Interestingly, conditional knockout of rank in BMSCs with Prx1-Cre mice leads to a higher bone mass and increased trabecular bone formation independent of osteoclasts. In addition, rank: Prx1-Cre mice show resistance to ovariectomy-(OVX) induced bone loss. Thus, our results reveal that RANKL signaling regulates both osteoclasts and osteoblasts by inhibition of osteogenic differentiation of BMSCs and promotion of osteoclastogenesis.

Osteogenic Differentiation of Bone Mesenchymal Stem Cells Regulated by Osteoblasts under EMF Exposure in a Co-culture System

This study examined the osteogenic effect of electromagnetic fields （EMF） under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells （BMSCs） and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit （CCK-8）. For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase （ALP） staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure （2 h/day） could obviously promote prolifera- tion of BMSCs and osteoblasts, while long-term EMF （8 h/day） could promote osteogenic differen- tiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when ceils were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differ- entiation of BMSCs.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

Comparison of biological characteristics of marrow mesenchymal stem cells in hepatitis B patients and normal adults

AIM: To establish a culture system of marrow mesenchymal stem cells (MSCs)from hepatitis B patients and normal adults and to compare their biological characteristics. METHODS: MSCs were isolated from bone marrow in 34 male hepatitis B patients and 15 male normal adults and cultivated in vitro. Their biological characteristics including surface markers, shapes and appearances, growth curves, first passage time and passage generations were compared. RESULTS: Cultivation achievement ratio of hepatitis B patients was lower than that of normal adults, no statistical significance (82.35% vs 100%, P > 0.05). Compared with MSCs of normal adults, MSCs of hepatitis B patients presented a statistical lower growth curve, longer first passage time (13.0 ± 1.6 d vs 11.4 ± 1.5 d, P < 0.05), fewer passaging generation numbers (10.5 ± 1.4 generations vs 12.3 ± 1.7 generations, P < 0.05), though both shared same appearances, shapes and surface markers. MSCs in hepatitis B patients would expand, spread out and age more easily and there were more refractive particles in the cytoplasm. CONCLUSION: MSCs from hepatitis B patients can be cultured in vitro. Although their appearance, shape and surface marker are similar to those of MSCs from normal adults, there are differences in their biological characteristics.

Cell multiplication, apoptosis and p-Akt protein expression of bone mesenchymal stem cells of rat under hypoxia environment

Objective ：To elucidate whether cell multiplication, apoptosis, glucose intake and p-Akt protein expression of bone Mesenchyreal Stem Cells（MSCs） of rats is influenced by a hypoxic environment ex vivo. Methods ：Passage 3 of bone marrow MSCs taken from Wistar rats,were cultured in a culturing chamber with 94%N2,1%O2,5%CO2 at 37℃. At different hypoxia time points ,0,0.5, 1,4 and 8 h, glucose uptake was assayed by using radiation isotope ^3H-G, Apoptotic Rate（AR） and dead rate（DR） were analyzed by flow cytometry（FCM） after Annexin V/PI staining, cell multiplication（by MTr methods） and p-Akt protein by immunocytochemistry and western blot. Results ：Assay for CD29^± ,CD44^± ,CD71^± ,CD34^-, Tn T^±（after 5-azacytidine agent inducing） and ALP^±（after bone differentiation agent inducing） suggested these bone-derived cells were MSCs. The ^3H-G intaking ratio （CPM/ flask value：157 ± 11,110 ± 11,107 ± 13,103 ± 10,100 ± 9 and 98 ± 10） of MSCs at different hypoxia time points, significantly decreased compared to that of normoxia（P 〈 0.01） and tended to descend slowly with hypoxia time duration, for which there was no statistical significance（P 〉 0.05）. The AR（0.09 ± 2.03%,12.9 ± 1.72%,13.7 ± 2.26%,13.8 ± 3.01%,14.1 ± 2.78% and 14.7 ± 4.01% at 0,0.5,1,4 and 8 h,respectively,P 〈 0.01） and DR （0.04, ± 1.79% ,0.93 ± 1.85% ,3.11 ± 2.14% ,4.09 ± 2.36% ,4.72 ± 2.05% and 4.91 ± 3.72% at 0,0.5,1,4 and 8 h, respectively, P 〈 0.05） at different hypoxia time points significantly increased compared to those time in normoxia; The AR further went up with time （P 〈 0.05）, however there was no statistical significance （P 〉 0.05） for the DR. Optical absorption value of MTr methods at different hypoxia time points significantly decreased compared to those with a corresponding normoxia time （P 〈 0.01） and degraded with time （in an hypoxic environment -P 〈 0.01）. IOD of p-Akt protein of MSCs at different hypoxia time points significantly increased （0.367 ± 0.031,0.556 ± 0.023,0.579 ± 0.013, 0.660 ± 0.024, 0.685 ± 0.039 and 0.685 ± 0.011, respectively） compared to their equivalents in normoxia （P〈0.05）, however, there was no statistical significance （P 〉 0.05） for different hypoxia time points. Hypoxia may result in ultramicrostructure changes, such as defluvium of Microvilli, apoptotic body, ＂margination＂ and so on and are further aggravated with hypoxia time stretching. Conclusion： Hypoxia may lead to a depression of MSCs intaldng glucose, creep of cell multiplication, upregulation of p-Akt protein and apoptosis of MSCs ex vivo.

Biological Characteristics of Human Bone Marrow Mesenchymal Stem Cell Cultured in Vitro

Some biological characteristics of human bone marrow mesenchymal stem cells (MSCs) cultured in vitro were observed. hMSCs were isolated from bone marrow and purified by density gradient centrifugation method, and then cultured in vitro. The proliferation and growth characteristics of hMSCs were observed in primary and passage culture. MSCs of passage 3 were examined for the purify by positive rate of CD29 and CD44 through flow cytometry. Human bone marrow MSCs showed active proliferation capacity in vitro. The purify of MSCs separated by our method was higher than 90 %. It was concluded that hMSCs have been successfully cultured and expanded effectively. It provided a foundation for further investigation and application of MSCs.

Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model

Sodium nitrite（Na NO2） is an inorganic salt used broadly in chemical industry. Na NO2 is highly reactive with hemoglobin causing hypoxia. Mesenchymal stem cells（MSCs） are capable of differentiating into a variety of tissue specific cells and MSC therapy is a potential method for improving brain functions. This work aims to investigate the possible therapeutic role of bone marrow-derived MSCs against Na NO2 induced hypoxic brain injury. Rats were divided into control group（treated for 3 or 6 weeks）, hypoxic（HP） group（subcutaneous injection of 35 mg/kg Na NO2 for 3 weeks to induce hypoxic brain injury）, HP recovery groups N-2 w R and N-3 w R（treated with the same dose of Na NO2 for 2 and 3 weeks respectively, followed by 4-week or 3-week self-recovery respectively）, and MSCs treated groups N-2 w SC and N-3 w SC（treated with the same dose of Na NO2 for 2 and 3 weeks respectively, followed by one injection of 2 × 106 MSCs via the tail vein in combination with 4 week self-recovery or intravenous injection of Na NO2 for 1 week in combination with 3 week self-recovery）. The levels of neurotransmitters（norepinephrine, dopamine, serotonin）, energy substances（adenosine monophosphate, adenosine diphosphate, adenosine triphosphate）, and oxidative stress markers（malondialdehyde, nitric oxide, 8-hydroxy-2′-deoxyguanosine, glutathione reduced form, and oxidized glutathione） in the frontal cortex and midbrain were measured using high performance liquid chromatography. At the same time, hematoxylin-eosin staining was performed to observe the pathological change of the injured brain tissue. Compared with HP group, pathological change of brain tissue was milder, the levels of malondialdehyde, nitric oxide, oxidized glutathione, 8-hydroxy-2′-deoxyguanosine, norepinephrine, serotonin, glutathione reduced form, and adenosine triphosphate in the frontal cortex and midbrain were significantly decreased, and glutathione reduced form/oxidized glutathione and adenosine monophosphate/adenosine triphosphate ratio were significantly increased in the MSCs treated groups. These findings suggest that bone marrow-derived MSCs exhibit neuroprotective effects against Na NO2-induced hypoxic brain injury through exerting anti-oxidative effects and providing energy to the brain.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Characteristics of Mesenchymal Stem Cells under Hypoxia

Mesenchymal stem cells (MSC) are considered non-hematopoietic multipotent stem cells with self-renewal properties and the ability to differentiate into a variety of mesenchymal tissues. Optimal conditions for the culture of these cells have been the subject of investigation for several years. In particular, ideal oxygen tension levels have not been established in the literature. In physiological environments, oxygen tension may vary from 12% in peripheral blood to 1% in the deep zone of cartilage regions. In any case, oxygen tension is considerably lower in vivo when compared with the normal atmosphere of standard cell culture conditions (21%). The objective of this study was to review the literature available on MSC characteristics (cell cycle, survival, proliferation, differentiation, morphology, immunophenotype, cytogenetics) when cultured under hypoxic conditions. Our focus on optimal culture conditions is justified by the key role currently played by these cells in regenerative medicine.

Effects of neurotrophin-3 intervention on on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis

Objective:To study the effects of neurotrophin-3 (NT-3) intervention on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis. Methods: Bone marrow mesenchymal stem cells were cultured and divided into control group, 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group, they were treated with different doses of NT-3 for 24 h, and then osteoblast marker gene, cell proliferation gene and apoptosis gene expression were determined.Results: RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression in 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group were significantly higher than those in control group whereas Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression were significantly lower than those in control group, and the larger the dose of NT-3, the higher the RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression whereas the lower the Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression.Conclusion: NT-3 intervention in bone marrow mesenchymal stem cells can promote osteoblast differentiation and cell proliferation and inhibit apoptosis.

Conditions to improve expansion of human mesenchymal stem cells based on rat samples

AIM: To improve the isolation and expansion of human marrow-derived mesenchymal stem cells (MSCs) based on rat samples. METHODS: Based on the fact that rat MSCs are relatively easy to obtain from a small aspirate, bone marrow-derived MSCs from rat were cultured and characterized to set up the different protocols used in this study. Then, accordingly, almost the same protocols were performed on human healthy bone marrow samples, after obtaining approval of the ethics committee and gaining informed consent. We used different protocols and culture conditions, including the type of basal media and the culture composition. The MSCs were characterized by immunophenotyping and differentiation. RESULTS: There was no difference in morphology and proliferation capacity between different culture media at the first passage. During the 5-7th passages, the cells gradually lost their morphology and proliferation potential on Dulbecco’s modified Eagle’s medium (DMEM) high glucose and α modified Eagle’s medium. Although the cells expanded rapidly for up to 10 passages on DMEM low glucose containing 10% to 15% fetal calf serum (FCS), their proliferation was arrested without change in morphology and differentiation capacity at the third passage on 5% FCS. Flow cytometric analysis and functional tests confirmed that more than 90% of marrow cells which were isolated and expanded by our selective protocols were MSCs. CONCLUSION: We improved the isolation and expansion of human bone marrow derived MSCs, based on rat sample experiments, for further experimental and clinical use.

Effects of normobaric cyclic hypoxia exposure on mesenchymal stem-cell differentiation–pilot study on bone parameters in elderly

BACKGROUND Mesenchymal stem cells(MSC)of bone marrow are the progenitor of osteoblasts and adipocytes.MSC tend to differentiate into adipocytes,instead of osteoblasts,with aging.This favors the loss of bone mass and development of osteoporosis.Hypoxia induces hypoxia inducible factor 1αgene encoding transcription factor,which regulates the expression of genes related to energy metabolism and angiogenesis.That allows a better adaptation to low O2 conditions.Sustained hypoxia has negative effects on bone metabolism,favoring bone resorption.Yet,surprisingly,cyclic hypoxia(CH),short times of hypoxia followed by long times in normoxia,can modulate MSC differentiation and improve bone health in aging.AIM To evaluate the CH effect on MSC differentiation,and whether it improves bone mineral density in elderly.METHODS MSC cultures were induced to differentiate into osteoblasts or adipocytes,in CH(3%O2 for 1,2 or 4 h,4 d a week).Extracellular-matrix mineralization and lipid-droplet formation were studied in MSC induced to differentiate into osteoblast or adipocytes,respectively.In addition,gene expression of marker genes,for osteogenesis or adipogenesis,have been quantified by quantitative real time polymerase chain reaction.The in vivo studies with elderly(>75 years old;n=10)were carried out in a hypoxia chamber,simulating an altitude of 2500 m above sea level,or in normoxia,for 18 wk(36 CH sessions of 16 min each).Percentages of fat mass and bone mineral density from whole body,trunk and right proximal femur(femoral,femoral neck and trochanter)were assessed,using dual-energy X-ray absorptiometry.RESULTS CH(4 h of hypoxic exposure)inhibited extracellular matrix mineralization and lipid-droplet formation in MSC induced to differentiate into osteoblasts or adipocytes,respectively.However,both parameters were not significantly affected by the other shorter hypoxia times assessed.The longest periods of hypoxia downregulated the expression of genes related to extracellular matrix formation,in MSC induced to differentiate into osteoblasts.Interestingly,osteocalcin(associated to energy metabolism)was upregulated.Vascular endothelial growth factor an expression and low-density lipoprotein receptor related protein 5/6/dickkopf Wnt signaling pathway inhibitor 1(associated to Wnt/β-catenin pathway activation)increased in osteoblasts.Yet,they decreased in adipocytes after CH treatments,mainly with the longest hypoxia times.However,the same CH treatments increased the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio in both cell types.An increase in total bone mineral density was observed in elderly people exposed to CH,but not in specific regions.The percentage of fat did not vary between groups.CONCLUSION CH may have positive effects on bone health in the elderly,due to its possible inhibitory effect on bone resorption,by increasing the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio.

The effect of bone marrow mesenchymal stem cell transplantation on hypoxic pulmonary hypertension in rats

Objective:To study the influence of bone marrow mesenchymal stem cells(MSCs)transplantation on hypoxic pulmonary hypertension(HPH)in rats.Methods:MSCs in SD rats were separated,cultivated,identified in vitro,and labeled by the green fluorescence protein(GFP)adenovirus.Healthy male SD rats were randomly divided into four groups:normal control group(NC group)and HPH group,with eight rats in each group respectively;HPH+mesenchymal stem cell transplantation group(MSCs group)and HPH+vascular endothelial growth factor+mesenchymal stem cell transplantation group(VEGF+MSCs group),with twenty-four rats in each group respectively.In this experiment,intermittent normobaric hypoxia was employed to establish the pulmonary hypertension rat models,with stem cells transfected and transplanted.The mean pulmonary artery pressure(mPAP)was observed in rats to calculate the right ventricular hypertrophy index(RVHI);the morphological changes of pulmonary arterioles in each group of rats were observed under the microscope;the distribution and manifestation of MSCs fluorescently labeled by adenovirus transfection were observed in pulmonary arterioles under the fluorescence microscope at the set time points of 7 d,14 d and 28 d after the transplantation of stem cells.Results:For NC group,the mPAP(mmHg)was 15.5±1.5 at 28 d,while the mPAP in HPH,MSCs and VEGF+MSCs groups were 26.1±1.9,21.6±2.7 and 20.1±2.9 respectively which were apparently higher than that in NC group(p<.01).Compared with HPH group(p<.01),the mPAP was obviously decreased in MSCs and VEGF+MSCs groups.There was no significant difference between MSCs and VEGF+MSCs groups.At 28 d,RVHI for NC group was 0.28±0.02,while the RVHI in HPH,MSCs and VEGF+MSCs groups were 0.43±0.07,0.34±0.03 and 0.35±0.01 respectively which were apparently higher than that in NC group(p<.01).In comparison with HPH group,RVHI was significantly decreased in MSCs and VEGF+MSCs groups(p<.05).There was no significant difference between MSCs and VEGF+MSCs groups.For HPH group,at 28 d,pulmonary arterioles were apparently thickened,with luminal stenosis&obliteration and incomplete endothelial cells.Compared with HPH group,pulmonary arterioles in MSCs group became thinning,with the lumen unobstructed and the integrity of endothelial cells improved.The changes in the manifestation of MSCs and VEGF+MSCs groups were not significant.Conclusions:The transplantation of MSCs can improve the remodeling of pulmonary arterioles to partially reverse the progress of HPH;the combined transplantation of VEGF and MSCs doesn’t improve the effect of MSC transplantation.

Dynamic culture of a thermosensitive collagen hydrogel as an extracellular matrix improves the construction of tissue-engineered peripheral nerve

Tissue engineering technologies offer new treatment strategies for the repair of peripheral nerve injury, hut cell loss between seeding and adhesion to the scaffold remains inevitable. A thermosensitive collagen hydrogel was used as an extracellular matrix in this study and combined with bone marrow mesenchymal stem cells to construct tissue-engineered peripheral nerve composites in vitro. Dynamic culture was performed at an oscillating frequency of 0.5 Hz and 35° swing angle above and below the horizontal plane. The results demonstrated that bone marrow mesenchymal stem cells formed membrane-like structures around the poly-L-lactic acid scaffolds and exhibited regular alignment on the composite surface. Collagen was used to fill in the pores, and seeded cells adhered onto the poly-L-lactic acid fibers. The DNA content of the bone marrow mesenchymal stem cells was higher in the composites constructed with a thermosensitive collagen hydrogel compared with that in collagen I scaffold controls. The cellular DNA content was also higher in the thermosensitive collagen hydrogel composites constructed with the thermosensitive collagen hydrogel in dynamic culture than that in static culture. These results indicate that tissue-engineered composites formed with thermosensitive collagen hydrogel in dynamic culture can maintain larger numbers of seeded cells by avoiding cell loss during the initial adhe-sion stage. Moreover, seeded cells were distributed throughout the material.

In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system

Background Cartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells （BMSCs） is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide （PLGA） scaffolds.Methods The BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat＇s last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A,whereas the goat＇s BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining,Alcian blue staining, collagen Ⅱ immunofluoresence, collagen Ⅱ immunochemical staining, collagen Ⅰ, collagen Ⅱ, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.Results Cells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen Ⅱ immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.Conclusions The Transwell co-culture system and the PLGA scaffolds co-culture system can promote the chondrogenic differentiation of the goat＇s BMSCs, while up-regulated osteopontin gene expression in the Transwell co-culture system implies the osteogenic potential of BMSCs.

Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.