Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation

Physical loading is a potent stimulus required to maintain bone homeostasis, partly through the renewal and osteogenic differentiation of mesenchymal stem cells (MSCs). However, the mechanism by which MSCs sense a biophysical force and translate that into a biochemical bone forming response (mechanotransduction) remains poorly understood. The primary cilium is a single sensory cellular extension, which has recently been shown to demonstrate a role in cellular mechanotransduction and MSC lineage commitment. In this study, we present evidence that short periods of mechanical stimulation in the form of oscillatory fluid flow (OFF) is sufficient to enhance osteogenic gene expression and proliferation of human MSCs (hMSCs). Furthermore, we demonstrate that the cilium mediates fluid flow mechanotransduction in hMSCs by maintaining OFF-induced increases in osteogenic gene expression and, surprisingly, to limit OFF-induced increases in proliferation. These data therefore demonstrate a pro-osteogenic mechanosensory role for the primary cilium, establishing a novel mechanotransduction mechanism in hMSCs. Based on these findings, the application of OFF may be a beneficial component of bioreactor-based strategies to form bone-like tissues suitable for regenerative medicine and also highlights the cilium as a potential therapeutic target for efforts to mimic loading with the aim of preventing bone loss during diseases such as osteoporosis. Furthermore, this study demonstrates a role for the cilium in controlling mechanically mediated increases in the proliferation of hMSCs, which parallels proposed models of polycystic kidney disease. Unraveling the mechanisms leading to rapid proliferation of mechanically stimulated MSCs with defective cilia could provide significant insights regarding ciliopathies and cystic diseases. Stem Cells2012;30:2561–2570

SummaryThe gap in knowledge of the molecular mechanisms underlying differentiation of human pluripotent stem cells (hPSCs) into the mesenchymal cell lineages hinders the application of hPSCs for cell-based therapy. In this study, we identified a critical role of muscle segment homeobox 2 (MSX2) in initiating and accelerating the molecular program that leads to mesenchymal stem/stromal cell (MSC) differentiation from hPSCs. Genetic deletion of MSX2 impairs hPSC differentiation into MSCs. When aided with a cocktail of soluble molecules, MSX2 ectopic expression induces hPSCs to form nearly homogeneous and fully functional MSCs. Mechanistically, MSX2 induces hPSCs to form neural crest cells, an intermediate cell stage preceding MSCs, and further differentiation by regulating TWIST1 and PRAME. Furthermore, we found that MSX2 is also required for hPSC differentiation into MSCs through mesendoderm and trophoblast. Our findings provide novel mechanistic insights into lineage specification of hPSCs to MSCs and effective strategies for applications of stem cells for regenerative medicine.

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells.

We synthesized biomimetic hydrogels modified with an osteopontin-derived peptide (ODP) and used them as a substrate for in vitro culture of marrow stromal cells (MSCs) to investigate the effect of the biomimetic surface on differentiation of MSCs into osteoblasts. Proliferation and biological assays for 16 days proved that MSCs became differentiated into osteoblasts secreting osteogenic phenotypic markers such as alkaline phosphatase (ALP), osteopontin, and mineralized calcium. In addition, there was an additive effect of the cell-binding peptide on differentiation and mineralization of MSCs cultured in the presence of soluble osteogenic supplements in cell culture media. For example, calcium content at day 16 on peptide-modified hydrogels was significantly higher than on tissue culture polystyrene. Two general trends were observed: (1) proliferation of MSCs decreased as the amount of differentiation markers increased, and (2) higher peptide concentrations accelerated the differentiation of MSCs. On the hydrogel modified with ODP, ALP activity exhibited a maximum value of 36.7 +/- 4.2 pmol/cell/h at day 10 for the concentration of 2 micromol/g while the culture time needed for maximum ALP activity occurred on day 13 for the lower concentrations. On the same hydrogel, the calcium content at day 10 was 21.4 +/- 2.3 ng/cell for the peptide concentration of 2 micromol/g and 1.0 +/- 0.3 ng/cell for 1.0 micromol/g. We used Gly-Arg-Gly-Asp-Ser (GRGDS) for modification of the hydrogel as a comparison to the results with ODP. However, osteoblast development was not significantly affected by the nature of the binding peptide sequences. These results suggest that MSC function can be modulated by variation of the peptide concentration in biomimetic hydrogels used for scaffold-based bone tissue engineering.

Regulation of insulin-like growth factor binding protein-5, four and a half lim-2, and a disintegrin and metalloprotease-9 expression in osteoblasts

Effect of La3+ on osteoblastic differentiation of rat bone marrow stromal cells

Primary Cilia is appendage that extrudes from cell surface into the extracellular matrix .These organelles play a sensor role for mechanical stimulation in the cell and due to stretch ion channels in its base, play critical role in induce osteogenic differentiation of stem cells. Primary cilia deflected under fluid flow passing through the surface of the cell, which deflection causes tensile ion channels to be opened. In this study, cilia is assumed as linear elastic. The innovative aspect of this research is exerting of oscillatory fluid flow to the primary cilia and evaluating the response of cilia to the fluid flow. The results show that under conditions of exerting the oscillatory fluid flow, maximum strain occur in the base of the cilia which is experienced by tensile ion channels, is 0.5 and in the condition of steady flow is 0.3, Accordingly, mechanical stimuli are sensed by the tensile ionic channels during oscillatory flow higher than steady flow. osteogenic differentiation of stem cells, in addition, the result showed that by using the oscillatory fluid flow the mechanical stimulation better senses by cilia and It is anticipated that exerting oscillatory fluid flow facilitate osteogenic differentiation of stem cell.

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative stress may play a major role in the development of osteoporosis in part by inhibiting osteoblastic differentiation of bone marrow stromal cells (MSCs). Some evidence suggested that antioxidant selenium could prevent osteoporosis, but the underlying mechanism remains unclear. In this work, the effect of sodium selenite on H₂O₂-induced inhibition of osteoblastic differentiation of primary rat bone MSCs and the related mechanisms were examined. Pretreatment with selenite inhibited the adverse effect of H₂O₂ on osteoblastic differentiation of MSCs, based on alkaline phosphatase activity, gene expression of type I collagen and osteocalcin, and matrix mineralization. In addition, selenite pretreatment also suppressed the activation of extracellular signal-regulated kinase (ERK) induced by H₂O₂. The above effects were mediated by the antioxidant effect of selenite. Selenite enhanced the gene expression and activity of glutathione peroxidase, reversed the decreased total antioxidant capacity and reduced glutathione, and suppressed reactive oxygen species production and lipid peroxidation level in H₂O₂-treated MSCs. These results showed that selenite protected MSCs against H₂O₂-induced inhibition of osteoblastic differentiation through inhibiting oxidative stress and ERK activation, which provided, for the first time, the mechanistic explanation for the negative association of selenium status and risk of osteoporosis in terms of bone formation.

Ectopic bone formation in rat marrow stromal cell/titanium fiber mesh scaffold constructs: Effect of initial cell phenotype

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

In order to raise an abundant and accessible reservoir for Schwann cells (SCs), which are used as seed cells for constructing tissue-engineered nerve grafts, we investigated the feasibilty of in vivo differentiation of bone marrow stromal cells (MSCs) into SC-like cells. In this study, MSCs were harvested from adult rats' bone marrow, culture-expanded, and characterized. Subcultured MSCs were then labeled with Hoechst 33342, followed by transplantation into the nerve regeneration chamber, which was made of a silicone tube bridging the sciatic nerve defect of the rats. Four weeks after surgery, some of the differentiated MSCs turned into SC-like cells immunopositive to S-100 protein, accompanied by myelination of the regenerated nerve fibers. Walking-track analyses provided evidence that transplantation of MSCs contributed to reconstruction of the sciatic nerve and reinnervation of target tissues. The experimental results suggest that MSCs are capable of differentiating into SC-like cells in vivo, making them a promising candidate for cell transplantation in peripheral nerve repair.

Objective To study the effects of serum of the Zhuartgguqiang jin tablets on the proliferation and osteogenic differentiation of bone marrow stromal stem cells (BMSCs) of aged rats.Methods The BMSCs were obtained from male SD rats of 18 months.The cell surface markers CD34,CD31,CD29 were detected by flow cytometry.The proliferation of BMSCs treated by different concentration of medicated serum (2.5％,5％,10％) at 24,48,72hours was detected by CCK-8 method.The BMSCs were divided into 4 groups,which were osteogenic induction group,the Chinese medicated osteogenic induction group,Chinese medicine group,the blank group.After the BMSCs were cultured for 7 days,alkaline phosphatase (ALP) vitality was detected.After the BMSCs were cultured for 14 days,the BMSCs were stained by alizarin red,the mRNA of ALP and osteocalcin(OC) was detected.Results The expressionof CD29 was positive,and the expression of CD31 and CD34 were negative.2.5％,5％ of the medicated serum could promote cell proliferation.The ALP vitality of osteogenic induction group[202.76 ± 15.44(U/gprot)],the Chinese medicated osteogenic induction group [240.48 ± 18.55 (U/gprot)],Chinese medicine group [178.87 ± 17.29 (U/gprot)]were significantly higher than that of blank group [111.24 ± 20.71 (U/gprot)] (t =22.50,7.985,3.535,all P ＜0.01).The ALP activity of Chinese medicated osteogenic induction group was higher than that of osteogenic induction group (t =3.103,P ＜ 0.05).The ALP gene relative OD value of osteogenic induction group (0.40 ± 0.20) and Chinese medicated osteogenic induction group (0.60 ± 0.06) were higher than the blank group (0.09 ± 0.03) (t =2.372,9.547,all P ＜0.01).The ALP gene expression of Chinese medicated osteogenic induction group was obviously higher than that of the osteogenic induction group(P ＜0.05).The OC gene relative OD value of osteogenic induction group(0.58 ± 0.09) and Chinese medicated osteogenic induction group(0.76 ± 0.11) were higher than the blank group(0.41 ± 0.02) (P ＜ 0.05,P ＜ 0.01).The OC gene expression of Chinese medicated osteogenic inductiongroup was obviously higher than that of the osteogenic induction group(t =2.673,P ＜ 0.05).Conclusion Medicated serum of Zhuangguqiangjin tablets can promote the proliferation and osteogenic differentiation of BMSCs of aged rats,which may be the mechanism of prevention senile osteoporosis. Key words: Chinese medicine; Bone marrow stromal stem cells; Osteoblasts; Cell differentiation

Although human bone marrow stromal cells (MSCs) have been used for clinical bone reconstruction, how the physiological status of patients and culture conditions of MSCs affect the result of bone reconstruction must be clarified to use MSCs in a clinical stage. If in vitro parameters of the status of MSCs may be correlate with in vivo bone formation capability, the better cells for clinical bone reconstruction can be defined by the parameters. In order to explore the parameters and define the optimum cells for clinical use, the proliferation and differentiation capabilities in vitro and the in vivo bone formation capability of MSCs were analyzed. An age-related proliferation capability was found. The in vitro alkaline phosphatase activity of bone formation finding groups was higher than that of the no bone formation group. This may be provide a parameter to obtain the optimum cells for clinical use to benefit improving the cure efficiency. In this study, it is preferable that MSCs of passage 1 have stronger osteogenic potential than those of passage 2 and 3 in vitro, and might be suitable for clinical application to bone tissue engineering.

BackgroundOscillatory fluid flow (OFF)-induced shear stress leads to positive bone remodeling through pro-formative and anti-resorptive effects on bone cells. In this study, the effects of high frequency OFF on expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), two important regulators of osteoclast differentiation, were investigated.MethodsCells were exposed to 1 Pa peak shear stress using three loading frequencies (1, 10, and 20 Hz) widely employed in cell, animal, and clinical studies of bone remodeling. Two separate experiments were performed where either the total number of cycles (3600 cycles) or the total loading time (60 min) was kept constant. Real-time RT-PCR was used to quantify mRNA levels of RANKL, OPG.Results3600 cycles of OFF at 1 Hz and 10 Hz loading decreased RANKL/OPG ratio. Interestingly, these results were due to different mechanisms where at 1 Hz the decrease was due to an increase in OPG mRNA, whereas at 10 Hz the decrease was due to a decrease in RANKL mRNA.ConclusionAlthough high frequency OFF does not appear to further enhance the decrease in the RANKL/OPG ratio, these results suggest a potential to differentially control the change in either RANKL or OPG mRNA expression by applying different loading frequencies.

The in vitro and in vivo osteoblastic differentiation of rat bone marrow stromal cells (MSCs) was assessed on hydroxyapatite disks with 3 different porosities: 30%, 50%, and 70% (HA30, HA50, and HA70, respectively). MSCs obtained by 10-day culture of fresh bone marrow cells were subcultured for 2 weeks on 3 kinds of porous HA disks in the presence and absence of dexamethasone (Dex). After 2 weeks of subculture, alkaline phosphatase (ALP) activity and osteocalcin production of MSCs/HA composites with Dex were higher than those without, and increased with increasing porosity. The resultant bone tissue grafts "cultured-bone/HA constructs" were implanted subcutaneously into the backs of syngeneic rats, and harvested 1, 2, and 4 weeks after implantation. At 1 week, only cultured-bone/HA70 constructs exhibited expanded bone formation. At 2 and 4 weeks, active osteoblasts and progressive bone formation were observed morphologically in both cultured-bone/HA50 and HA70 constructs. At 4 weeks, bone tissue was observed even in cultured-bone/HA30 constructs. ALP activity and osteocalcin production also increased with increasing porosity and time after implantation. In this in vivo model, different scaffold porosity with similar crystal morphology of the apatite phase demonstrated marked differences in ability to support osteogenesis by implanted rat MSCs.