Differentiation Of Bone Marrow Stromal Cells Research Articles

Naringin promotes osteoblast differentiation and effectively reverses ovariectomy-associated osteoporosis

BackgroundOsteoporosis is a common pathological condition that influences 20% of women over 50years of age. This condition decreases bone strength and increases the risk of bone fracture. Naringin is a major flavonoid found in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae). Studies have shown that naringin possesses many pharmacological effects. The current study evaluated the influence of naringin on osteoblastic cell differentiation and proliferation, and assessed its therapeutic effects on a rat osteoporosis model. MethodThe proliferation, differentiation, and function of rat bone marrow stromal cells (BMSCs) were determined following treatment with various concentrations of naringin. Ovariectomy (OVX)-induced osteoporotic rats were orally administered naringin daily at low, medium, and high dosages, while a control group received PBS for 2months. Femoral X-ray images and microCT scans were used for bone mineral density (BMD) and BV/TV (bone volume/total volume) analyses, and histological assessments of left tibiae were employed to check for changes in trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the groups. ResultsNaringin was effective at enhancing the proliferation and osteogenic differentiation of BMSCs, and a concentration of 10μg/ml prompted the highest levels of osteocalcin expression among the in vitro study groups. There appeared to be a delayed response pattern of BMSCs to the naringin treatment. Naringin also effectively reversed OVX-induced bone loss via increasing BMD, bone volume, and trabecular thickness. The medium dose (300mg/kg) appeared to be the optimal dosage for delivering satisfactory therapeutic effects. ConclusionNaringin promotes the proliferation and differentiation of BMSCs, and increases osteocalcin expression. Naringin also effectively reverses ovariectomy-induced osteoporosis in rats. The study suggests that naringin administration may represent an effective treatment for osteoporosis.

Curculigoside promotes osteogenic differentiation of bone marrow stromal cells from ovariectomized rats

Curculigoside, a natural compound isolated from the medicinal plant Curculigo orchioides has been reported to prevent bone loss in ovariectomized rats. However, the underlying molecular mechanisms are largely unknown. This study investigated the effects of curculigoside on proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). The toxicity, proliferation and osteogenic differentiation of BMSCs cultured with various concentrations (0 as control, 10, 100 and 500 µm) of curculigoside were measured by viability assay, MTT analysis, alkaline phosphatase (ALP) activity assay, alizarin red staining and mineralization assay, real-time PCR analysis on osteogenic genes including ALP, type I collagen (Col I), osteocalcin (OCN) and osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2), as well as OPG enzyme-linked immunosorbent assay. No significant cytotoxicity was observed for BMSCs after supplementation with curculigoside. The proliferation of BMSCs was enhanced after administration of curculigoside, especially 100 µm curculigoside. Moreover, the osteogenic gene expression was significantly enhanced with 100 µm curculigoside treatment. Importantly, curculigoside significantly increased OPG secretion. The data indicate that curculigoside could promote BMSC proliferation and induce osteogenic differentiation of BMSCs. The most profound response was observed with 100 µm curculigoside. These findings may be valuable for understanding the mechanism of the effect of curculigoside on bone, especially in relation to osteoporosis.

Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro

Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE2) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE2 receptors (Ptger4 and Ptger2 KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE2 to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE2 but also BMMs. Sufficient PGE2 to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE2 was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger4, but not Ptger2, in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE2 also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE2, acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This suppression of OB differentiation could contribute to the bone loss seen with continuous PTH in vivo.

Transplantation of differentiated bone marrow stromal cells promotes motor functional recovery in rats with stroke

Objective: To investigate the effects of transplantation into rats with stroke of bone marrow-derived mesenchymal stem cells (MSCs) induced by brain-derived neurotrophic factor (BDNF). Materials and methods: MSCs were harvested from 6-week-old Sprague–Dawley male rats, and transplanted into adult Sprague–Dawley male rats with transient middle cerebral artery occlusion. At 48 hours after stroke, the adult rats, weighing 250–280 g, were injected via the tail vein with 1×107 MSCs or MSCs induced by BDNF (BDNF-MSCs) suspended in 1 ml phosphate-buffered saline. All animals underwent the neurological function test for 14 days. Infarct volume and blood–brain barrier permeability assay were assessed on day 14 post-middle cerebral artery occlusion. Western blotting and immunohistochemical staining were used to detect the protein expression of neuron-specific enolase (NSE). Results: Both BDNF-MSCs and MSCs produced improvement in neurological deficits compared with vehicle controls on day 14 (P<0·05). In particular, the group transplanted with BDNF-MSCs exhibited significant recovery of motor function compared with the group transplanted with MSCs alone (P<0·05). Both BDNF-MSCs and MSCs, but particularly the former, reduced infarct volume, improved blood–brain barrier dysfunction, reduced serum NSE levels, activated the NSE activity, and inhibited neuronal apoptosis in the ischemic boundary zone. Conclusions: BDNF-MSCs might contribute to the motor function improvement partly by reducing neuronal damage via upregulating the NSE expression level and inhibiting neuronal apoptosis.

In silico investigations of potential anabolic treatments in multiple myeloma-induced bone disease

No anabolic drugs are currently approved to treat multiple myeloma (MM)-induced bone disease and the anti-MM agent bortezomib exhibits the anabolic effects in the clinic. In this study, we focus on investigating potential anabolic treatments of MM-induced bone disease using our previously proposed MM–bone model, with the goal for clarifying the underlying molecular/cellular mechanisms. Firstly, a variety of virtual drug treatments are explored by the parametric study to clarify the anabolic-related molecular/cellular mechanisms. The real drug (i.e., bortezomib) treatments are further examined by developing an integrated model with bortezomib to validate the clarified anabolic-related molecular/cellular mechanisms. The simulated responses to the bortezomib treatments that are validated by the clinical data are consistent with the simulated responses to the virtual drug treatments. Our study clarifies that the anabolic effects in the treatment of MM-induced bone disease are associated with promoting the differentiation of bone marrow stromal cells (BMSC) and inhibiting the apoptosis of active osteoblasts, while promoting the differentiation of osteoblast precursors is instead suggested to be associated with the anti-catabolic effects. Compared with the individual anabolic therapies, the anabolic therapies that promote the differentiation of BMSC in combination with the anti-MM/anti-catabolic therapies are found to induce a greater increase in the bone volume, while the anabolic therapies that inhibit the apoptosis of active osteoblasts in combination with the anti-MM/anti-catabolic therapies induce a lower increase in the bone volume. The simulations also suggest that the direct inhibition of bortezomib on the osteoclast activity is probably a redundant mechanism.

Low Oxygen Tension Maintains Multipotency, Whereas Normoxia Increases Differentiation of Mouse Bone Marrow Stromal Cells

Optimization of mesenchymal stem cells (MSC) culture conditions is of great importance for their more successful application in regenerative medicine. O2 regulates various aspects of cellular biology and, in vivo, MSC are exposed to different O2 concentrations spanning from very low tension in the bone marrow niche, to higher amounts in wounds. In our present work, we isolated mouse bone marrow stromal cells (BMSC) and showed that they contained a population meeting requirements for MSC definition. In order to establish the effect of low O2 on cellular properties, we examined BSMC cultured under hypoxic (3% O2) conditions. Our results demonstrate that 3% O2 augmented proliferation of BMSC, as well as the formation of colonies in the colony-forming unit assay (CFU-A), the percentage of quiescent cells, and the expression of stemness markers Rex-1 and Oct-4, thereby suggesting an increase in the stemness of culture when exposed to hypoxia. In contrast, intrinsic differentiation processes were inhibited by 3% O2. Overall yield of differentiation was dependent on the adjustment of O2 tension to the specific stage of BMSC culture. Thus, we established a strategy for efficient BMSC in vitro differentiation using an initial phase of cell propagation at 3% O2, followed by differentiation stage at 21% O2. We also demonstrated that 3% O2 affected BMSC differentiation in p53 and reactive oxygen species (ROS) independent pathways. Our findings can significantly contribute to the obtaining of high-quality MSC for effective cell therapy.

The support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds

Nanofiber scaffolds are effective for tissue engineering since they emulate the fibrous nanostructure of native extracellular matrix (ECM). Although electrospinning has been the most common approach for fabricating nanofiber scaffolds, airbrushing approaches have also been advanced for making nanofibers. For airbrushing, compressed gas is used to blow polymer solution through a small nozzle which shears the polymer solution into fibers. Our goals were 1) to assess the versatility of airbrushing, 2) to compare the properties of airbrushed and electrospun nanofiber scaffolds and 3) to test the ability of airbrushed nanofibers to support stem cell differentiation. The results demonstrated that airbrushing could produce nanofibers from a wide range of polymers and onto a wide range of targets. Airbrushing was safer, 10-fold faster, 100-fold less expensive to set-up and able to deposit nanofibers onto a broader range of targets than electrospinning. Airbrushing yielded nanofibers that formed loosely packed bundles of aligned nanofibers, while electrospinning produced un-aligned, single nanofibers that were tightly packed and highly entangled. Airbrushed nanofiber mats had larger pores, higher porosity and lower modulus than electrospun mats, results that were likely caused by the differences in morphology (nanofiber packing and entanglement). Airbrushed nanofiber scaffolds fabricated from 4 different polymers were each able to support osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs). Finally, the differences in airbrushed versus electrospun nanofiber morphology caused differences in hBMSC shape where cells had a smaller spread area and a smaller volume on airbrushed nanofiber scaffolds. These results highlight the advantages and disadvantages of airbrushing versus electrospinning nanofiber scaffolds and demonstrate that airbrushed nanofiber scaffolds can support stem cell differentiation.

The effect of silicate ions on proliferation, osteogenic differentiation and cell signalling pathways (WNT and SHH) of bone marrow stromal cells.

Silicon (Si) is a trace element, which plays an important role in human bone growth. Si has been incorporated into biomaterials for bone regeneration in order to improve their osteogenic potential, both in vitro and in vivo. Little is known, however, as to how Si ions elicit their biological response on bone-forming cells. The aim of this study was to investigate the effect of Si ions on the proliferation, differentiation, bone-related gene expression and cell signalling pathways of bone marrow stromal cells (BMSCs) by comparing the BMSC responses to different concentrations of NaCl and Na2SiO3, while taking into account and excluding the effect of Na ions. Our study showed that Si ions at a concentration of 0.625 mM significantly enhanced the proliferation, mineralization nodule formation, bone-related gene expression (OCN, OPN and ALP) and bone matrix proteins (ALP and OPN) of BMSCs. Furthermore, Si ions at 0.625 mM could counteract the effect of the WNT inhibitor (W.I.) cardamonin on the osteogenic genes expression, (OPN, OCN and ALP), WNT and SHH signalling pathway-related genes in BMSCs. These results suggest that Si ions by themselves play an important role in regulating the proliferation and osteogenic differentiation of BMSCs, with the involvement of WNT and SHH signalling pathways. Our study provides evidence to explain possible molecular mechanisms whereby Si ions released from Si-containing biomaterials can acquire enhanced bioactivity at desired concentration.

Rat Bone Marrow Stromal Cell–Conditioned Medium Promotes Early Osseointegration of Titanium Implants

To enhance the stability of titanium (Ti) implants using conditioned medium (CM) derived from rat bone marrow stromal cell (BMSC). BMSCs were isolated from rat femurs and grown in culture, and the culture medium was used as CM. The CM was immobilized on the surface of Ti implants with calcifying solution. The topology of the Ti implants after immobilization of CM was observed by scanning electron microscopy (SEM). The Ti-immobilized CM was analyzed by liquid chromatography with tandem mass spectrometry. The adhesiveness and the osteogenic differentiation of BMSCs grown on CM-coated discs were analyzed by reverse-transcription polymerase chain reaction. Ti implants with specimen-immobilized CM labeled with quantum dots (QDs) were placed into rat femurs. The localization of the CM was detected by in vivo imaging at 1, 7, 14, and 28 days after implantation. The removal torque test and histologic bone implant contact (BIC) were also analyzed. Rat BMSC-CM was successfully immobilized on Ti implants. The immobilized CM contained about 2000 proteins, including collagen type I, bone sialoprotein, fibronectin, and vascular endothelial growth factor that are important in new bone formation. CM promoted cell adhesion and osteocalcin gene expression of rat BMSCs. The labeled CM remained associated with the Ti implant at 1, 7, 14, and 28 days postimplantation. The removal torque value and BIC of Ti implants with immobilized CM were higher than those of control implants on days 1, 7, and 14 after implantation. Immobilized CM components on the surface of Ti implants promoted integration into bone during an early stage.

Inhibition of JNK and ERK pathways by SP600125- and U0126-enhanced osteogenic differentiation of bone marrow stromal cells

Bone marrow stromal cells (BMSCs) can undergo osteogenesis when treated with an osteogenic stimulus, which makes them a potential cell source for bone tissue engineering. MAPK signaling pathways involved in osteogenesis have shown opposing effects. The aim of this study was to characterize the MAPK signaling pathways involved in the osteogenic differentiation of BMSCs. Cells were treated or non-treated with osteogenic differentiation medium (ODM) and specific inhibitors of JNK, ERK, p38. Cell proliferation, alkaline phosphatase (ALP) activity, calcium content, expression levels of osteogenic markers and mineralization were measured to assess osteogenic differentiation of BMSCs. ALP activity, calcium content, expression of ALP, osteopontin and osteocalcin, and calcium deposition were significantly enhanced by blocking the JNK or ERK pathway with SP600125 and U0126, respectively but they were strongly down-regulated by inhibiting the p38 pathway with SB203580, indicating that SP600125 and U0126 enhanced osteogenic differentiation of BMSCs, whereas SB203580 suppressed osteogenic differentiation of BMSCs. In addition, Western Blot and immunofluorescent analysis showed that treatment with the p38 inhibitor resulted in an increase in the activated form of ERK, whereas treatment with the ERK inhibitor resulted in an increase in the activated form of p38. These findings suggest that the MAPK signaling pathways play a regulatory role in osteogenesis, in which, JNK and ERK pathways are repressors of osteogenesis, whereas p38 pathway is an enhancer of osteogenesis of BMSCs. A cross-talk between ERK and p38 signaling pathways in BMSCs by their specific inhibitors was observed. SP600125 and U0126 may be good candidates for promoting osteogenesis during bone formation.

Adenosine Regulates Bone Metabolism Via A1, A2A and A2B Receptors in Bone Marrow Cells From Normal and Patients with Multiple Myeloma

Abstract 4977 Background:Multiple myeloma is characterized by osteolytic bone lesions, wherein coupled bone remodeling is disrupted with increased osteoclast activation and decreased osteoblast differentiation. We have previously demonstrated that adenosine, acting via A2A receptors, diminishes human and murine osteoclast formation and others have reported that adenosine, acting at A2Breceptors, promotes osteoblast differentiation in murine osteoblast precursors and cell lines. In this study, we examined the effect of adenosine on osteoblast and osteoclast differentiation derived from multiple myeloma (MM) patients. Methods:Human bone marrow was collected from multiple myeloma patients. Bone marrow stromal cells (BMSCs) and bone marrow derived mononuclear (BMMs) cells were isolated and osteoblasts and osteoclastswere cultured, respectively. Adenosine A1 receptor agonist CHA and antagonist Rolofylline, A2A receptor agonist CGS and antagonist ZM, and A2b receptor agonist BAY and antagonist MRS 1754, A3receptor agonist IB-MECA and antagonist MRS 1191; and dipyridamole, a nucleoside transport inhibitor, were added to the culture media. Alkaline phosphatase (ALP) activity assay was used to quantitate the osteoblast differentiation. In vitro osteoblast calcification was determined by alizarin red staining. TRAP+ staining was used to examine the osteoclast differentiation and bone resorption assay was used to study the osteoclast activity. Results:We found that A1R blockade by rolofyllineand A2aR ligation by CGS21680 inhibited differentiation of both normal and MM BMMs into TRAP+ multinucleated cells (IC50= 1nM for A1R, IC50= 10μM for A2AR;p<0. 001, n=3 for both). The inhibition of osteoclast differentiation by Rolofylline was also seen in bone resorption assay (Pit formation assay). The A2A receptor antagonist completely reversed the effects of CGS21680 on osteoclast differentiation. Moreover, enhanced adenosine accumulation in the presence of dipyridamole (0. 5μM) and A2BR activation promoted the differentiation of BMSCs from myeloma patients into osteoblasts shown byArlizarin red staining and ALP activity assay (by 1. 8 ± 0. 41 and 1. 57 ± 0. 26 fold, respectively, p<0. 05, compared with osteogenic media only, n=3 for both). Conclusions:These results indicate that adenosine A2 receptors may be useful targets for the treatment and prevention of MM-induced bone disease. Disclosures:No relevant conflicts of interest to declare.

Repair of mandibular defects by bone marrow stromal cells expressing the basic fibroblast growth factor transgene combined with multi-pore mineralized Bio-Oss

The aim of the present study was to evaluate the effect of combining Bio‑Oss with bone marrow stromal cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene on bone regeneration during mandibular distraction of rabbits. BMSCs obtained from rabbits were transfected with bFGF gene‑encoding plasmids and proliferation rate and the differentiation marker alkaline phosphatase activity were measured. Following seeding into Bio‑Oss collagen and 9‑day culture invitro, the surface morphology of the Bio‑Oss was assessed using scanning electron microscopy analysis. Three mandibular defects were induced in the lower border of the bilateral mandibular ramus in each New Zealand white rabbit (total n=6). Three scaffolds, groupA (seeded with BMSCs/bFGF), B (seeded with BMSCs/pVAX1) and C (cell‑free), which had been cultured invitro under standard cell culture conditions for 18days, were implanted into mandibular defects under sterile conditions. Animals were sacrificed by anesthesia overdose 12weeks following surgery and the scaffolds were extracted for bone mineral density and histological analyses. Results indicate that bFGF was successfully transfected into BMSCs. Proliferation and osteoblast differentiation of BMSCs were stimulated by bFGF invitro. No differences were identified in surface morphology for Bio‑Oss loaded with variable groups of cells. At week12 following implantation of Bio‑Oss scaffolds, mineralization of BMSCs in Bio‑Oss scaffolds was observed to be increased by bFGF. New bone and cartilage formation was revealed in hematoxylin and eosin‑stained sections in Bio‑Oss scaffolds and was most abundant in groupA (BMSCs transfected with bFGF). In the current study, the bFGF gene was transfected into BMSCs and expressed successfully. bFGF promoted proliferation and differentiation of BMSCs invitro and implantation of bFGF‑expressing BMSCs combined with Bio‑Oss enhanced new bone regeneration more effectively than traditional methods.

Electrical stimulation enhances neurogenin2 expression through β-catenin signaling pathway of mouse bone marrow stromal cells and intensifies the effect of cell transplantation on brain injury

Bone marrow stromal cells (BMSCs) have received significant attention for its use in neural regeneration. However, neural replacement by transplanted BMSCs was not very effective. Recently, the gene transfection method has improved the capability of cell transplantation; however, this method results in canceration and immune rejection. We induced the differentiation of mouse BMSCs into neural cells using electrical stimulation and transplanted the cells into traumatic brain injury (TBI) model mice. We found that the electrically stimulated cells have good potential to differentiate into neural cells and contribute to recovery from TBI without differentiating into astrocytes. In addition, we found that electrical stimulation enhanced neurogenin2 (Ngn2) expression. Ngn2 is involved in neural differentiation and inhibits astrocytic differentiation during cell growth. Furthermore, we found that this enhancement of Ngn2 expression occurred through β-catenin signaling pathway. This study may contribute to the use of BMSCs for neural replacement in central nervous system diseases.

Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells

Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells

Bone material properties in hypoparathyroidism

Bone material properties in hypoparathyroidism

The effects of thiazolidinediones on human bone marrow stromal cell differentiation in vitro and in thiazolidinedione-treated patients with type 2 diabetes

Thiazolidinedione (TZD) therapy has been associated with an increased risk of bone fractures. Studies in rodents have led to a model in which decreased bone quality in response to TZDs is due to a competition of lineage commitment between osteoblasts (OBs) and adipocytes (ADs) for a common precursor cell, resulting in decreased OB numbers. Our goal was to investigate the effects of TZD exposure on OB-AD lineage determination from primary human bone marrow stromal cells (hBMSCs) both invitro and invivo from nondiabetic subjects and patients with type 2 diabetics. Our experimental design included 2 phases. Phase 1 was an invitro study of TZD effects on the differentiation of hBMSCs into OBs and ADs in nondiabetic subjects. Phase 2 was a randomized, placebo-controlled trial to determine the effects of 6-month pioglitazone treatment invivo on hBMSC differentiation using AD/OB colony forming unit assays in patients with type 2 diabetes. Invitro, TZDs (pioglitazone and rosiglitazone) enhanced the adipogenesis of hBMSCs, whereas neither altered OB differentiation or function as measured by alkaline phosphatase activity, gene expression, and mineralization. The ability of TZDs to enhance adipogenesis occurred at a specific time/stage of the differentiation process, and pretreating with TZDs did not further enhance adipogenesis. Invivo, 6-month TZD treatment decreased OB precursors, increased AD precursors, and increased total colony number in patients with type 2 diabetes. Our results indicate that TZD exposure invitro potently stimulates adipogenesis but does not directly alter OB differentiation/mineralization or lineage commitment from hBMSCs. However, TZD treatment in type 2 diabetic patients results in decreased osteoblastogenesis from hBMSCs compared with placebo, indicating an indirect negative effect on OBs and suggesting an alternative model by whichTZDs might negatively regulate bone quality.

Phosphatidylcholine-specific phospholipase C/heat shock protein 70 (Hsp70)/transcription factor B-cell translocation gene 2 signaling in rat bone marrow stromal cell differentiation to cholinergic neuron-like cells

Although bone marrow stromal cells (BMSCs) can differentiate into neuron-like cells, the mechanisms underlying neuronal differentiation are not well understood. We recently found that inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC) by its inhibitor D609 promoted BMSCs’ differentiation into cholinergic neuron-like cells. Using the effective small molecule D609 and gene microarray technology, we investigated the change of gene expression profile to identify key mediators involved in the neuronal differentiation. We selected heat shock protein 70 (Hsp70) and transcription factor B-cell translocation gene 2 (Btg2) that were maximally up-regulated for further study. We found that functional suppression of Hsp70 blocked D609-induced increase of Btg2 expression and cholinergic neuronal differentiation of BMSCs. These results demonstrated that Hsp70 was the pivotal factor in PC-PLC-medicated neuronal differentiation of BMSCs, and Btg2 might be its downstream target. Our findings provide new clues for controlling BMSCs’ differentiation into cholinergic neuron-like cells and provide a putative strategy for neurodegenerative diseases therapies.

The mineralization inducing peptide derived from dentin sialophosphoprotein for bone regeneration

Dentin sialophosphoprotein (DSPP) has been shown to play a primary role in the formation and growth of hydroxyapatite crystals in an extracellular matrix of hard tissue such as bone and teeth. We hypothesized that the mineralization ability of DSPP might depend on a specific domain within it. Three peptides, which have hydroxyapatite (HA) binding affinity, denoted as mineralization inducing peptide (MIP1, MIP2, and MIP3) were identified from DSPP. The both of MIP2 and MIP3 had HA nucleation activity demonstrated by XRD. Among three MIPs, MIP3 significantly supported the human bone marrow stromal cell differentiation into osteoblastic cells. An immunoblot with antibodies specific for the phosphorylated forms of ERK was conducted with cells treated by MIP3. MIP3 transduced intracellular signals via the ERK pathways and was able to induce osteoblastic differentiation, as seen by high expression of ALP, type 1 collagen, OC, OPN, and Runx2 in accordance with applied MIP3 concentration. The Asp, Glu, and Ser residues in MIP3 play important roles for the affinity of calcium in HA bone mineral. Further animal experiment with MIP3 in combination with hydroxyapatite mineral induced marked new bone formation for 4 weeks at rabbit calvarial defect model. The new bone area was much higher in test group, implying that the peptide modified group had excellent biocompatibility when compared with the unmodified group. Taken together, the MIP from DSPP has potential to enhance mineralization followed by to enhance osteoblastic differentiation and bone regeneration.

Sodium-dependent vitamin C transporter SVCT2: Expression and function in bone marrow stromal cells and in osteogenesis

Ascorbic acid (Vitamin C) has a critical role in bone formation and osteoblast differentiation, but very little is known about the molecular mechanisms of ascorbic acid entry into bone marrow stromal cells (BMSCs). To address this gap in knowledge, we investigated the identity of the transport system that is responsible for the uptake of ascorbic acid into bone marrow stromal cells (BMSCs). First, we examined the expression of the two known isoforms of the sodium-coupled ascorbic acid transporter, namely SVCT1 and SVCT2, in BMSCs (Lin−ve Sca1+ve) and bone at the mRNA level. Only SVCT2 mRNA was detected in BMSCs and bone. Uptake of ascorbic acid in BMSCs was Na+-dependent and saturable. In order to define the role of SVCT2 in BMSC differentiation into osteoblasts, BMSCs were stimulated with osteogenic media for different time intervals, and the activity of SVCT2 was monitored by ascorbic acid uptake. SVCT2 expression was up-regulated during the osteogenic differentiation of BMSCs; the expression was maximal at the earliest phase of differentiation. Subsequently, osteogenesis was inhibited in BMSCs upon knock-down of SVCT2 by lentivirus shRNA. We also found that the expression of the SVCT2 could be negatively or positively modulated by the presence of oxidant (Sin-1) or antioxidant (Ascorbic acid) compounds, respectively, in BMSCs. Furthermore, we found that this transporter is also regulated with age in mouse bone. These data show that SVCT2 plays a vital role in the osteogenic differentiation of BMSCs and that its expression is altered under conditions associated with redox reaction. Our findings could be relevant to bone tissue engineering and bone related diseases such as osteoporosis in which oxidative stress and aging plays important role.

Canonical Wnt signaling is required for Panax notoginseng saponin-mediated attenuation of the RANKL/OPG ratio in bone marrow stromal cells during osteogenic differentiation

Panax notoginseng saponins (PNS) are known to regulate the osteogenic differentiation of bone marrow stromal cells (BMSCs). In the present study, we investigated whether PNS could promote the osteogenic differentiation of BMSCs through modulating the Wnt/β-catenin signaling pathways, which are implicated in BMSCs osteogenesis. We found that PNS enhanced the mRNA expression of OPG, β-catenin, and cyclin D1 while decreased the mRNA expression of RANKL and PPARγ2. The actions of PNS on BMSCs were reversed (or partially) by DKK-1, a classical inhibitor of Wnt/β-catenin signaling. These results suggest that PNS stimulating bone formation by promoting the proliferation and osteogenic differentiation of BMSCs, and could also protect the skeletal system by decreasing bone resorption through reduction of RANKL/OPG expression via Wnt/β-catenin signaling pathways.