Mouse Bone Mesenchymal Stem Cells Research Articles

Properties and Cytocompatibility of Anti-Washout Calcium Phosphate Cement by Introducing Locust Bean Gum

The washout resistance of injectable calcium phosphate cement (CPC) is highly requisite for more widely clinical applications. In this work, locust bean gum (LBG) was used as the anti-washout agent to improve the washout resistance of CPC. The results indicated that the washout resistance was greatly improved, and meanwhile the injectability, setting time and compressive strength slightly decreased when the content of LBG was no more than 1.0%. Additionally, the CPC with 1.0% LBG exhibited good cell compatibility of the mouse bone mesenchymal stem cells (mBMSCs). Therefore, the 1.0% LBG content was proposed to serve as a useful additive in CPC as a result of its ability to promote washout resistance, which may play an important role in clinical applications.

Facile fabrication of poly(L-lactic acid) microsphere-incorporated calcium alginate/hydroxyapatite porous scaffolds based on Pickering emulsion templates

In this study, we develop a facile one-pot approach to the fabrication of poly(L-lactic acid) (PLLA) microsphere-incorporated calcium alginate (ALG-Ca)/hydroxyapatite (HAp) porous scaffolds based on HAp nanoparticle-stabilized oil-in-water Pickering emulsion templates, which contain alginate in the aqueous phase and PLLA in the oil phase. The emulsion aqueous phase is solidified by in situ gelation of alginate with Ca2+ released from HAp by decreasing pH with slow hydrolysis of d-gluconic acid δ-lactone (GDL) to produce emulsion droplet-incorporated gels, followed by freeze-drying to form porous scaffolds containing microspheres. The pore structure of porous scaffolds can be adjusted by varying the HAp or GDL concentration. The compressive tests show that the increase of HAp or GDL concentration is beneficial to improve the compressive property of porous scaffolds, while the excessive HAp can lead to the decrease in compressive property. Moreover, the swelling behavior studies display that the swelling ratios of porous scaffolds reduce with increasing HAp or GDL concentration. Furthermore, hydrophobic drug ibuprofen (IBU) and hydrophilic drug bovine serum albumin (BSA) are loaded into the microspheres and scaffold matrix, respectively. In vitro drug release results indicate that BSA has a rapid release while IBU has a sustained release in the dual drug-loaded scaffolds. In vitro cell culture experiments verify that mouse bone mesenchymal stem cells can proliferate on the porous scaffolds well, indicating the good biocompatibility of porous scaffolds. All these results demonstrate that the PLLA microsphere-incorporated ALG-Ca/HAp porous scaffolds have a promising potential for tissue engineering and drug delivery applications.

Construction and properties of poly( lactic-co-glycolic acid )/calcium phosphate cement composite pellets with microspheres-in-pellet structure for bone repair

Granular-type grafts can be easily filled in complex bone defect sites, and the interconnected voids formed by stacking of granules can provide spaces and channels for the ingrowth of new bone tissues and blood vessels. In this study, poly( lactic-co-glycolic acid )/calcium phosphate cement composite (PLGA/CPC) pellets with sea island structure were fabricated by the freeze-dripping method. The phase composition, compressive stress, morphology, in vitro degradation and cytology behavior of PLGA/CPC pellets were investigated. The results indicated that the earlier compressive stress of CPC pellets was reinforced by the addition of PLGA microspheres and the in situ pores were generated inside pellets along with degradation of PLGA microspheres. Besides, the proliferation and alkaline phosphatase (ALP) activity of mouse bone mesenchymal stem cells (mBMSCs) cultured on PLGA/CPC pellets was comparable than CPC pellets. The PLGA/CPC pellets are promising to be used in bone defect repair.

MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a direct interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation.

Osteogenesis of rat mesenchymal stem cells and osteoblastic cells on strontium-doped nanohydroxyapatite-coated titanium surfaces.

The aim of this study was to compare the promotion of osteogenesis in vitro on three types of titanium surfaces: a strontium-hydroxyapatite (Sr-HA)-coated surface, a nano-HA-coated surface, and an uncoated roughened surface. Sr-HA and HA were placed on disks with a roughened titanium surface by electrochemical deposition. MC3T3-E1 preosteoblast cells and rat bone mesenchymal stem cells were cultured on the Sr-HA, HA-coated, and uncoated roughened disks, and cell adhesion, proliferation, viability, osteogenic differentiation, and formation of mineralized nodules were measured at various time points. The Sr-HA coating produced by a simple electrochemical deposition treatment evidently enhanced the attachment, spreading, alkaline phosphatase activity, and extracellular matrix calcium mineralization of mouse bone mesenchymal stem cells and MC3T3-E1 cells compared with an untreated roughened titanium surface and a nano-HA-coated surface. This study suggests that a Sr-doped nano-HA coating produced through electrochemical deposition improves the osteoconductivity of a microrough titanium surface.

Neurotrophine-3 may contribute to neuronal differentiation of mesenchymal stem cells through the activation of the bone morphogenetic protein pathway.

We investigated whether neurotrophin-3 (NT-3) can promote differentiation of mouse bone mesenchymal stem cells (MSCs) into neurons via the bone morphogenetic protein pathway. MSCs were prepared from rat bone marrow and either transfected with pIRES2-EGFP or pIRES2-EGFP-NT-3 or treated with bone morphogenetic protein 4. The pIRES2-EGFP-NT-3-transfected MSCs further underwent noggin treatment or siRNA-mediated knockout of the TrkC gene or were left untreated. Immunofluorescence staining, real-time PCR and Western blot analyses were performed to evaluate the transcription and expression of neural-specific genes and BMP-Smad signaling. MSCs were efficiently transduced by the NT-3 gene via pIRES2-EGFP vectors. pIRES2- EGFP-NT-3 could initiate the transcription and expression of neural-specific genes, including nestin, NSE and MAP-2, and stimulate BMP-Smad signaling. The transcription and expression of neural-specific genes and BMP-Smad signaling were significantly suppressed by siRNA-mediated knockdown of the TrkC gene of MSCs. These findings suggest that the BMP signaling pathway may be a key regulatory point in NT-3-transfected neuronal differentiation of MSCs. The BMP and neurotrophin pathways contribute to a tightly regulated signaling network that directs the precise connections between neuronal differentiation of MSCs and their targets.

Facile fabrication of poly(L-lactic acid)-grafted hydroxyapatite/poly(lactic-co-glycolic acid) scaffolds by Pickering high internal phase emulsion templates.

Porous scaffolds consisting of bioactive inorganic nanoparticles and biodegradable polymers have gained much interest in bone tissue engineering. We report here a facile approach to fabricating poly(l-lactic acid)-grafted hydroxyapatite (g-HAp)/poly(lactide-co-glycolide) (PLGA) nanocomposite (NC) porous scaffolds by solvent evaporation of Pickering high internal phase emulsion (HIPE) templates, where g-HAp nanoparticles act as particulate stabilizers. The resultant porous scaffolds exhibit an open and rough pore structure. The pore structure and mechanical properties of the scaffolds can be tuned readily by varying the g-HAp nanoparticle concentration and internal phase volume fraction of the emulsion templates. With increasing the g-HAp concentration or decreasing the internal phase volume fraction, the pore size and the porosity decrease, while the Young's modulus and the compressive stress enhance. Moreover, the in vitro mineralization tests show that the bioactivity of the scaffolds increases with increasing the g-HAp concentration. Furthermore, the anti-inflammatory drug ibuprofen (IBU) is loaded into the scaffolds, and the drug release studies indicate that the loaded-IBU exhibits a sustained release profile. Finally, in vitro cell culture assays prove that the scaffolds are biocompatible because of supporting adhesion, spreading, and proliferation of mouse bone mesenchymal stem cells. All the results indicate that the solvent evaporation based on Pickering HIPE templates is a promising alternative method to fabricate NC porous scaffolds for potential bone tissue engineering applications.

Gene expression profile study on osteoinductive effect of natural hydroxyapatite

The aim of this study was to investigate the osteoinductive effect of natural hydroxyapatite (NHA). NHA was extracted from pig bones and prepared into disk-like samples. Then, proliferation of mouse bone mesenchymal stem cells (MSCs) cultured on NHA was assessed by the methylthiazoltetrazolium (MTT) assay. Furthermore, microarray technology was applied to obtain the gene expression profiles of MSCs cultured on NHA at 24, 48, and 72 h. The gene expression profile was then comprehensively analyzed by clustering, Gene Ontology (GO), Gene Microarray Pathway Profiler (GenMAPP) and Ingenuity Pathway Analysis (IPA). According to the results of microarray experiment, 8992 differentially expressed genes were obtained. 90 differential expressed genes related to HA osteogenic differentiation were determined by GO analysis. These genes included not only 6 genes related to HA osteogenic differentiation as mentioned in the literatures but also newly discovered 84 genes. Some important signaling pathways (TGF-β, MAPK, Wnt, etc.) were influenced by these genes. Gene interaction networks were obtained by IPA software, in which the scoring values of two networks were highest, and their main functions were related to cell development. The comprehensive analysis of these results indicate that NHA regulate some crucial genes (e.g., Bmp2, Spp1) and then activate some pathways such as TGF-β signaling pathway, and ultimately osteogenic differentiation was induced.

MicroRNA-9 promotes differentiation of mouse bone mesenchymal stem cells into neurons by Notch signaling

MicroRNAs are members of the family of noncoding small RNAs that regulate gene expression either by inhibiting mRNA translation or by promoting mRNA degradation at the posttranscriptional level. They play an important role in the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into neurons. However, the role of microRNAs in this process remains to be poorly understood. Studies have shown that Notch signaling is involved in regulating MSC differentiation. Here, we found that microRNA-9 could promote MSC neuronal differentiation. Using immunocytochemistry, western blotting, and reverse transcription-PCR analyses, we showed that the expression of the neural cell specific marker, microtubule-associated protein 2, increased during the process, while the expression of Notch-1 decreased. This study suggests that microRNA-9 might promote MSC neuronal differentiation by modulating the Notch signaling pathway.