Differentiation Of Bone Marrow Stromal Cells Research Articles

Zinc Silicate/Nano-Hydroxyapatite/Collagen Scaffolds Promote Angiogenesis and Bone Regeneration via the p38 MAPK Pathway in Activated Monocytes.

Recent studies show that biomaterials are capable of regulating immune responses to induce a favorable osteogenic microenvironment and promote osteogenesis and angiogenesis. In this study, we investigated the effects of zinc silicate/nanohydroxyapatite/collagen (ZS/HA/Col) scaffolds on bone regeneration and angiogenesis and explored the related mechanism. We demonstrate that 10ZS/HA/Col scaffolds significantly enhanced bone regeneration and angiogenesis in vivo compared with HA/Col scaffolds. ZS/HA/Col scaffolds increased tartrate-resistant acid phosphatase (TRAP)-positive cells, nestin-positive bone marrow stromal cells (BMSCs) and CD31-positive neovessels, and expression of osteogenesis (Bmp-2 and Osterix) and angiogenesis-related (Vegf-α and Cd31) genes increased in nascent bone. ZS/HA/Col scaffolds with 10 wt % ZS activated the p38 signaling pathway in monocytes. The monocytes subsequently differentiated into TRAP+ cells and expressed higher levels of the cytokines SDF-1, TGF-β1, VEGF-α, and PDGF-BB, which recruited BMSCs and endothelial cells (ECs) to the defect areas. Blocking the p38 pathway in monocytes reduced TRAP+ differentiation and cytokine secretion and resulted in a decrease in BMSC and EC homing and angiogenesis. Overall, these findings demonstrate that 10ZS/HA/Col scaffolds modulate monocytes and, thereby, create a favorable osteogenic microenvironment that promotes BMSC migration and differentiation and vessel formation by activating the p38 signaling pathway.

β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2.

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.

Bio 3D Conduits Derived from Bone Marrow Stromal Cells Promote Peripheral Nerve Regeneration

We previously reported that a nerve conduit created from fibroblasts promotes nerve regeneration in a rat sciatic nerve model. This study aims to determine whether a nerve conduit created from bone marrow stromal cells (BMSCs) can promote nerve regeneration. Primary BMSCs were isolated from femur bone marrow of two Lewis rats, and cells at passages 4–7 were used. We created seven Bio 3D nerve conduits from BMSCs using a Bio-3D Printer. The conduits were transplanted to other Lewis rats to bridge 5-mm right sciatic nerve gaps (Bio 3D group, n = 7). We created two control groups: a silicone group (S group, n = 5) in which the same nerve gap was bridged with a silicone tube, and a silicone cell group (SC group, n = 5) in which the gap was bridged with a BMSC injection. Twelve weeks after transplantation, nerve regeneration was evaluated functionally and morphologically. In addition, PKH26-labeled BMSCs were used to fabricate a Bio 3D conduit that was transplanted for cell trafficking analysis. Electrophysiological study, kinematic analysis, wet muscle weight, and morphological parameters showed significantly better nerve regeneration in the Bio 3D group than in the S group or SC group. In immunohistochemical studies, sections from the Bio 3D group contained abundant S-100-positive cells. In cell trafficking analysis, PKH26-positive cells stained positive for the Schwann cell markers S-100, p75NTR, and GFAP. Bio 3D nerve conduits created from BMSCs can promote peripheral nerve regeneration in a rat sciatic nerve model through BMSC differentiation into Schwann-like cells.

Neuroblastoma-secreted exosomes carrying miR‐375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells

ABSTRACT Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Design of hydroxyapatite bioceramics with micro-/nano-topographies to regulate the osteogenic activities of bone morphogenetic protein-2 and bone marrow stromal cells.

Biomimicking the nanostructure of natural bone apatite to enhance the bioactivity of hydroxyapatite (HA) biomaterials is an eternal topic in the bone regeneration field. In the present study, we designed four kinds of HA bioceramics with micro- to nanosized grains and investigated the effects of bioceramic topographies on the structures of bone morphogenetic protein-2 (BMP-2) and the effects on the responses of bone marrow stromal cells (BMSCs). Compared to the samples with submicron-scale crystalline particles, HA bioceramics with grain sizes of 104.6 ± 27.8 nm exhibited increased roughness, improved hydrophilicity and enhanced mechanical properties. The synergistic effects of these surface characteristics could well maintain the conformation of BMP-2, facilitate cell adhesion and spreading, and activate the osteogenic differentiation of BMSCs. Furthermore, SBF immersion and in vivo canine intramuscular implantation confirmed that the HA bioceramics with nanotopography also processed excellent bone-like apatite forming ability and outstanding osteoinductivity. In summary, these findings suggest that the nanotopography of HA bioceramics is a critical factor to enhance their bioactivity and osteoinductivity.

Accumulation of kynurenine elevates oxidative stress and alters microRNA profile in human bone marrow stromal cells.

Kynurenine, a metabolite of tryptophan breakdown, has been shown to increase with age, and plays a vital role in a number of age-related pathophysiological changes, including bone loss. Accumulation of kynurenine in bone marrow stromal cells (BMSCs) has been associated with a decrease in cell proliferation and differentiation, though the exact mechanism by which kynurenine mediates these changes is poorly understood. MiRNAs have been shown to regulate BMSC function, and accumulation of kynurenine may alter the miRNA expression profile of BMSCs. The aim of this study was to identify differentially expressed miRNAs in human BMSCs in response to treatment with kynurenine, and correlate miRNAs function in BMSCs biology through bioinformatics analysis. Human BMSCs were cultured and treated with and without kynurenine, and subsequent miRNA isolation was performed. MiRNA array was performed to identify differentially expressed miRNA. Microarray analysis identified 50 up-regulated, and 36 down-regulated miRNAs in kynurenine-treated BMSC cultures. Differentially expressed miRNA included miR-1281, miR-330-3p, let-7f-5p, and miR-493-5p, which are important for BMSC proliferation and differentiation. KEGG analysis found up-regulated miRNA targeting glutathione metabolism, a pathway critical for removing oxidative species. Our data support that the kynurenine dependent degenerative effect is partially due to changes in the miRNA profile of BMSCs.

Multiple myeloma-derived exosomes inhibit osteoblastic differentiation and improve IL-6 secretion of BMSCs from multiple myeloma

Bone marrow stromal cells (BMSCs) play a critical role in multiple myeloma (MM) pathogenesis by cell contact, and secretion of cytokines, growth factors and extracellular vesicles. Exosomes are secreted by...

Blockade of adrenergic β-receptor activation through local delivery of propranolol from a 3D collagen/polyvinyl alcohol/hydroxyapatite scaffold promotes bone repair in vivo.

ObjectivesActivation of the sympathetic system and adrenergic β‐receptors following traumatic bone defects negatively impairs bone regeneration. Whether preventing β‐receptor activation could potentially improve bone defect repair is unknown. In this study, we investigated the effect of systematic administration and local delivery of propranolol through composite scaffolds on bone healing.Materials and methodsCollagen/PVA/propranolol/hydroxyapatite（CPPH）composite scaffolds were fabricated with 3D printing technique and characterized by scanning electron microscope (SEM). Micro‐CT analysis and bone formation histology were performed to detect new bone formation. Osteogenic differentiation of bone marrow stromal cells (BMSCs) and osteoclastogenesis of bone marrow monocytes cultured with scaffolds extract were performed for further verification.ResultsIntraperitoneal injection of propranolol did not significantly improve bone repair, as indicated by micro‐CT analysis and bone formation histology. However, CPPH scaffolds exhibited sustained release of propranolol in vitro and significantly enhanced bone regeneration compared with vehicle collagen/PVA/hydroxyapatite (CPH) scaffolds in vivo. Moreover, in vitro experiments indicated the scaffolds containing propranolol promoted the osteogenic differentiation and migration of rat BMSCs and inhibited osteoclastogenesis by preventing β‐receptor activation.ConclusionsThis study demonstrates that local adrenergic β‐receptor blockade can effectively enhance the treatment of bone defects by stimulating osteogenic differentiation, inhibiting osteoclastogenesis and enhancing BMSCs migration.

Exosomes derived from human CD34+ stem cells transfected with miR-26a prevent glucocorticoid-induced osteonecrosis of the femoral head by promoting angiogenesis and osteogenesis

BackgroundDamaged endothelial cells and downregulated osteogenic ability are two key pathogenic mechanisms of glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Recent studies suggested that transplantation of CD34+ stem cell-derived exosomes (CD34+-Exos) can treat ischemic diseases by promoting neovascularization and that miR-26a is an important positive regulator of osteogenesis. Moreover, the biological effect of exosomes is closely related to their cargo miRNAs. However, it is not clear whether increasing the abundance of miR-26a in CD34+-Exos will inhibit the progress of GC-induced ONFH.MethodsMiR-26a was overexpressed in CD34+-Exos (miR-26a-CD34+-Exos) to increase their osteogenic potential. The angiogenic potential of miR-26a-CD34+-Exos was then examined through evaluations of migration and tube-forming capacities in vitro. In addition, in order to observe the osteogenic effect of miR-26a-CD34+-Exos on bone marrow stromal cells (BMSCs), Alizarin red staining, alkaline phosphatase (ALP) activity assays, and qPCR were carried out. Finally, miR-26a-CD34+-Exos were injected into a GC-induced ONFH rat model to prevent the progress of GC-induced ONFH. The biological effects of miR-26a-CD34+-Exos on the ONFH model were evaluated by micro-CT, angiography, and histological staining.ResultsOur data showed that miR-26a-CD34+-Exos enhanced human umbilical vein endothelial cell migration and tube-forming capacities. Furthermore, miR-26a-CD34+-Exos strengthened the osteogenic differentiation of BMSCs under the influence of GCs in vitro. Finally, the miR-26a-CD34+-Exos increased the vessel density and trabecular bone integrity of the femoral head in the GC-induced ONFH rat model, which inhibited the progress of ONFH.ConclusionsMiR-26a-CD34+-Exos protect the femoral head from damage caused by GCs by strengthening angiogenesis and osteogenesis. The biological effect of miR-26a-CD34+-Exos make them suitable for application in the prevention of GC-induced ONFH.

LncRNA-MALAT1 promotes osteogenic differentiation through regulating ATF4 by sponging miR-214: Implication of steroid-induced avascular necrosis of the femoral head

ObjectiveTo study roles oflncRNA-MALAT1 and miR-214 in steroid-induced avascular necrosis of the femoral head (SANFH). MethodsMALAT1, miR-214 andosteogenic-relatedgenes(Runx2, ALP, andOCN)expressions were determined in SANFH tissue samples and human bone marrow stromal cells (BMSC) by RT-qPCR. BMSCs were verifiedbyflowcytometry. The ATF4 level was determined by western blotting and RT-qPCR. Osteogenesis inducedbyosteogenic medium (OM) in BMSCs and dexamethasone (DEX) was used to inhibit osteogenesis. The alkaline phosphatase (ALP) activity was measured and ALP staining and alizarin red staining were conducted for evaluation of osteogenic differentiation. MTT assay was used for cell proliferation. The dual luciferase reporter assay was used to confirm binding between MALAT1 and miR-214, as well as miR-214 and ATF4. ResultsMALAT1 was down-regulated and miR-214 was up-regulated in SANFH tissues. DEX inhibited osteogenic differentiation of BMSC in a dose-dependent manner, leading to decreased MALAT1, increased miR-214, as well as reduced ALP activity and decreased expression of RUNX2, ALP and OCN. Either overexpression of MALAT1 or inhibition of miR-214 improved DEX-induced inhibition of BMSC osteogenic differentiation. The overexpression of miR-214 reversed the effects by MALAT1. MALAT1 directly sponged miR-214 and miR-214 directly targeted ATF4. ConclusionMALAT1 was down-regulated, while miR-214 was elevated in SANFH tissues. MALAT1 promoted osteogenesis differentiation by sponging miR-214 to upregulate ATF4.

Early transplantation of growth differentiation factor 11-silenced bone marrow stromal cells promotes osteogenesis in necrotic femoral head induced by steroid in rats

Objective To study the effect of growth differentiation factor (GDF) 11-silenced bone marrow stromal cells (BMSCs) on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats. Methods After GDF11 expression in BMSCs was inhibited by siRNA, the knockdown efficiency and transfection cytotoxicity were detected. The further experiments both in vitro (n=3) and in vivo (n=8) were divided into 4 groups respectively: blank control group (without any intervention), model group (glucocorticoid treatment), experimental group (siRNA transfection and glucocorticoid treatment) and negative control group (negative control transfection and glucocorticoid treatment). The BMSCs were induced into osteogenic differentiation. Alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction (qRT-PCR) was employed to detect the relative expression levels of osteogenic markers. The osteogenesis in the necrotic femoral head was evaluated by microCT, H&E staining, immunohistochemistry staining and biomechanical test. Results No transfection cytotoxicity was found (P>0.05). The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group. At the level of mRNA, the relative expression of ALP, runt-related transcription factor (Runx) 2, osteocalcin (OCN) and type Ⅰ collagen (α1) (COL1A1) in the blank control group (1.00±0.09, 1.02±0.23, 1.03±0.30 and 1.02±0.25, respectively) were significantly higher than those in the model group (0.46±0.11, 0.50±0.11, 0.35±0.01 and 0.57±0.02, respectively) but significantly lower than those in the experimental group (1.97±0.30, 0.94±0.19, 1.50±0.18 and 1.28±0.37) (all P<0.05). MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group (P<0.05). Histological examination showed better bone regeneration and higher expression of Runx2 and COL1 in the necrotic femoral head in the experimental group than in the model group. Improved biomechanical properties were shown in the experimental group compared with the model group (P<0.05). Conclusions Silence of GDF11 expression may alleviate the inhibitory effect of glucocorticoid on osteogenic differentiation of BMSCs. Early transplantation of GDF11-silenced BMSCs may promote osteogenesis in the necrotic femoral head in rats. Key words: Femoral head necrosis; Bone marrow cells; Growth differentiation factor 11; Glucocorticoids; Osteogenic differentiation

Detecting oxygen in growing tissues

Biomaterials Cells require sufficient oxygen for survival and differentiation, for example, during bone tissue regeneration. With the aid of probe molecules, two-photon phosphorescence lifetime microscopy (2PLM) can image oxygen distributions, although not in tissues lacking vasculature. Schilling et al. use electrospin polymer meshes by employing a coaxial approach to embed 2PLM probes into the cores of fibers. The fibers support bone marrow stromal cell growth and differentiation, while enabling real-time high-resolution probing of partial pressures of oxygen during the early stages of tissue repair both in vitro and in vivo. ACS Appl. Mater. Interfaces 10.1021/acsami.9b08341 (2019).

Electrospun Fiber Mesh for High-Resolution Measurements of Oxygen Tension in Cranial Bone Defect Repair.

Tissue oxygenation is one of the key determining factors in bone repair and bone tissue engineering. Adequate tissue oxygenation is essential for survival and differentiation of the bone-forming cells and ultimately the success of bone tissue regeneration. Two-photon phosphorescence lifetime microscopy (2PLM) has been successfully applied in the past to image oxygen distributions in tissue with high spatial resolution. However, delivery of phosphorescent probes into avascular compartments, such as those formed during early bone defect healing, poses significant problems. Here, we report a multifunctional oxygen-reporting fibrous matrix fabricated through encapsulation of a hydrophilic oxygen-sensitive, two-photon excitable phosphorescent probe, PtP-C343, in the core of fibers during coaxial electrospinning. The oxygen-sensitive fibers support bone marrow stromal cell growth and differentiation and at the same time enable real-time high-resolution probing of partial pressures of oxygen via 2PLM. The hydrophilicity of the probe facilitates its gradual release into the nearby microenvironment, allowing fibers to act as a vehicle for probe delivery into the healing tissue. In conjunction with a cranial defect window chamber model, which permits simultaneous imaging of the bone and neovasculature in vivo via two-photon laser scanning microscopy, the oxygen-reporting fibers provide a useful tool for minimally invasive, high-resolution, real-time 3D mapping of tissue oxygenation during bone defect healing, facilitating studies aimed at understanding the healing process and advancing design of tissue-engineered constructs for enhanced bone repair and regeneration.

Groove structure of porous hydroxyapatite scaffolds (HAS) modulates immune environment via regulating macrophages and subsequently enhances osteogenesis.

Researches have revealed the vital roles of the generated immune environment via the response of immune cells growing on biomaterial surfaces in the bone healing process. HAS and novel constructed microgrooved patterns of HAS (HAS-G) are widely used as biocompatible ceramic, especially as a mimic of the natural bone matrix. However, it is unclear whether osteoimmune response induced by HAS and HAS-G affects the osteogenic differentiation of bone marrow stromal cells (BMSCs). RAW264.7 cells were seeded on different surface of materials and cytokines released by macrophages were detected by enzyme-linked immunosorbent assay. The cell viability and mitochondrial function of macrophages seeded on different surface of materials were detected. Then, the effects of modified inflammatory microenvironment by macrophages on osteogenesis of BMSCs were measured by performing ALP staining, Alizarin Red S staining, and western blot. We confirmed that HAS-G is more favorable for RAW cell attaching and subsequently regulated the expression and release of cytokines/chemokines. Decrease in interleukin-6 (IL-6) release was further confirmed for contributing significantly to improve mitochondrial function in RAW cells. HAS-G-conditioned medium promoted osteogenic differentiation in BMSCs and was reversed by IL-6 addition. Decrease in IL-6 contributes to downregulation of miR-214 and subsequently upregulated p38/JNK pathway, which is potentially contributes to osteogenic promotion by HAS-G. This study is the first report to reveal the effects of HAS-G on osteogenesis via immune response, which could lead to a new insight into novel material for the advantage of biomaterials for tissue engineering applications.

20(S)-hydroxycholesterol and simvastatin synergistically enhance osteogenic differentiation of marrow stromal cells and bone regeneration by initiation of Raf/MEK/ERK signaling.

Previous studies have demonstrated the significant roles of simvastatin (SVA) and oxysterols in the osteogenesis process. In this study, we evaluate the effect of a combination of SVA and 20(S)-hydroxycholesterol (20(S)OHC) on the cell viability and osteogenic differentiation of bone marrow stromal cells (BMSCs). After treatment with a control vehicle, SVA (0.025, 0.10, 0.25 or 1.0 μM), 20(S)OHC (5 μM), or a combination of both (0.25 μM SVA + 5 μM 20(S)OHC), the proliferation, apoptosis, ALP activity, mineralization, osteogenesis-related gene expression and Raf/MEK/ERK signaling activity in BMSCs were measured. Our results showed that high concentrations of SVA (0.25 and 1.0 μM) enhanced osteogenesis-related genes expression while attenuating cell viability. The addition of 5 μM 20(S)OHC induced significantly higher proliferative activity, which neutralized the inhibitory effect of SVA on the viability of BMSCs. Moreover, compared to supplementation with only one of the additives, combined supplementation with both SVA and 20(S)OHC induced significantly enhanced ALP activity, calcium sedimentation, osteogenesis-related genes (ALP, OCN and BMP-2) expression and Raf/MEK/ERK signaling activity in BMSCs; these enhancements were attenuated by treatment with the inhibitor U0126, indicating a significant role of Raf/MEK/ERK signaling in mediating the synergistically enhanced osteogenic differentiation of BMSCs by combined SVA and 20(S)OHC treatment. Additionally, histological examination confirmed a synergistic effect of SVA and 20(S)OHC on enhancing bone regeneration in a rabbit calvarial defect model. This newly developed SVA/20(S)OHC formulation may be used as an osteoinductive drug to enhance bone healing.

Sesamin Promotes Osteoblastic Differentiation and Protects Rats from Osteoporosis.

BackgroundOsteoporosis is a common osteopathy, resulting in fractures, especially in elder people. Sesamin has many pharmacological effects, including supplying calcium. However, how sesamin might prevent osteoporosis is still under study.Material/MethodsBone marrow stromal cells (BMSCs) extracted from rat femur were induced for osteoblastic differentiation. Cell proliferation, alkaline phosphatase (ALP), osterix (OSX), SRY-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), β-catenin, low density lipoprotein receptor-related protein 5 (LRP5), and glycogen synthase kinase-3β (GSK-3β) levels in BMSCs were detected in the presence or absence of sesamin (1 μM or 10 μM). In addition, FH535 (1 μM) was used to silence Wnt/β-catenin in vitro. Ovariectomized (OVX) rats were established and intragastrically administrated sesamin (80 mg/kg), and then the rat bones were analyzed by micro-computed tomography. Osteocalcin and collagen type I were measured in the rat femurs.ResultsSesamin had no influence on BMSC proliferation. Higher sesamin concentration promoted Wnt/β-catenin activity and enhanced more expressions of ALP, OSX, SOX9, RUNX2, and OCN, gradually and significantly (P<0.05). Silencing Wnt/β-catenin weakened the enhancement on RUNX2 and OCN expression. Sesamin (80 mg/kg) promoted bone structure in ovariectomized rats, and significantly enhanced osteocalcin and collage type I expression (P<0.05).ConclusionsSesamin promoted osteoblastic differentiation of rat BMSCs by regulating the Wnt/β-catenin pathway, and improved rat bone structure. Sesamin could have therapeutic and preventive effects on osteoporosis.

Investigation of osteoblast-like cells cultured on nano-hydroxyapatite/chitosan based composite scaffold in the treatment of bone defects and limited mobility

Objective(s): Design and construction of biocompatible and biodegradable scaffolds are among the main goals of tissue engineering. Recently, use of nano-hydroxyapatite as a bioactive bioceramic agent with high similarity to the mineral phase of the human bone tissue, in combination with biodegradable polymers and implant coatings has attracted the attention of researchers in the field of biomaterial sciences. The present study aimed to assess the differentiation of bone marrow stromal cells (BMSCs) in osteoblast-like cells on the chitosan/polyethylene oxide (PEO)/nano-hydroxyapatite scaffold in mature rats.Materials and Methods: Chitosan and PEO solution with the weight ratio of 80:20 and 70:30 were prepared, and 2% weight of nano-hydroxyapatite was added. Nanofibers were prepared using the electrospinning method, and the morphology was studied using scanning electron microscopy (SEM). Afterwards, the BMSCs of mature rats were cultured on nanofibers and differentiated by adding a differentiation medium. The survival of the differentiated cells was evaluated at the end of the first, second, and third week using acridine orange staining, and the morphology of the differentiated cells exposed to nanofibers was assessed using SEM. Results: The mean diameter of the nanofibers with the ratio of 80:20 was 150±17 nanometers. The differentiation of BMSCs into the osteoblast-like cells on nanofibers was confirmed using Alizarin red staining. The results indicated a significant decrease in the survival of the differentiated cells in the nanofiber groups by the end of the third week of differentiation compared to the control samples.Conclusion: According to the results, BMSCs could be differentiated into osteoblast-like cells in the presence of the chitosan/PEO nanofibers containing nano-hydroxyapatite.

Regulation of Osteosclerosis by Inoculated Cd133+ PC3 Cells in Bone-marrow Microenvironmental Niches.

ABSTRACTBone is the most common site of prostate cancer (PC) metastasis. Studies suggest that cancer stem cells (CSCs) are associated with stemness characteristics, providing some support for the concept that CSCs act as osteosclerotic precursors in bone microenvironmental niches. Here, we asked whether ectopic overexpression of CD133 maintains stability of CSCs in human PC cell lines and induces the changes of molecular features in the bone microenvironment. Ectopic overexpression of CD133 in PC3 or DU145 cells led to increased expression of ALDHA1, OCT4, and NANOG, enhanced colony‐forming ability, and increased ALDH activity. In addition, micro‐CT imaging, confocal microscopy, and H&E staining of mouse tissue confirmed that CD133 overexpression in PC3 and DU145 led to marked osteolytic bone tumor. However, expression of osteoblastic markers such as collagen type I, bone sialoprotein, and osteocalcin (OC) at the tumor margin of CD133‐overexpressing PC3 tumors in mouse tibiae was higher than that of CD133‐overexpressing DU145 tumors with osteosclerotic molecular features. In addition, expression of osteopontin (OPN) mRNA/protein by CD133‐overexpressing PC3 cells was higher than that by DU145 cells. Especially, conditioned medium (CM) from PC3CD133+ cells increased osterix (OSX) activity in bone marrow stromal cells (BMSCs), resulting in increased expression of OC mRNA/protein resulted in increased staining of mineralized matrix by Alizarin red. However, CM from OPN silenced PC3CD133+ cells led to a reduction of OC mRNA and protein expression through OSX activity resulted in reduced amount of mineralized matrix. In conclusion, these findings suggest that CD133 plays a functional role in regulating CSC characteristics in PCs and modulates their abilities in which induce the osteosclerosis of BMSCs. In addition, OPN from CSCs acts as a niche component that promotes osteosclerosis by supporting osteoblastic differentiation of BMSCs. © 2019 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Purification of alginate improves its biocompatibility and eliminates cytotoxicity in matrix for bone tissue engineering

There is a growing interest about using natural polymers from renewable sources as biomaterials for applications in tissue engineering. In the present work, alginates were extracted from Undaria pinnatifida, a brown sea weed invasive in Argentinian coast. The isolated alginate was structurally characterized by IR and 1H NMR spectroscopies, intrinsic viscosity and TGA. For comparison purposes, commercial sodium alginate was purified and characterized using the same protocol as for raw material. Toxicity and biocompatibility of sodium alginate obtained from algae were studied using a murine macrophage-like cell line (RAW 264.7) and bone marrow stromal cells (BMSC), respectively. The presence of impurities inhibited both RAW 264.7 and bone marrow stromal cell proliferation and increased nitric oxide production from macrophages, while inhibited osteoblastic differentiation of BMSC. All these effects were reverted by the purification of alginate. In conclusion, alginate purification improves biocompatibility and osteo-induction while decreases its toxicity.

A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.

Nocturnin (NOCT) belongs to the Mg2+ dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ. In this study, we characterized the skeletal and adipose tissue phenotype when we globally overexpressed Noct in vivo. After 12 weeks of Noct overexpression, transgenic male mice had lower fat mass compared to controls, with no significant differences in the skeleton. Based on the presence of a mitochondrial target sequence in NOCT, we determined that mouse NOCT protein localizes to the mitochondria; subsequently, we found that NOCT overexpression led to a significant increase in the preadipocytes ability to utilize oxidative phosphorylation for ATP (adenosine triphosphate) generation. In summary, the effects of NOCT on adipocytes are likely through its novel role as a mediator of mitochondrial function.