Nephronectin (NPNT) is a novel extracellular matrix protein and a new ligand of integrin α8β1. Recent studies showed that NPNT is highly expressed in kidney, lung, thyroid, etc, and it may play an important role in many pathological conditions. NPNT is involved in the process of kidney development and acute kidney injury, regulates proliferation and differentiation of osteoblast, and induces the vasculogenesis in vitro. NPNT may play a key role in pathological osteoporosis and therefore be a new therapeutic target of bone diseases. NPNT gene variants are not only associated with lung function, but also potentially implicated in chronic airway diseases development. Moreover, NPNT is also an important factor that mediates pathology of cardiac, epidermis, breast, liver and teeth diseases. In this paper, we reviewed some research progresses on the structure, distribution, physiological and pathophysiological functions of NPNT.
Cell Differentiation ; Cell Proliferation ; Extracellular Matrix Proteins ; physiology ; Humans ; Kidney ; physiology ; Osteoblasts ; cytology ; Osteoporosis

OBJECTIVETo establish osteoblast-osteoclast cell co-culture system in a Transwell chamber, and detect cell viability of osteoblasts and osteoclasts in system.

METHODSOsteoblast MC3T3-E1 and mouse monocytes RAW264.7 were cultivated in vitro. RANKL-induced mouse RAW264.7 monocytes differentiated into mature osteoclasts, osteoblast-osteoclast cell co-culture system was established in Transwell chamber. Cell activity of osteoblasts and osteoclasts were detected by CCK-8 experimenting, Alizarin Red staining, TRAP staining. The expression of OPG, ALP, RANKL, TGF-b1 gene and RANKL protein in osteoblast MC3T3-E1 were detected by PCR, Western-Blot methods. Also, the expression of RANK, NF-κB in gene and protein level in osteoclast were measured through the same method respectively.

RESULTSThe co-culture system of Mouse MC3T3-E1 cells and RAW264.7 cell were established in Transwell chamber. Co-culture system affected cell division activities of osteoblasts and osteoclasts. Differentiation of osteoblasts were increased, while differentiation of osteoclast division were slight decreased under microscope observation. OPG (0.65±0.08) and ALP (0.16±0.01) gene expression of co-culture system were less than single culture OPG(1.00±0.08) and ALP (1.01±0.16); TGF-b1(4.42±0.21) and RANKL(4.12±1.04) of osteoblasts in co-culture system were higher than TGF-b1(1.00±0.10) and RANKL(1.00±0.09) under single culture. However, gene expression of RANK(0.63±0.06) and NF-κB(0.64±0.08) in co-culture system were decreased than RANK(1.00±0.08) and NF-κB(1.00±0.09), in single culture, and had significant differences. Similarly, protein expression of OPG(0.43±0.05) and NF-κB(0.59±0.05) of co-culture system were less than OPG(0.84±0.06) and NF-κB(1.13±0.03) of single culture. While RANKL protein expression (0.54±0.03)of co-culture system was more than single culture RANKL(0.31±0.03), and had statistically differences, which was in agreement of the trend of gene expression change.

CONCLUSIONSCo-culture system of mouse MC3T3-E1 cells and RAW264.7 cell was viable in Transwell chamber, and the activity of osteoblasts is higher than osteoclasts in co-culture system.

3T3 Cells ; Animals ; Cell Differentiation ; Coculture Techniques ; Mice ; NF-kappa B ; metabolism ; Osteoblasts ; cytology ; Osteoclasts ; cytology ; Osteoprotegerin ; metabolism ; RANK Ligand ; metabolism ; RAW 264.7 Cells ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Objective: To compare the effects of 50 Hz 1.8 mT sinusoidal magnetic field (SEMF) and 50 Hz 0.6 mT pulsed electromagnetic field(PEMF) on the maturation and mineralization of rat calvaria osteoblasts. Methods: Primary cultured rat calvarial osteoblasts were divided into 3 groups:blank control group, SEMF group and PEMF group. The rats in SEMT and PEMT groups were treated with 50 Hz 1.8 mT SEMF or 50 Hz 0.6 mT PEMF for 90 min/d, respectively. Western blotting and Real-time RT-PCR were used to detect the protein and mRNA expressions of Collagen-1, bone morphogenetic protein 2 (BMP-2), osterix (OSX) and Runt-associated transcription factor 2(Runx-2). The alkaline phosphatase(ALP) activity was detected by ALP test kits at d6 and d9 after treatment, and by ALP staining using azo coupling at d10 after treatment. The formation of calcium nodules was observed by alizarin red staining. Results: Compared with blank control group, the protein and mRNA expressions of Collagen-1, BMP-2, OSX and Runx-2 in SEMT and PEMT groups were significantly increased (P <0.01 or P <0.05); while the mRNA expressions of Collagen-1 and BMP-2 in PEMF group were significantly higher than those in SEMF group. After 6 days treatment, the activity of ALP in PEMF group was significantly higher than that in blank control group (P<0.05), while such difference was not observed in SEMF group (P0.05); after 9 days treatment, the activities of ALP in both PEMF and SEMP groups were significantly higher than that in blank control group (all P<0.05), but the difference between PEMF and SEMF groups was not significant (P0.05). After 10 days treatment, ALP staining was increased in both PEMF and SEMF groups compared with that in blank control group (all P<0.01), and the stained area was bigger in PEMF group than that in SEMF group (P<0.05). After 12 days treatment, calcium nodules were increased in PEMF and SEMF groups compared with that in blank control group (all P<0.01), and more calcium nodules were observed in PEMF group than SEMF group (P<0.05). Conclusion: Both 50 Hz 1.8 mT that in SEMF and 50 Hz 0.6 mT PEMF can promote the maturation and mineralization of osteoblasts, and the effect of PEMF is more marked.
Animals ; Calcification, Physiologic ; drug effects ; Cell Differentiation ; Cells, Cultured ; Electromagnetic Fields ; Gene Expression Regulation, Developmental ; radiation effects ; Magnetic Fields ; Osteoblasts ; cytology ; radiation effects ; Rats ; Skull ; drug effects

Objective: To investigate the effect of icaritin on maturation and mineralization of mouse osteoblast MC3T3-E1 cells and its mechanism. Methods: The cultured MC3T3-E1 cells were divided into blank control group, CXC chemokine receptor type 4 (CXCR4) inhibitor (AMD3100) group, icaritin group, and icaritin plus AMD3100 group. The expression of CXCR4, stromal cell-derived factor 1 (SDF-1) and osteogenesis-related genes and proteins were detected by real-time RT-PCR and Western blotting after drug treatment for 24 h. The alkaline phosphatase (ALP) activity was determined with ALP kit on d3 and d6; calcium nodules were detected by alizarin red staining after drug treatment for 14 d. Results: Real time RT-PCR showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related genes in icaritin group were significantly increased (P<0.05 or P<0.01); After AMD3100 treatment, the relative expression of CXCR4 gene was decreased (P<0.05). Western blot showed that compared with the blank control group, relative expressions of CXCR4, SDF-1 and osteogenesis-related proteins in the icaritin group were significantly increased (all P<0.01), but were decreased after AMD3100 was added (all P<0.01). The ALP activity of icaritin group was significantly higher than that of blank control group (all P<0.01) on d3 and d6 after drug treatment, while the activity of ALP was significantly decreased after AMD3100 treatment (all P<0.01). At d14 after drug treatment, compared with the blank control group, the area of alizarin red staining was increased in the icaritin group, while it was significantly reduced after the addition of AMD3100. Conclusion: Icaritin may promote maturation and mineralization of mouse osteoblast MC3T3-E1 cells through CXCR4/SDF-1 signaling pathway.
3T3 Cells ; Animals ; Calcification, Physiologic ; drug effects ; Chemokine CXCL12 ; metabolism ; Flavonoids ; pharmacology ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Osteoblasts ; cytology ; drug effects ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; drug effects

To investigate the effects of Danshensu on bone formation in ovariectomized rats.Thirty female SD rats were randomly divided into three groups with 10 rats in each:blank control group, model control group and Danshensu group. The osteoporosis model was induced by bilateral ovariectomy and rats in Danshensu group were fed with Danshensu 12.5 mg·kg·dby gavage after ostroporosis model induced. All animals were sacrificed after 90 days. The bone mineral density (BMD) of the whole body, femur and lumbar vertebra was measured by dual energy X-ray absorptiometry. The biomechanical properties of femur were measured by AG-IS mechanical universal testing machine. Serum osteocalcin and bone alkaline phosphates (BALP) levels were measured by ELISA. The number of osteoblasts of proximal femoral metaphysis was counted with light microscopy after HE staining.Compared with blank control group, BMD, biomechanical properties of femur, serum osteocalcin and BALP levels and the number of osteoblasts were decreased in model control group (<0.05 or<0.01). While compared with model control group, BMDs of the whole body, femur and lumbar vertebra, the elastic modulus, maximum load, yield strength, breaking point load of femur, the serum levels of osteocalcin and BALP, and the number of osteoblasts were significantly improved in Danshensu group (<0.05 or<0.01).Danshensu can improve bone quality by increasing bone density, improving biomechanical properties, promoting the expression of osteogenesis-related factors, and increasing the number of osteoblasts.
Alkaline Phosphatase ; blood ; drug effects ; Animals ; Biomechanical Phenomena ; drug effects ; Bone Density ; drug effects ; Cell Count ; Female ; Femur ; anatomy & histology ; cytology ; drug effects ; Lactates ; pharmacology ; Lumbar Vertebrae ; anatomy & histology ; drug effects ; Osteoblasts ; drug effects ; Osteocalcin ; blood ; drug effects ; Osteogenesis ; drug effects ; Osteoporosis ; drug therapy ; Ovariectomy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo understand the mechanism by which tenascin-C regulates osteoblast differentiation and the role of tenascin-C in osteoporosis.

METHODSTenascin-C protein expression in femoral spongy bone of mice with or without osteoporosis was analyzed using Western blotting. In MC3T3-E1 osteoblasts with or without tenascin-C depletion by a specific siRNA targeting tenascin-C, alkaline phosphatase activity and Dickkopf-1 (DKK-1) expression were determined using quantitative RT-PCR and Western blotting, and the transcriptional activity of Wnt signaling pathway was analyzed using a luciferase reporter assay. The possible interaction of tenascin-C with DKK-1 predicted by STRING software was verified by immunoprecipitation.

RESULTSs Tenascin-C was markedly down-regulated in hemoral spongy bone of mice with osteoporosis as compared with the control mice. Osteoblastic differentiation was markedly suppressed in MC3T3-E1 osteoblast after tenascin-C depletion, and was significantly reversed by simultaneous β-catenin over-expression. siRNA-mediated knockdown of tenascin-C, which bound DKK-1, up-regulated the expression of DKK-1 and consequently lowered the transcriptional activity of Wnt pathway.

CONCLUSIONTenascin-C knockdown attenuates its negative control on DKK-1 to suppress the transcriptional activity of Wnt pathway, which in turn suppresses osteoblastic differentiation and promotes osteoporosis.

Animals ; Cell Differentiation ; Gene Knockdown Techniques ; Mice ; Osteoblasts ; cytology ; Osteogenesis ; Osteoporosis ; physiopathology ; RNA, Small Interfering ; genetics ; Tenascin ; genetics ; metabolism ; Up-Regulation ; Wnt Signaling Pathway ; beta Catenin ; metabolism

OBJECTIVETo explore the effect of MicroRNA-146a (miR-146a) on the ability of BM-MSC to differentiate into adipocytes and osteoblasts.

METHODSBM-MSC were isolated from the bone marrow of healthy donors. The differentiation of BM-MSC into adipocytes and osteoblasts cells were done in vitro. After transfection with miR-146a inhibitor or mimics, the expression of miR-146a in BM-MSC was detected by real time quantitative PCR. The effect of MicroRNA-146a on the differentiation potential of BM-MSC was evaluated after transfection.

RESULTSBM-MSC possessed the ability to differentiate into adipocytes and osteoblasts cells when cultured in the induction medium. The expression of miR-146a was correspondingly down-regulated and up-regulated in BM-MSC after transfection. Compared with the control group, the expression of miR-146a was down-regulated (P < 0.01) after transfection with miR-146a inhibitor, while after transfection with miR-146a mimics it was significantly up-regulated. This study proved that the transfection with miR-146a inhibitor can inhibit BM-MSC differentiate into adipocytes (P < 0.01), while transfection with miR-146a mimics can promote differentiation of BM-MSC into adipocytes (P < 0.01). No effect of miR-146a inhibitor or miR-146a mimics on osteogenic differentiation of BM-MSC was observed (P > 0.05).

CONCLUSIONBM-MSC possess the ability to differentiate into adipocytes and osteoblasts. The miR-146a can promote BM-MSC to differentiate into adipocytes.

Adipocytes ; cytology ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteoblasts ; cytology ; Osteogenesis ; Transfection

OBJECTIVETo investigate the modulatory effect of the MSC derived from low attaching culture systems (suspending MSC) on T lymphocytes and the related mechanisms.

METHODSThe suspending MSC were generated from mouse compact bones by using low attaching plates and adherent cell culture flasks, respectively. The morphology of suspending MSC was observed under the inverted microscope and the cells were induced to differentiate into osteoblasts and adipocytes. Further, the surface antigen profile of MSC was analyzed with flow cytometry. In addition, the culture medium (CM) of suspending MSC and adherent MSC was collected and added into the activated T cell cultures before detection of the proliferation by CFSE assay. Moreover, the modulaory effects of the CM on the T cell-derived cytokines were detected by quantitative PCR. Also, the mRNA expression of cytokines of MSC was detected.

RESULTSThe suspending MSC grew in floating cell spheres and differentiated into osteoblasts and adipocytes in the induction medium. Furthermore, the suspending MSC shared the typical immuno-phenotype with their adherent counterparts. In addition, the results of CFSE assay demonstrated that suspending MSC derived CM suppressed ConA induced T cell proliferation. The results of quantitative PCR revealed that suspending MSC expressed transforming factor β1 and interleukin-6 at a higher level and suppressed the T cell expressing interferon γ and interleukine-17A.

CONCLUSIONThe suspending MSC exerted an unique modulatoy effect on T cells, which is quite different to adherent MSC.

Adipocytes ; cytology ; Animals ; Cell Adhesion ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; Flow Cytometry ; Immunophenotyping ; Interleukin-6 ; metabolism ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Osteoblasts ; cytology ; T-Lymphocytes ; cytology ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo evaluate the biocompatibility and viability of nonionic triblock copolymer Pluronic F-127 as a cell scaffold for osteogenic differentiation of dental pulp stem cells(DPSC).

METHODSDPSC were obtained via enzymatic digestion method and purified bylimited dilution method. The freeze dried hydrogel of 20% Pluronic F-127 was prepared and itsstructurewas observed usingscanning electron microscopy(SEM). After the encapsulation of cells of passage 3 in Pluronic F-127, the effects of hydrogel on the proliferations of DPSC were assessed with methyl thiazolyl terazolium(MTT) after one day and 3, 5, 7 days of incubations, respectively. On day 14, osteogenic abilities of DPSC encapsulated in the hydrogel were estimated by means of alizarin red S, immunocytochemical staining and real-time quantitative PCR(RT-qPCR).

RESULTSDPSC were isolated and cultured successfully in the present study. SEM observations showed that porous structures which might be suitable for cell culture. A570 values of MTT were then normalized. A570 values of the cells in 2D cultures were 0.30±0.06, 0.30±0.17, 0.35±0.04 and 0.25±0.06 and A570 values of DPSC in 3D cultures were 0.36±0.06, 0.54±0.18, 0.70±0.10 and 0.32±0.10 on day 1, 3, 5 and 7, respectively. A570 value peaks were found on day 5 in both groups. The proliferation of 3D cultured DPSC was higher than that of 2D cultured cells(P<0.05). After 14 days of osteogenic induction, there were no calcium nodules observed in the control group and the numbers of calcium nodulesin the 2D and 3D groups had no significant difference(P>0.05). Inmmunocytochemical staining demonstrated strong expression of osteoblast marker Runt-related transcription factor 2(RUNX2), type Ⅰ collagen(Col-Ⅰ) and relatively low expression of osteocalcin(OCN). Moreover, RT-qPCR showed no differences between the relative expression of ALP, RUNX-2, OCN in the 2D and 3D groups (P>0.05), but a higher relative expression of Col-Ⅰ was observed in the 3D group(P=0.023).

CONCLUSIONSPluronic F-127 is a promising cell scaffold or cell carrier for the osteobalst differentiation of dental pulp stem cells.

Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dental Pulp ; cytology ; Humans ; Hydrogel, Polyethylene Glycol Dimethacrylate ; Osteoblasts ; metabolism ; Osteocalcin ; metabolism ; Osteogenesis ; Poloxamer ; Stem Cells ; cytology ; Tissue Scaffolds

BACKGROUNDSeveral studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro.

METHODSADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay.

RESULTSSecondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05).

CONCLUSIONSSecondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs.

Adipocytes ; cytology ; metabolism ; Adipose Tissue ; cytology ; Animals ; Cell Differentiation ; physiology ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Chondroitin ABC Lyase ; metabolism ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteoblasts ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley