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Osteoblasts
Kinds of Osteoblasts Terms modified by Osteoblasts Selected AbstractsStromal cells promote bone invasion by suppressing bone formation in ameloblastomaHISTOPATHOLOGY, Issue 4 2008G S A Sathi Aims:, To study the stromal variation and role of stromal,tumour cell interaction in impaired bone formation as well as enhanced bone resorption in ameloblastoma. Methods and results:, Four types of stroma were observed histologically; fibrous, desmoplastic, myxoid and myxoid with hyalinization. Osteoblast and osteoclast were counted using haematoxylin and eosin sections and immunohistochemistry with CD68. After histomorphometric analysis, only fibrous and myxoid types of stroma were distinctly identified. Secreted frizzled-related peptide (sFRP)-2, transforming growth factor-beta 1 and receptor activator of nuclear factor-,B ligand (RANKL) revealed strong expression in myxoid type compared with the normal stroma. Bone morphogenetic protein (BMP)-2 was negative in myxoid type, but positive in normal stroma. Fibrous-type stroma showed weak expression of all antigens except RANKL compared with myxoid type. Conclusions:, The results suggest that stroma does not act only in bone resorption, but also in the suppression of new bone formation. sFRP-2 is the main factor for impaired bone formation. The expression of markers related to osteoclastogenesis and suppression of osteoblast formation is higher in myxoid-type than in fibrous-type stroma. Tumour cells create a favourable environment for impaired bone formation by secreting sFRP-2 as well as bone resorption by secreting RANKL and interleukin-6. [source] Ferritin ferroxidase activity: A potent inhibitor of osteogenesisJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2010Abolfazl Zarjou Abstract Hemochromatosis is a known cause of osteoporosis, and iron overload has deleterious effects on bone. Although iron overload and its association with osteoporosis has long been recognized, the pathogenesis and exact role of iron have been undefined. Bone is an active tissue with constant remodeling capacity. Osteoblast (OB) development and maturation are under the influence of core binding factor ,-1 (CBF-,1), which induces expression of OB-specific genes, including alkaline phosphatase, an important enzyme in early osteogenesis, and osteocalcin, a noncollagenous protein deposited within the osteoid. This study investigates the mechanism by which iron inhibits human OB activity, which in vivo may lead to decreased mineralization, osteopenia, and osteoporosis. We demonstrate that iron-provoked inhibition of OB activity is mediated by ferritin and its ferroxidase activity. We confirm this notion by using purified ferritin H-chain and ceruloplasmin, both known to possess ferroxidase activity that inhibited calcification, whereas a site-directed mutant of ferritin H-chain lacking ferroxidase activity failed to provide any inhibition. Furthermore, we are reporting that such suppression is not restricted to inhibition of calcification, but OB-specific genes such as alkaline phosphatase, osteocalcin, and CBF-,1 are all downregulated by ferritin in a dose-responsive manner. This study corroborates that iron decreases mineralization and demonstrates that this suppression is provided by iron-induced upregulation of ferritin. In addition, we conclude that inhibition of OB activity, mineralization, and specific gene expression is attributed to the ferroxidase activity of ferritin. © 2010 American Society for Bone and Mineral Research [source] Osteoblast-Derived TGF-,1 Stimulates IL-8 Release Through AP-1 and NF-,B in Human Cancer Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008Yi-Chin Fong Abstract Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-,1 is associated with osteolytic bone diseases. Materials and Methods: IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-,1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c- fos, c- jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-,B, and activator protein (AP)-1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-,B promoter in human cancer cells. Osteoblasts were transfected with TGF-,1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-,1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-,1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-,B DNA-protein binding and MAPKs after TGF-,1 treatment was shown, and TGF-,1,induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-,1, BMP-2, and IGF-1. TGF-,1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-,B on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis. [source] Perspective: Quantifying Osteoblast and Osteocyte Apoptosis: Challenges and Rewards,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2007Robert L Jilka Abstract Since the initial demonstration of the phenomenon in murine and human bone sections ,10 yr ago, appreciation of the biologic significance of osteoblast apoptosis has contributed greatly not only to understanding the regulation of osteoblast number during physiologic bone remodeling, but also the pathogenesis of metabolic bone diseases and the pharmacology of some of the drugs used for their treatment. It is now appreciated that all major regulators of bone metabolism including bone morphogenetic proteins (BMPs), Wnts, other growth factors and cytokines, integrins, estrogens, androgens, glucocorticoids, PTH and PTH-related protein (PTHrP), immobilization, and the oxidative stress associated with aging contribute to the regulation of osteoblast and osteocyte life span by modulating apoptosis. Moreover, osteocyte apoptosis has emerged as an important regulator of remodeling on the bone surface and a critical determinant of bone strength, independently of bone mass. The detection of apoptotic osteoblasts in bone sections remains challenging because apoptosis represents only a tiny fraction of the life span of osteoblasts, not unlike a 6-mo -long terminal illness in the life of a 75-yr -old human. Importantly, the phenomenon is 50 times less common in human bone biopsies because human osteoblasts live longer and are fewer in number. Be that as it may, well-controlled assays of apoptosis can yield accurate and reproducible estimates of the prevalence of the event, particularly in rodents where there is an abundance of osteoblasts for inspection. In this perspective, we focus on the biological significance of the phenomenon for understanding basic bone biology and the pathogenesis and treatment of metabolic bone diseases and discuss limitations of existing techniques for quantifying osteoblast apoptosis in human biopsies and their methodologic pitfalls. [source] Activation of Sirt1 Decreases Adipocyte Formation During Osteoblast Differentiation of Mesenchymal Stem Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2006Carl-Magnus Bäckesjö PhD Abstract In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation. Introduction: Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor ,2 (PPAR,2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPAR,2. Materials and Methods: We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining. Results: We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPAR,-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization. Conclusions: In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering. [source] The effect of deproteinized bovine bone on osteoblast growth factors and proinflammatory cytokine productionCLINICAL ORAL IMPLANTS RESEARCH, Issue 6 2010Paolo Amerio Abstract Objective: To test the ability of Bio-Oss® in inducing growth factors and proinflammatory cytokines that may have a role in inflammation after grafting, bone resorption, remodeling and in the homeostasis of osteoblasts. Material and methods: Normal human osteoblasts were seeded in Petri dishes containing granules of Bio-Oss®, cells were harvested after confluency and RNA was extracted. Reverse transcriptase polymerase chain reaction was performed using specific primers for osteonectin, bone sialoprotein (BSP), bone morphogenetic protein (BMP)-2 and BMP-7, platelet-derived growth factor (PDGF), interleukin 6 (IL-6), tumor necrosis factor , (TNF-,) and integrin ,1. Glycerol-3-phosphate dehydrogenase was used as the housekeeping gene and normal human osteoblasts grown on Petri dishes without Bio-Oss® granules were used as negative controls. Results: Osteoblast grown on Bio-Oss® showed a normal RNA expression of osteonectin, integrin ,1 and PDGF. However, compared with control osteoblasts it showed a reduced expression of BSP, BMP-2 and BMP-7, IL-6 and TNF-,. Conclusions: Our findings further support the evidence that Bio-Oss® is an excellent biomaterial that does not enhance the production of proinflammatory cytokines. To cite this article: Amerio P, Vianale G, Reale M, Muraro R, Tulli A, Piattelli A. The effect of deproteinized bovine bone on osteoblast growth factors and proinflammatory cytokine production. Clin. Oral Impl. Res. 21, 2010; 650,655. doi: 10.1111/j.1600-0501.2009.01881.x [source] Interaction of Osteoblasts with Macroporous Scaffolds Made of PLLA/PCL Blends Modified with Collagen and Hydroxyapatite,ADVANCED ENGINEERING MATERIALS, Issue 8 2009Halil Murat Aydin To mimic natural bone, a tissue engineering scaffold was developed that combines inorganic and organic components of natural bone, its pore diameter, and its interconnected structure. Collagen was coated onto a PLLA/PCL scaffold and hydroxyapatite particles were delivered throughout the polymer matrix much more easily than with other techniques thanks to the porosity-forming method of combining two porogens, namely, salt leaching and supercritical CO2 extraction. Compared with other coating techniques, this procedure can be performed readily and homogeneous 3D hydroxyapatite coating was achieved. [source] Control of surface free energy in titanium doped phosphate based glasses by co-doping with zincJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2009Ensanya Ali Abou Neel Abstract To significantly improve the biocompatibility of titanium doped phosphate based glasses, codoping with zinc has been attempted. This study investigated the effect of doping a quaternary 15Na2O:30CaO:5TiO2:50P2O5 glass with zinc oxide (1, 3, and 5 mol %) on bulk, structural, surface, and biological properties; the results were compared with glasses free from ZnO and/or TiO2. ZnO as adjunct to TiO2 was effective in changing density, interchain bond forces, degradation behavior, and ions released from the degrading glasses. Incorporation of both TiO2 and ZnO in T5Z1, T5Z3, and T5Z5 glasses reduced the level of Zn2+ release by two to three orders of magnitude compared with glasses containing ZnO only (Z5). 31P NMR results for T5Z1, T5Z3, and T5Z5 glasses showed the presence of Q3 species suggesting that the TiO2 is acting as a network former, and the phosphate network becomes slightly more connected with increasing ZnO incorporation. Regardless of their relative lower hydrophilicity and surface reactivity compared with the control glass free from TiO2 and ZnO (T0Z0), these glasses have significantly higher surface reactivity compared with Thermanox®. This has been also reflected in the maintenance of >98% viable Osteoblasts, proliferation rate, and expression level of osteoblastic marker genes in a comparable manner to Thermanox® and T5 glasses, particularly T5Z1 and T5Z3 glasses. However, T0Z0 and Z5 glasses showed significantly reduced viability compared to Thermanox®. Therefore, it can be concluded that ZnO doped titanium phosphate glasses, T5Z1 and T5Z3 in particular, can be promising substrates for bone tissue engineering applications. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [source] Sustained BMP Signaling in Osteoblasts Stimulates Bone Formation by Promoting Angiogenesis and Osteoblast Differentiation,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2009Fengjie Zhang Abstract Angiogenesis and bone formation are tightly coupled during the formation of the skeleton. Bone morphogenetic protein (BMP) signaling is required for both bone development and angiogenesis. We recently identified endosome-associated FYVE-domain protein (endofin) as a Smad anchor for BMP receptor activation. Endofin contains a protein-phosphatase pp1c binding domain, which negatively modulates BMP signals through dephosphorylation of the BMP type I receptor. A single point mutation of endofin (F872A) disrupts interaction between the catalytic subunit pp1c and sensitizes BMP signaling in vitro. To study the functional impact of this mutation in vivo, we targeted expression of an endofin (F872A) transgene to osteoblasts. Mice expressing this mutant transgene had increased levels of phosphorylated Smad1 in osteoblasts and showed increased bone formation. Trabecular bone volume was significantly increased in the transgenic mice compared with the wildtype littermates with corresponding increases in trabecular bone thickness and number. Interestingly, the transgenic mice also had a pronounced increase in the density of the bone vasculature measured using contrast-enhanced ,CT imaging of Microfil-perfused bones. The vessel surface and volume were both increased in association with elevated levels of vascular endothelial growth factor (VEGF) in osteoblasts. Endothelial sprouting from the endofin (F872A) mutant embryonic metatarsals cultured ex vivo was increased compared with controls and was abolished by an addition of a VEGF neutralizing antibody. In conclusion, osteoblast targeted expression of a mutant endofin protein lacking the pp1c binding activity results in sustained signaling of the BMP type I receptor, which increases bone formation and skeletal angiogenesis. [source] Osteoblast-Derived TGF-,1 Stimulates IL-8 Release Through AP-1 and NF-,B in Human Cancer Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2008Yi-Chin Fong Abstract Introduction: The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-,1 is associated with osteolytic bone diseases. Materials and Methods: IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-,1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c- fos, c- jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-,B, and activator protein (AP)-1 activity. Results: Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-,B promoter in human cancer cells. Osteoblasts were transfected with TGF-,1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-,1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-,1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-,B DNA-protein binding and MAPKs after TGF-,1 treatment was shown, and TGF-,1,induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. Conclusions: In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-,1, BMP-2, and IGF-1. TGF-,1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-,B on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis. [source] Negative Regulation by p70 S6 Kinase of FGF-2,Stimulated VEGF Release Through Stress-Activated Protein Kinase/c- Jun N-Terminal Kinase in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007Shinji Takai Abstract To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2,stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2,activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2,stimulated VEGF release and VEGF mRNA expression. The FGF-2,induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2,induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2,stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2,stimulated VEGF release and phosphorylation of SAPK/JNK. Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2,stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts. [source] Bone Morphogenetic Protein 2 Induces Cyclo-oxygenase 2 in Osteoblasts via a Cbfa1 Binding Site: Role in Effects of Bone Morphogenetic Protein 2 In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2005Daichi Chikazu Abstract We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfa1) consensus sequence (5,-AACCACA-3,) at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfa1 site was inhibited or supershifted by specific antibodies to Cbfa1. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2+/+ mice but not in cells from COX-2,/, mice. In vivo, BMP-2 (10 ,g/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (,CT), was decreased by 78% in COX-2,/, mice compared with COX-2+/+ mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfa1 binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo. [source] Focal Adhesion Kinase pp125FAK Interacts With the Large Conductance Calcium-Activated hSlo Potassium Channel in Human Osteoblasts: Potential Role in Mechanotransduction,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2003Roger Rezzonico Abstract Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. Introduction: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. Methods: Interaction of FAK with the C terminus of the hSlo ,-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. Results: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. Conclusions: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts. [source] Fluid Flow Induction of Cyclo-Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal-Regulated Kinase Signaling Pathway,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2002Sunil Wadhwa Abstract Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with ,371/+70 base pairs (bp) of the COX-2 5,-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to10 dynes/cm2, induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at ,8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by ,80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway. [source] Cloning and Functional Analysis of a Family of Nuclear Matrix Transcription Factors (NP/NMP4) that Regulate Type I Collagen Expression in OsteoblastsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2001Pasutha Thunyakitpisal Abstract Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen ,1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4- COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter-reporter constructs containing 3.5 kilobases (kb) of COL1A1 5, flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR-106 osteoblast-like cells. Several full-length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COL1A1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4- COL1A1 binding activity. Overexpression of specific clones in UMR-106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT-hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas-interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling. [source] Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein KinaseJOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2000Haruhiko Tokuda Abstract We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O -aminophinoxy)-ethane- N,N,N,N -tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase. [source] Thiazide Diuretics Affect Osteocalcin Production in Human Osteoblasts at the Transcription Level Without Affecting Vitamin D3 ReceptorsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2000D. Lajeunesse Abstract Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1,100 ,M) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]- induced OC release by these cells (maximal effect, ,40,50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (,47.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 ,M HCTZ). HCTZ (100 ,M) also stimulated calcium (Ca2+) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(,-amino ethyl ether)- N,N,N',N' -tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, ,22.5 ± 6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1,100 ,M) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate. (J Bone Miner Res 2000;15:894,901) [source] Insulin-Like Growth Factor I Production Is Essential for Anabolic Effects of Thyroid Hormone in Osteoblasts,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2000Bill K. Huang Abstract Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both ,IR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses. (J Bone Miner Res 2000;15:188,197) [source] Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat modelJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 3 2006U. Kneser Abstract Introduction: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. Methods: Discs of PBCB (Tutobone®) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. Results: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. Discussion: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects. [source] Promotion of osteogenesis in tissue-engineered bone by pre-seeding endothelial progenitor cells-derived endothelial cellsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 8 2008Haiying Yu Abstract In addition to a biocompatible scaffold and an osteogenic cell population, tissue-engineered bone requires an appropriate vascular bed to overcome the obstacle of nutrient and oxygen transport in the 3D structure. We hypothesized that the addition of endothelial cells (ECs) may improve osteogenesis and prevent necrosis of engineered bone via effective neovascularization. Osteoblasts and ECs were differentiated from bone marrow of BALB/c mice, and their phenotypes were confirmed prior to implantation. Cylindrical porous polycaprolactone (PCL)-hydroxyapatite (HA) scaffolds were synthesized. ECs were seeded on scaffolds followed by seeding of osteoblasts in the EC-OB group. In the OB group, scaffolds were only seeded with osteoblasts. The cell-free scaffolds were denoted as control group. A 0.4-cm-long segmental femur defect was established and replaced with the grafts. The grafts were evaluated histologically at 6 weeks postimplantation. In comparison with the OB group, the EC-OB group resulted in a widely distributed capillary network, osteoid generated by osteoblasts and absent ischemic necroses. Pre-seeding scaffold with ECs effectively promoted neovascularization in grafts, prevented the ischemic necrosis, and improved osteogenesis. The integration of bone marrow-derived ECs and osteoblasts in porous scaffold is a useful strategy to achieve engineered bone. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1147,1152, 2008 [source] Prostaglandin D2 pathway and peroxisome proliferator-activated receptor ,-1 expression are induced by mechanical loading in an osteoblastic cell lineJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2006Chitpol Siddhivarn Objective:, The hypothesis underlying the current study was that the arachidonic acid cascade, specifically activation of the prostaglandin (PG) D2 pathway in osteoblasts, is an anabolic signal induced by mechanical loading. Background:, Previous studies have shown that mechanical loading of osteoblasts triggers cyclooxygenase (COX)-2, PGE2 and prostacyclin (PGI2) synthesis. Since modest mechanical loading of osteoblasts promotes bone formation, we sought to determine whether mechanical stress activates the osteoblastic PGD2 pathway resulting in the synthesis of osteogenic cyclopentenones, including ,12PGJ2. Methods:, Osteoblast monolayers were stretched using a Bioflex apparatus at a frequency of 1 Hz with 1% elongation. Cells and cell media were collected at various time points: 5, 10, 15, 30 min; and 1, 4, 16, 24 h. RNA was extracted for quantitative reverse transcriptase,polymerase chain reaction (RT,PCR). In certain experiments, cells were pre-labeled with 14C arachidonic acid prior to stretching. Radiolabeled metabolites in cell media were identified by reverse-phase high performance liquid chromatography (RP-HPLC). Osteoblasts were evaluated for an induction in bone nodule formation by stretching. Results:, Mechanical strain significantly increased mRNA expression of COX-1, COX-2, PGD2 synthase and peroxisome proliferator-activated receptor (PPAR) ,-1, but not of PPAR,-2 as compared to control unstretched cells (p < 0.05). Mechanical loading stimulated the release of PGE2, PGD2 and the PGD2 metabolite ,12PGJ2. Mechanical strain resulted in the induction of bone nodules. Conclusions:, This report indicates that mechanical loading of osteoblasts results in activation of PGD2 and the concomitant expression of transcription factor PPAR,-1 mRNA. The coordinated synthesis of ,12PGJ2, a natural ligand for PPAR,-1, with the increased expression of PPAR,-1, suggests that biomechanical transduction pathways that initially involve the activation of cyclooxygenases may also involve the activation of the ,12PGJ2,PPAR pathway. [source] Osteoblasts stimulated with pulsed electromagnetic fields increase HUVEC proliferation via a VEGF-A independent mechanism,BIOELECTROMAGNETICS, Issue 3 2009Richard A. Hopper Abstract The clinically beneficial effect of low frequency pulsed electromagnetic fields (ELF-PEMF) on bone healing has been described, but the exact mechanism of action remains unclear. A recent study suggests that there is a direct autocrine mitogenic effect of ELF-PEMF on angiogenesis. The hypothesis of this study is that ELF-PEMF also has an indirect effect on angiogenesis by manipulation of vascular endothelial growth factor (VEGF)-A-based paracrine intercellular communication with neighboring osteoblasts. Conditioned media experiments measured fetal rat calvarial cell (FRC) and human umbilical vein endothelial cell (HUVEC) proliferation using tritiated thymidine uptake. We demonstrate that ELF-PEMF (15 Hz, 1.8 mT, for 8 h) has an indirect effect on the proliferation rate of both endothelial cells and osteoblasts in vitro by altering paracrine mediators. Conditioned media from osteoblast cells stimulated with ELF-PEMF increased endothelial proliferation 54-fold, whereas media from endothelial cells stimulated with ELF-PEMF did not affect osteoblast proliferation. We examined the role of the pro-angiogenic mediator VEGF-A in the mitogenic effect of ELF-PEMF-stimulated osteoblast media on endothelial cells. The production of VEGF-A by FRC as measured by ELISA was not changed by exposure to PEMF, and blocking experiments demonstrated that the ELF-PEMF-induced osteoblast-derived endothelial mitogen observed in these studies was not VEGF-A, but some other soluble angiogenic mediator. Bioelectromagnetics 30:189,197, 2009. © 2008 Wiley-Liss, Inc. [source] Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineeringBIOELECTROMAGNETICS, Issue 7 2007Ming-Tzu Tsai Abstract Bone tissue engineering is an interdisciplinary field involving both engineers and cell biologists, whose main purpose is to repair bone anatomical defects and maintain its functions. A novel system that integrates pulsed electromagnetic fields (PEMFs) and bioreactors was applied to bone tissue engineering for regulating osteoblast proliferation and differentiation in'vitro. Osteoblasts were acquired from the calvaria of newborn Wistar rats and isolated after sequential digestion. Poly(DL -lactic-co-glycolic acid) (PLGA) scaffolds were made by the solvent merging/particulate leaching method. Osteoblasts were seeded into porous PLGA scaffolds with 85% porosity and cultured in bioreactors for the 18-day culture period. Cells were exposed to PEMF pulsed stimulation with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT amplitude. The resulting induced electric field waveform consisted of single, narrow 300 µs quasi-rectangular pulses with a repetition rate of 7.5'Hz. The results showed that PEMF stimulation for 2 and 8 h at .13 mT increased the cell number on days 6 and 12, followed by a decrease on day 18 using 8 h stimulation. However, ALP activity was decreased and then increased on days 12 and 18, respectively. On the other hand, PEMF-treated groups (irrespective of the stimulation time) at 0.32 mT inhibited cell proliferation but enhanced ALP activity during the culture period. These findings suggested that PEMF stimulation with specific parameters had an effect on regulating the osteoblast proliferation and differentiation. This novel integrated system may have potential in bone tissue engineering. Bioelectromagnetics 28:519,528, 2007. © 2007 Wiley-Liss, Inc. [source] Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3-E1 osteoblast-like cellsCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2009Magdalena Coetzee Abstract Osteoblasts in culture can differentiate into mature mineralizing osteoblasts when stimulated with osteogenic agents. Clinical trials and in vivo animal studies suggest that specific polyunsaturated fatty acids (PUFAs) may benefit bone health. The aim of this study was to investigate whether arachidonic acid (AA) and docosahexaenoic acid (DHA) affect osteogenesis in osteoblasts and the transdifferentiation into adipocytes. Results from this study show that long-term exposure to AA inhibited alkaline phosphatase (ALP) activity in these cells, which might be prostaglandin E2 (PGE2)-mediated. DHA exposure also inhibited ALP activity which was evident after both short- and long-term exposures. The mechanism whereby DHA inhibits ALP activity is not clear and needs to be investigated. Although long-term exposure to PUFAs inhibited ALP activity, the mineralizing properties of these cells were not compromised. Furthermore, PUFA exposure did not induce adipocyte-like features in these cells as evidenced by the lack of cytoplasmic triacylglycerol accummulation. More research is required to elucidate the cellular mechanisms of action of PUFAs on bone. Copyright © 2008 John Wiley & Sons, Ltd. [source] Buried alive: How osteoblasts become osteocytesDEVELOPMENTAL DYNAMICS, Issue 1 2006Tamara A. Franz-Odendaal Abstract During osteogenesis, osteoblasts lay down osteoid and transform into osteocytes embedded in mineralized bone matrix. Despite the fact that osteocytes are the most abundant cellular component of bone, little is known about the process of osteoblast-to-osteocyte transformation. What is known is that osteoblasts undergo a number of changes during this transformation, yet retain their connections to preosteoblasts and osteocytes. This review explores the osteoblast-to-osteocyte transformation during intramembranous ossification from both morphological and molecular perspectives. We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial). We summarize which morphological and molecular changes could be used as characters (or markers) to follow the transformation process. Developmental Dynamics 235:176,190, 2006. © 2005 Wiley-Liss, Inc. [source] Development of renal bone diseaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2006A. Ferreira Abstract Renal osteodystrophy (ROD) develops as the early stages of chronic renal failure (CRF) and covers a spectrum of bone changes observed in the uraemic patient, which extend from high remodelling bone disease (frequently known as osteitis fibrosa) to low turnover, or adynamic disease. Between these two extremes there are also cases of bone mineralization compromised in variable degrees, as is the case of ,mixed bone disease' and osteomalacia. The dynamic process of bone remodelling is compromised in CRF, and a positive or negative bone balance can be observed in uraemic patients. In addition to the classic modulators of bone remodelling, like parathyroid hormone, calcitriol and calcitonin, other factors were recently identified as significant modulators of osteoblast and osteoclast activation in uraemic patients. In fact, different cytokines and growth factors, acting at an autocrine or paracrine level, seem to play a relevant role in the bone and mineral changes observed in uraemia. Recently, observations have been made of the development of more sensitive and specific techniques to assay different biochemical markers of bone turnover and mineral metabolism. Analogously, new contributions of conventional bone histology, bone immunocytochemistry and molecular biology, which enabled the understanding of some etiopathogenic mechanisms of ROD, were observed. [source] Inside Front Cover (Adv. Eng.ADVANCED ENGINEERING MATERIALS, Issue 8 2010Mater. Intense mobility of an osteoblast (actin, green) on a plasma activated surface leaving footprints of fibronectin (red). [source] Physical and Biological Properties of a Novel Hydrogel Composite Based on Oxidized Alginate, Gelatin and Tricalcium Phosphate for Bone Tissue Engineering,ADVANCED ENGINEERING MATERIALS, Issue 12 2007K. Cai A novel hydrogel composite is reported in this study, which was derived from oxidized alginate, gelatin and tricalcium phosphate (TCP). The physical and biological properties of these hydrogel composites prepared with oxidized sodium alginate with different oxidation degrees were investigated. The drug delivery potential of this hydrogel composite as a carrier was evaluated by using Vitamin B2 as a model drug as well. An in vitro investigation with encapsulation of osteoblast revealed that these composites were biocompatible. This hydrogel composite presented here may be utilized for the fabrication of potential injectable systems for tissue engineering, drug delivery and other medical applications. [source] In vitro evaluation of porous poly(L -lactic acid) scaffold reinforced by chitin fibersJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 2 2009Xiaoming Li Abstract In this study, the previously reported porous three-dimensional poly(L -lactic acid) (PLLA) scaffolds reinforced by the chitin fibers (PLLA/CF) with and without the link were evaluated in vitro. Firstly, pH value of the phosphate buffered saline lixiviums of the PLLA/CF with different content of the chitin fibers was measured to get an appropriate content of the chitin fibers in the PLLA/CF. Then, the cell functions (attachment, proliferation, alkaline phosphatase per unit cell, total protein per unit cell, and osteonectin, osteopontin, and osteocalcin gene expression) of human osteoblast-like cells (SaOS2) cultured on the PLLA/CF with the link, PLLA/CF without the link and PLLA scaffold were compared. The results showed that the link treatment did not significantly influence the pH value of the lixiviums of the scaffolds, 30% volume content might be an appropriate content of the chitin fibers in PLLA/CF scaffold to keep the pH value of the lixiviums of the scaffolds between 7.0 and 7.2 during the lixiviation time of 16 weeks, the PLLA/CF scaffold was significantly better for the attachment, proliferation, differentiation, and mineralization of the osteoblast than PLLA, the link treatment did not significantly influence these cells activities, which further suggested that PLLA/CF with the link treatment might be an appropriate scaffold for tissue engineering. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009 [source] Growth of osteoblast-like cells on biomimetic apatite-coated chitosan scaffoldsJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2008I. Manjubala Abstract Porous scaffold materials that can provide a framework for the cells to adhere, proliferate, and create extracellular matrix are considered to be suitable materials for bone regeneration. Interconnected porous chitosan scaffolds were prepared by freeze-drying method, and were mineralized by calcium and phosphate solution by double-diffusion method to form nanoapatite in chitosan matrix. The mineralized chitosan scaffold contains hydroxyapatite nanocrystals on the surface and also within the pore channels of the scaffold. To assess the effect of apatite and porosity of the scaffolds on cells, human osteoblast (SaOS-2) cells were cultured on unmineralized and mineralized chitosan scaffolds. The cell growth on the mineralized scaffolds and on the pure chitosan scaffold shows a similar growth trend. The total protein content and alkaline phosphatase enzyme activity of the cells grown on scaffolds were quantified, and were found to increase over time in mineralized scaffold after 1 and 3 weeks of culture. The electron microscopy of the cell-seeded scaffolds showed that most of the outer macropores became sealed off by a continuous layer of cells. The cells spanned around the pore wall and formed extra cellular matrix, consisting mainly of collagen in mineralized scaffolds. The hydroxyproline content also confirmed the formation of the collagen matrix by cells in mineralized scaffolds. This study demonstrated that the presence of apatite nanocrystals in chitosan scaffolds does not significantly influence the growth of cells, but does induce the formation of extracellular matrix and therefore has the potential to serve for bone tissue engineering. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2008 [source] | |||||||||||||||||||