Primary Cultures Of Human Osteoblasts Research Articles

Recombinant irisin prevents cell death and mineralization defects induced by random positioning machine exposure in primary cultures of human osteoblasts: A promising strategy for the osteoporosis treatment.

Spaceflight exposure, like prolonged skeletal unloading, is known to result in significant bone loss, but the molecular mechanisms responsible are still partly unknown. This impairment, characterizing both conditions, suggests the possibility of identifying common signalling pathways and developing innovative treatment strategies to counteract the bone loss typical of astronauts and osteoporotic patients. In this context, primary cell cultures of human osteoblasts derived from healthy subjects and osteoporotic patients were exposed to random positioning machine (RPM) to reproduce the absence of gravity and to exacerbate the pathological condition, respectively. The duration of exposure to RPM was 3 or 6days, with the aim of determining whether a single administration of recombinant irisin (r-irisin) could prevent cell death and mineralizing capacity loss. In detail, cellular responses were assessed both in terms of death/survival, by MTS assay, analysis of oxidative stress and caspase activity, as well as the expression of survival and cell death proteins, and in terms of mineralizing capacity, by investigating the pentraxin 3 (PTX3) expression. Our results suggest that the effects of a single dose of r-irisin are maintained for a limited time, as demonstrated by complete protection after 3days of RPM exposure and only partial protection when RPM exposure was for a longer time. Therefore, the use of r-irisin could be a valid strategy to counteract the bone mass loss induced by weightlessness and osteoporosis. Further studies are needed to determine an optimal treatment strategy based on the use of r-irisin that is fully protective even over very long periods of exposure and/or to identify further approaches to be used in a complementary manner.

Hydroxyapatite/tricalcium phosphate (HA/beta-TCP) scaffold combined with bone and endothelial cells as a potential candidate for oral and maxillofacial bone regeneration.

Cell-based scaffolds have been proposed as useful for tissue repair and bone regeneration in oral and maxillofacial tissue engineering. However, vascularization, a key player in bone regeneration, is often inadequate. To improve this, the use of biphasic calcium phosphate biomaterial (30% hydroxyapatite (HA)-70% tricalcium phosphate (TCP) mixture) HA/beta-TCP-scaffold with defined micron-channel sizes and pore sizes capable of driving angiogenesis, may prove helpful. Therefore, our study aimed to combine the HA/beta-TCP scaffold with in vitro differentiated cells, especially endothelial cells, well-known players in angiogenesis. The HA/beta-TCP-scaffold was combined with primary cultures of human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells (Human Umbilical Vein Endothelial Cells, HUVECs). Cells were co-cultured with HA/beta-TCP-scaffold in normoxia (20% oxygen tension) for 14 days. Then, we evaluated: cell adhesion/morphology (Scanning Electron Microscopy and Confocal Microscopy); cell viability (Calcein AM/Propidium Iodide Assay); the presence of multinucleated hOCs (Tartrate Resistant- Acid Phosphatase assay); expression of specific markers of both osteo- and vasculogenic differentiation (Immunohistochemistry). All cell types were viable and well adhered to the entire scaffold surface. Interestingly, bone matrix formation and newly formed vessel-like structures were both detected. Although further investigation is needed, our data proved that differentiated cells combined in vitro with HA/beta-TCP-scaffold prior to implantation could represent a useful strategy for future applications in bone regeneration. Especially, the use of customized micro-vascularized cell-based scaffold would fall within the innovative "personalized medicine” approach.

Modulatory activity of a bovine hydrolyzed collagen-hydroxyapatite food complex on human primary osteoblasts after simulating its gastrointestinal digestion and absorption

Introduction: osteoporosis is the most prevalent bone disease and one of the main causes of chronic disability in middle and advanced ages. Conventional pharmacological treatments are still limited, and their prolonged use can cause adverse effects that motivate poor adherence to treatment. Nutritional strategies are traditionally based on supplementing the diet with calcium and vitamin D. Recent studies confirm that the results of this supplementation are significantly improved if it is accompanied by the intake of oral hydrolyzed collagen. Objective: to evaluate the possible in vitro osteogenic activity of a peptide-mineral complex formed by bovine hydrolyzed collagen and bovine hydroxyapatite (Phoscollagen®, PHC®). Methods: the digestion and absorption of PHC® were simulated using the dynamic gastrointestinal digester of AINIA and Caco-2 cell model, respectively. Primary cultures of human osteoblasts were treated with the resulting fraction of PHC® and changes were evaluated in the proliferation of preosteoblasts and in the mRNA expression of osteogenic biomarkers at different stages of osteoblast maturation: Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen (ColA1). Results: an increase in preosteoblastic proliferation was observed (p ≤ 0,05). No changes were detected in the biomarkers of osteoblasts with 5 days of differentiation, but were with 14 days, registering an increase in Runx2 (p = 0.0008), ColA1 (p = 0.035), OC (p = 0.027) and ALP (without significance). Conclusion: these results show that the PHC® peptide-mineral complex stimulates the activity of mature osteoblasts, being capable of promoting bone formation.

Cyanogenic glycoside amygdalin influences functions of human osteoblasts in vitro

Amygdalin has been promoted as an alternative cancer cure. However, it is still unclear how this cyanogenic glycoside affects non-cancer cells including bone cells. This study first investigated the impact of amygdalin on viability, morphology and expression of important genes in human osteoblasts in vitro. Primary human osteoblast cultures were exposed to amygdalin at concentrations 0; 0.1; 1 and 10 mg/mL in growth medium for 72 h. Cell viability, osteoblasts morphology and expression of 10 genes associated with osteoblast-specific pathways, oxidative stress and cell death were determined. Osteoblasts viability was significantly decreased (–27.26%) and their size was reduced (–23.20%) at the highest concentration of amygdalin (10 mg/mL). This concentration of amygdalin down-regulated the expression of COL1A1 and ALPL genes, whereas the expression of BGLAP, TNFSF11 and WNT5A genes was increased. The osteoblast cultivation with 0.1 mg/mL amygdalin caused down-regulation of COL1A1 gene. No changes in expression were determined for RUNX2, BAX, CASP1, SOD1 and GPX1 genes among all tested concentrations of amygdalin. In conclusion, amygdalin in a high concentration negatively affected mineralization of extracellular matrix, increased bone resorption and decreased osteoblast viability. These changes were accompanied by modified expression profiles of responsible genes.

SUN-347 Glucagon-like Peptide 1 (GLP-1) Acts Directly On Human Osteoclasts To Increase Differentiation And Bone Resorptive Activity

Glucagon-like peptide 1 (GLP-1) is an intestinal hormone released in response to nutrient intake that promotes glucose-dependent insulin secretion by acting upon the pancreatic GLP-1 receptor (GLP-1R). GLP-1R agonists (GLP-1RAs) are widely used in treatment of type 2 diabetes. Preclinical data indicate that GLP-1RAs could be repurposed to treat low bone mass as GLP-1R-depleted mice have higher bone resorption and thinner cortical bones, while insulinopenic and insulin resistant rats have improved bone formation and reduced bone mass deterioration when treated with GLP-1 or GLP-1RAs. However, the effect of GLP-1 and GLP-1RAs on human bone cells remains undetermined. We aimed to elucidate the effect of GLP-1 on primary human osteoclast (OC) and osteoblast (OB) cultures. OCs were differentiated over 10 days from human blood-derived CD14+ monocytes and OBs over 4–6 weeks from human bone. Cells were seeded on bovine bone slices and studies performed using fetal bovine serum, MCSF and RANKL (OC monocultures) or MCSF only (OB monocultures and OB+OC co-cultures). We first investigated the effect of GLP-1 on bone resorptive activity of mature OCs on bovine bone slices. GLP-1 increased the eroded bone surface percentage compared to vehicle in both OC monocultures (1nM P=0.002; 10nM P=0.023; n=8 donors) and OC+OB co-cultures (1nM P=0.013; 10nM P=0.012; n=8 donors). We then tested the effects of GLP-1 on osteoblast activity in OC+OB co-cultures by measuring alkaline phosphatase (ALP). We found that GLP-1 increased ALP in OC+OB cultures (1nM, P=0.049; 10nM, P=0.019) and these effects were reversed by the GLP-1R antagonist exendin 9–39 (1nM, P=0.93, 10nM, P=0.64). However, in OB monocultures GLP-1 had no effects on ALP (1nM P=0.93, 10nM P=0.64) indicating a GLP-1-driven increase in osteoblast activity through osteoclast-osteoblast coupling. We then assessed the effect of GLP-1 on OC differentiation by assessing TRAcP activity. Although there was a trend towards increased TRAcP activity upon stimulation with GLP-1 on day 10 of osteoclastogenesis, this was not statistically significant (1nM P=0.12; n=8 donors; 10nM P=0.29, n=4 donors). Our studies indicated GLP-1 may have a direct effect on osteoclasts, and we therefore sought to characterise GLP-1-mediated signalling in these cells. We assessed the effect of GLP-1 on cAMP signalling using LANCE assays and assessed phosphorylation of ERK proteins by Western blot analysis in human OC cultures. OCs treated with 10nM GLP-1 for 30 minutes had increased cAMP signaling (P=0.004, n=12 bone slices from 2 donors) when compared to vehicle. Furthermore, 10nM GLP-1 induced rapid increases in phosphorylated ERK (P=0.03 following 2 minutes exposure, n=4 blots). In conclusion, our studies reveal that GLP-1 increases activity in primary mature human OCs, and OBs, via OCs. Our signaling studies in OCs indicate this is mediated by direct action of GLP-1 on human bone cells.

Effects of Novel Laser Dental Implant Microtopography on Human Osteoblast Proliferation and Bone Deposition

The aim of this study was to compare how two innovative laser titanium surfaces and sandblasted and acid-etched surfaces influence human osteoblast behavior during osteogenesis and the initial phases of bone deposition. Human osteoblasts from human adipose stem cells were sorted by flow cytometric analysis and induced to differentiate. After 40 days, the osteogenic differentiation was detected by alizarin red staining, and the alkaline phosphatase (ALP) was evaluated with western blot (WB) and real-time reverse transcriptase-polymerase chain reaction (RT-PcR) analysis. After confluence, human osteoblasts were cultured onto two different innovative laser-obtained titanium surfaces (L1 and L2) and compared with one sandblasted and acid-etched (SBAE) surface as the control. At different times, human osteoblast behavior was evaluated with cell proliferation viability assay (MTT), scanning electron microscopy (SEM), energy-dispersive x-rays (EDAX), osteogenic markers with RT-PcR, and WB analysis of matrix extracellular phosphoglycoprotein (MEPE), ALP, and osteocalcin (OCN). After cell sorting, the human osteoblasts from human adipose stem cells showed increasing values of ALP mRNA and protein expression. The osteogenic differentiation was confirmed by quantitative alizarin red staining assay. Profilometric and SEM analysis showed relevant differences between SBAE, L1, and L2 specimens. After 20 days of culture onto titanium samples, SEM evaluation showed a small number of human osteoblasts and isolated sites of bone matrix deposition in SBAE specimens. At the same time, on L1 surfaces, only an osteoblast mono-layer with initial bone deposition was found, while on L2 specimens, there was a thick network with flattened large stellate cells, many cellular interconnections with strong titanium adhesion, and a large complex mineralized structure of crystal bone. After 20 days, for all titanium samples, human osteoblasts culturing EDAX analysis showed the absence of impurities and a higher bone matrix deposition in L2 specimens compared with L1 and SBAE samples. The innovative microtopography and nanotopography laser-induced surface showed high biocompatibility with primary human osteoblast cultures and the absence of impurities. The innovative laser texture was capable of influencing the osteogenic process, confirming the critical role of titanium surface characteristics in the cell adhesion and bone deposition during the early phases of osseointegration. The association of human adipose stem cells and titanium surfaces laser-induced with an innovative procedure could generate promising improvements and developments in orthopedics, maxillofacial, and dental implant surgery.

Inflammatory factors in the circulation of patients with active rheumatoid arthritis stimulate osteoclastogenesis via endogenous cytokine production by osteoblasts.

The combination of cytokines present in the circulation of patients with active rheumatoid arthritis might contribute to the generalized bone loss that commonly occurs in these patients, by directly inhibiting osteoblast proliferation and differentiation, but especially by enhancing endogenous cytokine (i.e., receptor activator of nuclear factor-kappa B ligand (RANKL) and interleukin-6 (IL)-6) production by osteoblasts, thereby stimulating osteoclastogenesis. Generalized bone loss, as occurs in patients with rheumatoid arthritis (RA), is related to elevated levels of circulating cytokines. Individual cytokines have deleterious effects on proliferation and differentiation of osteoblast cell lines, but little is known about the effect of the interaction between inflammatory factors in the circulation of patients with active RA on human osteoblast function, including their communication towards other bone cells. We investigated whether serum from patients with active RA enhances cytokine production by osteoblasts, thereby effectively altering osteoblast-stimulated osteoclastogenesis. Serum was obtained from 20 patients with active RA (active RA sera) and from the same patients in clinical remission (remission RA sera). To determine osteoclastogenesis, RA serum-pretreated primary human osteoblast cultures were established in direct contact with human osteoclast precursors in the presence or absence of osteoprotegerin (OPG) or IL-6 inhibitor. Compared to remission RA sera, active RA sera inhibited osteoblast proliferation and differentiation in vitro as demonstrated by a reduced DNA content and gene expression of KI-67, collagen type 1, osteopontin, and osteocalcin. Active RA sera inhibited OPG expression and enhanced RANKL and IL-6 expression but did not alter IL-8 expression in osteoblasts. IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) expression were undetectable. In coculture, active RA sera treatment of osteoblasts stimulated while addition of OPG or IL-6 inhibitory antibodies significantly reduced the number of osteoclasts. Active RA sera contain circulating factors, likely cytokines and chemokines, that might contribute to bone loss by directly inhibiting osteoblast proliferation and differentiation, but especially, these factors modulate endogenous cytokine production by osteoblasts, thereby affecting osteoclastogenesis.

Osteocytes: Regulating the Mineral Reserves?

Osteocytes are increasingly recognized as multifunctional cells that help orchestrate bone and mineral metabolism— and are currently one of the hottest topics in the field. In addition to their putative role as detectors/transducers of mechanical strain in bone, osteocytes are a major source of sclerostin, a key inhibitor of Wnt signaling and bone formation, and appear to be important regulators of osteoclast function via their production of NF‐kB ligand (RANKL) and osteoprotegerin. It has also long been believed that osteocytes could remove or even replace mineralized bone around their lacunae and canaliculi. Thanks to a number of studies and reviews in recent years, this once‐ controversial concept of “osteocytic osteolysis” is now verymuch back on the agenda. In this issue of the Journal of Bone and Mineral Research, Kogawa and colleagues present evidence that sclerostin could act to stimulate the dissolution of bone mineral by osteocytes by increasing their expression of some of the same resorptive machinery used by osteoclasts. The authors show that treatment of cultured MLO‐Y4 and primary human osteocyte‐like cells with recombinant human sclerostin increased the expression of mRNA for a number of resorptionmarkers, most notably carbonic anhydrase 2 (CA2). The authors found that sclerostin caused both intracellular and extracellular acidification in cultures of the osteocyte‐like cells, albeit in a non–dose‐dependentmanner. The classic CA inhibitor, acetazolamide decreased the sclerostin‐ stimulated extracellular acidification in MLO‐Y4 cells, whereas antisense knockdown of CA2 led to an extracellular alkalinization, with elimination of the effect of acetazolamide. Sclerostin additionally caused a slight stimulation of calcium release from mineralized layers of primary osteocyte‐like cells, an effect that was also blocked by acetazolamide. Stimulation of mRNA for CA2, tartrate‐resistant acid phosphatase (TRAP), chloride channel 7 (CLCN7), and cathepsin K by sclerostin in the MLO‐Y4 cell line was inhibited by small interfering RNA (siRNA) constructs for Lrp4, Lrp5, or Lrp6, suggesting the possible involvement of Wnt signaling. Last, sclerostin treatment increased the plan area of osteocyte lacunae by about 25% in cultured samples of trabecular bone taken from patients undergoing hip surgery, an effect that was again abrogated by acetazolamide. The concept of osteocytic osteolysis was first mooted in 19th century, although is perhaps now most closely associated with Belanger. He suggested that either parathyroid hormone (PTH) or a low‐calciumdiet can induce osteocytes to enlarge their lacunae. In its broadest sense, the term osteocytic osteolysis implies dissolution of not only the mineral component of bone adjacent to lacunae and canaliculi but of the collagenous matrix, too. These ideas provoked vigorous rebuttals but began to gain traction again following reports that osteocyte lacunar area wasmarkedly increased in rats continuously infused with PTH for 4 weeks and in mice infused with prednisolone for 3 weeks. Moremodest increases in lacunar sizewere also observed inmice subjected to ovariectomy. More recently, some of the coauthors of the paper by Kogawa and colleagues have presented a comprehensive study of the effects of lactation on osteocyte lacunae in mice. Lactation was shown to be associated with increases in lacunar area of about 20–40% in tibias and lumbar vertebrae; these increases were reversed with 7 days of cessation of lactation. The effects were mimicked by PTH‐related protein (PTHrP) and abrogated by targeted deletion of PTH receptor 1. Increased expression of mRNAs for osteoclast‐specific markers (including cathepsin K, which cleaves collagen) was also noted in “osteocyte‐enriched” bones of lactating animals. These reversible changes in osteocyte lacunar size have been referred to as “remodeling,” although this process clearly differs from bone remodeling by osteoclasts and osteoblasts, which involves obvious turnover of substantial quantities of both mineral and organic matrix. Despite suggestions that removal and replacement of collagenous matrix around osteocytes might occur in extreme pathophysiological situations, such as in the ultra‐high turnover bone of egg‐ laying hens, there seems to be little wider evidence for such a process. Removal of mineral alone might be sufficient to account for the enlargement of osteocyte lacunae observed by light or scanning electron microscopy if the resulting thin layer of demineralized collagenous matrix shrinks back against the lacunar walls during dehydration for specimen preparation. In vivo, such a layer of demineralized (hydrated) matrix adjacent to the osteocyte could presumably serve as a scaffold to facilitate

Unique microstructural design of ceramic scaffolds for bone regeneration under load

During the past two decades, research on ceramic scaffolds for bone regeneration has progressed rapidly; however, currently available porous scaffolds remain unsuitable for load-bearing applications. The key to success is to apply microstructural design strategies to develop ceramic scaffolds with mechanical properties approaching those of bone. Here we report on the development of a unique microstructurally designed ceramic scaffold, strontium–hardystonite–gahnite (Sr–HT–gahnite), with 85% porosity, 500μm pore size, a competitive compressive strength of 4.1±0.3MPa and a compressive modulus of 170±20MPa. The in vitro biocompatibility of the scaffolds was studied using primary human bone-derived cells. The ability of Sr–HT–gahnite scaffolds to repair critical-sized bone defects was also investigated in a rabbit radius under normal load, with β-tricalcium phosphate/hydroxyapatite scaffolds used in the control group. Studies with primary human osteoblast cultures confirmed the bioactivity of these scaffolds, and regeneration of rabbit radial critical defects demonstrated that this material induces new bone defect bridging, with clear evidence of regeneration of original radial architecture and bone marrow environment.

Vitamin D activation of functionally distinct regulatory miRNAs in primary human osteoblasts

When bound to the vitamin D receptor (VDR), the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of osteoblast transcription. Less clear is the impact of 1,25D on posttranscriptional events in osteoblasts, such as the generation and action of microRNAs (miRNAs). Microarray analysis using replicate (n = 3) primary cultures of human osteoblasts (HOBs) identified human miRNAs that were differentially regulated by >1.5-fold following treatment with 1,25D (10 nM, 6 hours), which included miRNAs 637 and 1228. Quantitative reverse transcription PCR analyses showed that the host gene for miR-1228, low-density lipoprotein receptor-related protein 1 (LRP1), was coinduced with miR-1228 in a dose-dependent fashion following treatment with 1,25D (0.1-10 nM, 6 hours). By contrast, the endogenous host gene for miR-637, death-associated protein kinase 3 (DAPK3), was transcriptionally repressed by following treatment with 1,25D. Analysis of two potential targets for miR-637 and miR-1228 in HOB, type IV collagen (COL4A1) and bone morphogenic protein 2 kinase (BMP2K), respectively, showed that 1,25D-mediates suppression of these targets via distinct mechanisms. In the case of miR-637, suppression of COL4A1 appears to occur via decreased levels of COL4A1 mRNA. By contrast, suppression of BMP2K by miR-1228 appears to occur by inhibition of protein translation. In mature HOBs, small interfering RNA (siRNA) inactivation of miR-1228 alone was sufficient to abrogate 1,25D-mediated downregulation of BMP2K protein expression. This was associated with suppression of prodifferentiation responses to 1,25D in HOB, as represented by parallel decrease in osteocalcin and alkaline phosphatase expression. These data show for the first time that the effects of 1,25D on human bone cells are not restricted to classical VDR-mediated transcriptional responses but also involve miRNA-directed posttranscriptional mechanisms.

Expression of vascular endothelial growth factor in normal, osteoarthritic and osteoporotic osteoblasts

To evaluate vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis in primary human osteoblast cultures from healthy, osteoporotic and osteoarthritic subjects. Normal primary human osteoblast cultures were obtained from healthy subjects undergoing surgery for the reduction in traumatic fractures. Pathological osteoblasts were obtained from patients undergoing to total hip replacement for osteoporotic hip fracture or advanced osteoarthritis. VEGF mRNA expression and protein synthesis were evaluated in cultured cells, by semiquantitative real-time PCR and ELISA, respectively, both under basal conditions than after vitamin D3 stimulation. Osteoarthritic osteoblasts showed a significantly higher VEGF expression compared to the normal and OP osteoblasts, both under basal conditions than in the presence of vitamin D3, whereas no difference was found between osteoporotic and normal osteoblast. Vitamin D3 significantly enhanced VEGF expression in normal and pathological osteoblasts. This preliminary study supports the hypothesis that VEGF is involved in the pathogenic mechanisms underlying the bone alterations typical of osteoarthritis and confirms the crucial role of vitamin D3 supplementation in metabolic bone diseases.

Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase

Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. Integrins regulate cell-ECM (extracellular matrix) adhesion and must associate with phosphorylating proteins such as ILK (integrin-linked kinase). Dysregulation of ILK expression is associated with anchorage-independent growth, cell survival and inhibition of apoptosis. Glucocorticoids influence differentiation and adhesion of osteoblasts and can affect bone protein synthesis. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts, together with its influence on the expression of ILK and β1 integrin. For this, primary cultures of human osteoblasts were exposed to DEX at 10-9 M (physiological dose) and 10-6 M (pharmacological dose) for 24 and 48 h. Cell viability, apoptosis and cell adhesion were analysed, as well as protein expression of β1 integrin and ILK. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of β1 integrin was significantly lower. Both proteins under study were co-localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased β1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti-apoptotic role.

Calcification of primary human osteoblast cultures under flow conditions using polycaprolactone scaffolds for intravascular applications

Total atherosclerotic occlusion is a leading cause of death. Recent animal models of this disease are devoid of cell-mediated calcification and arteries are often not occluded gradually. This study is part of a project with the objective of developing a new model featuring the above two characteristics, using a tissue-engineering scaffold. The amount and distribution of calcium deposits in primary human osteoblast (HOB) cultures on polycaprolactone (PCL) scaffolds under flow conditions were investigated. HOBs were cultured on PCL scaffolds with TGF-β1 loadings of 0 (control), 5 and 50 ng. HOB-PCL constructs were cultured in spinner flasks. Under flow conditions, cell numbers present in HOB cultures on PCL scaffolds increased from day 7 to day 14, and most calcification was induced at day 21. TGF-β1 loadings of 5 and 50 ng did not show a significant difference in ALP activity, cell numbers and amount of calcium deposited in HOB cultures, but calcium staining showed that 50 ng TGF-β1 had higher calcium deposited on both days 21 and 28 under flow conditions compared with 5 ng of loading. Amount of calcium deposited by HOBs on day 28 showed a decrease from their levels on day 21. PCL degradation may be a factor contributing to this loss. The results indicate that cell-induced calcification can be achieved on PCL scaffolds under flow conditions. In conclusion, TGFβ1-HOB loaded PCL can be applied to create a model for total atherosclerotic occlusion with cell-deposited calcium in animal arteries.

Alendronate and raloxifene affect the osteoprotegerin/RANKL system in human osteoblast primary cultures from patients with osteoporosis and osteoarthritis

The osteoprotegerin/RANKL system modulates bone remodelling. Alendronate and raloxifene are anti-resorptive drugs effective in osteoporotic disease. They reduce fracture risk, the activity of bone remodelling and increase bone mineral density. It is not known if they can exert a direct effect in osteoblasts via the osteoprotegerin/RANKL system. Our objective was to assess the effects of alendronate and raloxifene among osteoprotegerin production (ELISA), as well as osteoprotegerin and RANKL expression (RT-PCR), in primary cultures of human osteoblasts (hOB). We compared 17 osteoporotic patients with 16 patients affected by osteoarthritis in basal conditions and after incubation with alendronate (10 −6 M), raloxifene (10 −7 M) or 17-β estradiol (10 −7 M) for 24 h. The statistical analysis was determined by ANOVA. Osteoprotegerin protein secretion in hOB cultures was higher in patients with osteoporosis than osteoarthritis. Osteoprotegerin secretion levels remained unchanged after each treatment. The osteoporotic group was more sensitive to treatment. Both raloxifene (34%) and estradiol (37%) increased osteoprotegerin mRNA expression, and alendronate (118%) and raloxifene (61%) increased the mRNA expression of RANKL. The RANKL/osteoprotegerin mRNA ratio was higher in osteoporotic than osteoarthritic patients. In the osteoporotic group, the RANKL/osteoprotegerin mRNA ratio was significantly increased after treatment with alendronate (112%) and after treatment with raloxifene (60%). These results indicate a direct action of alendronate and raloxifene on hOB cultures from osteoporotic patients, and the cited drugs are able to modulate the osteoprotegerin/RANKL system.

MiR-29 Modulates Wnt Signaling in Human Osteoblasts through a Positive Feedback Loop

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.

In Vitro Effects of PDGF Isoforms (AA, BB, AB and CC) on Migration and Proliferation of SaOS-2 Osteoblasts and on Migration of Human Osteoblasts

PDGF is a major constituent of platelet rich plasma (PRP), responsible of chemotactic and possibly of mitogenic effects of PRP on osteoblasts. PDGF family includes 5 isoforms: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC and PDGF-DD, all expressed in platelets except PDGF-DD. Aim of this study was to analyze the effect of recombinant hPDGF-A, -AB, -B and -C, on migration and proliferation of a human osteoblastic cell line, SaOS-2. Preliminary observations on cell migration were also done in primary cultures of human osteoblasts. In vitro microchemotaxis and 3H-thymidine mitogenic assays were used. While PDGF-AB is active at concentrations present in PRP, PDGF-AA and BB are chemotactic only at much higher doses. PDGF-C is totally inactive alone or together with the active isoforms. PDGF-AA, PDGF-BB and PDGF-C stimulate SaOS-2 proliferation only at the highest dose tested, while PDGF-AB is ineffective. Primary osteoblasts are less sensitive than SaOS-2 and progressively lose responsiveness with increasing passages in culture, in line with loss of cell differentiation. The different PDGF isoforms act differentially on osteoblasts, the-AB isoform appearing the major responsible of the PRP chemiotaxis. PDGF, at the concentrations present in PRP, does not affect cell proliferation.

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E 2 in human bone

Bone loss is an important clinical issue in patients with cystic fibrosis (CF). Whether the cystic fibrosis transmembrane conductance regulator (CFTR) plays a direct role in bone cell function is yet unknown. In this study, we provide evidence that inhibition of CFTR-Cl − channel function results in a significant decrease of osteoprotegerin (OPG) secretion accompanied with a concomitant increase of prostaglandin (PG) E 2 secretion of primary human osteoblast cultures ( n = 5). Our data therefore suggest that in bone cells of CF patients, the loss of CFTR activity may result in an increased inflammation-driven bone resorption (through both the reduced OPG and increased PGE 2 production), and thus might contribute to the early bone loss reported in young children with CF.

Antigenic Phenotype and Phagocytic Capacity of MG‐63 Osteosarcoma Line

Human osteoblasts isolated from bone tissue samples have their own specific antigen profile but also share expression of antigens that are characteristic of other immunocompetent cells. Given that these findings come from studies performed in primary cultures of human osteoblasts, it was decided to test whether these antigen profiles and functional characteristics are retained in a characterized osteoblast cell line (MG-63). We show that some of these characteristics are also found in the MG-63 osteosarcoma cell line. We have demonstrated, using monoclonal antibodies and cytometry, that these cells expressed CD10, CD13, CD44, and CD54 antigens but were negative for CD69 and HLA-DR antigens. Functionally, 100% of MG-63 cells showed phagocytic capacity with a high phagocytic index. This study corroborates that osteoblastic cells have an immunological profile.

The effect of platelet‐rich plasma on osteoblast and periodontal ligament cell migration, proliferation and differentiation

Platelet-rich plasma is used to deliver growth factors, in a safe and convenient manner, for enhancing bone and periodontal regeneration. However, conflicting reports regarding its effectiveness suggest that further study of the relevant cellular mechanisms is required. The aim of this study was to investigate the in vitro effect of platelet-rich plasma on osteoblasts and periodontal ligament cell function. Various concentrations of platelet-rich plasma (100, 50, 20 and 10%) and platelet-poor plasma, obtained from human donors, were applied to primary cultures of human osteoblasts and periodontal ligament cells. [(3)H]-Thymidine incorporation, crystal violet staining and MTT assays were utilized to assess DNA synthesis and proliferation. Migration was determined by assessing the cell response to a concentration gradient, while differentiation was assessed using Alazarin Red staining. Platelet-rich plasma and platelet-poor plasma had stimulatory effects on the migration of both human osteoblasts and periodontal ligament cells. At 24 h, DNA synthesis was suppressed by the application of the various concentrations of platelet-rich plasma, but over a 5-d period, a beneficial effect on proliferation was observed, especially in response to 50% platelet-rich plasma. Platelet-poor plasma resulted in the greatest enhancement of cellular proliferation for both cell types. At a concentration of 50%, platelet-rich plasma and platelet-poor plasma facilitated differentiation of both cell types. Platelet-rich plasma can exert a positive effect on osteoblast and periodontal ligament cell function, but this effect is concentration specific with maximal concentrations not necessarily resulting in optimal outcomes. Platelet-poor plasma also appears to have the ability to promote wound healing-associated cell function.

Modifying RANKL/OPG mRNA Expression in Differentiating and Growing Human Primary Osteoblasts

The OPG/RANKL system in primary cultures of human osteoblasts has been studied by different authors. However, very few studies have been performed on gene expression of RANKL and OPG at different stages of maturation on human osteoblast cultures. The effect of 17- beta-estradiol and 1,25dihydroxyvitamin D3 on the OPG/RANKL system is not known during the different states of cellular maturation. In this work we quantified OPG and RANKL protein levels (ELISA) and the mRNA of OPG, RANKL, collagen type I, alkaline phosphatase, and osteocalcin (semi-quantitative RT-PCR) in human osteoblasts. We analyzed these in basal conditions and after incubation with 17- beta-estradiol and 1,25dihydroxyvitamin D3 in the first and second phases. We found that OPG secretion and expression levels increased throughout cellular growth. RANKL proteins were detected only in the first stage, and the expression increased throughout the first phase. Thus, the RANKL/OPG ratio was higher in immature osteoblasts than in mature osteoblasts. The evolution of RANKL gene expression was related to collagen I and alkaline phosphatase, while OPG was related to osteocalcin. We observed no modifications after estradiol and 1,25dihydroxyvitamin D3 treatment. Our results suggest that the OB is a positive stimulator at precocious stages of differentiation on osteoclastogenic modulates.