Impairment of mineralization by metavanadate and decavanadate solutions in a fish bone-derived cell line

Vanadium, a trace metal known to accumulate in bone and to mimic insulin, has been shown to regulate mammalian bone formation using in vitro and in vivo systems. In the present work, short- and long-term effects of metavanadate (containing monomeric, dimeric, tetrameric and pentameric vanadate species) and decavanadate (containing decameric vanadate species) solutions on the mineralization of a fish bone-derived cell line (VSa13) were studied and compared to that of insulin. After 2 h of incubation with vanadate (10 μM in monomeric vanadate), metavanadate exhibited higher accumulation rates than decavanadate (6.85 ± 0.40 versus 3.95 ± 0.10 μg V/g of protein, respectively) in fish VSa13 cells and was also shown to be less toxic when applied for short periods. In longer treatments with both metavanadate and decavanadate solutions, similar effects were promoted: stimulation of cell proliferation and strong impairment (75%) of extracellular matrix (ECM) mineralization. The effect of both vanadate solutions (5 μM in monomeric vanadate), on ECM mineralization was increased in the presence of insulin (10 nM). It is concluded that chronic treatment with both vanadate solutions stimulated fish VSa13 cells proliferation and prevented ECM mineralization. Newly developed VSa13 fish cells appeared to be appropriate in the characterization of vanadate effects on vertebrate bone formation, representing a good alternative to mammalian systems. Daniel M. Tiago and Vincent Laizé1 contributed equally to this work.     

Development of two bone-derived cell lines from the marine teleost<Emphasis Type="Italic"> Sparus aurata</Emphasis>; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitzs L-15 medium and a polygonal phenotype and rapid growth in Dulbeccos modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.A.R. Pombinho and V. Laizé contributed equally to this workThis work was partially funded with grants from the Portuguese Science and Technology Foundation PRAXIS/BIA/11159/98, POCTI/34668/Fis/2000 and POCTI/BCI/48748/2002. V.L., S.M.P.M and A.P. were the recipients of a postdoctoral fellowship (BPD/1607/2000 and BPD/9403/2002) and a CCMAR/University of Algarve fellowship, respectively     

Identification of an osteopontin-like protein in fish associated with mineral formation

Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.     

Development of a serum‐free system to expand dental‐derived stem cells: PDLSCs and SHEDs

Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum‐free media (K‐M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum‐containing media (FBS‐M) with cells cultured in four different serum‐free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS‐M. Additional proliferation assays were performed using pre‐coated fibronectin (FN) tissue culture plates and of the four serum‐free medium, only K‐M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS‐M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K‐M or FBS‐M, and, additionally, cells retained their multipotency in K‐M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K‐M or FBS‐M. Taken together, the data suggest that K‐M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency. J. Cell. Physiol. 226: 66–73, 2010. © 2010 Wiley‐Liss, Inc.     

Differential induction of cell-mediated mineralization in rat marrow stroma by sera from women of low and high risk for vertebral fracture

The purpose of this study was to analyze the ability of sera to reflect the state of bone metabolism by testing the osteogenic response of mesenchymal cells in culture. Sera of 20 peri- and postmenopausal women were tested before the initiation of hormone replacement therapy. The responding cells were osteoprogenitors (OPC) of rat marrow stroma which normally respond to dexamethasone (DEX) and β-glycerophosphate (βGP) by proliferation, differentiation, and mineralization in culture. Instead of DEX, diluted sera (1:50) were applied to rat stromal cell cultures for analysis of their ability to affect cell proliferation, specific alkaline phosphatase (ALP) activity, and cell-mediated mineralization. The results were compared individually with the respective values of vertebral bone mineral density (BMD), expressed as the number of standard deviations above or below the mean BMD of reference populations (positive or negative Z-score). Serum donors were divided in two; the group with positive Z-scores was considered to have a low risk, and that with negative Z-scores was considered to have a higher risk for vertebral fractures. No significant difference was found between the two groups in the ability of their sera to induce cell proliferation or specific ALP activity. However, sera representing negative Z-scores induced sixteenfold less mineralization than those of positive Z-scores. The scatter of individual mineralization values was highly discriminatory between the two groups (α < 0.00). These results indicate that the serum-induced, cell-mediated mineralization in culture might be suitable for initial evaluation of fracture risk and thus deserve further investigation. © 1996 Wiley-Liss, Inc.     

Expression of Gla‐rich protein (GRP) in newly developed cartilage‐derived cell cultures from sturgeon (Acipenser naccarii)

Sturgeons are representative of an ancient fish group, and present mainly an internal cartilaginous skeleton, with bone found essentially in the ganoid plaques forming the exogenous skeleton. Because of its archaic genetics, sturgeon represents an important model organism to understand the role of bone and cartilage‐related Gla proteins and determine if their molecular mechanisms of action were maintained throughout evolution. Of particular relevance is understanding the regulation, in sturgeon, of those proteins known to be involved in tissue mineralization in mammals, as well as unveiling the function of newly identified calcification‐related genes such as the one encoding the recently discovered Gla‐rich protein (GRP), thus contributing to understand the poor calcification observed in sturgeon endoskeleton. However, regulation of gene expression and promoter functional analysis of sturgeon cartilage and bone‐specific genes has been hampered by lack of suitable in vitro cell systems. We have recently developed the first sturgeon vertebra (VAn2H) and branchial arches (BAAn1F) derived cell cultures, and here we report their inability to mineralize their ECM under mineralizing culture conditions, as detected by von Kossa staining. Although a more extensive characterization of these systems is ongoing, our first data indicate that these cells represent a valuable tool for expression analysis of sturgeon bone and cartilage genes.     

Green tea polyphenol (−)‐epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3‐E1 cells

Recently, various physiological effects of the tea polyphenol catechin for alleviating diseases such as cancer, arteriosclerosis, hyperlipidaemia and osteoporosis have been reported. However, the physiological effect of catechin on bone metabolism remains unclear. We examined the physiological effect of EGCG [(?)‐epigallocatechin‐3‐gallate], which is the main component of green tea catechin, on osteoblast development using the precursor cell line of osteoblasts, MC3T3‐E1, and co‐culture of the osteoblasts from mouse newborn calvaria and mouse bone marrow cells. Although EGCG did not affect the viability and proliferation of MC3T3‐E1 cells, EGCG inhibited the osteoblast differentiation. Furthermore, EGCG did not affect the mineralization of differentiated MC3T3‐E1 cells, and reduced osteoclast formation in co‐culture. These results suggest that EGCG can effectively suppress bone resorption, and can be used as an effective medicine in the treatment of the symptoms of osteoporosis.     

Collagen peptides modulate the metabolism of extracellular matrix by human dermal fibroblasts derived from sun‐protected and sun‐exposed body sites

Clinical data published in recent years have demonstrated positive effects of collagen hydrolysate (CH) on skin aging clinical signs. CH use as food supplement has a long history; however, few studies have addressed the underlying purpose of CH on the cellular and molecular biology of skin cells that could elucidate clinical improvement findings. Wide diversity of characteristics has been reported for dermal fibroblasts derived from different body sites and it is unknown whether collagen peptides could modulate differently cells from chronological aged and photoaged skin areas. This study investigated the influence of CH on the extracellular matrix metabolism and proliferation of human dermal fibroblasts (HDFs) derived from chronological aged (sun‐protected) and photoaged (sun‐exposed) body sites. CH treatment did not affect cellular proliferation of either cell cultures, but notably modulated cell metabolism in monolayer model, increasing the content of dermal matrix precursor and main protein, procollagen I and collagen I, respectively. These effects were confirmed in the human dermal equivalent model. The increase in collagen content in the cultures was attributed to stimulation of biosynthesis and decreased collagen I metabolism through inhibition of metalloproteinase activity (MMP) 1 and 2. Modulation of CH in dermal metabolism did not differ between cells derived from sun‐protected and sun‐exposed areas, although lower concentrations of CH seemed to be enough to stimulate sun‐exposed‐derived HDFs, suggesting more pronounced effect in these cells. This study contributes to understanding the biological effects of CH on skin cells and viability of its use as a functional ingredient in food supplements.     

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Hydroxychloroquine decreases human MSC‐derived osteoblast differentiation and mineralization in vitro

We recently showed that patients with primary Sjögren Syndrome (pSS) have significantly higher bone mineral density (BMD) compared to healthy controls. The majority of those patients (69%) was using hydroxychloroquine (HCQ), which may have favourable effects on BMD. To study the direct effects of HCQ on human MSC‐derived osteoblast activity. Osteoblasts were cultured from human mesenchymal stromal cells (hMSCs). Cultures were treated with different HCQ doses (control, 1 and 5 µg/ml). Alkaline phosphatase activity and calcium measurements were performed to evaluate osteoblast differentiation and activity, respectively. Detailed microarray analysis was performed in 5 µg/ml HCQ‐treated cells and controls followed by qPCR validation. Additional cultures were performed using the cholesterol synthesis inhibitor simvastatin (SIM) to evaluate a potential mechanism of action. We showed that HCQ inhibits both MSC‐derived osteoblast differentiation and mineralization in vitro. Microarray analysis and additional PCR validation revealed a highly significant up‐regulation of the cholesterol biosynthesis, lysosomal and extracellular matrix pathways in the 5 µg/ml HCQ‐treated cells compared to controls. Besides, we demonstrated that 1 µM SIM also decreases MSC‐derived osteoblast differentiation and mineralization compared to controls. It appears that the positive effect of HCQ on BMD cannot be explained by a stimulating effect on the MSC‐derived osteoblast. The discrepancy between high BMD and decreased MSC‐derived osteoblast function due to HCQ treatment might be caused by systemic factors that stimulate bone formation and/or local factors that reduce bone resorption, which is lacking in cell cultures.     

Sclerostin is expressed in osteoclasts from aged mice and reduces osteoclast‐mediated stimulation of mineralization

Osteoclast‐mediated bone resorption precedes osteoblast‐mediated bone formation through early adulthood, but formation fails to keep pace with resorption during aging. We previously identified several factors produced by osteoclasts that promote bone formation. In this study, we determined if osteoclast‐produced factors contribute to the impaired bone formation with aging. We previously found that mice between the ages of 18 and 22 months develop age‐related bone loss. Bone marrow‐derived pre‐osteoclasts were isolated from 6‐week, 12‐month, and 18‐ to 24‐month‐old mice and differentiated into osteoclasts in vitro. Conditioned media were collected and compared for osteoblast mineralization support. Conditioned medium from osteoclasts from all ages was able to support mineralization of bone marrow stromal cells. Concentrating the conditioned medium from 6‐week‐old and 12‐month‐old mouse marrow cells‐derived osteoclasts enhanced mineralization support whereas concentrated conditioned medium from 18‐ to 24‐month‐old mouse marrow‐derived osteoclasts repressed mineralization compared to base medium. This observation suggests that an inhibitor of mineralization was secreted by aged murine osteoclasts. Gene and protein analysis revealed that the Wnt antagonist sclerostin was significantly elevated in the conditioned media from 24‐month‐old mouse cells compared to 6‐week‐old mouse cells. Antibodies directed to sclerostin neutralized the influences of the aged mouse cell concentrated conditioned media on mineralization. Sclerostin is primarily produced by osteocytes in young animals. This study demonstrates that osteoclasts from aged mice also produce sclerostin in quantities that may contribute to the age‐related impairment in bone formation. J. Cell. Biochem. 114: 1901–1907, 2013. © 2013 Wiley Periodicals, Inc.     

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell‐based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non‐invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)‐free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin‐based Cultispher^®S microcarriers and xeno‐free culture medium for the expansion of umbilical cord matrix (UCM)‐derived MSC. This system enabled the production of 2.4 (±1.1) x10⁵ cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)‐fold increase in cell number. The established protocol was then implemented in a stirred‐tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier‐based stirred culture system, using xeno‐free culture medium that suits the intrinsic features of UCM‐derived MSC represents an important step towards a GMP compliant large‐scale production platform for these promising cell therapy candidates.     

Evidence for direct effects of prolactin on human osteoblasts: Inhibition of cell growth and mineralization

Hyperprolactinemia is one of the risk factor of decrease in bone mass which has been believed to be mediated by hypogonadism. However, the presence of prolactin receptor in human osteosarcoma cell line and primary bone cell culture from mouse calvariae supported the hypothesis of a direct prolactin (PRL) action on bone cells. Therefore, the aim of this study was to investigate the role of PRL and its signal transduction pathway in the regulation of bone metabolism via osteoblast differentiation. Human pre‐osteoblasts (SV‐HFO) that differentiate in a 3‐week period from proliferating pre‐osteoblasts (days 2–7) to extracellular matrix producing cells (days 7–14) which is eventually mineralized (days 14–21) were used. Concentration of PRL mimicked a lactating period (100 ng/ml) was used to incubate SV‐HFO for 21 days in osteogenic medium. Human prolactin receptor mRNA and protein are expressed in SV‐HFO. PRL significantly decreased osteoblast number (DNA content) which was due to a decrease in proliferation. PRL increased osteogenic markers, RUNX2 and ALP in early stage of osteoblast differentiation while decreasing it later suggesting a bi‐directional effect. Calcium measurement and Alizarin red staining showed a reduction of mineralization by PRL while having neither an effect on osteoblast activity nor RANKL/OPG mRNA ratio. We also demonstrated that PRL action on mineralization was not via PI‐3 kinase pathway. The present study provides evidence of a direct effect of prolactin on osteoblast differentiation and in vitro mineralization. J. Cell. Biochem. 107: 677–685, 2009. © 2009 Wiley‐Liss, Inc.     

Fish bone‐derived cell lines: an alternative in vitro cell system to study bone biology

Human bone diseases represent a major health problem worldwide and effective therapies have still to be developed. Despite numerous studies using mammalian systems, cellular and molecular processes governing bone and cartilage homeostasis in vertebrates are still not fully understood. Recently, fish have emerged as a suitable model and a promising alternative to the classical mammalian systems to study vertebrate development, in particular skeletogenesis. To complement in vivo developmental studies and identify signalling pathways involved in development processes, fish cell lines have been developed, in particular bone‐derived cells. This work intends to review what is presently known about fish bone‐derived cell lines, focusing on their relevance for bone biology studies.     

Direct inhibitory and indirect stimulatory effects of RAGE ligand S100 on sRANKL‐induced osteoclastogenesis

Diabetes results in increased fracture risk, and advance glycation endproducts (AGEs) have been implicated in this pathophysiology. S100 proteins are ligands for the receptor of AGEs (RAGE). An intracellular role of the S100 family member S100A4 (Mts1) to suppress mineralization has been described in pre‐osteoblastic MC3T3‐E1 cells. However, S100 proteins could have additional effects on bone. The goal of the current study was to determine effects of increased extracellular S100 on osteoclastogenesis. We first determined the direct effects of S100 on pre‐osteoclast proliferation and osteoclastic differentiation. RANKL‐treated RAW 264.7 cell proliferation and TRAP activity were significantly inhibited by S100, and the number and size of TRAP‐positive multinucleated cells were decreased. We then determined whether S100 could affect osteoclastogenesis by an indirect process by examining effects of conditioned media from S100‐treated MC3T3‐E1 cells on osteoclastogenesis. In contrast to the direct inhibitory effect of S100, the conditioned media promoted RAW 264.7 cell proliferation and TRAP activity, with a trend toward increased TRAP‐positive multinucleated cells. S100 treatment of the MC3T3‐E1 cells for 14 days did not significantly affect alkaline phosphatase, M‐CSF, or OPG gene expression. RANKL was undetectable in both untreated and treated cells. The treatment slightly decreased MC3T3‐E1 cell proliferation. Interestingly, S100 treatment increased expression of RAGE by the MC3T3‐E1 cells. This suggested the possibility that S100 could increase soluble RAGE, which acts as a decoy receptor for S100. This decrease in availability of S100, an inhibitor of pre‐osteoclast proliferation, could contribute to osteoclastogenesis, ultimately resulting in increased bone resorption. J. Cell. Biochem. 107: 917–925, 2009. © 2009 Wiley‐Liss, Inc.