The Sulfated Polysaccharide Fucoidan Stimulates Osteogenic Differentiation of Human Adipose-Derived Stem Cells

Abstract

Human adipose-derived stem cells (hADSCs) are an attractive source for cell therapies, because they can be obtained from aspirated adipose tissues with the capacity of proliferation and differentiation into several mesenchymal lineages under certain conditions. Sulfated polysaccharides, including heparin, modulate osteogenic differentiation of stem cells through the regulation of growth factor binding and signaling pathways. Here, we examined the effects of the sulfated polysaccharide fucoidan on osteogenic differentiation of hADSCs. Strikingly, fucoidan treatment resulted in increased alkaline phosphatase (ALP) activity and alizarin red and von Kossa staining. At the molecular level, fucoidan treatment enhanced the expression of osteogenesis-specific marker genes, including ALP, osteopontin, type I collagen, Runt-related transcription factor 2, and osteocalcin. Furthermore, fucoidan also promoted the osteogenic differentiation of another mesenchymal cell lineage, human amniotic fluid stem cells. These findings strongly suggest that fucoidan enhances osteogenic differentiation of hADSCs and possibly other mesenchymal cell lineages, indicating that it may be a potential candidate for promoting bone regeneration.

Introduction

H uman mesenchymal stem cells are an attractive source for tissue engineering and regenerative medicine, because they have the capacity of self-regeneration and differentiation into several lineages [1 –3]. These cells are found in several tissues, including bone marrow, skeletal muscle, and adipose tissue. Adipose tissue offers an abundant and easily accessible pool of stem cells. Adipose stem cells (ADSCs) obtained from lipoaspirates also have the multi-lineage potential to differentiate into adipogenic, chondrogenic, myogenic, and osteogenic cells [4 –8]. Among the several lineages, osteogenic differentiation of human adipose-derived stem cells (hADSCs) is important for bone repair and regeneration in human diseases such as osteoporosis [9 –13].

Differentiation of hADSCs into osteocytes is regulated by endogenous and environmental factors [14 –16]. Recently, several reports have demonstrated that sulfated polysaccharides, including heparin/heparan sulfate and chondroitin sulfate, influence osteogenesis [17,18]. These polysaccharides were able to stimulate fibroblast proliferation and extracellular matrix deposition in vitro. In particular, some reports have shown the modulation of osteogenic differentiation by heparin. For instance, in a recent study, Jiao et al. demonstrated that heparin sulfate modulates BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts [19].

Fucoidan is a sulfated, fucosylated polysaccharide found mainly in various species of brown seaweed that exhibits some heparin/heparan sulfate properties, including cell proliferation and differentiation [20 –23]. Many of these effects can be attributed to stimulation of hADSC differentiation ability. However, scant data are available on the osteogenic differentiation effect of fucoidan, which has a molecular structure and functions similar to that of heparin. In the recent study by Changotade et al., it was reported that low-molecular-weight (LMW) fucoidan extracts showed potential effects on bone biomaterial osteoconductive properties [24]. However, the functions and mechanisms in osteogenic differentiation of hADSCs by fucoidan treatment have not been previously reported.

On the basis of these findings, the present study was undertaken to investigate the potential effects of fucoidan on hADSC differentiation into osteoblasts through the analysis of matrix deposition and osteogenic marker gene expression.

Materials and Methods

Fucoidan

High-molecular-weight fucoidan (>30 kDa) was purchased from Haewon Biotech, Inc. (Seoul, Korea, www.fucoidan.co.kr) and Sigma-Aldrich Korea Ltd. (Fluka, catalog #, FK47865, Yongin-city, Korea). Fucoidan contains at least 25% organic sulfate and 60% polysaccharide content. Fucoidan was dissolved in DMSO (10 mg/mL), filtered by using a 0.2 μm filter, and then stored at −20°C.

Isolation and cultivation of hADSCs and amniotic fluid stem cells

hADSCs were isolated from subcutaneous adipose tissue based on standard protocols. Tissue specimens were obtained using a protocol approved by the IRB of the Kangbuk Samsung Hospital. The raw adipose tissue was processed according to established methodologies to obtain a stromal vascular fraction. To isolate ADSCs from adipose tissue, samples were digested at 37°C for 30 min with 0.075% type I collagenase (Sigma, St. Louis, MO), followed by inactivation with an equal volume of complete medium containing dulbecco's modified eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and 1% penicillin/streptomycin (Gibco BRL). After centrifugation for 10 min at 1200g, cells were resuspended in complete medium and filtrated through a 100 μm strainer to remove debris. The hADSCs were maintained at 37°C in a humidified atmosphere containing 5% CO₂. To prevent spontaneous differentiation, cells were maintained at subconfluent levels.

Amniotic fluid (AF) samples were collected from women undergoing routine amniocentesis for genetic testing from July 2001 to November 2005. After centrifugation of AF (10 mL) at 1800 rpm for 10 min, the AF pellet was added to 2 mL of CHANG media (Irvine Scientific, Santa Ana, CA) in a culture dish. Informed consent was obtained from all participants.

Immunophenotype of hADSCs

The specific surface antigens of ADSCs in the cultures of passages 5–6 were characterized by flow cytometry analysis. Cells were trypsinized and then incubated with fluorochrome-conjugated antibody on ice for 40 min in the dark. After incubation, the cells were rinsed twice with cold PBS containing 4% FBS and 0.03g/L sodium azide and then analyzed on an FACS Calibur device (BD Biosciences, San Jose, CA). A total of 10⁴ events for each sample were acquired. The following human antibodies were used: HLA-DR (FITC), CD34 (PE), CD45 (PE), CD73 (PE), CD90 (FITC), CD105 (FITC), and CD166 (FITC) (BD Biosciences).

In vitro differentiation of hADSCs

To confirm the differentiation capacity of hADSCs, we induced differentiation into each lineage. For in vitro osteogenic differentiation, cells were incubated in osteogenic medium (OS) consisting of low glucose Dulbecco's modified Eagle's medium (LG-DMEM) supplemented with 10% FBS, 10 mM β-glycerol phosphate (Sigma), 100 nM dexamethasone (Dex; Sigma), and 0.5 μM ascorbate 2-phosphate (Sigma). For in vitro adipogenic differentiation, cells were incubated in adipogenic medium consisting of LG-DMEM supplemented with 10% FBS, 5 μg/mL insulin (Sigma), 1×10⁻⁶ M Dex, and 0.5 μM 3-isobutyl-1-methylxanthine (Sigma). Medium was changed twice a week for 14 days. For in vitro myogenic differentiation, cells were incubated in MCDB131 medium (Welgene, Daegu, Korea) supplemented with 1% FBS and 100 U/mL heparin (Sigma).

Treatment

For fucoidan treatment, hADSCs obtained from 5 passages were replated at 4×10³ cells/cm² in 12-well and 100 mm culture plates. When culture plates became 80% confluent, the culture medium of each group were replaced with each respective lineage induction medium. Fucoidan was added to fuco-groups at a final concentration of 100μg/mL in osteogenic or culture medium, whereas the control group was maintained without fucoidan. We removed and replaced experimental and control media every 3–4 days.

For Dex treatment, after primary culture in the control medium and expansion for 2 passages, the cells were trypsinized and replated into 6-well tissue culture plates at a density of 10⁵ cells per well. The cells were incubated in the control medium for 24 h to adhere them to the plates and then placed in osteogenic medium containing 100 nmol/L of Dex. The osteogenic medium was changed every 2–3 days.

Mineralization assays

For the alkaline phosphatase (ALP) activity assay, cells were fixed with formaldehyde and stained with naphtol AS-BI alkaline solution (Sigma) for 15 min at room temperature. Extracellular matrix mineralization was confirmed with alizarin red S (ARS) staining. Briefly, cells were fixed with 70% ice-cold ethanol for 1 h at 4°C and then stained with 40 mM alizarin red (pH 4.2) for 10 min at room temperature. Von Kossa (VK) staining was used to confirm calcium deposits. Briefly, cells were fixed with 100% ice-cold ethanol, washed in distilled water, and then covered with 5% silver nitrate for 1 h under light. After extensive washing in distilled water, the cells were exposed to 5% sodium carbonate solution for 1 min. The area of mineralization appeared black.

RT-PCR analysis of osteogenic marker genes

About 10⁶ cells were harvested from each group by trypsinization after incubation for 14 days, and the harvested cells were washed with cold PBS solution and centrifuged at 800 g for 10 min at 4°C (more than thrice). We then isolated the RNA from hADSCs and differentiated cells using an RNeasy kit according to the manufacturer's instructions (Qiagen, Valencia, CA). For RNA analysis, equivalent amounts of total RNA served as the template for cDNA synthesis using reverse transcriptase (Invitrogen, Carlsbad, CA). The first strand of cDNA was synthesized from 1 μg of total RNA using a SuperScript^™ III First Strand Kit (Invitrogen) according to the manufacturer's instructions. Gene expression was determined by reverse transcription-PCR (RT-PCR). We confirmed the expression of osteogenic-specific marker genes, including ALP, osteopontin (OPN), type I collagen (Col I), and osteocalcin (OCN). Primers used for PCR reactions are shown in Table 1.

Table 1.

Primer Sequences Used in This Study

Name	Forward sequence	Reverse sequence
ALP	5′- ACCATTCCCACGTCTTCACATTTG-3′	5′- AGACATTCTCTCGTTCACCGCC-3′
OPN	5′- AGCCAGGACTCCATTGACTCGAAC-3′	5′-GTTTCAGCACTCTGGTCATCCAGC-3′
Col I	5′-GGACACAATGGATTGCAAGG-3′	5′-TAACCACTGCTCCACTCTGG-3′
OCN	5′-ATGAGAGCCCTCACACTCCTC-3′	5′-GCCGTAGAAGCGCCGATAGGC-3′
α-SMA	5′-CCCCATCTATGAGGGCTATGC-3′	5′-CGGCCAGCCAGATCC-3′
PPAR-γ	5′-GCTGTTATGGGTGAAACTCTG-3′	5′-TCGCAGGCTCTTTAGAAACTC-3′
Runx2	5′-TTACTTACACCCCGCCAGTC-3′	5′-TATGGAGTGCTGCTGGTCTG-3′
GAPDH	5′-CGGAGTCAACGGATTTGGTCGTAT-3′	5′-AGCCTTCTCCATGGTGGTGAAGAC-3′

ALP, alkaline phosphatase; OPN, osteopontin; Col I, type I collagen; OCN, osteocalcin; α-SMA, α-smooth muscle actin; PPAR-γ, peroxisome proliferator-activated receptor gamma; Runx2, runt-related transcription factor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Results

hADSCs have immunophenotypes and differentiation capacities of typical mesenchymal stem cells

The expression of cell surface antigens was evaluated by flow cytometry on hADSCs. hADSCs showed positive expression of the mesenchymal stem cells (MSC)-specific surface markers CD73, CD90, CD105, and CD166, but did not express HLA-DR, CD34, and CD45, which are hematopoietic cell specific markers (Fig. 1A). It shows that hADSCs express the typical bone marrow-derived MSC immunophenotypes. Additionally, three of the hADSC cultures were tested for their ability to differentiate into other cell types. Beyond doubt, hADSCs were capable of differentiating into multiple lineages, including osteogenic, adipogenic, and myogenic cells. Moreover, we confirmed the specific marker gene expression [OCN for osteogenesis, α-smooth muscle actin (α-SMA) for myogenesis, and peroxisome proliferator-activated receptor-γ (PPAR-γ) for adipogenesis] of each lineage through an RT-PCR assay (Fig. 1B). These data indicated that hADSCs showed multi potentiality for differentiation to other cell types.

FIG. 1.

Stemness of hADSCs. (A) The marker phenotype of ADSC cultures from human subcutaneous adipose tissue was analyzed using direct flowcytometry. Cells were stained with various antibodies. (B) The differentiation capacity of hADSCs. Cells were incubated in osteogenic medium, myogenic medium, and adipogenic medium. After 14 days, total RNA was isolated from cells, and cDNA was synthesized for RT-PCR. hADSCs, human adipose-derived stem cells. RT-PCR, reverse transcription-polymerase chain reaction.

Fucoidan promotes osteogenic differentiation

Fucoidan is a highly branched, sulfated polysaccharide based mainly on L-fucose units with a similar chemical structure to heparin [25]. The ordered fucoidans mostly contain a linear backbone composed of (1→2)-α-L-fucose, (1→3)-α-L-fucose, and (1→4)-α-L-fucose [26,27]. A disaccharide repeat unit in the structure of fucoidan and heparin is diagrammed in Figure 2A.

FIG. 2.

Fucoidan induced osteogenic differentiation of hADSCs. Cells were cultured in DMEM control medium or osteogenic medium (OS) in the presence or absence of fucoidan (Fuco) for 14 days. (A) Schematic structures of fucoidan and heparin disaccharide repeat units. (B) Histochemical staining of hADSCs. From top to bottom: Alkaline phosphatase (ALP), Alizarin red S (ARS), and von Kossa (VK) staining. (C) hADSCs expressed osteogenic marker genes. Total RNA was isolated from cells, and cDNA was synthesized for RT-PCR. Scale bars:100 μm. DMEM, Dulbecco's modified Eagle's medium. Color images available online at www.liebertonline.com/scd

To investigate whether fucoidan has an influence on osteogenic differentiation of hADSCs, we performed various staining assays associated with osteogenic differentiation. As shown in Figure 2B, hADSCs cultured with fucoidan in osteogenic medium (OS+Fuco) were strongly positive for ALP, ARS, and VK staining assays, as compared with hADSCs cultured in osteogenic medium without fucoidan (OS). These data indicated that fucoidan treatment prominently induced osteogenic differentiation of hADSCs, showing strong boosting effects of fucoidan on osteogenic differentiation. hADSCs cultured in DMEM with fucoidan (ADSC+Fuco) almost did not show specific reactions in any staining assays. In contrast, hADSCs highly expressed osteogenic marker genes, including ALP, Col I, OPN, and Runt-related transcription factor 2 (Runx2), when cultured with fucoidan in osteogenic medium (OS+Fuco) for 14 days, as compared with hADSCs cultured in osteogenic medium without fucoidan (Fig. 2C). These data further supported the results just cited.

In addition, we examined whether fucoidan has a synergistic effect on hADSC osteogenic differentiation when co-treated with Dex in osteogenic medium. Dex has been known to be an inducer for osteogenic differentiation of hADSCs [8]. Fucoidan-induced osteogenic differentiation showed no difference in the absence (OS−Dex) or presence of Dex (OS+Dex) (Fig. 3). Cells cultured in fucoidan-containing osteogenic medium (OS) without Dex (OS−Dex) for 4 weeks were markedly positive in both ARS and ALP staining assays, compared with the case in Dex-containing osteogenic medium (OS+Dex) in the absence of fucoidan. These data indicate that fucoidan may be an ideal substitute for Dex in osteogenic differentiation of hADSCs.

FIG. 3.

Effects of fucoidan (Fuco) on matrix deposition and ALP activity of hADSCs. Cells were cultured in DMEM medium either with or without fucoidan (100 μg/mL) or dexamethasone (Dex) (0.1 μM)/fucoidan-containing or not osteogenic medium (OS) for 14 or 28 days. hADSCs were stained by ARS or ALP for extracellular matrix mineralization. Color images available online at www.liebertonline.com/scd

Fucoidan also promotes osteogenic differentiation of other mesenchymal cell lineage, human amniotic fluid stem cells

Next, to confirm the beneficial effect of fucoidan on osteogenic differentiation of stem cells, we conducted the VK staining assay in human amniotic fluid stem cells (hAFCs). As shown in Figure 4A, hAFCs cultured with fucoidan in osteogenic medium (OS) were strongly positive in the VK staining assay over time, as compared with AF stem cells in osteogenic medium without fucoidan (Fig. 4A). These data suggested that fucoidan may promote osteogenic differentiation regardless of stem cell type. Additionally, to investigate the effect of fucoidan on differentiation into other lineages of hAFCs, we conducted the RT-PCR assay using either a myogenic α-SMA or an adipogenic PPAR-γ marker gene. As shown in Figure 4B, AF stem cells cultured in each induction medium containing fucoidan (Induction+Fuco) expressed the α-SMA gene to a similar level and the PPAR-γ gene to a slightly reduced level compared with AF stem cells in induction media without fucoidan (Induction). These results suggested that fucoidan may also induce osteogenic differentiation of other mesenchymal cell lineages.

FIG. 4.

Fucoidan also enhanced osteogenic differentiation of human amniotic fluid stem cells (hAFCs). (A) hAFCs were cultured in DMEM control medium or osteogenic medium (OS) in the presence or absence of fucoidan (100 μg/mL) for 7 (1 week), 14 (2 weeks), and 28 days (4 weeks). Cells were stained by VK for calcium deposition. (B) Additionally, hAFCs were cultured in DMEM control medium, myogenic/adipogenic medium (Induction), or fucoidan-containing myogenic/adipogenic medium (Induction+Fuco) for 14 days. Total RNA was extracted, and cDNA was synthesized for RT-PCR. Scale bars: 100 μm.

Discussion

The potential of the sulfated polysaccharide fucoidan on hADSC differentiation into osteogenic lineages

Our results showed that fucoidan significantly induced osteogenic differentiation of hADSCs, increasing expression of osteogenic marker genes, including ALP, Col I, OPN, and Runx2. hADSCs from adipose tissue are defined as multipotential adult stem cells and can be differentiated into various types of osteoblasts, chondrocytes, adipocytes, muscle cells, and neural cells [4 –8]. Our data were consistent with the reports just provided (Fig. 1).

Sulfated polysaccharides such as glucosamine sulfate, heparin, and heparin sulfate are known to be macromolecules connected with the cell surface and the extracellular matrix [18,19,28]. These polysaccharides have been shown to interact directly with numerous growth factors, including BMPs, and to stimulate fibroblast proliferation [19]. Fucoidan is one of the representative sulfated polysaccharides extracted from marine brown algae. Recent evidence has shown that fucoidan has numerous biological properties, such as antioxidative, anticoagulant, anti-thrombotic, anti-inflammatory, antithrombotic, and anti-tumoral activities [27,29 –31]. Furthermore, fucoidan was reported to induce osteoblastic cell differentiation, and LMW fucoidan promotes human osteoblast proliferation and collagen fibrillation [24]. Here, we have demonstrated that high-molecular-weight fucoidan strongly influences osteogenic differentiation of hADSCs. hADSCs treated with fucoidan were markedly positive in ALP, ARS, and VK staining, and additionally significantly expressed all the osteogenic marker genes (Fig. 2C). ALP plays a crucial role as a phenotypic marker for early stage bone formation, and increased ALP expression correlates with increased bone formation [32]. Col I is an early osteogenic marker and the most abundant ECM protein found in osteoblasts. Additionally, OPN is a bone-related marker gene during osteoblastic differentiation. Runx2 has been identified as the primary transcription factor for osteoblastic differentiation [33]. In vitro studies have shown that Runx2 positively controls Col I, OCN, and OPN expression [34]. These reports infer that Runx2 might play a very important role in osteogenic induction processes of hADSCs.

Mineralization of extracellular matrix, which is the last phase in bone formation, was assessed by ARS and VK staining assays. According to these results (Figs. 2 and 3), fucoidan increased mineralized extracellular matrix and Ca²⁺ deposition. These findings indicate that fucoidan-stimulated hADSCs differentiate into osteoblasts. Dex-containing osteogenic medium is usually used for osteoblastic differentiation of MSCs and hADSCs [8]. In this study, we found that fucoidan further induced osteogenic differentiation of hADSCs (Fig. 3), compared with Dex. Recently, researchers have wondered about the efficacy of glucocorticoids such as Dex, which mediates the osteogenic process of skeletal progenitor cells and processed lipoaspirate cells [7,35]. In this regard, fucoidan might be an ideal inducer, enabling Dex to excel in osteogenic differentiation of hADSCs.

The efficacy of fucoidan on osteogenic differentiation using human amniotic fluid-derived stem cells

In addition, we also confirmed the efficacy of fucoidan on osteogenic differentiation using hAFCs. hAFCs cultured with fucoidan in osteogenic medium were strongly positive in the VK staining assay. These findings suggested that fucoidan may induce osteogenic differentiation regardless of cell type.

As shown in Figure 4B, fucoidan did not induce other differentiation pathways of AFCs, although it slightly suppressed adipogenesis. Kim et al. showed that fucoidan suppressed adipogenesis via the mitogen-activated protein kinase pathway in 3T3-L1 preadipocytes, a finding that is in part consistent with our current data [36].

In conclusion, we could demonstrate that fucoidan plays an important role in osteogenic differentiation of adult stem cells. On the basis of these findings, this study suggests that fucoidan could be possible source as a therapeutic agent for bone repair and regeneration.

Footnotes

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2009-0076696). The authors are grateful to Professor Il Hwan Oh (Catholic University, Korea) for helping with the flow cytometry analysis of hADSCs.

Author Disclosure Statement

The authors have declared that no competing interests exist.

References

De Ugarte

, Morizono

, Elbarbary

, Alfonso

, Zuk

, Zhu

, Dragoo

, Ashjian

, Thomas

et al. 2003. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs, 174:101–109.

Oishi

, Noguchi

, Yukawa

, Hayashi

. 2009. Differential ability of somatic stem cells. Cell Transplant, 18:581–589.

Satija

, Gurudutta

, Sharma

, Afrin

, Gupta

, Verma

, Singh

, Tripathi

. 2007. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 16:7–23.

Bunnell

, Estes

, Guilak

, Gimble

. 2008. Differentiation of adipose stem cells. Methods Mol Biol, 456:155–171.

Meliga

, Strem

, Duckers

, Serruys

. 2007. Adipose-derived cells. Cell Transplant, 16:963–970.

Strem

, Hicok

, Zhu

, Wulur

, Alfonso

, Schreiber

, Fraser

, Hedrick

. 2005. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med, 54:132–141.

Zuk

, Zhu

, Ashjian

, De Ugarte

, Huang

, Mizuno

, Alfonso

, Fraser

, Benhaim

, Hedrick

. 2002. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 13:4279–4295.

Zuk

, Zhu

, Mizuno

, Huang

, Futrell

, Katz

, Benhaim

, Lorenz

, Hedrick

. 2001. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 7:211–228.

Elabd

, Chiellini

, Massoudi

, Cochet

, Zaragosi

, Trojani

, Michiels

, Weiss

, Carle

et al. 2007. Human adipose tissue-derived multipotent stem cells differentiate in vitro and in vivo into osteocyte-like cells. Biochem Biophys Res Commun, 361:342–348.

10.

Hattori

, Sato

, Masuoka

, Ishihara

, Kikuchi

, Matsui

, Takase

, Ishizuka

, Kikuchi

, Fujikawa

. 2004. Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source. Cells Tissues Organs, 178:2–12.

11.

Weinzierl

, Hemprich

, Frerich

. 2006. Bone engineering with adipose tissue derived stromal cells. J Craniomaxillofac Surg, 34:466–471.

12.

Zhu

, Kohan

, Bradley

, Hedrick

, Benhaim

, Zuk

. 2009. The effect of age on osteogenic, adipogenic and proliferative potential of female adipose-derived stem cells. J Tissue Eng Regen Med, 3:290–301.

13.

Liao

, Li

, Wang

, Yanoso

, Niyibizi

. 2008. Distribution of murine adipose-derived mesenchymal stem cells in vivo following transplantation in developing mice. Stem Cells Dev, 17:303–314.

14.

Jethva

, Otsuru

, Dominici

, Horwitz

. 2009. Cell therapy for disorders of bone. Cytotherapy, 11:3–17.

15.

Panetta

, Gupta

, Longaker

. Bone regeneration and repair. Curr Stem Cell Res Ther, 5:122–128.

16.

Seong

, Kim

, Park

, Kwon

, Mantalaris

, Hwang

. Stem cells in bone tissue engineering. Biomed Mater, 5:062001.

17.

Blanquaert

, Saffar

, Colombier

, Carpentier

, Barritault

, Caruelle

. 1995. Heparan-like molecules induce the repair of skull defects. Bone, 17:499–506.

18.

Manton

, Leong

, Cool

, Nurcombe

. 2007. Disruption of heparan and chondroitin sulfate signaling enhances mesenchymal stem cell-derived osteogenic differentiation via bone morphogenetic protein signaling pathways. Stem Cells, 25:2845–2854.

19.

Jiao

, Billings

, O'Connell

, Kaplan

, Shore

, Glaser

. 2007. Heparan sulfate proteoglycans (HSPGs) modulate BMP2 osteogenic bioactivity in C2C12 cells. J Biol Chem, 282:1080–1086.

20.

Durand

, Helley

, Al Haj Zen

, Dujols

, Bruneval

, Colliec-Jouault

, Fischer

, Lafont

. 2008. Effect of low molecular weight fucoidan and low molecular weight heparin in a rabbit model of arterial thrombosis. J Vasc Res, 45:529–537.

21.

, Lu

, Wei

, Zhao

. 2008. Fucoidan: structure and bioactivity. Molecules, 13:1671–1695.

22.

Matou

, Helley

, Chabut

, Bros

, Fischer

. 2002. Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro. Thromb Res, 106:213–221.

23.

Senni

, Gueniche

, Foucault-Bertaud

, Igondjo-Tchen

, Fioretti

, Colliec-Jouault

, Durand

, Guezennec

, Godeau

, Letourneur

. 2006. Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis. Arch Biochem Biophys, 445:56–64.

24.

Changotade

, Korb

, Bassil

, Barroukh

, Willig

, Colliec-Jouault

, Durand

, Godeau

, Senni

. 2008. Potential effects of a low-molecular-weight fucoidan extracted from brown algae on bone biomaterial osteoconductive properties. J Biomed Mater Res A, 87:666–675.

25.

Berteau

, Mulloy

. 2003. Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology, 13:29R–40R.

26.

Sithranga Boopathy

, Kathiresan

. 2010. Anticancer drugs from marine flora: an overview. J Oncol, 2010:214186.

27.

Chevolot

, Mulloy

, Ratiskol

, Foucault

, Colliec-Jouault

. 2001. A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae. Carbohydr Res, 330:529–535.

28.

Ling

, Murali

, Dombrowski

, Haupt

, Stein

, van Wijnen

, Nurcombe

, Cool

. 2006. Sulfated glycosaminoglycans mediate the effects of FGF2 on the osteogenic potential of rat calvarial osteoprogenitor cells. J Cell Physiol, 209:811–825.

29.

Cumashi

, Ushakova

, Preobrazhenskaya

, D'Incecco

, Piccoli

, Totani

, Tinari

, Morozevich

, Berman

et al. 2007. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology, 17:541–552.

30.

Maruyama

, Tamauchi

, Iizuka

, Nakano

. 2006. The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu) Planta Med, 72:1415–1417.

31.

Mourao

. 2004. Use of sulfated fucans as anticoagulant and antithrombotic agents: future perspectives. Curr Pharm Des, 10:967–981.

32.

Walsh

, Jefferiss

, Stewart

, Jordan

, Screen

, Beresford

. 2000. Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4. Bone, 27:185–195.

33.

Komori

, Yagi

, Nomura

, Yamaguchi

, Sasaki

, Deguchi

, Shimizu

, Bronson

, Gao

et al. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 89:755–764.

34.

Ducy

, Zhang

, Geoffroy

, Ridall

, Karsenty

. 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell, 89:747–754.

35.

Cooper

, Hewison

, Stewart

. 1999. Glucocorticoid activity, inactivity and the osteoblast. J Endocrinol, 163:159–164.

36.

Kim

, Lee

. 2010. Fucoidan, a sulfated polysaccharide, inhibits adipogenesis through the mitogen-activated protein kinase pathway in 3T3-L1 preadipocytes. Life Sci, 86:791–797.