REGULAR ARTICLE

Chitosan as a potential osteogenic factor comparedwith dexamethasone in cultured macaque dental pulp stromalcells

Lisa R. Amir & Dewi F. Suniarti & Sri Utami & Basril Abbas

Received: 5 September 2013 /Accepted: 3 June 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Chitosan, a natural biopolymer derived from chitin,is considered a promising scaffold material for bone tissueengineering. The ability of chitosan to promote the osteogenicdifferentiation of dental pulp stromal/stem cells (DPSCs) isunknown. We have evaluated the potential of chitosan toinduce the osteogenic differentiation of macaque DPSCs incomparison with that of dexamethasone. DPSCs were culturedin mineralizing medium supplemented with 5 or 10 !g/mlchitosan or with 1 or 10 nM dexamethasone. The metabolicactivity of DPSCs was measured by MTT assay. Their osteo-genic differentiation was determined by the number of tran-scripts of RUNX2, alkaline phosphatase (ALP), and COL1A1by using real-time polymerase chain reaction, by alizarin redstaining for mineral deposition, and by the ALP activity re-leased into the medium for their ability to supportbiomineralizaton. Addition of chitosan to the mineralizingmedium significantly increased DPSCs metabolism after 7and 14 days of culture (P!0.0001). Chitosan at 5 !g/ml alsosignificantly enhanced RUNX2 and ALP mRNA but notCOL1A1 mRNA; chitosan tended to increase the release ofALP hydrolytic enzyme activity into the medium during thefirst week. Dexamethasone upregulated the osteogenicmarkers tested. Mineral deposition was similar in the chitosanand dexamethasone groups and was not statistically different

from that of the mineralizing control group. Thus, the potentialof chitosan to stimulate DPSCs proliferation and early osteo-genic differentiation is comparable with that of dexametha-sone, but mineralization remains unaffected by chitosan treat-ment. In addition to its role as a three-dimensional scaffold forosteogenic cells in vivo, chitosan might also stimulate DPSCsproliferation and early osteogenic differentiation in vitro.

Keywords Chitosan . Dexamethasone . Dental pulp stromalcells . Osteogenic supplement . Macaque

Introduction

Tissue engineering is currently being developed to treat largebone defects. The technique involves the seeding of progenitorcells onto a scaffold material that serves as a temporary extra-cellular matrix to facilitate attachment, proliferation, and differ-entiation of the cells to form the desired tissues (Howard et al.2008). The ideal scaffold should be biocompatible, biodegrad-able at a similar rate to new tissue formation and should mimicthe natural environment of the tissue in vivo (Yang et al. 2001;Sachlos and Czernuszka 2003). Chitosan is now being activelyinvestigated as a promising natural biopolymer scaffold mate-rial (Lee et al. 2000; Seol et al. 2004; Cho et al. 2004).

Chitosan is a partially deacetylated form of chitin, apolysaccharide that biomineralizes and forms the shell ofcrustaceans. It appears to be biocompatible and is degradedby enzymes into oligosaccharides that are rapidly resorbed. Itforms an insoluble complex with connective tissue moleculessuch as collagen and glycosaminoglycans to form a porousinterconnected three-dimensional (3D) structure, making thisbiomolecule attractive as a scaffolding material for tissueengineering purposes (Mi et al. 2006; Yamada et al. 2007).Chitosan is highly versatile and useful for a wide range ofapplications: as injectable particles in solution, as solid porous

This study was financially supported by a Universitas IndonesiaResearch Excellence Grant (DRPM/RUUI Unggulan/2010/I/4150).

L. R. Amir (*) :D. F. Suniarti : S. UtamiDepartment of Oral Biology, Faculty of Dentistry, UniversitasIndonesia, Salemba Raya No.4, Jakarta Pusat 10430, Indonesiae-mail: lisa.amir@gmail.com

L. R. Amire-mail: lisa.amir@ui.ac.id

B. AbbasCenter for Application of Isotope and Radiation Technology,National Atomic Energy Agency, Jakarta, Indonesia

Cell Tissue ResDOI 10.1007/s00441-014-1938-1

3D structures, or as flat 2D membranes (Lee et al. 2000; Parket al. 2000a, 2000b; Amaral et al. 2005; Jiang et al. 2006). Thefeasibility of utilizing this biopolymer alone or in combinationwith other biomaterials as a scaffold for the purpose ofbone/cartilage tissue engineering has been demonstrated(Hoemann et al. 2005, 2007; Jiang et al. 2006). Integratingchitosan with other biomaterials is important in order to im-prove its biological and mechanical properties suitable forclinical application. Chitosan has been used as a carrier forgrowth factor delivery (Lee et al. 2002; Park et al. 2000a,2000b). The sequential release of bone morphogeneticprotein-2 (BMP-2) followed by the sustained delivery ofinsulin-like growth factor-1 in a chitosan gel/gelatin micro-sphere increases early osteogenic differentiation (Park et al.2000b). Other groups have reported the incorporation of chito-san with calcium-phosphate-based ceramic materials in thedevelopment of composite scaffolds. Mineralized tissue forma-tion has been observed in chitosan/tricalcium phosphate spongeseeded with fetal rat calvarial osteoblasts (Lee et al. 2000). Theapplication of chitosan powder onto bone fractures in an animalmodel increases the regeneration process and stimulates osteo-genesis in vivo (Khanal et al. 2000). Similar results have beenobserved in studies in vitro (Seol et al. 2004; Amaral et al.2006; Jiang et al. 2006; Wang et al. 2008). The differentiationof an osteoblast cell line is identical or even increased whencells are cultured on chitosan-coated disks, chitosan sponges, orchitosan membrane (Seol et al. 2004; Amaral et al. 2006).

The numerous reports of the chemical and biological prop-erties of chitosan demonstrate the increasing interest of thisbiopolymer for its potential use in bone tissue regeneration.However, whether chitosan can be used as an osteogenicfactor to stimulate the differentiation of dental pulp stromal/stem cells (DPSCs) into an osteoblastic lineage is not currentlyclear. DPSCs consist of a stroma-cell-like population thatdisplays multi-differentiation potential including that ofbone-forming cells (Gronthos et al. 2000). DPSCs have greatclinical potential as a source of mesenchymal stromal cells(MSCs) that can be relatively easily harvested. MSCs can bestimulated into the osteogenic lineage by using glucocorticoid,BMP, and other growth factors (Cheng et al. 1994; Lee et al.2000; Gharibi and Hughes 2012). Typical osteogenic differ-entiation medium contains dexamethasone for transcriptionalactivation, ascorbic acid for collagen synthesis, and "-glycerophosphate supplementation for mineralization(Geesin et al. 1991; Igarashi et al. 2004). Dexamethasoneis a synthetic glucocorticoid that is frequently used toinduce the differentiation of osteoblasts (Guzmán-Morales et al. 2009). Whether chitosan can be used asan alternative for dexamethasone in osteogenic differen-tiation media for DPSCs is unknown. The aim of thestudy has been to compare the effect of chitosan withdexamethasone with respect to their ability to induceosteogenic differentiation in DPSCs.

Materials and methods

Shrimp chitosan was obtained from the Center for Applicationof Isotope and Radiation Technology, Indonesia NationalAtomic Energy Agency. Briefly, chitosan was prepared bythe alkaline deacetylation of chitin with 50 % sodium hydrox-ide at 90ºC for 8 h and precipitated with 30 % hydrochloricacid. The degree of deacetylation was approximately 80 % asmeasured by Fourier transform infrared spectroscopy.Chitosan was sterilized by 25 kGy of gamma irradiation.The procedure reduced the molecular weight of chitosan froman initial 75 kDa to 34.8 kDa following gamma irradiation asdetermined by measuring its intrinsic viscosity (Kasaai et al.2000). Chitosan particles (10 mg) were dissolved in 10 ml1 % v/v acetic acid (0.1 M) on a rotary mixer for 8 h and werekept as a stock solution (1 mg/ml). Final chitosan concentra-tions of 5 and 10 !g/ml were diluted in cell culture medium.Our pilot study indicated that 5 !g/ml was the minimumchitosan concentration that induced metabolic activity inDPSCs. Dexamethasone (Sigma, USA; 1 mg) was dissolvedin 1 ml ethanol (2.5 mM) and further diluted 25! in sterilemedium to give a stock solution (100 !M). The final concen-trations of dexamethasone used in this study were 1 nM and10 nM: the common doses of dexamethasone to inducein vitro osteogenic differentiation (Guzmán-Morales et al.2009; Chadipiralla et al. 2010). The final ethanol concentra-tion did not exceed 0.05 % v/v ethanol (Castañeda and Kinne2000). The "-glycerol phosphate, ascorbate-2-phosphate, andalizarin red were purchased from Sigma-Aldrich. Dulbecco’smodified Eagle medium, fetal bovine serum, penicillin-streptomycin, fungizone, trizol, and collagenase I were pur-chased from Gibco (USA). We obtained real-time polymerasechain reaction (PCR) primers (Table 1) from Invitrogen, USA,dispase from Roche (Japan), CD70, CD90, and CD45 fromBD Biosciences (USA), an alkaline phosphatase (ALP) kitfrom Bioassay systems (USA), a cDNA synthesis kit fromThermoFisher Scientific (Lithuania), and Sybr Green fromApplied Biosystems (UK).

DPSC isolation and identification

DPSCs were collected from threeMacaque nemestrima upperand lower first incisors (6 teeth) at the Primate ResearchCenter, Bogor Agricultural University, Indonesia. This studywas a survival study, whereby all animals remained in thefacility following tooth extraction. The procedure receivedethical clearance from the Animal Care and Use Committee(ACUC No.11-B005-IR). Dental pulp tissues were removedfrom the root canal by an extirpation needle and finelychopped with a sterile scalpel followed by enzymatic degra-dation with 3 mg/ml collagenase I and 4 mg/ml dispase at37 °C under 5 % CO2 in an incubator for 1 h. The tissue wasdissociated by serial pipetting every 15–20 min. Cells were

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subsequently cultured at 37 °C, under 5 % CO2 in air, in anincubator until they reached about 90 % confluency.Subsequently, 107 cells were prepared for cell sorting byfluorescence-activated cell sorting (FACS) in an FACSAria(BD Biosciences, USA) by using CD71, CD90, and CD45antibodies (BD Biosciences). CD71- and CD90-positive cellsand CD45-negative cells indicated the presence of DPSCs.Passages 4–6 were used for the experiments.

Cell culture

DPSCs (1 ! 104) were cultured in 24-well plates (Nunc,Roskilde, Denmark) in mineralizing and experimental mediaas described above. Mineralizing medium (MM) consisted ofDMEM supplemented with 10 % FBS, penicillin 100 IU/ml,100 !g/ml streptomycin, 1.25!g/ml fungizone, 100 !g ascor-bic acid, and 10 mM "-glycerophosphate, and cells grown inthis medium served as the control group. The experimentalgroups consisted of cells grown in MM with the addition ofchitosan (5, 10 !g/ml) or dexamethasone (1, 10 nM). DPSCswere incubated in MM, in chitosan-containing medium, or indexamethasone-containing medium for 7 and 14 days, and themedia were refreshed twice a week. Cell culture media werethen collected for protein analysis, whereas the attached cellswere kept in TRIzol for 24 h in a freezer at "80 °C for RNAisolation.

MTT assay

Cell metabolism was analyzed by using the MTT (3–[4,5-dimethylthiazol-2yl]"2,5-diphenyl-2H-tetrazolium bromide)assay and was used as an indirect measure of the cellproliferation of the cells. DPSCs (1 ! 103) were plated in 96-well plates and cultured in the control or experimental mediumas described above for 7 and 14 days. MTT assay was per-formed by adding 15 !l MTTsolution (5 mg/ml; Sigma, USA)to each well, followed by incubation for 3 h at 37 °C under 5 %CO2. Next, isopropopanol was added, followed by incubationfor 1 h on a shaker at room temperature to dissolve the formedformazan crystals. The optical densities (OD) of the sampleswere determined by using a microplate reader (Benchmark,

Biorad) at 655 nm. Data were corrected for blank valueswithout cells. Samples were examined in quadruplicate, andthe experiments were repeated twice.

ALP activity assay

ALP activity in DPSC culture media was detected by measur-ing the release of p-nitrophenol (pNP) from p-nitrophenylphosphate (pNPP; QuantiChrom Alkaline PhosphataseAssay Kit; Bioassay Systems, USA). Total protein in thecollected medium was measured by the Bradford assay (Bio-Rad protein assay kit; Bio-Rad, USA), and the absorbancevalue was calculated at 595 nm by using a microplate reader(Benchmark, Bio-Rad). Total protein was standardized at40 !g/ml and transferred to 96-well plates. The reaction mixconsisted of assay buffer (pH 10.5), 5 mMmagnesium acetate,and 10 mM pNPP liquid substrate was added to each of thesample wells. Spectrophotometric quantification of pNPP wasperformed in a microplate reader at 405 nm. ALP activity ofthe samples is presented in International units per liter (IU/l).

Alizarin red staining

At day 14, mineral deposition was examined by alizarin redstaining. Cells in 24-well plates were washed with phosphate-buffered saline and fixed with cold ethanol for 30 min. Thefixed cells were incubated in 2 % alizarin red in Milli-Q water(pH 4.2) for 20 min at room temperature and rinsed twice toremove non-specific staining. Images were captured by usinga digital camera (Canon, Japan). Red staining representingmineralized nodules was quantified by using ImageJ 1.45r(National Institute of Health, Bethesda, Md., USA). Sampleswere measured quadruplicate, and the experiments were re-peated twice.

RNA isolation and real-time PCR

Total RNA from cultured cells was isolated by using TRIzolreagent (Gibco, USA); 5 !g glycogen (Invitrogen, USA) wasadded to isopropanol to increase RNA yield. The RNA con-tent was determined by measuring the absorbance in water at

Table 1 Primers used for real-time polymerase chain reaction Gene Primer Sequence (5! to 3!)

ALP (alkaline phosphatase) Forward gCTTCAAACCgAgATACAAgCA

Reverse gCTCgAAgAgACCCAATAggTAgT

RUNX2 (Runt-related transcription factor-2) Forward ATgCTTCATTCgCCTCAC

Reverse ACTgCTTgCAgCCTTAAAT

COL1A1 (collagen type 1A1) Forward TCCAACgAgATCgAgATCC

Reverse AAgCCgAATTCCTggTCT

GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) Forward ATggggAAggTgAAggTCg

Reverse TAAAAgCAgCCCTggTgACC

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260 nm with a spectrophotometer (Ultrospec 4300 pro,Amersham Pharmacia Biotech, UK). cDNA synthesis wasperformed by PCR (iCycler Biorad) with 2 !g total RNA ina 20-!l final reaction volume consisting of 5 !M randomhexamer primer, 5 !M Oligo (dT)18 primer, 5! reactionbuffer, 20 U/!l RiboLock RNase Inhibitor, 1 nM dNTPMix, 200 U/!l RevertAid M-MuLV reverse transcriptase(RT). The reverse transcription step was performed by incu-bating the RNA at 70ºC for 5 min, followed by addition ofM-MuLVRTmix and incubation at 37ºC for 1 h and then at 70ºCfor another 10 min to deactivate the enzyme.

Real-time PCR was performed on a StepOne Real-TimePCR System (Applied Biosystems, USA). The gene for thehousekeeping enzyme glyceraldehyde-3-phosphate dehydro-genase (GAPDH) served as the endogenous reference to nor-malize RUNX2, ALP, and COL1A1 expression (Table 1). Foramplification of the PCR product, 10 ng cDNAwas added tothe PCR Master Mix consisting of SYBR Green I Dye and300 nM of each primer in a final volume of 15 !l. The cycleconditions were as follows: denaturation at 95 °C for 15 sfollowed by an annealing and extension step at 60ºC for 1 minfor 45 cycles. Relative expression was calculated by using thecomparative CT method. Relative expressions of the targetgenes were expressed as 2–(##CT) as described by Livak andSchmittgen (2001). RT-PCR products were analyzed byelectophoresis in 2 % agarose gels and visualized by geldocumentation (Quantity One Universal Hood; Bio-Rad,USA).

Statistical analysis

Data were calculated by using GraphPad Prism 6 for Mac OSX and are presented as means and standard deviation. Themean values were analyzed for their normality by using aShapiro-Wilk normality test. All data passed the normalitytest and were tested for significant differences by using aone-way analysis of variance, except for the data concerningthe relative expression of RUNX2 mRNA at day 7 for whichwe used the non-parametric Kruskal-Wallis test. Significancewas accepted at P!0.05.

Results

Effect of chitosan and dexamethasone on DPSCs metabolism

First, we analyzed chitosan metabolism as an indirect measureof DPSCs proliferation. The metabolism of DPSCs was mark-edly increased in the chitosan group compared with the con-trol group (P!0.05; Fig. 1a, b). Both chitosan concentrationsenhanced MTT values to the same degree for 2 weeks ofculture. Dexamethasone gave the same values as both

chitosan concentrations at 1 n,M but significantly lowervalues at 10 nM in comparison with the 5 !g/ml chitosangroup (Fig. 1a).

Fig. 1 Metabolism of dental pulp stromal cells (DPSCs) after incubationwith chitosan (Dex dexamethasone, MM mineralizing medium). a Cellmetabolism at day 7. b Cell metabolism at day 14. Note the higher cellmetabolism in the chitosan group at days 7 and 14.Means±SD; *P!0.05,**P!0.01, ****P!0.0001

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Effect of chitosan and dexamethasone on osteogenicdifferentiation of DPSCs

Osteogenic differentiation of DPSCs was analyzed by mea-suring the normalized expression of RUNX2, ALP, andCOL1A1 mRNA and the activity of ALP in the culture medi-um (Figs. 2, 3). The activity of ALP in the medium had atendency to increase in both the chitosan and dexamethasonegroups compared with the mineralizing control group, al-though this was not statistically significant. ALP activitywas found to be highest in the 5 !g/ml chitosan group, witha six-fold increase, and the least in the dexamethasone groupwith a three-fold increase compared with the mineralizingcontrol group (P>0.05).

The expression patterns of RUNX2, ALP, and COL1A1mRNA are presented in Figs. 2c, d, 3, 4. ALP mRNA expres-sion and that of the osteoblast transcription factor for RUNX2mRNAwere significantly upregulated in the 5 !g/ml chitosan

group (P!0.05, P!0.001, Figs. 2c, 3a). Similarly, 10 nMdexamethasone induced RUNX2 mRNA expression after1 week of culture, and much higher expression was foundafter 2 weeks. The lower concentration of chitosan used in thisstudy seemed to induce higher RUNX2 mRNA expression.Dexamethasone at 10 nM upregulated COL1A1 mRNA ex-pression four-fold (P!0.01, Fig. 3c, d). A two- and three-foldincrease in COL1A1 mRNA expression was found in thechitosan group after 1 week of culture (P>0.05). Treatmentwith chitosan particles and dexamethasone combined neitherincreased ALP activity nor upregulated COL1A1 mRNA ex-pression, and even downregulated RUNX2mRNA expressionin comparison with treatment with chitosan or dexamethasonealone (data not shown). A small number of mineralizationnodules was detected in the 10 !g/ml chitosan group and inthe dexamethasone group. Quantification of the staining in-tensity revealed comparable values in all groups (P>0.05,Fig. 5a, b).

Fig. 2 Alkaline phosphatase(ALP) activity of DPSCs afterincubation with chitosan (Dexdexamethasone, MMmineralizing medium). a ALPactivity at day 7. bALP activity atday 14. c ALP mRNA relativeexpression at day 7. d ALPmRNA relative expression at day14. Note the higher ALP mRNAexpression in medium containing5 !g/ml chitosan at day 7 (P!0.05) and a tendency for higherALP mRNA activity in both thechitosan and dexamethasonegroups. Means±SD

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Discussion

In the past decade, DPSCs have been studied as an alternativesource of MSCs given the ease of harvesting the tissue.Despite numerous reports of the potential of chitosan as a

scaffold material for bone tissue engineering, the influencesof chitosan on the metabolism and differentiation of DPSCsinto the osteoblastic lineage in vitro remain unclear. We havetested the hypothesis that, in addition to acting as a scaffoldmaterial, chitosan can also serve as an osteogenic factorin vitro. The proliferation of macaque DPSCs has been shownto be enhanced by treatment with chitosan, in agreement withdata presented by others using different cells and testingdifferent forms of chitosan (Cai et al. 2008; Pang et al. 2005;Wang et al. 2008). Chitosan-induced proliferative responses inhuman periodontal ligament fibroblasts (hPDLF) reach a pla-teau at 100 !g/ml chitosan (Pang et al. 2005). Chitosan-coatedtitanium surfaces stimulate neonatal rat calvaria osteoblasts toproliferate compared with uncoated titanium surfaces (Caiet al. 2008). However, no significant changes in DNA contenthave been observed in bone-marrow-derived mesenchymalstromal cells (BMSCs) exposed to chitosan (Guzmán-Morales et al. 2009). Differences in the properties and

Fig. 3 Osteogenic markersexpressed by DPSCs (Dexdexamethasone, MMmineralizing medium). a Runt-related transcription factor-2(RUNX2) mRNA relativeexpression at day 7. b RUNX2mRNA relative expression at day14. c Collagen type 1A1(COL1A1) mRNA relativeexpression at day 7. d COL1A1mRNA relative expression at day14. Means±SD; *P!0.05,**P!0.01, ***P!0.001

Fig. 4 Gel electrophoresis of products from the reverse transcription pluspolymerase chain reaction (Chit chitosan, Dex dexamethasone, MMmineralizing medium, NTC no template control, GAPDH D-glyceralde-hyde-3-phosphate dehydrogenase)

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characteristics of chitosan or the sensitivity of BMSCs forchitosan might explain these different results. In support ofthis, one of the earliest reports of DPSCs showed morecolony-forming cells at various plating densities and morecells that could be labeled with bromodeoxyuridine than werefound for BMSCs, indicating that, in a population of DPSCs,more clonogenic mesenchymal stem cells exist than in apopulation of BMSCs (Gronthos et al. 2000). Our data sug-gesting that dexamethasone reduces the proliferation ofDPSCs are in agreement with the report that dexamethasoneinduces cell-cycle arrest leading to the osteogenic differentia-tion of human BMSCs in vitro (Guzmán-Morales et al. 2009).

The osteogenic differentiation ofMSCs can be achieved byincubating MSCs in standard culture medium supplementedwith dexamethasone and 1$,25-dihydroxyvitamin D3, incombination with "-glycerophosphate and ascorbic acid(Cheng et al. 1994; Lee et al. 2000; Gharibi and Hughes2012). The differentiation of MSCs toward the osteoblasticlineage is initiated with the expression of osteoblast-specificRUNX2, the main osteoblast transcription factor, followed bythe expression of ALP, COL1A1, and other osteoblast-specificdifferentiation genes that play roles in bone formation(Karsenty and Wagner 2002). Our data show that the lowestused concentration of chitosan stimulates RUNX2 transcrip-tion significantly in the first week and in the second week ofculture, reaching far higher levels in the second week than inthe first week. The stimulation of DPSCs metabolism and theupregulation of RUNX2mRNA have been observed, followedby a transient increase in ALP mRNA expression in the cells

and a tendency for higher ALP activity in the culture medium.Notably, despite the stimulation of RUNX2 and ALP mRNAexpression in DPSCs cultured with chitosan,COL1A1mRNAexpression and mineral deposition in DPSCs cultured withchitosan remain comparable with those of the dexamethasonegroup and the mineralizing control group. Collectively, thesedata suggest that chitosan is able to induce the proliferationand early osteogenic differentiation of DPSCs in a similar wayto dexamethasone, but that the stimulation ofCOL1A1mRNAexpression by dexamethasone is not detectable in the chitosangroup. A longer incubation period might be necessary in orderto observe any stimulating effects of chitosan on extracellularmatrix maturation and on the mineralization process.

The data seem to contradict previous reports that demon-strate enhanced mineralization following treatment with chi-tosan (Ambre et al. 2013; Lim et al. 2009; Wang et al. 2011).However, these studies have shown that human bone-marrow-derived mesenchymal stromal cells (hBMSCs) secrete ahigher calcium content in the mineral deposit whenhBMSCs are cultured with a chitosan-hydroxyapatite com-posite scaffold. The osteoconductive property of hydroxyap-atite has been widely studied. Thus, the positive stimulationon mineralization might be related to the incorporation ofchitosan with this osteoconductive material (Ambre et al.2013; Lim et al. 2009; Wang et al. 2011). Our results arepartially consistent with the study reported by Guzmàn-Morales and colleagues (2009) concerning the osteogeniceffect of chitosan in hBMSCs culture compared with that ofdexamethasone. The authors have shown that the addition ofchitosan particles in the mineralizing medium has no effect oncollagen deposition and interferes with the mineralized matrixdeposition (Guzmán-Morales et al. 2009).

Dexamethasone induces osteoblast differentiation throughthe activation of the osteoblast transcription factor (Chenget al. 1994). The positive effects of dexamethasone onMSCs differentiation are believed to be mediated by FHL2(four-and-a-half LIM domains protein 2), a member of theLIM (Lin11, Isl-1, Mec-3) protein superfamily. Short-RNA-mediated FHL2 silencing reduces the expression of osteoblastmarker genes and diminishes dexamethasone-induced upreg-ulation of Runx2 and Col1a1, two major phenotypic osteo-blast genes (Hamidouche et al. 2008). Another report hasdemonstrated that dexamethasone induces osteoblast differen-tiation through the reduction of inflammatory factors such asinterleukin-1$ and the increase of cell attachment (Guzmán-Morales et al. 2009). Whether a similar mechanism occurs inthe osteogenic differentiation of DPSCs stimulated by chito-san remains unknown. Further studies are needed in order toverify the underlying mechanism of the osteogenic differenti-ation of MSCs by chitosan.

In conclusion, under the present in vitro conditions, thisstudy shows the potential stimulating ability of chitosan inDPSCs proliferation and early osteogenic differentiation

Fig. 5 Alizarin red staining on DPSCs cultured with mineralized medi-um (MM), supplemented with dexamethasone (Dex) or chitosan. a Aliz-arin red staining of culture plates. b Quantification of staining intensity

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comparable with that of dexamethasone, but no significantstimulation on mineral deposition. In addition to its role as a3D scaffold for osteogenic cells in vivo, chitosan might alsostimulate DPSCs proliferation and early osteogenic differen-tiation in vitro.

Acknowledgments The authors thank Dr. Antonius Bronckers (Aca-demic Center for Dentistry Amsterdam, The Netherlands) for criticallyreading the manuscript and facilitating the FACS analysis.

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