biosilica porous microspheres promote the osteogenic

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Copyright copy 2018 American Scientific PublishersAll rights reservedPrinted in the United States of America

Journal ofBiomaterials and Tissue Engineering

Vol 8 258ndash266 2018

Biosilica Porous Microspheres Promote the OsteogenicOdontogenic Differentiation of Human Dental Pulp Cells

Wenhao Zhu1 Xuejun Gao2 Xiaoying Zou2 Werner E G Muumlller3 Shunfeng Wang3Yuguang Wang4 and Yihong Liu1lowast

1Department of General Dentistry Peking University School and Hospital of Stomatology Beijing 100081 China2Department of Cariology and Endodontology Peking University School and Hospital of Stomatology Beijing 100081 China

3ERC Advanced Investigator Grant Research Group at the Institute for Physiological ChemistryUniversity Medical Center of the Johannes Gutenberg University Mainz 55128 Germany

4Center of Digital Dentistry Peking University School and Hospital of Stomatology Beijing 100081 China

Silicatein is a unique enzyme in sponges that serves as a template and catalyzes biosilica formationfrom silica and therefore may be useful for bone regeneration In this study we investigated theeffects of novel biosilica porous microspheres consisting of poly(lactic-co-glycolic acid)-tricalciumphosphate coated with silica and silicatein on the growth and osteogenicodontogenic differentiationof human dental pulp cells (hDPCs) Viability and morphology were assessed using Cell Count-ing Kit-8 assays laser confocal microscopy and scanning electron microscopy Marker expressionwas evaluated by immunofluorescence staining quantitative reverse transcription-polymerase chainreaction (collagen type I and dentin sialophosphoprotein) and alkaline phosphatase activity quan-tification Compared with poly(lactic-co-glycolic acid)-tricalcium phosphate (a control material) thenovel biomicrospheres did not affect viability supported the attachment and expansion of hDPCsupregulated the expression of collagen type I and dentin sialophosphoprotein and promoted alka-line phosphatase secretion in the osteoinductive environment These findings demonstrate thatthese biosilica porous microspheres have potential clinical applications as novel carriers of inductivematerials for dentin regeneration

Keywords Biosilica Porous Scaffold Human Dental Pulp Cell Osteogenic DifferentiationOdontogenic Differentiation

1 INTRODUCTIONDentin is a calcified tissue that makes up most of the toothaccordingly it is a key component in tissue engineeringfor the tooth structure1 Tissue engineering is an emergingfield that combines stem cells growth factors and scaf-folds to regenerate diseased or lost tissues Scaffolds areindispensable in tissue regeneration as they serve as car-riers to facilitate the delivery of stem cells andor growthfactors to a local receptor site2 Ideally scaffold materi-als should be biodegradable and biocompatible promotecellular interactions and tissue development and possessproper mechanical properties3

To regenerate dentin several types of scaffolds havebeen tested using dental pulp stem cells (DPSCs) bothin vitro and in vivo including those made of colla-gen polylactic acid (PLA) polyglycolic acid (PGA) a

lowastAuthor to whom correspondence should be addressed

copolymer of PGA and PLA poly(lactic-co-glycolic acid) (PLGA)and bioceramics4ndash6 Composite scaffolds can be used toexploit the advantages of multiple individual materials278

In PLGAtricalcium phosphate (TCP) composites PLGAmay cause a localized pH reduction due to the relativeacidity of its hydrolytically degraded by-products and thedegradation products of TCP can potentially neutralizethis acidic pH thereby reducing the mild inflammationassociated with polymer degradation2 Blaker et al9

demonstrated improved adhesion spreading and viabil-ity of cells grown on polymerndashbioglass composites andfurther confirmed the high bioactivity and biocompatibil-ity of the material for hard tissue repair A PLGATCPcomposite scaffold is appropriate for the proliferation anddifferentiation of DPSCs and was found to be superiorto a pure PLGA scaffold for tooth tissue regenerationespecially for dentin formation10 PLGATCP containingsilver can increase alkaline phosphatase (ALP) expressionin human dental pulp cells (hDPCs)11 Although these

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Zhu et al Biosilica Porous Microspheres Promote the OsteogenicOdontogenic Differentiation of Human Dental Pulp Cells

materials promote the proliferation and differentiation ofDPCs few are able to regenerate the complete dentintissue12 and in some cases the newly formed dentinis very different from primary dentin13 Moreover noneof the materials developed to date has been tested ina clinical trial Therefore it is necessary to develop asuitable scaffold material for dentin regeneration

Recently Professor Muumlller and colleagues synthesizedPLGA-TCP microspheres coated with silica and sil-icatein as a new type of bioactive and biodegradablematerial14 Silicateins are exclusively found in sponges(phylum Porifera) and serve as a template and catalyze thepolymerization of nanoscale silicate to biosilica the mainmineral component of sponge skeletal elements (siliceousspicules) Both silicate and biosilica can induce boneregeneration15ndash17 The microspheres (diameterasymp 800 m)were prepared by encapsulation of -TCP supplementedwith silica and silicatein in PLGA The PLGA-TCPmicrospheres coated with silica and silicatein showed notoxicity in a cell viability assay using human osteogenicsarcoma cells (SaOS-2 cells) Moreover mineralization ofSaOS-2 cells increased when -TCP or silica was added tothe microspheres and mineralization was relatively weakin microspheres without -TCP However the additionof silicatein to the spheres did not affect mineralizationFurthermore an additional increase in mineralization wasobserved when silicatein-containing spheres were mixed ata 11 ratio with silica-containing spheres14

hDPCs can differentiate toward the osteoblast lineageand contribute to the dentinal regeneration process18

accordingly several studies have employed hDPCs as tar-get cells to evaluate the hard tissue regeneration ability ofvarious biomaterials19 Moreover the environment of thetransplantation site influences the fate of stemprogenitorcells in vivo That is the fate of implanted stemprogenitorcells may be determined by the site rather than by theorigin of cells with a tendency to differentiate into theodontoblast phenotype when transplanted into the pulpchamber but into other cell lineages when transplantedto other target locations3 Since the bone and teeth areboth apatite-like hard tissues a material that promotes theosteogenic differentiation of hDPCs in vitro has potentialbenefits for the odontogenesis of hDPCs

Based on its characteristics and function in boneregeneration we speculated that a 11 mixture of thenovel biosilica microspheres (PLGA-TCPsilica-microPLGA-TCPsilicatein-micro) may be beneficial for thegrowth and differentiation of hDPCs and is a potentialnew scaffold material for dentin regeneration Thereforethe aim of this study was to investigate the effect ofPLGA-TCP microspheres coated with both silica andsilicatein on the growth and osteogenicodontogenic dif-ferentiation of hDPCs in comparison with the effects ofuncoated PLGA-TCP microspheres

2 MATERIALS AND METHODS21 Material PreparationMicrospheres were prepared by the encapsulation of-TCP supplemented with silica and silicatein intoPLGA -TCP (sim10 ww) sim5 silica (ww) and05 gmg silicatein were entrapped into PLGA micro-spheres (diameter asymp 800 m) according to a previouslyreported procedure14 The average hardness of the micro-spheres was 005 GPa and the average elastic moduluswas 278 GPa The PLGA-TCPsilica-silicatein micro-spheres were used as the experimental material and thePLGA-TCP microspheres were used as a control mate-rial Each material was molded into disks (diameter =10 mm thickness= 13 mm weight= 38 mg) to preparethe scaffolds used in subsequent cell experiments Prior touse all materials were treated with ultraviolet light for 1 h

22 Cell CultureNormal human dental pulp tissues were obtained fromthe third molars of three patients (18ndash25 years of age)at Peking University School and Hospital of Stomatologyafter obtaining informed consent from the donors and per-mission from the ethics committee (reference no PKUS-SLA2014193) The extracted pulp tissues were mincedand digested in a solution containing 3 mgml collage-nase type I (Worthington Biochemical Lakewood NJUSA) and 4 mgml dispase (Roche Molecular Biochemi-cal Mannheim Germany) for 1 h at 37 C The hDPCswere then transferred to alpha-minimal essential medium(-MEM) supplemented with 10 fetal bovine serum(GIBCO Waltham MA USA) 100 Uml penicillin G100 gml streptomycin (Invitrogen Carlsbad CA USA)and 2 mM L-glutamine (Invitrogen) in a humidified incu-bator with 5 CO2 at 37 C The culture medium waschanged every 3 days and the cells were subcultured at70 confluence hDPCs at passages 4ndash6 were used forall cellular analyses All experiments were repeated threetimes using hDPCs from the three patients

23 Effects of PLGA-TCPSilica-Silicatein on theGrowth Behavior of hDPCs

231 Cell Viability AssayThe effect of PLGA-TCPsilica-silicatein on the viabilityof hDPCs was determined in vitro using the Cell Count-ing Kit-8 (CCK-8 Dojindo Kumamoto Japan) PLGA-TCPsilica-silicatein and PLGA-TCP were pre-wettedin the culture medium for 24 h in a 24-well plate Thenthe hDPCs (2times 104) were seeded onto each scaffold andincubated at 37 C 5 CO2 For the negative controlgroup hDPCs were cultured without any materials Theculture medium was changed every 2 days After 1 35 7 and 9 days of cell culture cells were further incu-bated with the counting reagent for 1 h according tothe manufacturerrsquos instructions The relative cell numberwas determined by measuring absorbance of the formazan

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Biosilica Porous Microspheres Promote the OsteogenicOdontogenic Differentiation of Human Dental Pulp Cells Zhu et al

dye product in the cultures at a wavelength of 450 nmThe results are expressed as optical density values Therelative cell numbers in the PLGA-TCPsilica-silicateingroup PLGA-TCP group and negative control groupwere compared

232 Confocal Laser-Scanning MicroscopyThe adhesion and growth of hDPCs on the scaf-fold surfaces were observed by confocal laser-scanningmicroscopy The hDPCs (2times104) were seeded onto eachscaffold and incubated at 37 C 5 CO2 After 3 5 7 9and 11 days of culture the cell-scaffold constructs wererinsed in phosphate-buffered saline (PBS) and fixed in 4paraformaldehyde for 30 min The constructs were thenstained with 5 gml fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma St Louis MO USA) for 1 hincubated in 1 gml 6-diamidino-2-phenylindole (DAPISigma) solution for 5 min at room temperature (24ndash26 C)and viewed under a confocal Zeiss Axiovert 650 micro-scope (Carl Zeiss Microimaging Oberkochen Germany)using a laser with wavelengths of 488 nm (green FITC-labeled phalloidin) for F-actin and 405 nm (blue DAPI)for nuclei

233 Scanning Electron Microscopy (SEM)The scaffold surfaces were examined by SEM to con-firm the attachment of hDPCs The hDPCs (2times104) wereseeded onto each scaffold and incubated at 37 C 5CO2 Initially the cell-scaffold constructs were washedwith PBS and fixed in 4 paraformaldehyde for 30 minafter culturing for 7 days The specimens were dehydratedwith a graded series of ethanol mounted onto aluminumstubs sputter-coated with gold and viewed under field-emission SEM (EVO 18 Zeiss Wetzlar Germany) Themicrospheres without cells were also observed

24 Effects of PLGA-TCPSilica-Silicatein on theOsteogenicOdontogenic Differentiation of hDPCs

The hDPCs (2times 104) were seeded onto the two scaf-folds and incubated at 37 C 5 CO2 in regular cul-ture medium for 2 days followed by incubation inosteogenic-inducing medium for 14ndash28 days which com-prised fresh -MEM containing 10 fetal bovine serum100 Uml penicillin G 100 mgml streptomycin 2 mML-glutamine 100 nM dexamethasone (Sigma) 10 mM-glycerophosphate (Sigma) and 02 mM L-ascorbic acid(Sigma) The effects of PLGA-TCPsilica-silicatein onthe osteogenicodontogenic differentiation of hDPCs wereevaluated by immunofluorescence staining to observethe expression of osteogenicodontogenic-related proteinsImmunofluorescence staining to detect collagen type I(COLI) was performed on the 14th day and dentinsialophosphoprotein (DSPP) staining was performed onthe 28th day after osteoinduction The protocol wasas follows cell-scaffold constructs were fixed in 4

paraformaldehyde for 20 min at room temperature fol-lowed by post-fixation in 01 Triton X-100 for 15 minThe constructs were washed three times in PBS and incu-bated with 1100 primary antibodies (COLI ProteintechRosemont IL USA DSPP Santa Cruz BiotechnologySanta Cruz CA USA) overnight at 4 C The constructswere then washed again and incubated in 1200 secondaryantibodies (Proteintech) for 1 h at room temperature fol-lowed by staining with 5 gml FITC-labeled phalloidinfor 1 h and incubation in 1 gml DAPI solution for5 min at 37 C Following three additional washings withPBS the specimens were viewed under the confocal ZeissAxiovert 650 microscope using a laser with wavelengths of594 nm (red COLI and DSPP) 488 nm (green F-actin)and 405 nm (blue nuclei)

25 Effects of Biosilica on the OsteogenicOdontogenicDifferentiation of hDPCs

To evaluate the effect of biosilica released from the micro-spheres on cell differentiation hDPCs were co-culturedwith the scaffolds rather than being seeded directly onthe scaffolds The ALP activity and gene expression lev-els of COLI and DSPP in adherent cells were analyzedThe hDPCs (5times 104) were seeded in 12-well plates withregular culture medium for 2ndash3 days Upon reaching70ndash80 confluence the regular culture medium wasreplaced with osteogenic-inducing medium the scaffolds(PLGA-TCPsilica-silicatein and PLGA-TCP) wereadded to each well simultaneously and co-culturing wasconducted for 7ndash21 days In the negative control group thehDPCs were cultured in the osteogenic-inducing mediumwithout any additional materials

251 ALP Activity QuantificationThe ALP activity and protein levels of hDPCs were mea-sured on the 7th 14th and 21st day of osteoinductionusing the ALP Kit (JianCheng Bioengineering InstituteNanjing China) and the BCA Protein Assay Kit (ComWinBiotech Co Ltd Beijing China) respectively accordingto the manufacturersrsquo instructions Absorbance was mea-sured at 520 nm and ALP levels were normalized againstthe total protein content

252 Quantitative Reverse Transcription-PolymeraseChain Reaction (qRT-PCR)

The expression levels of osteogenicodontogenic-relatedgenes in hDPCs were evaluated by qRT-PCR Total cellularRNAs were isolated at 7 14 and 21 days after osteoin-duction using the TRIzol reagent (Invitrogen) and usedfor first-strand cDNA synthesis using the PrimeScript RTReagent Kit (TaKaRa Bio Otsu Japan) Gene transcriptswere quantified by qRT-PCR using a SYBRreg Premix ExTaq II Kit (TaKaRa) and an ABI PRISM 7500 SequenceDetection System (Applied Biosystems Foster City CAUSA) The primer sets used to detect COLI DSPP and

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Table I Primer sequences for qRT-PCR

Genes Primer sequence

COLI (F) 5prime-AAAAGGAAGCTTGGTCCACT-3prime

(R) 5prime-GTGTGGAGAAAGGAGCAGAA-3prime

DSPP (F) 5prime-ATATTGAGGGCTGGAATGGGGA-3prime

(R) 5prime-TTTGTGGCTCCAGCATTGTCA-3prime

GAPDH (F) 5prime-GAGTCAACGGATTTGGTCGT-3prime

(R) 5prime-GACAAGCTTCCCGTTCTGAG-3prime

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) arelisted in Table I GAPDH was used as a housekeepinggene to normalize the mRNA expression levels The cyclethreshold (Ct) values were used to calculate the fold differ-ences by the Ct method The qRT-PCR conditions wereas follows denaturation at 95 C (10 min) and 40 cyclesat 95 C (15 s) and 60 C (1 min annealing)

26 Statistical AnalysisQuantitative results are reported as meansplusmn standard devi-ations (n= 3) Results were evaluated by one-way analysisof variance for the CCK-8 assay and ALP activity analysisand by t-tests for qRT-PCR data using SPSS 190 (SPSSInc Chicago IL USA) A value of P lt 005 was consid-ered statistically significant

3 RESULTS AND DISCUSSION31 Structures of MicrospheresSEM shows that microspheres were bonded together Thesurfaces of PLGA-TCP microspheres were fairly smooth

Fig 1 Scanning electron microscopy images of microspheres The surfaces of PLGA-TCP microspheres were smooth (AndashC) and those of PLGA-TCPsilica-silicatein microspheres were microporous (DndashF)

(Figs 1(AndashC)) The surfaces of PLGA-TCPsilica-silicatein microspheres were covered with micropores withdiameters ranging from 5 m to 8 m and nanopores(Figs 1(DndashF)) Compare to the smooth surface porous sur-face are favorable for the adhesion and growth of DPCs20

32 Effects of PLGA-TCPSilica-Silicatein onhDPC Growth

321 PLGA-TCPSilica-Silicatein Does NotAffect the Viability of hDPCs

The effect of PLGA-TCPsilica-silicatein microsphereson the viability of hDPCs was assessed by CCK-8 assaysAs shown in Figure 2 there were showed no statisticallysignificant differences (P gt 005) in the relative numberof hDPCs among the PLGA-TCPsilica-silicatein groupPLGA-TCP group and negative control group through-out the experimental period (0ndash9 days) The relative cellcount increased by 25-fold on the 5th day and by 53-foldon the 9th day after cell seeding (Fig 2)The cytotoxicity of materials used in dentinal regener-

ation is an important practical concern Materials shouldeither improve cell viability or be biologically neutral21

Given that PLGA-TCPsilica-silicatein did not causeany significant changes in the viability of hDPCs com-pared to the viability of control cells at the early stageof cell culture this material could provide a suitablesubstrate for the initial adherence spreading and fur-ther multiplication of cells Thus these results indicatedthat PLGA-TCPsilica-silicatein is a biocompatible scaf-fold material without cytotoxicity consistent with previous




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Fig 2 A CCK-8 assay showed no statistically significant differences(P gt 005) in the viability of hDPCs among the PLGA-TCPsilica-silicatein group PLGA-TCP group and negative control group atdays 0ndash9

analyses of the effects of this material on the viability ofSaOS-2 cells14

322 PLGA-TCPSilica-Silicatein Supports theAttachment and Expansion of hDPCs

Based on observations of microspheres by confocal laser-scanning microscopy as shown in Figures 3(A) and (C)hDPCs grew well on the surfaces of both kinds of scaf-folds The hDPCs gradually covered the microsphere sur-face reaching 70ndash80 confluence at the end of the7 days The microspheres were completely covered withhDPCs by the 11th day after cell seedingSEM was used to observe the fine structures of cells

to assess the effect on hDPC attachment As shownin Figures 3(B) and (D) the porous topography of thePLGA-TCPsilica-silicatein surface allowed hDPCs toform a blanket and flourish The cells bridged micro-spheres via their pseudopodia (Figs 3(B D) arrows)Moreover the hDPCs cultured on PLGA-TCPsilica-silicatein exhibited more clearly defined cytoplasmicextensions which projected from the cells to the surround-ing surface or adjacent cells than those on the surface ofPLGA-TCPThe attachment and spread of hDPCs on material

surfaces are important for subsequent cellular functionincluding differentiation22 The results described aboveindicated that the silicon and porous microspheres areappropriate for the growth of hDPCs consistent with pre-vious findings19202324 The geometry of porous scaffoldsused for tissue engineering has recently been shown to sig-nificantly influence the cellular response Surface (nano-)topography could determine stem cell fate facilitatingstem cell differentiation towards the osteogenic lineage25

Small pores may be beneficial in vitro because a higherfluid velocity means that cells have less time for attach-ment to the surface of the scaffold The cell seeding

efficiency may therefore decrease as pore size increases26

The pores on the surface of PLGA-TCPsilica-silicateinwere smaller than the cellular dimensions providing achannel for the release of silicon while simultaneouslyincreasing the roughness of the microspheres Microscaf-folds aid in the rapid expansion of cells20 Owing totheir geometry porous microscaffolds provide a large sur-face area thereby minimizing the utilization of mediaand can effectively mimic the in vivo microenviron-ment for cell growth2023 The pores formed between thebonded microspheres and the nanopores on the surfaceof microspheres result in the formation of multiple three-dimensional porous structures that can transport oxygennutrients and metabolites which facilitate cell attachmentand growth inside the structuresBiosilica is composed of nanoparticles of 50ndash70 nm in

diameter which is suitable for uptake by cells via endocy-tosis Indeed biosilica has been shown to promote the pro-liferation of SaOS-2 cells16 The positive effects of siliconon cell proliferation and adhesion are achieved by alteringthe functional presentation of the major integrin-bindingdomains of adsorbed proteins and consequently by mod-ulating integrin binding localization and specificity24

Moreover a high silicon content can trigger the enhance-ment of total integrin and COLI expression thereby affect-ing integrin expression profiles via collagen-binding andfibronectin-binding subintegrin on human primary cells19

Given this background the PLGA-TCPsilica-silicatein microspheres appear to provide a suitablemicroenvironment for the growth and subsequent differ-entiation of hDPCs

33 PLGA-TCPSilica-Silicatein Promotes theExpression of COLI and DSPP in hDPCs

As shown in Figure 4 hDPCs seeded onPLGA-TCPsilica-silicatein exhibited COLI-positivestaining after 14 days of osteoinduction and DSPP-positivestaining after 28 days of osteoinduction However nopositive staining for either marker was detected in thecells seeded on PLGA-TCP (Fig 4)A high porosity of a scaffold is thought to aid in osteo-

conductivity since the increased surface area supportsenhanced bone growth and exposes cells to greater surfacereactivity27 When mesenchymal stem cells were seededon a three-dimensional PLA porous scaffold in a previ-ous study the expression levels of osteoblast-related genesincluding collagen type I osteocalcin and bone sialopro-tein were upregulated after 14 days of culture The authorsattribute this to the adherence of cells to a surface with acomplex three-dimensional pore geometry and the benefi-cial effect of the surrounding curved surface for their sub-sequent spread and migration leading to further osteogenicdifferentiation23

In addition a previous study has shown that biosil-ica could induce SaOS-2 cells to generate more




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Fig 3 Confocal laser-scanning microscopy and scanning electron microscopy images of the hDPC-microsphere construct The whole microspheresof PLGA-TCP (A) and PLGA-TCPsilica-silicatein (C) were completely covered with hDPCs on the 11th day after cell seeding (magnification40times) Nuclei were stained blue and F-actin was green SEM showed that hDPCs cultured on PLGA-TCPsilica-silicatein (D) exhibited better definedcytoplasmic extensions (arrows) that projected from the cells to the surrounding surface or adjacent cells than those on the surface of PLGA-TCP(B) (magnification 200times)

hydroxyapatite (HA) nodules and express higher levelsof bone morphogenetic protein 2 (BMP2) which inducesbone formation by directing the differentiation of bone-forming progenitor cells to mature osteoblasts while simul-taneously inhibiting the function of osteoclasts16 BMP2can also promote postnatal pulp stem cells to differen-tiate into odontoblast-like cells and induces reparativedentin formation in vivo28 Most studies of the effectsof biosilica on tooth mineralization have focused onenamel formation For example the biosilica-regulatedexpression levels of amelogenin and enamelin which areboth involved in enamel formation were investigated inSaOS-2 cells29 During the reaction of silicatein withthe substrate sodium metasilicate nanoscale biosilica lay-ers (50ndash150 nm) form on the dental surface to generateprotective biosilica layers on the teeth which could inturn reduce the risk of bacterial-induced cariescavities30

However no studies have evaluated the role of biosil-ica in dentin mineralization In the present study DSPPan odontogenic-related protein was selected to confirmthe effect of PLGA-TCPsilica-silicatein on hDPC odon-toblastic differentiation DSPP is highly expressed inodontoblasts but is expressed at very low levels in othertissues such as the bone and kidney DSPP is also one

of the most important non-collagenous proteins involvedin the nucleation and control of the HA mineral phaseduring dentin calcification31 Therefore DSPP expres-sion is a useful marker of mature odontoblasts Thusthe finding of positive DSPP expression in hDPCs onPLGA-TCPsilica-silicatein after 28 days of osteoinduc-tion with no such expression detected in the PLGA-TCPgroup provides the first demonstration that biosilica canpromote the odontogenesis of hDPCs However furtherevaluation in vivo is needed to confirm this resultCollagen particularly COLI has potent effects on

the proliferation and mineralization capacity of hDPCs32

COLI is associated with the extracellular matrix itis highly expressed during the proliferation stage andthen gradually declines prior to differentiation33 Cementwith a certain amount of silicon is beneficial for COLIadsorption and secretion34 Moreover COLI was foundto be preferable to fibronectin for hDPC adhesion24

When SaOS-2 cells were cultured with biosilica COLIwas upregulated along with the upregulation of BMP2expression35 Similarly in the present study hDPCs seededon PLGA-TCPsilica-silicatein microspheres showedstrong COLI protein expression indicating that the porousstructure of the microspheres along with the properties of




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Fig 4 Immunofluorescent staining for COLI (A) and DSPP (B) in hDPCs cultured on PLGA-TCPsilica-silicatein and PLGA-TCP (magnification400times) COLI and DSPP were stained red nuclei were blue and F-actin was green hDPCs seeded on PLGA-TCPsilica-silicatein exhibited COLI-positive staining after 14 days of osteoinduction and DSPP-positive staining after 28 days of osteoinduction

biosilica promoted the expression of COLI to enhance cellproliferation and attachment

34 Biosilica Promotes ALP Secretion and DSPPGene Expression in hDPCs

Early osteoblastic differentiation can be assessed by esti-mating the expression and activity of ALP36 Therefore toassess the specific effect of biosilica released from PLGA-TCPsilica-silicatein on the osteogenicodontogenic dif-ferentiation of hDPCs ALP activity and the expressionlevels of COLI and DSPP on adherent hDPCs co-culturedwith the scaffolds were analyzed ALP activity in the threegroups (PLGA-TCPsilica-silicatein PLGA-TCP andnegative control groups) increased continuously over timeafter osteoinduction reaching peak levels on the 14th dayand then began to decline on the 21st day (Fig 5(A))There were no significant differences in ALP activityamong the three groups (P gt 005) on days 7 and 14However the PLGA-TCPsilica-silicatein group showed

significantly greater ALP activity than that of the othertwo groups on the 21st day (P lt 005) ALP plays animportant role in mineral deposition and is also presentin the early differentiation stage37 Earlier studies haveshown that solutions containing a certain concentrationof silicon promote collagen production and osteogenesisin osteoblasts38ndash40 Although PLGA-TCPsilica-silicateindid not promote ALP secretion during the early stageof cell differentiation it resulted in a gradual and lesssubstantial decrease in ALP over time compared withALP secretion in the other two groups and relativelystrong activity was observed at the later stage of celldifferentiationSimilar to the results for ALP activity the PLGA

-TCPsilica-silicatein group showed significantly higher(15-fold) DSPP expression levels than those of thePLGA-TCP and negative control groups (P lt 005) onthe 21st day whereas there were no statistically signifi-cant differences among the three groups on the 7th and




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Fig 5 (A) ALP activity of hDPCs cultured with and without scaffoldsin osteogenic-inducing medium for 7 14 and 21 days (B and C) Relativegene expression levels (COLI and DSPP) of hDPCs cultured with andwithout scaffolds in osteogenic-inducing medium for 7 14 and 21 dayslowastSignificant difference at P lt 005

14th days (P gt 005 Fig 5(C)) The significant upregula-tion of DSPP expression at 21 days after cell osteoinduc-tion may be related to the mechanism of action of biosilicawhich is readily taken up by cells by endocytosis and maybe degraded to orthosilicate16 Therefore biosilica can pro-mote the osteogenicodontogenic differentiation of hDPCsin the early stage of cell culture however further studiesare needed to elucidate the underlying signal transductionpathways

There were no significant differences in the relativeexpression levels of the COLI gene among the threegroups at any time point (P gt 005 Fig 5(B)) HoweverhDPCs on PLGA-TCPsilica-silicatein microspheres hadstronger COLI protein expression after 14 days of osteoin-duction compared with expression levels in the negativecontrol groups This result indicated that the porous struc-ture of PLGA-TCPsilica-silicatein in combination withthe release of biosilica rather than the biosilica alone pro-moted the expression of COLI

4 CONCLUSIONSA PLGA-TCPsilica-silicatein nanoporous microspherescaffold can provide a suitable environment to supportthe growth of hDPCs The released biosilica can promotethe osteogenicodontogenic differentiation of hDPCs in theosteoinduction environment and the interaction betweenthe porous structure and biosilica can further enhance thiseffect This novel material thus shows good potential as acarrier of inductive materials for dentin regeneration Fur-ther in vivo experiments are needed to confirm its abilityto promote dentin formation

Acknowledgment This study was supported by grantsfrom the Natural Science Foundation of China (Projectnos 51772007 and 51361130032) We would like to thankEditage [wwweditagecn] for English language editing

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11 B Cvikl S C Hess R J Miron H Agis D Bosshardt T AttinP R Schmidlin and A Lussi Response of human dental pulp cellsto a silver-containing PLGATCP-nanofabric as a potential antibac-terial regenerative pulp-capping material BMC Oral Health 17 57(2017)

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16 M Wiens X Wang U Schlossmacher I Lieberwirth G GlasserH Ushijima H C Schroumlder and W E Muumlller Osteogenic potentialof bio-silica on human osteoblast-like (SaOS-2) cells Calcif TissueInt 87 513 (2010)

17 W E Muumlller H C Schroumlder Q Feng U Schlossmacher T Linkand X Wang Development of a morphogenetically active scaffoldfor three-dimensional growth of bone cells Biosilica-alginate hydro-gel for SaOS-2 cell cultivation J Tissue Eng Regen Med 9 E39(2015)

18 C C Chen M Y Shie and S J Ding Human dental pulp cellsresponses to new calcium silicate-based endodontic materials IntEndod J 44 836 (2011)

19 M Y Shie and S J Ding Integrin binding and MAPK signal path-ways in primary cell responses to surface chemistry of calcium sili-cate cements Biomaterials 34 6589 (2013)

20 R S Bhuptani and V B Patravale Porous microscaffolds for 3Dculture of dental pulp mesenchymal stem cells Int J Pharma515 555 (2016)

21 L Zhu J Yang J Zhang and B Peng A comparative study ofBioAggregate and ProRoot MTA on adhesion migration and attach-ment of human dental pulp cells J Endod 40 1118 (2014)

22 L M Lin and P A Rosenberg Repair and regeneration in endodon-tics Int Endod J 44 889 (2011)

23 H Y Lee G Z Jin U S Shin J H Kim and H W Kim Novelporous scaffolds of poly (lactic acid) produced by phase-separationusing room temperature ionic liquid and the assessments of biocom-patibility J Mater Sci Mater Med 23 1271 (2012)

24 J Sun L Wei X Liu J Li B Li G Wang and F Meng Influ-ence of ionic dissolution products of dicalcium silicate coating onosteoblastic proliferation differentiation and gene expression ActaBiomater 5 1284 (2009)

25 A A Zadpoor Bone tissue regeneration The role of scaffold geom-etry Biomater Sci 3 231 (2015)

26 S Van Bael Y C Chai S Truscello M Moesen G KerckhofsH Van Oosterwyck J P Kruth and J Schrooten The effect of poregeometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4 V bone scaf-folds Acta Biomater 8 2824 (2012)

27 A Aarvold J O Smith E R Tayton S A Lanham J BChaudhuri I G Turner and R O Oreffo The effect of porosity ofa biphasic ceramic scaffold on human skeletal stem cell growth anddifferentiation in vivo J Biomed Mater Res A 101 3431 (2013)

28 K M Galler The Dental Pulp edited by M Goldberg SpringerBerlin Heidelberg (2014) Vol 18 pp 251ndash265

29 W E Muumlller A Boreiko X Wang A Krasko W Geurtsen M RCustoacutedio T Winkler L Lukic-Bilela T Link and H C SchroumlderMorphogenetic activity of silica and biosilica on the expression ofgenes controlling biomineralization using SaOS-2 cells Calcif Tis-sue Int 81 382 (2007)

30 X Wang H C Schroumlder M Wiens U Schloszligmacher and W EMuumlller Biosilica Molecular biology biochemistry and function indemosponges as well as its applied aspects for tissue engineeringAdv Mar Biol 62 231 (2012)

31 S K Lee K E Lee D Jeon G Lee H Lee C U Shin Y JJung S H Lee S H Hahn and J W Kim A novel mutation inthe DSPP gene associated with dentinogenesis imperfecta type IIJ Dent Res 88 51 (2009)

32 N R Kim D H Lee P H Chung and H C Yang Distinct differ-entiation properties of human dental pulp cells on collagen gelatinand chitosan scaffolds Oral Surg Oral Med Oral Pathol OralRadiol Endod 108 e94 (2009)

33 N Al-Sharabi Y Xue M Fujio M Ueda C Gjerde K Mustafaand I Fristad Bone marrow stromal cell paracrine factors directosteoodontogenic differentiation of dental pulp cells Tissue EngPart A 20 3063 (2014)

34 M Y Shie H C Chang and S J Ding Composition-dependentprotein secretion and integrin level of osteoblastic cell on calciumsilicate cements J Biomed Mater Res A 102 769 (2014)

35 X Wang H C Schroumlder M Wiens H Ushijima and W E MuumlllerBio-silica and bio-polyphosphate Applications in biomedicine (boneformation) Curr Opin Biotechnol 23 570 (2012)

36 H S Ching N Luddin I A Rahman and K T Ponnuraj Expres-sion of odontogenic and osteogenic markers in DPSCs and SHEDA review Curr Stem Cell Res Ther 12 71 (2017)

37 S Zhang X Yang and M Fan BioAggregate and iRoot BP plusoptimize the proliferation and mineralization ability of human dentalpulp cells Int Endo J 46 923 (2013)

38 H W Li and J Y Sun Effects of dicalcium silicate coating ionicdissolution products on human mesenchymal stem-cell proliferationand osteogenic differentiation J Int Med Res 39 112 (2011)

39 M Y Shie H C Chang and S J Ding Effects of altering the SiCamolar ratio of a calcium silicate cement on in vitro cell attachmentInt Endod J 45 337 (2012)

40 M Honda K Kikushima Y Kawanobe T Konishi M Mizumotoand M Aizawa Enhanced early osteogenic differentiation by silicon-substituted hydroxyapatite ceramics fabricated via ultrasonic spraypyrolysis route J Mater Sci Mater Med 23 2923 (2012)

Received 25 October 2017 Accepted 10 December 2017




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materials promote the proliferation and differentiation ofDPCs few are able to regenerate the complete dentintissue12 and in some cases the newly formed dentinis very different from primary dentin13 Moreover noneof the materials developed to date has been tested ina clinical trial Therefore it is necessary to develop asuitable scaffold material for dentin regeneration

Recently Professor Muumlller and colleagues synthesizedPLGA-TCP microspheres coated with silica and sil-icatein as a new type of bioactive and biodegradablematerial14 Silicateins are exclusively found in sponges(phylum Porifera) and serve as a template and catalyze thepolymerization of nanoscale silicate to biosilica the mainmineral component of sponge skeletal elements (siliceousspicules) Both silicate and biosilica can induce boneregeneration15ndash17 The microspheres (diameterasymp 800 m)were prepared by encapsulation of -TCP supplementedwith silica and silicatein in PLGA The PLGA-TCPmicrospheres coated with silica and silicatein showed notoxicity in a cell viability assay using human osteogenicsarcoma cells (SaOS-2 cells) Moreover mineralization ofSaOS-2 cells increased when -TCP or silica was added tothe microspheres and mineralization was relatively weakin microspheres without -TCP However the additionof silicatein to the spheres did not affect mineralizationFurthermore an additional increase in mineralization wasobserved when silicatein-containing spheres were mixed ata 11 ratio with silica-containing spheres14

hDPCs can differentiate toward the osteoblast lineageand contribute to the dentinal regeneration process18

accordingly several studies have employed hDPCs as tar-get cells to evaluate the hard tissue regeneration ability ofvarious biomaterials19 Moreover the environment of thetransplantation site influences the fate of stemprogenitorcells in vivo That is the fate of implanted stemprogenitorcells may be determined by the site rather than by theorigin of cells with a tendency to differentiate into theodontoblast phenotype when transplanted into the pulpchamber but into other cell lineages when transplantedto other target locations3 Since the bone and teeth areboth apatite-like hard tissues a material that promotes theosteogenic differentiation of hDPCs in vitro has potentialbenefits for the odontogenesis of hDPCs

Based on its characteristics and function in boneregeneration we speculated that a 11 mixture of thenovel biosilica microspheres (PLGA-TCPsilica-microPLGA-TCPsilicatein-micro) may be beneficial for thegrowth and differentiation of hDPCs and is a potentialnew scaffold material for dentin regeneration Thereforethe aim of this study was to investigate the effect ofPLGA-TCP microspheres coated with both silica andsilicatein on the growth and osteogenicodontogenic dif-ferentiation of hDPCs in comparison with the effects ofuncoated PLGA-TCP microspheres

2 MATERIALS AND METHODS21 Material PreparationMicrospheres were prepared by the encapsulation of-TCP supplemented with silica and silicatein intoPLGA -TCP (sim10 ww) sim5 silica (ww) and05 gmg silicatein were entrapped into PLGA micro-spheres (diameter asymp 800 m) according to a previouslyreported procedure14 The average hardness of the micro-spheres was 005 GPa and the average elastic moduluswas 278 GPa The PLGA-TCPsilica-silicatein micro-spheres were used as the experimental material and thePLGA-TCP microspheres were used as a control mate-rial Each material was molded into disks (diameter =10 mm thickness= 13 mm weight= 38 mg) to preparethe scaffolds used in subsequent cell experiments Prior touse all materials were treated with ultraviolet light for 1 h

22 Cell CultureNormal human dental pulp tissues were obtained fromthe third molars of three patients (18ndash25 years of age)at Peking University School and Hospital of Stomatologyafter obtaining informed consent from the donors and per-mission from the ethics committee (reference no PKUS-SLA2014193) The extracted pulp tissues were mincedand digested in a solution containing 3 mgml collage-nase type I (Worthington Biochemical Lakewood NJUSA) and 4 mgml dispase (Roche Molecular Biochemi-cal Mannheim Germany) for 1 h at 37 C The hDPCswere then transferred to alpha-minimal essential medium(-MEM) supplemented with 10 fetal bovine serum(GIBCO Waltham MA USA) 100 Uml penicillin G100 gml streptomycin (Invitrogen Carlsbad CA USA)and 2 mM L-glutamine (Invitrogen) in a humidified incu-bator with 5 CO2 at 37 C The culture medium waschanged every 3 days and the cells were subcultured at70 confluence hDPCs at passages 4ndash6 were used forall cellular analyses All experiments were repeated threetimes using hDPCs from the three patients

23 Effects of PLGA-TCPSilica-Silicatein on theGrowth Behavior of hDPCs

231 Cell Viability AssayThe effect of PLGA-TCPsilica-silicatein on the viabilityof hDPCs was determined in vitro using the Cell Count-ing Kit-8 (CCK-8 Dojindo Kumamoto Japan) PLGA-TCPsilica-silicatein and PLGA-TCP were pre-wettedin the culture medium for 24 h in a 24-well plate Thenthe hDPCs (2times 104) were seeded onto each scaffold andincubated at 37 C 5 CO2 For the negative controlgroup hDPCs were cultured without any materials Theculture medium was changed every 2 days After 1 35 7 and 9 days of cell culture cells were further incu-bated with the counting reagent for 1 h according tothe manufacturerrsquos instructions The relative cell numberwas determined by measuring absorbance of the formazan




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biosilica porous microspheres promote the osteogenic

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