Cytocompatibility assessment of chemical surface treatments

Cytocompatibility assessment of chemical surface treatments for phosphate glass to improve adhesion between glass and polyester

M.S.Hasan,I.Ahmed,A.J.Parsons,G.S.Walker,C.A.Scotchford

Division of Materials,Mechanics and Structures,Faculty of Engineering,University of Nottingham,University Park, Nottingham NG72RD,United Kingdom

Received11August2012;revised20October2012;accepted1November2012

Published online26August2013in Wiley Online Library(0347fe8381c758f5f61f67f9).DOI:10.1002/jbm.a.34534

Abstract:Fully resorbable phosphate glass?ber reinforced polymer composites have shown real potential for replacing some of the existing metallic bone fracture?xation devices. However,some of these composites have not provided suita-ble mechanical strength pro?les over the required healing pe-riod for bone.Typically,it has been seen that these composites can lose up to50%or more of their strength within the?rst week of degradation.Functionalizing the glass surface to promote polymer adhesion or to introduce hydro-phobicity at the glass surface could potentially introduce con-trol over the mechanical properties of the composite and their retention.In this study eight chemical agents namely, Glycerol2-phosphate disodium salt;3-phosphonopropionic acid;3-aminopropyltriethoxy silane;etidronic acid;hexam-ethylene diisocyanate;sorbitol/sodium ended PLA oligomers and amino phosphonic acid,were selected to functionalise the bulk phosphate glass surface.Selected chemical agents had one functional group(A OH or O?C?N)to react with the glass and another functionality(either A OH,NH2,or Na)to react with the polymer matrix and/or produce hydrophobicity at the?ber surface.Bulk phosphate glass surface-treated with the above agents were assessed for the cytotoxicity of degradation products cell-material interaction in short-and long-term direct cytocompatibility studies.Results obtained from these cytocompatibility studies(using human osteosar-coma(MG63)and primary human osteoblast cell lines) revealed no cytotoxicity from the degradation products and a response comparable to controls in terms of cell functions (attachment,viability,metabolic activity,proliferation,and differentiation)and morphology.V C2013Wiley Periodicals,Inc.J Biomed Mater Res Part A:101A:3301–3310,2013.

Key Words:coupling agents,cytocompatibility,phosphate glass,interface,PLA

How to cite this article:Hasan MS,Ahmed I,Parsons AJ,Walker GS,Scotchford CA.2013.Cytocompatibility assessment of chemical surface treatments for phosphate glass to improve adhesion between glass and polyester.J Biomed Mater Res Part A 2013:101A:3301–3310.

INTRODUCTION

Resorbable phosphate glass?ber(PGF)reinforced polylactic acid(PLA)composites have shown real potential for replac-ing some of the existing metallic bone fracture?xation devi-ces.1–7However,these composites do not retain mechanical strength required for healing period of bone(which can vary from8to12weeks).In some cases a rapid decrease in mechanical properties has been observed(in the?rst week)which can be attributed to two phenomena;(i)early hydration and loss of the polymer-reinforcement interfacial properties1,2,8and(ii)polymer swelling during degradation generating hydrostatic forces that crack the reinforcement phase.2,9Introducing covalent bonds and/or hydrophobicity at the interface could potentially reduce the water uptake and delay hydration at the inter-phase region and decrease polymer swelling,helping to reduce the internal stress of the system.10Bonding strength between?ller(particles/?bers)and polymer matrix,also known as interfacial shear strength(IFSS)can be improved via surface modi?cation of the reinforcement phase with plasma(physical and chemical modi?cation)or chemical coupling agent treatments.Cou-pling agents are chemical substances capable of interacting with both the reinforcement phase and the polymer matrix within a composite,promoting a stronger bond at the inter-face.This enables effective stress transfer across the?ber-matrix interface,improving overall mechanical properties.11

A variety of coupling agents have been investigated in order to improve the?ber/matrix interface,characterized by IFSS measurements.4,12–18A common example is3-ami-nopropyltriethoxysilane(APS),which has been used both for silica based glass16as well as phosphate glass?bers (PGFs).12,14,17It was reported that APS improved the IFSS signi?cantly for silica-based glass conversely very little or no improvement was reported at0.043M concentration for PGFs.14However,when APS was used at higher concentra-tions(1–10wt%)within a PGF reinforced polycaprolactone

Additional Supporting Information may be found in the online version of this article.

Correspondence to:M.S.Hasan;e-mail:sami.hasan@dal.ca

Contract grant sponsor:‘‘Dean of Engineering Research Scholarship’’,University of Nottingham

V C2013WILEY PERIODICALS,INC.3301

(PCL)composite,the bending strength was reported to increase by30MPa(from55to85MPa).Although IFSS val-ues were not measured,the improvement was attributed to better IFSS.19Alternative treatments that have been consid-ered for a variety of reinforcements such as hydroxyapatite (HA),Bioglass V R and phosphate-based glasses(PBG)have included:phosphonic acids18;2-hydroxyethyl methacrylate4; bisphosphonates14,20;hexamethylene di-iso-cyanate(HDI)13 and more recently PLA oligomers.15The effect of these cou-pling agents on IFSS has been quanti?ed for PGFs.14,15How-ever,quanti?cation of IFSS values of particulate(e.g.,HA, TCP,or Bioglass V R)reinforced composites are not avail-able.13,18,21Each of the above agents has been reported to improve the mechanical properties of composites;however, the mechanism and extent of the improvement varies con-siderably.Silanes,HDI and phosphonic acids have been reported to create covalent bonds between polymer and reinforcement,10,13,16,18while PLA oligomers have been reported to improve IFSS by creating hydrogen bonds with the phosphate glass reinforcement.14,15

A comprehensive biocompatibility assessment is required for any material that aims to be used in medical applications.In terms of biocompatibility,the degradable implant material must ful?ll even more demanding require-ments;they have to go through cytotoxicity assessment as well as routine cytocompatibility tests for the bulk material including the degradation products.In a degradable com-posite material all three phases(reinforcement,matrix,and coupling agents)should be tested separately as well as synergistically.

The chemical agents used in this study were selected on the basis of their potential to improve IFSS by providing covalent bonding between PBG and PLA,with the emphasis here?rmly?xed on their cytocompatibility.For example, phosphonic acid-based chemicals have been employed as coupling agents by Phillips et al.,18Greish and Brown,21,22 and Tanaka et al.23These studies reported no cytotoxicity from the phosphonic acids used.Similarly,S a nchez-Vaquero et al.24and Jung et al.25reported favorable cell interaction with silane containing biomaterials.However,con?icting results regarding the cytotoxicity or cytocompatibility for hexamethylene diisocyanate(HDI)has been reported.13,26–28 In this study,the surfaces of bulk phosphate glass discs (9mm diameter and1.5mm thick)were chemically treated with the selected chemicals:Glycerol2-phosphate disodium salt(GP);3-phosphonopropionic acid(PPA);3-aminopropyl-triethoxy silane(APS);etidronic acid(EA);hexamethylene diisocyanate(HDI);sorbitol/sodium ended PLA oligomers (S/Na-PLA)and amino phosphonic acid(APA).After con-?rming the presence of these coupling agents on the modi-?ed glass surface(mPBG),these samples were subjected to comprehensive cytocompatibility tests.A three-step assess-ment program was applied;evaluation of cytotoxicity due to degradation products according to ISO10993-5;short-term direct contact tests and long-term direct contact studies. Cytotoxicity measurements and short-term direct contact of MG63osteosarcoma cells’interaction with chemically modi-?ed glass surfaces were used as a screening step.Finally,primary human osteoblasts(HOBs)were employed to assess cell-material interaction including attachment,prolif-eration,and differentiation via cell metabolic activity,early and late differentiation marker assays for osteoblast cells. Primary HOB morphology was also assessed at each time point using scanning electron microscope(SEM).

MATERIALS AND METHODS

Glass synthesis

Phosphate glass(45P2O516CaO24MgO11Na2O4Fe2O3in molar%)was prepared using the following precursors: NaH2PO4,CaHPO4,MgHPO4.3H2O,P2O5and FePO4.2H2O (Sigma Aldrich,UK).The precursors were weighed out and mixed into a Pt/5%Au crucible type BC18(Birmingham Metal Company,UK),which was then dried in a furnace at 350 C for30min before being transferred to another furnace at1100 C for90min.The molten glass was poured onto a steel plate and left to cool to room temperature.Hav-ing obtained the T g of the glasses via differential scanning calorimetry,they were remelted and poured into a graphite mould at5 C above the T g value and left to anneal for60 min.The furnace was then switched off and allowed to cool slowly to room temperature.The glass rods obtained from the mould were cut into4mm or2mm thick discs of9 mm diameter using a low speed diamond wheel saw(Model 650,South Bay Technology).

Surface treatment of phosphate glass

Eight different chemicals were selected as surface function-alization agents:Glycerol2-phosphate disodium salt(GP);3-phosphonopropionic acid(PPA);3-aminopropyltriethoxy sil-ane(APS);etidronic acid(EA);hexamethylene diisocyanate (HDI);sorbitol/sodium ended PLA oligomers(S/Na-PLA) and amino phosphonic acid(APA).All the chemicals except the PLA oligomers were bought from Sigma Aldrich(U.K) and used without further modi?cation.The PLA oligomers were synthesized following the protocol reported else-where.15The coupling agents were applied to the glass in a range of concentrations(0.05–0.25M)by dip coating.The concentrations were optimized for their effect on interfacial shear strength between phosphate glass and polylactic acid which were reported elsewhere.29,30

Cytotoxicity assessment for extractions products Cytotoxicity assessment of elution products was performed according to the ISO standard for biological evaluation of medical devices(ISO10993-52009).mPBG samples(9mm diameter,4mm thick)were eluted in Dulbecco’s Modi?ed Eagle Medium(DMEM)t10%(4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid(HEPES)for3days with a1mg/ mL weight to volume ratio.Sub-con?uent monolayers of MG63cells were exposed to the treated medium for24h, using a range of concentrations(10–100%eluent diluted in media).A neutral red uptake(NRU)assay was used to assess cell viability.Neutral red(NR)absorption was detected at an optical density of540nm using an ELx800 microplate colorimeter(BioTek Instruments).

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Short-term direct contact test

Cell culture.MG63cells(human osteosarcoma),obtained from the European Collection of Cell Cultures(ECACC),were cultured in complete Dulbecco’s Modi?ed Eagle Medium (CDMEM)consisting of DMEM supplemented with10%fetal calf serum(FCS),2%HEPES buffer,2%penicillin/streptomy-cin,1%glutamine,1%nonessential amino acids(Gibco Invi-trogen,UK)and0.85m M of ascorbic acid(Sigma Aldrich, UK).Cells were cultured in75cm3?asks(Falcon,Becton, Dickinson and Company;UK)at37 C in a humidi?ed atmos-phere with5%CO2.Once con?uent,the cells were dissociated from the?ask using trypsin-EDTA solution(a sterile,phos-phate-buffered saline solution(1x)containing0.025%trypsin and0.01%EDTA)and centrifuged at1200rpm for4min to produce a pellet,which was resuspended in fresh media.Cell concentrations were determined using a haemocytometer;via-ble cells were identi?ed using trypan blue exclusion.

Primary human osteoblasts were also obtained from the European Collection of Cell Cultures(ECACC)and cultured in osteoblast growth medium(417–500)(Cell Applications) following the same subculture protocol described for the MG63cell line.

Coated phosphate glass discs were sterilized using dry heat at190 C for15min and washed three times with sterilized PBS prior to cell culture.Tissue culture plastic (TCP)was used as a positive control for cell growth.Cells were seeded onto the disc sample surfaces at a concentra-tion of40,000cells/cm2and incubated at37 C in a humidi-?ed atmosphere with5%CO2.

Cell viability/metabolic activity

At designated time points,culture medium was removed from the wells and the samples were washed three times with warm PBS.Alamar Blue solution(1:9Alamar Blue: warm hanks balanced salt solution(HBSS))(1mL)was added to each well and incubated for90min.From each well100l L aliquots were transferred to a96-well plate in triplicate and?uorescence was measured at530nm excita-tion and590nm emission using a FLx800microplate reader(BioTek Instruments).

Proliferation

At designated time points cell culture media was removed and the samples were washed three times with warm PBS prior to the addition of1mL deionized water to each well. Cells were lysed using a freeze/thaw technique three times One hundred micro liter aliquots of cell lysate were transferred to a96-well plate.DNA standards were pre-pared using calf thymus DNA(Sigma,UK)and TNE buffer (10m M Tris,2M NaCl,and1m M EDTA in deionized water, adjusted to pH7.4)as a diluent.One hundred micro liter of Hoechst stain33258was added to each well(1mg of bis-benzimide33258in deionized water,further diluted to1:50 in TNE buffer)and the plate agitated.Fluorescence was measured at360nm excitation and460nm emission using a FLx800microplate?uorimeter(BioTek Instruments).DNA concentrations were derived from a standard curve gener-ated by the software(KCjunior).Alkaline phosphatase activity

Alkaline phosphatase activity was measured using the Gran-utest25alkaline phosphatase assay(Randox,UK).A50l L aliquot of cell lysate(as prepared for DNA quanti?cation assay)was added to a96-well plate along with50mL of the alkaline phosphatase substrate(p-nitrophenyl phosphate in diethanolamine HCI buffer,pH9.8),shaken gently and the absorbance measured at wavelengths of405and620 nm using a ELx800microplate colorimeter(BioTek Instruments).

Collagen quanti?cation

Collagen produced by primary human osteoblasts was quan-ti?ed using the Sircol collagen quanti?cation assay.Cell lysates,as prepared for DNA quanti?cation assay,were used as test solution.The assay was performed according to manufacturer’s instruction.Absorbance was measured against distilled water for the reagent blanks,standards, and test samples and collagen concentrations were calcu-lated from the standard curve.

Osteocalcin quanti?cation

Cell lysate was prepared using the protocol mentioned in DNA quanti?cation assay.Osteocalcin produced by primary human osteoblasts was quanti?ed by using a commercially available kit(KAQ1381(96tests)Invitrogen,UK).The assay was performed according to manufacturer’s instructions. Morphology

Samples were washed with warm PBS at37 C and?xed in 3%glutaraldehyde in0.1M Na-cacodylate buffer for30min. After30min the?xative was replaced by a7%sucrose so-lution.Fixed samples were than washed twice in0.1M Na-cacodylate buffer and post?xed in1%osmium tetroxide in PBS for45min in a fume cupboard.Samples were dehy-drated through a graded ethanol series(20,30,40,50,60, 70,80,90,96,and100%in water)for$5min each.Sam-ples were then dried via hexamethyldisilazine(HMDS) before being sputter coated in gold and viewed with a Phi-lips XL30scanning electron microscope operated at10kV. Statistical analyses

Average values and standard deviation were computed for two iterations of the experiments.Statistical analysis was performed using the Prism software package(version3.02, GraphPad Software,San Diego,CA,0347fe8381c758f5f61f67f9). Two-way analysis of variance(ANOVA)was calculated with the bonferroni post-test to compare the signi?cance of change in one factor with time.The error bars presented represent standard error of mean with n?6.

RESULTS

Cytotoxicity assessment for extraction products Cytotoxicity of extraction products from chemically modi?ed PBG surfaces was assessed by neutral red uptake(NRU),a cell viability marker,and presented in parallel with untreated PBG control sample results in Figure1.

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In accordance with the de?nition of cytotoxicity in the ISO standard (BSI-ISO 10993-52009)none of the samples exhibited cytotoxicity,since the cell viability remained over 70%of the nontoxic negative control (TCP)for all samples and all concentrations.However,Na-PLA,S-PLA,and HDI-treated samples did show reduced cell viability at higher concentrations.For all other coupling agents,no signi?cant difference (p <0.05)was observed between samples and controls over the range of concentrations investigated.

Short-term direct contact test (MG63)

Cell viability/metabolic activity.The Alamar blue assay was used to determine the effect of surface modi?cations of PBG on the metabolic activity of osteoblast-like cells.The cells were cultured for 7days (see Fig.2).

Metabolic activity was seen to increase over the 7-day culture period.The TCP control demonstrated an elevated

metabolic activity compared with other samples at later time points (96and 168h),which was statistically signi?-cant (p <0.001).For the last two time points APS-modi?ed glasses also demonstrated statistically signi?cantly higher (p <0.001)metabolic activity compared to control PBG and other mPBGs.TCP and untreated phosphate glass were used as controls;the value for metabolic activity after 7days was greatest for TCP control.

Proliferation

The DNA content of the cells cultured on samples was used as an indicator of cell population.The Hoechst 33258DNA assay was used to quantify the changing concentration of DNA with time for MG63osteosarcoma cells cultured on mPBG,TCP and untreated PBG were used as a positive con-trol (see Fig.3).

FIGURE 1.Viability of MG63osteosarcoma cells,as measured by the neutral red uptake assay.Cells cultured in culture medium with the extracts from modi?ed phosphate glasses.Error bar represents standard error of mean,n ?6.Effect of only neat aliquots on NRU is presented here,effect of 10-90%dilutions can be found online.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

FIGURE 2.Metabolic activity of MG63osteosarcoma cells,as meas-ured by the Alamar blue assay,cultured on modi?ed phosphate glasses,x -axis represents surface treatments.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]FIGURE 3.Proliferation of MG63osteosarcoma cells,as measured by the DNA (Hoechst 33258)assay,cultured on modi?ed phosphate glasses,x -axis represents surface treatments.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

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DNA concentration after 2h was $0.2l g mL à1for all surfaces with no signi?cant difference (p >0.05)observed.For all the sample surfaces,the levels of DNA gradually increased with time.There was no statistically signi?cant difference (p >0.05)seen between the ?nal DNA concen-trations on all tested mPBG surfaces.

Alkaline phosphatase activity

The effect of surface modi?cation on osteoblast-like pheno-type was analyzed by measuring the alkaline phosphatase activity of osteosarcoma cells cultured on mPBGs (see Fig.4).Data were normalized with the corresponding DNA con-centration at each time point.

For all surfaces,including the TCP positive control,the ALP activity was not detectable after 2,48,or 96h.How-ever,after 168h of culture,detectable amounts of ALP ac-tivity were observed on all mPBG surfaces,with notably higher values on EA and S-PLA mPBGs and a lower value for the Na-PLA sample.The ALP activity of cells cultured on S-PLA mPBG was signi?cantly higher (p<0.05)than other samples except for EA treated glass surface.However,there was no signi?cant difference (p >0.05)found between the other mPBG samples (APS,PPA,GP ,EA,Na-PLA,APA,and HDI)and the controls.

Long-term direct contact test

Based on their effect on IFSS 30and suitability for long-term study,selected coupling agents’(APS,GP ,EA,SPLA and HDI)treated samples were analyzed for their compatibility with primary human osteoblast cells.Effect of surface modi?ca-tion on cell proliferation,ALP activity,and collagen and osteocalcin production were monitored.

DNA concentration

The effect of chemical modi?cation of phosphate glass surfa-ces with selected coupling agents on the proliferation of pri-mary human osteoblasts was measured via the DNA content of the cells cultured over a culture period of 28days (see Fig.5).

For all the samples,including the controls,the DNA con-centrations were $0.24l g mL à1after 7days in culture.For all samples,a gradual increase in DNA concentration was observed from day 7to 21.However,no signi?cant change was observed after 21days as the DNA concentrations lev-eled out and remained at $0.35l g mL à1on all the surfa-ces.The trend was consistent for all samples and controls.Although DNA concentration appeared to be higher for the HDI mPBG surface,statistical analysis revealed no signi?-cance difference (p >0.05).

Alkaline phosphatase activity

The ALP activity of primary human osteoblasts was used as an early marker of osteoblastic differentiation.The effect of chemical modi?cation of phosphate glass on ALP activity of primary osteoblasts cultured on glass surfaces for up to 28days is presented in Figure 6.

For all but one of the mPBG surfaces and controls,an increase followed by a decrease in ALP activity was seen as the general trend.S-PLA mPBG surfaces seemed to demon-strate a continuous increase in ALP activity,although the

FIGURE 4.Alkaline phosphatase (ALP)activity of MG63osteosarcoma cells,as measured by ALP assay,cultured on modi?ed phosphate glasses,x -axis represents surface treatments.Data were normalized with corresponding DNA concentration for each inpidual sample.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]FIGURE 5.Proliferation of primary human osteoblasts,as measured by the DNA (Hoechst 33258)assay,cultured on modi?ed phosphate glasses,x -axis represents surface treatments.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

FIGURE 6.Alkaline phosphatase (ALP)activity of primary human osteoblasts,as measured by ALP assay,cultured on modi?ed phos-phate glasses,x -axis represents surface treatments.Data were modi-?ed with DNA values obtained for each inpidual sample.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

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difference between the last three time points was not statis-tically signi?cant (p >0.05).APS mPBG samples demon-strated the highest ALP activity and TCP showed the lowest.These differences were noted to be statistically signi?cant (p <0.001).

Collagen production

Collagen production by primary human osteoblasts cultured on selected modi?ed mPBG surfaces for up to 28days was assessed and quanti?ed via the Sircol collagen quanti?cation assay (Fig.7).

Collagen production was found to be highest on the APS and GP mPBG surfaces;however,concentrations were simi-lar for the remaining surfaces.Statistical analysis revealed no signi?cant difference (p <0.05)for the highest values found for each sample treatment and controls.Notably,a characteristic reduction of collagen production was observed for the TCP control.

Osteocalcin quanti?cation

Osteocalcin production was assessed in primary human osteoblasts cultured on selected modi?ed phosphate glass surfaces for up to 28days and was assessed and quanti?ed by an ELISA osteocalcin quanti?cation assay kit (Fig.8).

Levels of osteocalcin did not reveal any signi?cant differ-ences (p >0.05)between all sample surfaces investigated and between each time point.However,when osteocalcin concentration was normalized with DNA,a downward trend was observed for all samples including controls after 21days.Two-way ANOVA revealed a signi?cant difference (p <0.001)between control and mPBGs (excluding GP mPBG)samples at earlier time points (7and 14days)which were lowest on control PBG surface.

Morphology

The morphology of cells cultured on phosphate glass discs was visualized using SEM (see Fig.9and Supporting Infor-mation).Within each ?gure a representative image is presented of osteoblasts cultured on phosphate glass speci-

mens for 7,14,21,ad 28days.In general,the SEM images of human osteoblasts cultured on all glass surfaces showed a con?uent layer after 7days of culture.There were some very large clusters of osteoblast cells with lamellipodia extended to neighboring cells,resulting in the formation of a denser cell layer at later time points.However,no star-shaped clusters were spotted at any time point on the TCP control,where the cells were densely packed showing spin-dle-shaped cells.After 21days,layers that are much denser were observed and collagen ?brils could also be seen after 28days of culture.

DISCUSSION

Coupling agents to improve the interface between polymer matrix and reinforcing phosphate glass have been investi-gated for their cytocompatibility.A three-step screening approach was adopted.The study included an indirect or extraction study,a short-term direct contact cell viability,metabolic activity,and ALP activity assessment using an MG63osteosarcoma cell line and a long-term study using primary human osteoblasts in direct contact with mPBG surfaces to evaluate cell proliferation,differentiation,and morphological assessments.

To assess potential toxicity of the chemicals leaching out from the mPBG surfaces,combined degradation extracts from PBG as well as coupling agent layer were tested for cytotoxicity through neutral red uptake (NRU)(see Fig.1and Supporting Information).Greater NRU was observed for GP and EA mPBG surface compared with the PBG control;suggesting that the degradation products (mainly calcium and phosphates)may be supportive in cell metabolic activ-ity leading to greater cell number and hence a high NRU.This relatively higher cell metabolic activity was anticipated as b -glycerol phosphate is often used as a carbon source,essential for the expression of osteoblast phenotype expres-sion.A hydrolysis product of b -glycerol phosphate (phos-phates)are reported as positive effecter for induction of gene expression.31Bisphosphonate etidronic acid is also reported to be bene?cial for osteoblast growth,however,

FIGURE 7.Amount of collagen produced by primary human osteo-blasts,as measured by Sircol assay,cultured on modi?ed phosphate glasses.Data were modi?ed with DNA values obtained for each indi-vidual sample.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

FIGURE 8.Quantity of osteocalcin produced by of primary human osteoblasts,as measured by ELISA assay,cultured on modi?ed phos-phate glasses,x -axis represents surface treatments.All data were modi-?ed with DNA values obtained for each inpidual sample.Error bar represents standard error of mean,n ?6.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

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higher dosages of etidronic acid are known to impair normal skeletal mineralization.32

A signi?cantly lower NRU was observed for the oligomer treated samples which was suggested to be due to acidic degradation products from the PLA oligomers,as their molecular weight was very low (20,000Da)and therefore prone to rapid hydrolysis.Ignatius and Claes reported that high concentrations of degradation products from PLA had a toxic in?uence on the cell culture systems they studied using MTT and BrdU cytotoxicity assays.33Another study revealed that increased in?ammation was observed when the molecular weight of PLA/PGA copolymers decreased to between 10,000and 20,000Da.34Signi?cantly (p >0.01)lower NRU was also observed for HDI-treated surfaces.Dong et al.stated that during the process of polyurethane synthesis some of the toxic hexamethylene diamine (HDA)could be released.13Based on information on the chemical structure of HDI grafted HA (gHA),Dong et al.reported three possible mechanisms for release of HDA derivatives;(i)hydrolysis of the urea linkage in the extended chain;(ii)hydrolysis of urethane linkage between HA and HDI,and (iii)ionization of gHA.The results from this study are in agreement with Dong et al.28as the cytotoxicity was found to be dependent on the content ratios of treatment media in culture medium.

Because of the lack of evidence for cytotoxicity from any of the coupling agents investigated,all mPBGs were included in the next stage of cytocompatibility assessment,which included use of osteoblast-like MG63cells cultured in direct contact with the mPBG surfaces and assessed for cell viability,proliferation,and differentiation.

All samples including positive controls (PBG and TCP)revealed similar results for cell viability as measured via alamar blue assay,with no signi?cant differences observed

FIGURE 9.Representative SEM micrographs of primary human osteoblast cultured on control and surface modi?ed PBG for 28days.Mature cell matrix and clusters of cells were found on all the samples.SEM micrographs of cells cultured on sample surfaces for 7,14,and 21days can be found online.[Color ?gure can be viewed in the online issue,which is available at 0347fe8381c758f5f61f67f9.]

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between samples and/or controls at each time point(see Fig.2).However,for all surfaces investigated a linear increase in?uorescence values was seen from2h to7 days,which demonstrated that cells were metabolically active and there were no adverse effects from the surface chemistry on cell metabolic activity.

Alkaline phosphatase was detected after7days of MG63 culture for all samples investigated as expected(see Fig.4). Higher activity was observed for EA and S-PLA mPBG surfa-ces.EA as mentioned above encourages osteoblastic meta-bolic activity and sorbitol which is a hydrogenated carbohy-drate,is reported to regulate osteoblast differentiation35 which could be the reason for the higher ALP activity shown by these samples.

After this second stage cytocompatibility assessment, Na-PLA and PPA were excluded from the study.This deci-sion was based mainly on their fast degradation(not suita-ble for long-term study)and the relatively low cytocompati-bility of Na-PLA.However,as HDI showed to be the most effective surface treatment in terms of improving interfacial shear strength30it was also included in this long-term cyto-compatibility study.

Proliferation of primary human osteoblasts in terms of DNA content(see Fig.5),on coupling agent treated glass surfaces after each time point up to28days,were found to be comparable with the controls.After7days in culture media a con?uent layer was observed from SEM images for all mPBG and control sample surfaces.After7days the growth rate decreased,as expected.However,an increase in DNA content implies that once a con?uent layer is formed on the surface,proliferation is less dependent on the glass surface as further cell growth will occur on top of the con-?uent cell layer present,forming multi layers,which was observed from SEM images(see Fig.9and Supporting Information).

An increase in alkaline phosphatase concentration was also observed followed by a reduction in alkaline phospha-tase concentration for all mPBG surfaces investigated over the28day period,with the exception of SPLA mPBG,where a plateau was observed(Fig.6).Alkaline phosphatase activ-ity by osteoblasts in vitro was expected to be up-regulated as proliferation is retarded and down-regulated when osteo-blasts enter the mineralization phase.36,37Dias et al.38 reported an up regulation followed by a decrease in ALP ac-tivity with human bone marrow cells on calcium phosphate glass ceramics which correlated well with the results observed here.

Collagen production was detected after7days of osteo-blast culture on all mPBG surfaces including TCP(see Fig.

7).However,no signi?cant up or down regulation of colla-gen quantity was observed and only TCP showed a down regulation of collagen when normalized with DNA content. Similar trends(i.e.a plateau in the collagen quantity)were reported by Au et al.39with MG63cells cultured on TCP control and Consil V R bioactive glass particles and Bosetti et al.40,41with rat osteoblast-like cells cultured on bioactive glass powder surfaces.Leonardi et al.,also reported a signif-icant increase from day1to day7followed by constant col-lagen concentrations at days7and14using human bone marrow cells cultured on a resorbable phosphate based glass ceramic.42A50%increase in collagen concentration from day7to14followed by an almost constant concentra-tion up to28days in culture was also reported by Burl-ing.43Here,craniofacial osteoblast cells were cultured on ternary phosphate based glasses and TCP control.43The data presented by Burling also correlated well with the results obtained in this study.

Osteocalcin was found in detectable quantities on all mPBG surfaces including the positive controls(see Fig.8). The non-normalized osteocalcin concentrations were virtu-ally identical for all samples investigated and at each time point.However,when normalized with DNA concentrations, highest concentration of osteocalcin was found after7days of cell culture on all samples.On all samples osteocalcin concentration reduced after7days in culture,with the low-est values detected after28days.This trend was consistent for all mPBG surfaces examined.Osteocalcin,as reported by Owen et al.44and Stein et al.,36is a late marker of osteo-blastic differentiation which is detectable only after full mat-uration of cells and proliferation to con?0347fe8381c758f5f61f67f9parable trends to this study were reported by Carpenter et al.45Var-anasi et al.,reported a150%increase in proliferation rate for osteoblast-like MC3T3-E1cells supplemented by extracts from bioactive glass45S5and6P53-b.Collagen synthesis was enhanced and a higher expression of osteocalcin pro-tein was observed.It was reported that osteocalcin was only detectable after3days and highest values were obtained after6days with no signi?cant change in osteocal-cin quantity.46Attawia et al.analyzed media osteocalcin lev-els synthesized by osteoblasts cultured up to21days on the surface of PMA-ala:CPH(30:70)polymer matrices.It was reported that no signi?cant difference was found on the polymer surfaces.47When human bone marrow stromal cells were cultured on a resorbable P2O5–SiO2–CaO–MgO–Na2O–K2O phosphate glass ceramic,up-regulation in osteo-calcin/Glyceraldehyde3-phosphate dehydrogenase(GAPDH) gene expression was reported although this increment was statistically insigni?cant from day7to14and match with the results observed in this study.42

It can be deduced from the results obtained for collagen and osteocalcin quanti?cation that the degradation products from unmodi?ed phosphate glass and mPBG surfaces,espe-cially EA,GP,and APS,enhanced collagen production and assisted the up-regulation of downstream markers such as osteocalcin during osteoblast differentiation.

Phosphonic acid-based chemicals have been employed as coupling agents in the past.For example,Phillips et al. grafted allyl phosphonic acid onto calcium phosphate to produce a chemically bonded composite.18Tanaka et al. reported on the synthesis of surface modi?ed calcium hy-droxyapatite with pyrophosphoric acid.23All these studies reported no cytotoxicity from the phosphonic acids used which correlated and supported the results obtained in this study for PPA,APA,and EA.

S a nchez-Vaquero et al.prepared agarose hydrogels con-taining aminopropyl triethoxy silane(APTS)and evaluated

3308HASAN ET AL.CYTOCOMPATIBILITY ASSESSMENT OF CHEMICAL SURFACE TREATMENTS

them for adhesion and proliferation of mesenchymal stem cells(MSCs)and found that although adhesion was lower on APS rich scaffolds,the proliferation rate on these surfa-ces was higher.24In another study,Jung et al.,reported that type I collagen was immobilized on a HA surface via use of APS.It was reported that collagen-grafting on HA enhanced the?broblast adhesion due to the excellent biocompatibility of silane-treated HA.These studies support the results found in this study for APS mPBG and explain the relatively high MG63proliferation and human osteoblast ALP activity observed for APS mPBG surfaces.

In this study addition of Na or sorbitol functional groups to biocompatible PLA was hypothesized to not affect the cytocompatibility signi?cantly.As reported in a study by Mei et al.,35biocompatibility of a sorbitol-containing polyes-ter series was evaluated against a PCL control by measuring cell spreading and proliferation of a mouse?broblast3T3 cell line in vitro.It was found that sorbitol-containing poly-esters and PCL had comparable biocompatibility.

A number of studies have previously employed hexam-ethylene diisocyanate(HDI)in a variety of applications for medical use;such as a growth factor carrier,13,28a coupling agent in poly(ethylene glycol)-poly(-caprolactone)-poly(eth-ylene glycol)copolymers,48as a compatibiliser for bioactive glass nanoparticle reinforced poly(L-lactide)composites(g-BG/PLLA)26and as a coupling agent in a hydroxyapatite re-inforced PEG/PBT copolymer composites.27Dong et al. reported dose-dependent cytotoxicity for degradation prod-ucts from HDI grafted calcium hydrogen phosphate on rat osteoblast cell metabolic activity.Conversely,biocompatibil-ity with very low cytotoxicity was reported for PEG-PCL-PEG copolymers cross-linked with HDI.48Liu et al.reported that in-vitro testing of g-BG/PLLA composites in SBF showed that apatite was deposited easily on the surface of the composite scaffolds while in-vitro biocompatibility tests showed that introduction of BG or HDI compatibilized g-BG particles into the PLA matrix,attachment,and proliferation of mesenchymal stem cells was supported.26Therefore,due to lack of evidence on signi?cant cytotoxicity,from results observed in current study or reported in the literature,HDI is a rationale choice for surface treatment.However,care must be taken to remove all unreacted HDI from the system.

Eight different surface treatments were applied to a phosphate glass surface in order to make it adhere to the polymer strongly.All surface treatments investigated in this study were found non-toxic to MG63cells even at higher concentrations.Human osteosarcoma and primary human osteoblast exhibited normal cell functional(attachment,pro-liferation,differentiation)when cultured directly onto the surfaces of surface-treated phosphate glass.Morphology of cells cultured on treated glass surfaces was also found com-parable to the control(untreated phosphate glass). CONCLUSIONS

Results obtained for the cytotoxicity/cytocompatibility of human osteosarcoma and primary human osteoblast cell lines with surface treated phosphate glass revealed no cyto-toxicity from extraction products on osteosarcoma cells.A comparable response of MG63and primary human osteo-blast cells with unmodi?ed PG and TCP in terms of cell attachment,viability,metabolic activity,proliferation,and differentiation was also observed.Although some coupling agents investigated were relatively poor(e.g.,HDI,Na-PLA) others(e.g.,APS,GP,EA)were excellent in terms of cyto-compatibility.Variation observed in cytocompatibility could be attributed toward unreacted functional groups(e.g., O??C??N)or degradation products(e.g.,acidic products from PLA oligomers).However,it can be concluded that all the coupling agents investigated and their degradation prod-ucts were cytocompatible and had no adverse effect on cell functions and cell morphology.

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