IN VITRO BEHAVIOUR OF HUMAN OSTEOBLASTS ON DENTIN AND BONE

Cell Biology International 2002, Vol. 26, No. 4, 337–346doi:10.1006/cbir.2002.0860, available online at http://www.idealibrary.com on

IN VITRO BEHAVIOUR OF HUMAN OSTEOBLASTS ON DENTIN AND BONE

B. A. A. SCHEVEN, D. MARSHALL1 and R. M. ASPDEN

Department of Orthopaedic Surgery and 1Foresterhill Electron Microscopy Unit, Department of MedicalMicrobiology, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, Scotland, U.K.

Received 26 June 2001; accepted 10 January 2002

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culturepolystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material.Cellular attachment, viability and growth were monitored in parallel cultures of humanosteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells(HOBs). Culture plastic was either left untreated or roughened with abrasive paper of variousgrit sizes (4000–1200 grit) in order to obtain a level of roughness comparable to that of thedentin slices. Cell counting and intracellular BCECF staining showed that after an initialincubation of 2 h, the primary cells attached and spread out more quickly on the differentsubstrates than the three cell lines. The primary cells also showed a stronger mitochondrialstaining and viability on dentin. During subsequent culture morphological differences appearedwith the cells on dentin displaying more cellular extensions. All three cell lines proliferated moreslowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affectedin their growth by the different substrates. Total and specific alkaline phosphatase (AP) activityof the cell lines was not significantly affected by the different substrates after short-termadhesion, but it was increased for the primary cells on the dentin. However, after 2–3 days ofculture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasmatreatment of the roughened plastic did not alter cellular viability or AP activity, suggesting thatgrinding of the surface did not affect the property of the culture plastic to support cellattachment and growth. In conclusion, the results show that not only do osteoblastic cellsbehave differently on a natural calcified substrate surface than on standard culture plastic, butalso that differences were evident between the various cell types, in particular the primary HOBversus the continuous cell lines. � 2002 Elsevier Science Ltd. All rights reserved.

K: osteoblasts; cell culture; bone; dentin; substrate; surface roughness; culture plastic; cellattachment; viability; proliferation; differentiation.

Correspondence to: Dr B. A. A. Scheven; Tel: +44 1224 554910;Fax: +44 1224 685373; E-mail: [email protected]

INTRODUCTION

Although organ and cell cultures have long been acornerstone for basic research into the regulationof bone metabolism, it remains a concern how wellcell biological data obtained from isolated cells invitro relate to physiological and pathological pro-cesses as they occur within the skeleton. Followingan observation that osteoblasts appeared to pro-liferate more slowly and behaved differently when

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cultured on cut surfaces of bone than on the usualtissue culture-treated plastic, we were surprised tofind very little in the literature comparing thebehaviour of these cells in these two environments.Previous studies by Jones and Boyde (1977, 1979)focused on osteoblast behaviour in organ culturesusing SEM, and they showed that osteoblastsreadily coated and migrated onto various biologi-cal substrates irrespective of their shape and rough-ness. Interestingly, the authors noted that the cellsdisplayed an altered morphology on glass whenmigrated from the bone surface (Jones and Boyde,

� 2002 Elsevier Science Ltd. All rights reserved.

338 Cell Biology International, Vol. 26, No. 4, 2002

1977). Further work by Gray et al. (1996, 1998),using isolated rat calvarial osteoblasts, confirmedthat bone topography was an important factor inthe location and timing of bone formation inculture. Dentin slices in which resorption pits werecreated by prior cultivation with osteoclastic cells,increased MG63 cell proliferation and alkalinephosphatase activity (Schwartz et al., 2000), andrat calvarial osteoblasts produced new bonepatches initially in the resorption pits (Jones et al.,1994). Roughening of bovine cortical bone slicespromoted differentiation of rat osteoblasts as com-pared to those on ‘smooth’ bone surfaces, whichwas associated with an increased osteoclastic boneresorption (Matsunaga et al., 1999a,b).

Other studies have shown that osteoblastsrespond to different biomaterials and extracellularmatrices with modified cell differentiation andmatrix production depending on the substrate sur-face chemistry and topography (Boyan et al., 1996;Anselme, 2000). However, most studies havefocussed on assessing the biocompatibility ofartificial surfaces and engineered bone-derivedgrafts. A fundamental understanding of osteoblastbiology on natural bone surfaces is limited.

The present study started to address this bymeasuring the attachment and proliferation ofhuman osteoblasts on natural bone surfaces indirect comparison with similar cultures on tissueculture plastic. Preliminary work in our laboratoryusing thin slices from human and bovine corticalbone demonstrated a reduced cellular proliferationand an altered alkaline phosphatase in osteo-sarcoma cell cultures as compared to those onstandard culture plastic (unpublished obser-vations). However, the cortical bone slices con-tained relatively many irregularities and lacunaemaking it difficult to perform consistent compari-sons and microscopic observations. Therefore, forthe purpose and consistency of this study we showhere results from experiments employing dentinslices, which are more even and lack major lacunae.In addition to the obvious difference in surfacechemistry between these two substrates, the plasticsurface was much smoother. The surface roughnesswas made comparable by roughening the plastic, asit was found impossible to polish the dentin to thesmoothness of the plastic. However, because thiswould effectively remove the activated surface fromthe plastic, some roughened samples were plasmatreated to restore a hydrophilic surface. The resultsshowed that human osteoblastic cells from differentsources did show differences in behaviour whencultured on dentin compared with cells on standardculture plastic.

MATERIALS AND METHODS

Substrates

Slices were cut from small blocks of ivory/dentinusing a mineralogical saw (Accutom-2, Struers,Glasgow, U.K.) equipped with a 125 mm alu-minium oxide cut-off wheel. Subsequently, smallcircular slices (5 mm diameter to fit a well in a96-well-plate) were carefully punched out usinga standard paper punch. Thermanox coverslips(25 mm, Nunc) were used as control culture plastic.Plastic was roughened by grinding the surface withsilicon carbide paper of 4000, 2400 and 1200 grit(grain size 5, 10, and 15.2 �m, respectively), andcircular, 5 mm diameter slices were prepared fromthe coverslips with a punch (identical size as thebone slices). Care was taken to ensure that grindingwas done in a random pattern to avoid theformation of regular grooves which are known toaffect cellular behaviour. The slices were cleanedand stored in 70% ethanol. A New View 2003-D imaging surface structure analyser (ZygoCorporation, Connecticut, U.S.A.) was usedto visualise and measure the physical surfacecharacteristics of the prepared substrates.

Cell cultures and experimental approach

Human osteogenic sarcoma cell lines, designated asMG63, HOS TE85 and SaOS-2 were obtainedfrom the European Collection of Cell Cultures(Salisbury, U.K.), and propagated in DMEM sup-plemented with L-glutamine, antibiotics (penicillinand streptomycin) and 10% fetal bovine serum(FBS). Primary human osteoblast (HOB) cultureswere established from human femoral trabecularbone explants obtained from patients with estab-lished osteoarthritis undergoing total hip replace-ment (age range: 55–77; average age of 67.0�11.2years) according to routine procedures and main-tained in DMEM/10% FBS (e.g. Scheven et al.,1995). The osteoblast cultures have been shown toexpress osteoblastic features such as alkaline phos-phatase and osteocalcin production. Before reach-ing confluence, the cells were sub-cultured using0.05% trypsin/1 m EDTA digestion, and for theexperiments first to second passage cells were used.

Dentin slices were transferred into 35-mm petridishes or 6-well plates and air-dried. They wereplaced on a piece of parafilm that had been rinsedin 70% ethanol. This promoted the formation of adroplet when medium was pipetted onto the boneslice and prevented it flowing out onto the plasticof the culture plate. The plastic slices were placed

Cell Biology International, Vol. 26, No. 4, 2002 339

directly on the bottom of separate culture dishes.Cells were dissociated from the culture flasks bytrypsin treatment, counted and re-suspended intoculture medium to achieve the appropriate celldensity. A 20 �l drop of cell suspension containing3000 cells was then pipetted carefully onto eachslice, and the cells were allowed to attach in a5%-CO2 incubator at 37�C for 2 h. Subsequently,the cultures were gently rinsed in DMEM/FBS toremove the non-adherent cells, the parafilm wasremoved from the bone slice cultures, and extramedium was then added for further incubation. Atthis stage some cultures were used for biochemicalor morphological analysis (day 0), while otherswere incubated for different time periods (1–3days).

Cell attachment, viability and morphology

BCECF-AM (2�,7�-bis-(carboxyethyl)-5-carboxy-fluorescein acetomethylester; Molecular Probes)was used as a sensitive indicator of the number ofviable, attached cells (Sinha et al., 1994; Nordstrømet al., 1999). BCECF-AM is a non-fluorescentesterified dye that when internalised by living cells,is hydrolysed to a membrane-impermeable BCECFwhich is fluorescent (see also Molecular Probesprotocol sheet, http://www.probes.com). The cul-tures were rinsed in serum-free DMEM, and thenincubated in serum-free medium supplementedwith 2 � BCECF-AM. After 30 min, the cultureswere rinsed in PBS and the slices were transferredinto flat-bottomed 96-well plates and 200 �l PBSwas added per well. The fluorescence was quanti-fied using a Wallac multi-well spectrofluorimeter at485 nm excitation and 535 nm emission. Sliceswithout cells were used to measure backgroundfluorescence. Some slices were also examined forfluorescence using an inverted microscope (ZeissAxiovert S100) using a FITC filter to evaluate cellnumber and morphology. To visualise the cellnuclei, adherent cells were fixed with 70% ethanolor citrate-buffered acetone for 90 s, washed in PBS,and stained with DAPI (0.1 �mol/ml) for 2–3 min.The cultures were washed in PBS, and the numberof positive nuclei was assessed microscopicallyunder fluorescence with a DAPI filter. As allSaOS-2 cells contain high levels of alkaline phos-phatase (AP) activity, the number of cytochemicalAP positive stained cells was determined using alight microscope.

For scanning electron microscopy (SEM),the slices with or without adherent cells wererinsed gently with 0.89 isotonic phosphatebuffer (pH 7.2), followed by fixation in 2.5% (v/v)

glutaraldehyde and 2.5 MgCl2 in phosphatebuffer for 1 h at room temperature. The sampleswere rinsed in 0.1 phosphate buffer (pH 7.2), andpost-fixed in osmium tetroxide for 1 h. They werethen dehydrated through a series of graded ethanolsolutions, subjected to critical point drying, sputtercoated with 20 n platinum and examined with aJeol JSM-35CF SEM at an accelerating voltage of10 kV.

Cell proliferation assays

BrdU (5�-bromo-2�-deoxyuridine-5�-monophos-phate), a thymidine analogue, was used to visualiseand identify mitotically active, replicating, ‘S-phase’ cells using an immunocytochemical pro-cedure (Scheven et al., 1995). After 2–3 days,cultures were incubated with 50 � BrdU and50 � deoxycystidine (Sigma) for 2 h, then fixed incitrate-buffered acetone, and stained for cytochemi-cal AP (see below). Subsequently, the samples were(post)fixed with 70% ethanol for 15 min at roomtemperature, and further permeabilised by 20 mintreatment with 4 N HCl. After extensive rinsingwith PBS, the slices were incubated with aspecific mouse anti-BrdU antibody (BoehringerMannheim), 1:500 dilution, in PBS containing0.5% bovine serum albumin (BSA) and 0.1%Tween for 2 h at 37�C. After a PBS wash, thesamples were treated with a 1:100 dilution ofanti-mouse IgG conjugated with peroxidase(Sigma) in PBS with 0.1% Tween and 1% goatserum. The antibody labelling was visualised bystaining with DAB, and the nuclei were counter-stained with neutral red or hematoxylin.

Alamar Blue is a stable Redox indicator dye,which allows the monitoring of metabolic cellviability, which is directly correlated with cellnumbers (American Biotechnology Lab, 1993).Chemical reduction, related to cell growth, changesthe oxidised, non-fluorescent Alamar Blue into afluorescent, red form which can be measuredfluorometrically using an excitation wavelength of560 nm and emission of 590 nm, or spectrophoto-metrically at an absorbance of 570 nm. At the endof the culture period, the slices were transferredinto round-bottom 96-well plates containing 10%FBS/DMEM and 20 �l Alamar Blue (Serotec) wasadded per well (10% v/v). After 4 h incubation,medium was removed and transferred to anothermulti-well plate, and the absorbance and/or fluor-escence was determined using designated micro-plate readers. The remaining slices were rinsed inPBS and used for further biochemical analyses.


Alkaline phosphatase assays

Alkaline phosphatase (AP) was used as amarker for osteoblast phenotype and differen-tiation. Cytochemical detection was performed,after fixing the adherent cells with citrate-bufferedacetone for 90 s, by incubation for 15 min at roomtemperature in 0.2 mg/ml �AIBMX and 1 mg/mlFast Red-TR in 0.2 M Tris/HCl buffer, pH 8.5 (redstaining), or using a Sigma kit with Naphtol AS-BIas substrate and Fast Blue BB as coupler. Thenumber of positively stained cells was countedusing a light microscope.

Biochemical analysis of AP activity was carriedout as described previously (Scheven et al., 1995).Briefly, the cells were lysed in 0.1% Triton X-100,and incubated in 2-amino-2-methyl propan-1-olbuffer (pH 10.2) containing 20 m p-nitrophenylphosphate (pNPP) at 37�C. Production of PNP(p-nitrophenol) was quantified spectrophotometri-cally at an absorbance of 405 nm using an auto-matic plate reader. Protein content was measuredusing a micro-BioRad assay with albumin as stan-dard. DNA content of the cultures was assayedspectrofluometrically using Hoechst 33258 dye asDNA-binding fluorochrome (emission at 450 nm;excitation at 355 nm). Salmon testis DNA was usedas standard.

Statistical analysis

Data analysis and statistical comparisons weremade between groups using Student’s t-test, fortwo groups, and ANOVA for more than twogroups. Following ANOVA, Student-Newman-Keuls’ test was use to identify the groups whichwere significantly different.

RESULTS

Substrate surface and property analysis

Surface structure analysis showed that the Ra-value(the mean modulus of the height or depth of thesurface asperities) did not vary much between thedifferent cut bony surfaces and ranged between0.24 to 0.31 �m, whereas control culture plastic hadan average Ra-value of 0.026 �m. Roughening theplastic using silicon carbide paper with grit sizes of4000, 2400 and 1200 resulted in respective Ra-values of 0.071, 0.102, and 0.225 �m, showing thatthe 1200-grit roughened plastic had a comparableroughness to the dentin surface. Scanning electronmicroscope images of the substrates showed thedifferent physical surface characteristics of dentin

and plastic (Fig. 1). Despite similar average rough-ness, the 1200-grit plastic displayed a differentsurface texture than the bone.

Fig. 1. Scanning electron micrographs of the three substrates:A, Control plastic coverslip, B, coverslip roughened with1200-grit abrasive paper; and C, dentin/ivory slice cut using amineralogical Accutom saw (bar: 10 �m). Equivalent sliceswith primary HOB (D,E,F; bar: 10 �m) and MG63 (G,H; bar:10 �m) after 2 h-incubation. The HOBs are well spread on allsubstrates and the cells on the dentin slice display distinctextensions (F, arrows) but on the plastic have a smootherperimeter (D, arrows), whereas the MG63 cells are stillrounded up with few cellular extensions after 2 h-attachment(G,H).

Cell attachment, viability and morphology

Initial experiments with SaOS-2 cells showed thatthe cells adhered to the dentin and culture plastic inequal numbers and that a 2-h incubation wassufficient for maximum cell attachment (results notshown). Moreover, preliminary work confirmedthat quantitative microfluorescence measurementof BCEC-loaded cells correlated well with cellnumbers on the different substrates.

There were no measurable differences inattachment and viability between the transformedcell lines on the different substrates (Fig. 2A).However, the primary human osteoblastic cellsdisplayed a higher BCECF staining when attachedto the bone slices, which was associated with anincreased AP activity (Fig. 2A,B). This phenom-enon was not related to greater cell numbers, asthe number of DAPI-positive cells was similar onall substrates (Fig. 2C). Microscopic examinationindicated that after a short-term adhesion period(2 h), HOBs were already spread out on the differ-ent substrates, unlike the cell lines which at this


stage had a rounded morphology (Fig. 1). SEMalso revealed that the primary cells on dentin hadmany particular cellular extensions resemblingfocal adhesion points. After subsequent culture,the HOB on the dentin maintained a distinctivemorphology exemplified by more definite cellularextensions than the cells on plastic. The HOBs onroughened plastic (1200-grit) displayed a moreirregular shape, whereas the cells on control plastichad a ‘smoother’ appearance with few cellularextensions. This was also evident in SaOS-2 cellcultures, which displayed a smoother outline whencultured on plastic and distinct cellular extensionswhen cultured on bone (Fig. 3).

Cell proliferation (BrdU & Alamar Blue)

Immunocytochemical detection of BrdU labellingwas used to analyse the rate of cell replication,which also enabled visualisation of the proliferat-ing cells on the substrate surface (Fig. 3). Thetransformed cell lines had a significantly lowerproliferation rate on dentin, as shown by the

labelling index (LI), compared with cells on controlplastic (Fig. 4). Roughening the plastic had nosignificant effect. However, unlike the cell lines, theprimary cells did not show a difference in theBrdU-labelling, suggesting that proliferation ofthese cells was not affected by being on dentinrather than on plastic. Results obtained from thealamar blue method confirmed these findings in thecell lines; a decreased proliferation of the cell lineswas observed on dentin slices, but the roughness ofthe plastic had no effect (Fig. 5). Remarkably,the primary HOB on the natural bony slicesshowed a significant increase in Alamar Blue stain-ing suggesting an increased metabolic activity ofthese cells (Fig. 5).

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Fig. 2. Adhesion and activity of the various osteogenic cell lines (MG63, HOS TE85, SaOS-2) and primary osteoblast-like cells(HOB) (A) Spectrofluorimetric analysis of BCECF-loading, and (B) AP activity of the cells after attachment to the differentsubstrates for 2 h. (C) Adhesion of the primary HOB as determined by counting of nuclei of attached cells stained with DAPI.Mean�SEM of six replicate cultures. *P<0.05 vs control or 1200-grit coverslip.

Osteoblast differentiation

AP activity was used as a phenotypic marker forosteoblastic cells. AP, measured biochemically, wassignificantly decreased on bone slices for boththe continuous cell lines and primary cultures after3 days of culture (Fig. 6). AP activity was also


reduced on the roughened plastic, though to alesser degree than the cultures on bone. The bio-chemical data were confirmed by cytochemicalanalysis of the number of AP-positive cells in HOSTE85 cultures (data not shown).

Plasma treatment

Mechanical abrasion of the surface may haveaffected its ability to support cellular attach-ment and growth as it caused the plastic tobecome hydrophobic, shown by water dropletsrounding up rather than spreading. Subsequentplasma-treatment was used to restore surfacehydrophilicity or ‘wettability’. However, plasma-treatment of the roughened plastic had no effect onthe attachment or AP activity of the cells aftereither short-term incubation or 3 days in culturecompared with non-treated surfaces (results notshown).

Fig. 3. SaOS-2 and HOS TE85 cells after culture for 2 days oncontrol plastic or bone. BrdU was added to the cultures duringthe last 2 h, and was immuno-cytochemically detected incombination with AP staining as described in Materials andMethods. Block arrows point to BrdU-positive nuclei, and thethin arrows indicate the typical cellular extensions of the SaOScells on bone. BrdU labelling was markably reduced in the cellcultures on bone.

DISCUSSION

It is generally accepted that bone cells are sensitiveto the material composition and surface rough-ness of the substrate on which they are cultured(Boyan et al., 1996; Anselme, 2000). The particularresponse also depends on the cell type used and

its maturation stage (Hunter et al., 1995; Lohmannet al., 2000). However, the considerable volume ofresearch reporting on cellular interactions withbiomaterials has concentrated on the ability ofartificial substrates such as metallic alloys, poly-mers and ceramics, or bone-derived composites tosupport cell growth and function (Sinha et al.,1994; Zambonin and Grano, 1995; Hunter et al.,1995; Oreffo et al., 1998; Kue et al., 1999; Shahet al., 1999).

Surprisingly, few quantitative studies have dealtwith the in-vitro behaviour and growth of bone cellson their ‘natural’ biological substrate compared tocells on standard culture plastic, and the reliabilityof extrapolating from cell culture on plastic toa physiological environment. Culturing on bonyslices, mostly of dental origin, as dentin is acceptedto be a good substitute for bone, is an establishedand necessary approach to assess the function ofthe bone cell responsible for bone resorption, theosteoclast. Osteoclast-like cells maintained on cul-ture plastic lack certain specific features (e.g. ruffledborder) and the question remains whether thephysiology of these cells is changed on plastic.However, little is known about the response ofosteoblasts when seeded on bone or dentin ascompared to standard culture plastic. This studywas undertaken to address some of these issues,and experiments were designed to compare thebehaviour of osteoblast-like cells on dentin withsimilar cultures on standard tissue culture plastic.Our findings show that cultures of humanosteoblast-like cells from different sources responddifferently to the bony surface than to standardculture plastic and that these differences cannot beentirely accounted for by differences in surfaceroughness, though this may be a factor.

The rate of attachment of the human osteoblaststo the dentin slices was similar to that on theculture plastic, although the primary osteoblasts(HOBs) appeared to adhere and spread out morereadily on the different surfaces and in generaldisplayed a distinct morphology on dentin. It wasalso found that the viability of the primary cellswas enhanced when cultured on dentin slices.Moreover, the primary cells rounded up anddetached from the plastic surface when kept inserum-free medium for 30 min or longer, whereasmost cells on bone remained viable and well spreadout under these conditions (data not shown). Thesefindings suggest that the primary bone cells foundthe bone surface to be a more favourable (natural)environment than the culture plastic.

It has long been recognised that cellular growthis related to the roughness of the substrate, though


0Total

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Fig. 4. Effect of the dentin substrate on the cell replication as assessed by quantitative analysis of BrdU incorporation. Thenumber of cells positively labelled with BrdU (+/ve) and negative cells without the label (�/ve) were counted in at least threerandom microscope fields per slice, and the average calculated (5–6 slices in total). The labelling index (LI) denotes the percentageof positive cells of the total counted cell population. Results are mean�SD per microscope field/slice. *P<0.05, **P<0.01 vscontrols.

both inhibitory and stimulatory effects have beenreported in this respect (Martin et al., 1995; Boyanet al., 1996, 1998; Lincks et al., 1998; Matsuzakaet al., 1999; Degasne et al., 1999; Hatano et al.,1999; Deligianni et al., 2001). On the basis of cellcounting, Schwartz et al. (2000) compared MG63cell numbers on different dentin slices, and con-cluded that MG63 cell proliferation was equivalenton plastic and polished surfaces, but was increasedon dentin pretreated with tetracycline or bonemarrow cell-derived osteoclasts. Here we havefound that the rate of growth of the transformedcell lines was significantly reduced on bone or ivoryslices as assessed by BrdU incorporation and the‘alamar blue’ method, but was not consistentlyaffected in cultures on the roughened plastic. Itshould be noted, however, that cell numbersand DNA content of the cell lines after reachingconfluency were not different from those onplastic, indicating that the cultures on dentin dideventually catch up with the cultures on plastic.This emphasises that, for cell growth analysis on

different biomaterials, it is important that thestage of the culture should be taken into accountand that the rate of cellular replication (DNAsynthesis) should be assessed besides actual cellnumbers.

Unlike the cell lines, the growth of primaryosteoblasts was not adversely affected by the bonysurface. The human osteoblasts showed an elevatedalamar blue staining, which may be ascribed to agreater metabolic activity and/or viability, as actualcell numbers were not increased. Other studies havereported that primary human osteogenic cellsdisplayed an increased proliferation after exposureto substrate consisting of bone collagen matrixor rough hydroxyapatite-surfaces (Zambonin andGrano, 1995; Deligianni et al., 2001). Primary ratosteoblast-like cells showed an increased prolifer-ation rate on roughened bone as compared topolished slices as well as on roughened plastic ascompared to smooth plastic surfaces (Hatano et al.,1999; Matsunage et al., 1999a,b). Taken together,these data are suggestive that primary cells respond


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Fig. 5. Cell numbers and viability on the substrates as assessedby the alamar blue method after 2 days of culture. Results areexpressed as a percentage of control values (mean�SEM,n=6–12). *P<0.05 versus controls.

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Fig. 6. After 3 days in culture, cells on roughened plastic(1200-grit) and dentin have a reduced alkaline phosphatase(AP) activity. Results are expressed as percentage of con-trol values (mean�SEM; n=6–12). *P<0.05 vs controls;**P<0.001 versus controls.

differently to rough substrates than do transformedcells.

As reported elsewhere, alkaline phosphatase(AP), as a phenotypic marker for osteoblast differ-entiation is not only a sensitive parameter, but alsohighly variable in cultures on various biomaterials(e.g. Lincks et al., 1998; Lohmann et al., 1999;Deligianni et al., 2001). Though we also encoun-tered some variability in results, the generaltrends indicated that absolute and specific APactivity was decreased after 2 to 3 days in cellcultures on dentin, and to a lesser extent on 1,200-grit roughened plastic. Though inhibition ofosteoblastic AP on different substrates has beendescribed before, it was not necessarily correlatedwith a reduced osteoblast differentiation as deter-mined by additional markers such as osteocalcinand growth factor production (Lohmann et al.,2000).

Recently, it was reported by Schwartz et al.(2000) that cellular production of osteocalcin wassignificantly reduced in MG63 cultures on polisheddentin, however, AP activity was not affected,though it was increased in cultures on dentin slicespretreated with tetracycline or osteoclastic cells. AsAP is a membrane-bound, extracellularly exposedenzyme, it is possible that changes in AP wereprimarily caused by the surface topography of thesubstrate rather than processes involved in cellular(de-) differentiation. It is noteworthy, however,that after initial adhesion of the cells to the bonecellular AP activity was not significantly affected,although it was increased in the HOB cultures.However, after several days, it was remarkable thatdespite the considerable differences in proliferationrate, the primary osteoblasts displayed a similarlysuppressed AP activity on the bone substrate asthe transformed cell lines. This indicates that thereduced AP activity on bone is not the result of areduced cellular viability.

Like the bone slices, the control culture plastichad a hydrophilic nature, which became hydro-phobic following surface roughening. However,restoring the surface wettability of the grittedplastic by plasma-treatment did not affect theresponse of the cultured cells, suggesting that thesurface hydrophilicity itself is not a major factor inthe attachment and growth of cells.

It is beyond the scope of the present studyto elaborate on the mechanisms involved in thecellular responses to bone. The particular dentintopography may specifically influence expressionof cell adhesion molecules, signal transductionpathways and autocrine growth factors involvedin growth and differentiation (Kieswetter et al.,1996; Lohmann et al., 1999; Anselme, 2000).Nonetheless, differences in cell morphology andgrowth were still evident between cultures on den-tin and plastic roughened to a degree comparablewith that of the bone surface. This indicates thatroughness per se is not the only determinant, butthat substrate composition and/or surface textureare also important factors influencing cellularbehaviour. It is plausible that the bony substratepresented specific matrix molecules and/or releasedspecific growth factors, such as IGF-I/-II, TGF�and BMPs, which could modify the responses ofthe different cell types (Mohan and Baylink, 1991;Takata et al., 1998).

In conclusion, the results from this study,together with similar findings using human andbovine cortical bone slices (unpublished obser-vations), provide qualitative and quantitative evi-dence that human osteoblastic cells from different


sources behave differently when cultured on abony surface than on standard tissue culture plas-tic. Moreover, particular differences were notedbetween primary cells and the transformed osteo-genic sarcoma cell lines. Further work in ourlaboratory aims at investigating and comparing theresponsiveness of human cells from differentpatient groups and from healthy non-diseasedbone on different natural surfaces which may haveimplications for the interpretations of in vitro datafrom standard cell cultures and specific bone cellengineering applications.

ACKNOWLEDGEMENTS

This study was supported by the Medical ResearchCouncil (MRC) and RMA holds an MRC SeniorFellowship. The authors are grateful to J. Rowland(Neoligaments, Leeds, U.K.) for kindly under-taking the plasma-treatment of the slices, and Dr J.Muhl (University of Edinburgh) for the surfacestructure analysis. We also thank A. R. Marsh, S.M. Robertson, and F. G. Smith for performing thepreliminary studies for this investigation on humanand bovine bone, and the Orthopaedic Surgeons ofAberdeen Universities Hospital Trust for kindlyletting us use tissues from their patients.

REFERENCES

A K, 2000. Osteoblast behaviour on biomaterials.Biomaterials 21: 667–681.

B BD, H TW, D DD, S Z, 1996.Role of material surfaces in regulating bone and cartilagecell response. Biomaterials 17: 137–146.

B BD, B R, K K, L Y, C DL,S-M S, D DD, S Z, 1998.Titanium surface roughness alters responsiveness of MG63osteoblast-like cells to 1 alpha,25-(OH)2D3. J Biomed MaterRes 39: 77–85.

D I, B MF, D V, H́ G, L M,G B, M L, C D, 1999. Effects ofroughness, fibronectin and vitronectin on attachment,spreading, and proliferation of human osteoblast-like cells(Saos-2) on titanium surfaces. Calcif Tiss Int 64: 499–507.

D DD, K ND, K PG, MYF, 2001. Effect of surface roughness of hydroxyapatiteon human bone marrow cell adhesion, proliferation,differentiation and detachment strength. Biomaterials 22:87–96.

G C, B A, J SJ, 1996. Topographically inducedbone formation in vitro: Implications for bone implants andbone grafts. Bone 18: 115–123.

G C, 1998. Advanced bone formation in grooves in vitro isnot restricted to calcified biological materials. Tissue Eng 4:315–323.

H K, I H, K T, M T, T T,U C, U Y, 1999. Effect of surface roughnesson proliferation and alkaline phosphatase expression of ratcalvarial cells cultured on polystyrene. Bone 25: 439–445.

H A, A CW, W PS, B GW, 1995.Attachment and proliferation of osteoblasts and fibroblastson biomaterials for orthopaedic use. Biomaterials 16: 287–295.

J SJ, B A, 1977. The migration of osteoblasts. CellTissue Res 184: 179–193.

J SJ, B A, 1979. Colonization of various substratesby osteoblasts in vitro. Scann Electron Microsc 2: 529–538.

J SJ, G C, B A, 1994. Simulation of boneresorption-repair coupling in vitro. Anat Embryol 190: 339–349.

K K, S Z, H TW, C DL,S J, D DD, B BD, 1996. Surface roughnessmodulates the local production of growth factors andcytokines by osteoblast-like MG-63 cells. J Biomed MaterRes 32: 55–63.

K R, S A, N D, F C, H D,1999. Enhanced proliferation and osteocalcin production byhuman osteoblast-like MG63 cells on silicon nitride ceramicdiscs. Biomaterials 20: 1195–1201.

L J, B BD, B CR, L CH, L Y,C DL, D DD, S Z, 1998. Response ofMG63 osteoblast-like cells to titanium and titanium alloyis dependent on surface roughness and composition.Biomaterials 19: 2219–2232.

L CH, S RJ, S VL, C DL, DDD, B BD, S Z, 1999. Surface roughnessmodulates the response of MG63 osteoblast-like cells to1,25-(OH)(2)D(3) through regulation of phospholipase A(2)activity and activation of protein kinase A. J Biomed MaterRes 47: 139–151.

L CH, B LF, S MA, S VL,C DL, D DD, B BD, S Z, 2000.Maturation State Determines the Response of OsteogenicCells to Surface Roughness and 1,25-Dihydroxyvitamin D3.J Bone Miner Res 15: 1169–1180.

M JY, S Z, H TW, S DM,S J, L J J, D DD, C DL,B BD, 1995. Effect of titanium surface roughnesson proliferation, differentiation, and protein synthesis ofhuman osteoblast-like cells (MG63). J Biomed Mater Res 29:389–401.

M T, I H, K T, H K, T T,U C, U Y, 1999a. Increase in the potential ofosteoblasts to support bone resorption by osteoclasts in vitroin response to roughness of bone surface. Calcif Tissue Int65: 454–458.

M T, I H, K T, H K, T T,U C, U Y, 1999b. Disaggregated osteoclastsincrease in resorption activity in response to roughness ofbone surface. J Biomed Mater Res 48: 417–423.

M K, W XF, R JE, J JA,1999. The effect of poly-L-lactic acid with parallel surfacemicro groove on osteoblast-like cells in vitro. Biomaterials20: 1293–1301.

M S, B D, 1991. Bone growth factors. Clin OrthopRel Res 263: 30–48.

Nø T, K M, Nø E, L CA,1999. Microplate-based fluoromettric assay for monitor-ing human cancer cell attachment to cortical bone. AnalBiochem 267: 37–45.


O RO, D FC, P JA, T JT,1998. Growth and differentiation of human bone marrowosteoprogenitors on novel calcium phosphate cements.Biomaterials 19: 1845–1854.

S BAA, V MJ, D CA, LFPJG, R HJM, D SA, 1995. Effects ofmethotrexate on human osteoblasts in vitro. Modulationby 1,25-dihydroxyvitamin D3. J Bone Miner Res 10: 874–880.

S Z, L CH, W M, C DL,D DD, T M, B LF, B BD, 2000.Osteoblast proliferation and differentiation on dentinslices are modulated by pretreatment of the surface withtetracycline or osteoclasts. J Periodontol 71: 586–597.

S AK, S RK, H NJ, T RS, 1999. High-resolution morphometric analysis of human osteoblastic cell

adhesion on clinically relevant orthopedic alloys. Bone 24:499–506.

S RK, M F, S SA, T RS, 1994. Surfacecomposition of orthopaedic implant metals regulates cellattachment, spreading, and cytoskeletal organization ofprimary human osteoblasts in vitro. Clin Orthop Rel Res 305:258–272.

T T, D’E JA, A KB, B JE, SC, T RS, S MJ, 1998. Protein extractsof dentin affect proliferation and differentiation of osteo-progenitor cells in vitro. J Periodontol 69: 1247–1255.

Z G, G M, 1995. Biomaterials in orthopaedicsurgery: effects of different hydroxyapatites and demineral-ized bone matrix on proliferation rate and bone matrixsynthesis by human osteoblasts. Biomaterials 16: 397–402.

IN VITRO BEHAVIOUR OF HUMAN OSTEOBLASTS ON DENTIN AND BONE

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