Cell Seeding into Calcium Phosphate Cement

12 Pages·2004·1.18 MB·English

Cell seeding into calcium phosphate cement
Carl G. Simon, Jr.,1 William F. Guthrie,2 Francis W. Wang1
1Polymers Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8545
2Statistical Engineering Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
Received31January2002;revised26June2003;accepted29July2003
Abstract: To improve the effectiveness of calcium phos- CPC paste. Thus, cells were encapsulated in alginate beads
phate cement (CPC), we have developed a method to seed (3.6-mm diameter), mixed with CPC paste, and incubated
osteoblasts into the cement. CPC powder is mixed with overnight. Both vital staining (calcein-AM and ethidium
watertoformapastethatcanbeshapedtofitabonedefect homodimer-1) and the Wst-1 assay (measures dehydroge-
insitu.Thepastehardensin30min,reactstoformhydroxy- naseactivity)showedthatcellsurvivalinalginatebeadsthat
apatite, and is replaced with new bone. Reacted CPC is were mixed with CPC was similar to survival in untreated
biocompatible but unreacted CPC paste was found to have control beads. These results suggest that gel encapsulation
toxic effects when placed on cell monolayers (MC3T3-E1 could be used as a mechanism to protect cells for seeding
cells).Incontrast,whencellswereindirectlyexposedtoCPC intoCPC.©2004WileyPeriodicals,Inc.*JBiomedMaterRes
paste using a porous membrane or by placing a coverslip 68A:628–639,2004
containing adherent cells onto a bed of CPC paste, the
unreacted CPC was nontoxic. These results suggested that Keywords:alginate;bonegraft;calciumphosphatecement;
gel encapsulation of the cells might protect them from the cellseeding;tissueengineering
INTRODUCTION a bone graft would provide clinicians with an invalu-
able tool for treating patients.
Amaterialthatsuitsthispurposehasbeendesigned
It is estimated that approximately 1,000,000 bone
byourcolleaguesattheAmericanDentalAssociation
graftsareperformedeachyeartotreatosseousdefects
Health Foundation at the National Institute of Stan-
resulting from trauma and disease in the United
dardsandTechnology(NIST).Theyhavedevelopeda
States.1 Autografts are frequently used to treat these
moldable, self-setting calcium phosphate cement
defects but available bone can be inadequate and dif-
(CPC) that is used clinically as a bone graft to treat
ficulttoshape.Allograftsandxenograftsmustbepro-
non-load-bearingdentalandcraniofacialdefects.2The
cessed to eliminate the risk of disease transmission
cement powder, consisting of tetracalcium phosphate
which requires the removal of osteogenic cells and
and anhydrous dicalcium phosphate, is mixed with
reduces the osteoinductivity of the grafts.1 Thus, the
water to form a workable paste that can be shaped
developmentofasafeandeffectivealloplastforuseas
duringsurgerytofitthecontoursofabonedefect.The
cement, which reacts to form microcrystalline hy-
droxyapatite(HA),hardenswithin30min,isbiocom-
Certain commercial materials and equipment are identi-
patible, is resorbed by the body, and is replaced with
fiedinthisarticletospecifytheexperimentalprocedure.In
new bone over several years.2–5
noinstancedoessuchidentificationimplyrecommendation
or endorsement by the National Institute of Standards and To improve the effectiveness of CPC, we have de-
Technology or that the material or equipment identified is veloped a method for seeding osteoblasts into the
necessarilythebestavailableforthepurpose. cement. Recent research has shown that human bone
Correspondence to: F. W. Wang; e-mail: francis.wang@
marrow contains pluripotential mesenchymal stem
nist.gov
cells (MSCs) that can differentiate into bone in vitro
Contractgrantsponsor:NationalInstitutesofHealth;con-
tractgrantnumber:YI-DE-7006-01 and in vivo.6,7 These cells can be purified and ex-
Contract grant sponsor: National Institute of Standards pandedexvivoandretaintheirosteogenicpotential.In
andTechnology,AdvancedTechnologyProgram
addition, animal models have shown that isolated
MSCs can be expanded and used to repair bone de-
© 2004 Wiley Periodicals, Inc. *This article is a US Government
fects.7–9 These advances suggest that it may soon be
workand,assuch,isinthepublicdomainintheUnitedStatesof
America. feasible to treat a patient’s bone injuries using MSCs
CELL SEEDING INTO CALCIUM PHOSPHATE CEMENT 629
that have been isolated from their own marrow. mLofmedium.CellswerealsoplatedontoCPCdiscs(pre-
Longerrange,itmayalsobedesirabletotreatpatients pared as described above) by adding 50,000 cells to wells
with embryonic stem cells because these cells also containing a disc and medium. After an overnight incuba-
tion,cellswerewashedwithmediaand“Live-Dead”stained
possessthepotentialtoformboneandarebeingeval-
uatedforcelltherapies.10,11Towardthisaim,wehave inmediumcontaining2(cid:2)mol/Lcalcein-AMand2(cid:2)mol/L
ethidium homodimer-1 (both from Molecular Probes, Eu-
developed a method for seeding cells into CPC using
gene,OR).Afteraddingthestaintothecells,fourtreatments
the MC3T3-E1 murine osteoblast-like cell line as a
werestartedwhichlastedfrom2to4h:
model. Because our initial studies suggested that un-
1. threewellsservedascontrolsandwerestainedfor4h;
reacted CPC was toxic when mixed with cells, we
2. CPCpaste(0.25gofCPCplus0.063mLofwater)was
haveevaluatedgelencapsulationasaprotectivemea-
gentlyaddedontopofthecellsinthreewellsfor2h;
sure for seeding cells into the cement.
3. cellsthatwereplatedfor24hontopofthreeCPCdiscs
werestainedfor4h;and
4. aCPCdiscwasgentlyplacedoncellsinthreewellsfor
MATERIALS AND METHODS
4h.
After the treatments, the cells were observed by epifluo-
CPC
rescencemicroscopyandphotographed(Fig.1).
Tetracalcium phosphate [Ca (PO ) O] was prepared ac-
4 42
cording to Chow et al.12 and anhydrous dicalcium phos-
Cell culture insert experiments
phate(CaHPO )wasobtainedfromJ.T.BakerCo.(Phillips-
4
burg,NJ).CPCpowderwaspreparedbymixingequimolar
amounts of ground Ca (PO ) O (72.9% mass fraction) and Cellsweretrypsinizedandplatedintoasix-wellplate(BD
4 42
CaHPO (27.1%massfraction).2,13CPCpastewasmadeby Labware)at100,000cellsperwellin2mLofmedium.Cell
4
mixing four parts by mass of CPC powder (UV-sterilized) cultureinserts(0.4-(cid:2)mporesize,BDLabware)wereplaced
with one part of sterile-filtered water (0.25 g of CPC plus in each well and filled with 2 mL of medium. After an
0.063mLofwater)for30swithaspatula.WhenCPCdiscs overnightincubation,CPCpaste(0.5gofCPCplus0.125mL
wererequired,theCPCpastewasplacedinasteelmoldto ofwater)wasaddedintotheinsertofthreeofthewells[see
formadiscapproximately4.5mminthicknessand6.4mm Fig.2(A)foradiagram]andtheremainingthreewellswere
indiameter.Apressureof0.7MPawasappliedtothemold used as controls. After another 24-h incubation, the cells
usingadeviceforapplyingaconstantload.13Thediscswere were Live-Dead stained, observed by epifluorescence mi-
incubated at 37°C and 100% relative humidity overnight croscopy, and photographed (Fig. 3). The inverse of this
beforeremovalfromthemolds.CPCdiscsweresterilizedby experiment was also performed where cells (75,000) were
soaking them in 70% ethanol (mass fraction) for 15 min plated into the cell culture inserts (instead of onto the bot-
followedbyanovernightwashincellculturemedium. tom of the six-well plate) and the CPC paste was placed in
thebottomofthesix-wellplate[seeFig.2(B)foradiagram].
Cell culture
Coverslip experiments
MC3T3-E1 cells were used because they are a well-char-
acterized osteoblast-like cell line14 which can serve as a
Cells (50,000/well) were plated into a 24-well plate (BD
modelforendogenousosteoblasts.Establishedprotocolsfor
Labware) having round glass coverslips (12-mm diameter)
the culture and passage of MC3T3-E1 cells (murine osteo-
and 2 mL of medium in each well. After an overnight
blast-like cells) were followed.15 Cells were obtained from
incubation, three coverslips were placed in three separate
RikenCellBank(Hirosaka,Japan)andculturedinflasks(75
wells of a six-well plate containing 4 mL of serum-free
cm2surfacearea)at37°Cinafullyhumidifiedatmosphereat
medium and Live-Dead stain (untreated controls). Three
5%CO (volumefraction)in(cid:1)-modificationofEagle’smin-
2 othercoverslipswereplacedintoseparatewellsofasix-well
imum essential medium (BioWhittaker, Inc., Walkersville,
plate that had CPC paste (0.5 g of CPC plus 0.125 mL of
MD)supplementedwith10%(volumefraction)fetalbovine
water) and 4 mL of serum-free medium containing Live-
serum (Gibco, Rockville, MD) and kanamycin sulfate
Deadstain.Thecoverslipsweregentlyplacedontopofthe
(Sigma, Inc., St. Louis, MO). Medium was changed twice
CPCpastebutweresubmergedundermedium[seeFig.2(C)
weekly and cultures were passaged with 2.5 g/L trypsin
for a diagram]. The cells on the coverslips were incubated
(0.25% mass fraction) containing 1 mmol/L ethylenediami-
for 4 h and then observed by epifluorescence microscopy
netetraacetic acid (Gibco) once per week. Cultures of 90%
andphotographed(Fig.3).
confluentMC3T3-E1cellswereusedforallexperiments.
Tissue culture polystyrene (TCPS) experiments Determination of percent live cells
Cells were trypsinized and plated (50,000/well) into 24- The Live-Dead images taken for the TCPS, cell culture
well plates (BD Labware, Franklin Lakes, NJ) of TCPS in 2 insert,andcoverslipexperimentswereanalyzedtodeter-
630 SIMON, GUTHRIE, AND WANG
Figure1. TCPSexperiments,Live-Deadstain.PanelsAandB(TCPS):cellsplatedonTCPSfor24h.PanelsCandD(CPC
Paste):CPCpasteplacedoncellsfor2h.PanelsEandF(CellsonCPCDisc):cellsonaCPCdiscfor24h.PanelsGandH
(CellsunderCPCDisc):aCPCdiscplacedoncellsfor4h.Pairsofgreen(left)andred(right)panelsarethesamefieldviewed
withagreen(live)orred(dead)filter.ThesizebarinPanelAappliestoallpanels.[Colorfigurecanbeviewedintheonline
issue,whichisavailableatwww.interscience.wiley.com.]
mine the percent live cells present after each treatment Alginate encapsulation
(Fig.4).Twohundredcellswerecountedforeachwellof
each treatment and the percent live cells was determined Alginate was used as an encapsulating gel because it is
bydividingthenumberoflivecells(green)bythesumof biocompatible and easy to work with.16,17 A 1.2% sodium
the number of live cells (green) and dead cells (red). alginatesolution(massfraction)waspreparedbydissolving
CELL SEEDING INTO CALCIUM PHOSPHATE CEMENT 631
40% of the volume of CPC-cell-alginate-bead mixture. Me-
dium (8 mL) was added to the wells. After an overnight
incubation,thebeadswereremovedandLive-Deadstained
(Figs.5and6).
Preliminaryexperimentsrevealedadifferenceincellsur-
vival between beads that were completely covered by CPC
versus beads that were partially uncovered and had a por-
tion of their surface directly exposed to the medium. Thus,
asthecell-alginatebeadswereremovedfromtheCPC,they
wereclassifiedaseither“CPC-Exposed”or“CPC-Covered”
and analyzed separately. Exposed was defined as a bead
withanyportionofitssurfacevisiblebyeye(notcompletely
coveredbyCPC)atthetimeofremoval[seeFig.2(D)fora
diagram].ThedepthofcoveragewithCPCforCPC-Covered
beadswas(cid:1)2mm.A1-mm-thicksectionwascutfromthe
middle of each bead with a razor blade. The sections were
Live-Deadstainedandphotographedusingepifluorescence
microscopy (Fig. 5). The micrographs were used to deter-
mine the percentage of live cells present in Control, CPC-
Exposed,andCPC-Coveredbeadsasdescribedabove.Three
or more beads were analyzed for each well of each treat-
ment. Because this experiment was performed three times,
at least 27 beads were analyzed for each of the three treat-
ments(Control,CPC-Exposed,andCPC-Covered).
Seeding cell-alginate beads into silicone grease
Todeterminewhethercellsinalginatecansurvivefor24h
without access to media, one cell-alginate bead (105
cells/mLalginate)wasplacedinthebottomofeachoffour
sterile1.5-mLmicrofugetubes.Thebeadsinthetubeswere
Figure 2. Diagram of cell culture insert, coverslip, and
completelycoveredwith1mLofautoclavedsiliconegrease
CPC-cell-alginateexperiments.
suchthattheyhadnoaccesstoair.Thetubeswereleftopen
and placed in a cell incubator for 24 h without any media
1.2 g of Pronova UP LVG alginate (64% guluronic acid; present.Thenextday,thebeadsweresectioned,Live-Dead
MW (cid:3) 75,000–220,000 g/mol; Pronova Biomedical, Oslo, stained, and photographed using epifluorescence micros-
Norway)in100mLofsaline(155mmol/LNaCl).Cultures copy[Fig.5(G,H)].Themicrographswereusedtodetermine
of MC3T3-E1 cells (90% confluent) were trypsinized, thepercentageoflivecellspresentinthebeadsasdescribed
washed,andresuspendedinsterile-filteredalginate.Thecell above. Microfuge tubes were used for these experiments
concentration varied from 105 to 3 (cid:4) 106 cells/mL alginate because it was much easier to completely cover a cell-
dependingontheexperiment.Aliquots(0.5mL)ofthecell- alginatebeadwithsiliconegreasewhenitwasinthebottom
alginate suspension were released in droplets from a 1-mL of a tube than when it was in a 16-mm plastic ring in the
pipetteintosterile-filteredCaCl solution(100mmol/L)ina bottomofasix-wellplate.
2
six-wellplateandallowedtogelfor5min.After5min,the
cell-alginate beads (3.6 mm in diameter) were removed,
rinsedwithmedium,andusedinexperiments.
Seeding cell-alginate beads into CPC (Wst-1 assay)
Cell-alginate beads (3 (cid:4) 106 cells/mL alginate) were in-
Seeding cell-alginate beads into CPC (Live-Dead cubated overnight in 16-mm rings in six-well plates as de-
stain)
scribed above (four control wells and four CPC wells). The
nextday,thebeadswereanalyzedforcellviabilitywiththe
Asterileplasticring(16-mmdiameter;5-mmheight)was Wst-1assay(Fig7).TheWst-1assayisacolorimetricassay
placed in each well of a six-well plate and 12 cell-alginate of cellular dehydrogenase activity in which absorbance at
beads (105 cells/mL alginate) were placed into each of the 450 nm is proportional to the amount of dehydrogenase
plastic rings. Three wells were used as controls while CPC activityinthecells.18Sixcell-alginatebeadsfromeachofthe
paste(1gofCPCplus0.25mLofwater)wasplacedontop fourcontrolwells,sixCPC-Exposedbeadsfromeachofthe
of and pressed around the beads in three of the wells to fourCPCwellsandsixCPC-Coveredbeadsfromeachofthe
makeaconstructapproximately6-mmhigh[seeFig.2(D)for four CPC wells were placed in separate wells of a 24-well
adiagram].Thecell-alginatebeadsoccupiedapproximately plateandwashedwith1mLofTyrode’sHEPESbuffer(140
632 SIMON, GUTHRIE, AND WANG
Figure3. Cellcultureinsertandcoverslipexperiments,Live-Deadstain.PanelsAandB[Control(Insert)]:anemptyinsert
placedovercellsonTCPSfor24h.PanelsCandD[CPC(Insert)]:aninsertcontainingCPCpasteplacedovercellsonTCPS
for 24 h. Panels E and F [Control (Coverslip)]: a control coverslip covered by cells. Panels G and H [CPC (Coverslip)]: a
coverslipcoveredbycellsplacedonCPCpastefor4h.Pairsofgreen(left)andred(right)panelsarethesamefieldviewed
withagreen(live)orred(dead)filter.ThesizebarinPanelAappliestoallpanels.[Colorfigurecanbeviewedintheonline
issue,whichisavailableatwww.interscience.wiley.com.]
mmol/LNaCl,0.34mmol/LNa HPO ,2.9mmol/LKCl,10 clarified by centrifugation at 10,000 rcf for 30 s. A 0.2-mL
2 4
mmol/LHEPES,12mmol/LNaHCO ,5_mmol/Lglucose, aliquot from the supernatant of each tube was put in a
3
pH7.4).OnemilliliterofTyrode’sHEPESbufferand0.1mL 96-well plate and absorbance was measured with a plat-
of Wst-1 solution (5 mmol/L Wst-1 and 0.2 mmol/L 1-me- ereader (Wallac 1420 Victor2; PerkinElmer Life Sciences,
thoxyPMSinwater)werethenaddedtoeachwell.Aftera Gaithersburg, MD). Experiments were repeated with algi-
2-hincubationat37°C,0.5mLofeachreactionmixturewas nate beads that did not contain any cells. These no-cell-
CELL SEEDING INTO CALCIUM PHOSPHATE CEMENT 633
stained for epifluorescence microscopy and photography
(Fig.8).
Powder X-ray diffraction
Powder X-ray diffraction was used to confirm that CPC
converts to HA in our alginate experiments. Reacted CPC
(CPC paste mixed with cell-alginate beads and incubated
overnight) was ground to a fine powder and characterized
bypowderX-raydiffraction.X-raydiffractionpatternswere
takenwithmonochromatizedcopperK(cid:1)1radiation((cid:5)(cid:3)1.54
nm) generated at 40 kV and 25 mA on a Rigaku powder
diffractometer in the range 10–70° 2(cid:6). Spectra were taken
withacontinuousscanat1°/minandpercentconversionto
Figure4. PercentlivecellsintheTCPS,cellcultureinsert, HA was estimated to be between 88 and 100% based on
and coverslip experiments. The number of live and dead comparison of tetracalcium phosphate (29.2 and 29.8°) and
cellswascountedinmicrographs.(1)TCPS:cellsplatedon HA(25.9°)peakintensitiestopreviouslypublisheddata.19
TCPS.(2)CPCPaste:CPCpasteontopofcells.(3)OnDisc:
cells on a CPC disc. (4) Under Disc: a CPC disc placed on
cells. (5) Cells on Bottom, Control: an empty cell culture
insertinawellwithcellsonTCPS.(6)CellsonBottom,CPC: Statistics
acellcultureinsertcontainingCPCpasteinawellwithcells
onTCPS.(7)CellsonTop,Control:cellsinaninsertoveran
emptywell.(8)CellsonTop,CPC:cellsinaninsertinawell When a “standard deviation” is shown in the text as an
with CPC paste. (9) Coverslip, Control: a control coverslip errorbar,itreferstothe“standarddeviationofthemean,”
coveredbycells.(10)Coverslip,CPC:acoverslipcoveredby whichisthesameasthe“combinedstandarduncertaintyof
cells placed on CPC paste for 4 h. Bonferroni t tests to themean”forthepurposesofthiswork.Thedatafromthe
compare the four TCPS treatments (bars 1–4) showed that Live-Dead and Wst-1 assays were analyzed by computing
CPC-Paste was different from the other three treatments
simultaneousStudentttestsusingtheBonferroniinequality
(p (cid:7) 0.0083). t Tests to compare the CPC to Control for
toguaranteeaglobalconfidencelevelof95%.20Whenusing
“Inserts Cells on Bottom” (bars 5 and 6), “Inserts Cells on
theBonferroniprocedure,eachoftheindividualttestswas
Top” (bars 7 and 8), and “Coverslips” (bars 9 and 10)
performedatthe0.05/ksignificancelevelwhere“k”equals
showedthattheCPCandControltreatmentswerethesame
foreachoftheseexperiments(p(cid:8)0.05). the number of comparisons being made. All experiments
were performed three times and averages are presented in
thefigures.
alginatebeadswereusedasablankintheWst-1assayand
theirabsorbancevaluesweresubtractedfromthedata.The
absorbance value for Control was set at 100% and CPC-
Exposed and CPC-Covered were normalized to Control. RESULTS
Wst-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophe-
nyl)-2H-tetrazolium,monosodiumsalt]and1-methoxyPMS
Ourfirstattemptsatdevelopingamethodforseed-
(1-methoxy-5-methylphenazinium methylsulfate) were ob-
ingcellsintoCPCinvolvedusingacellsuspensionas
tainedfromDojindo(Gaithersburg,MD).
the liquid portion of the cement, mixing the cement,
Live-Dead staining the cement, and then looking for
the presence of cells by epifluorescence microscopy.
Glass slide experiment CellswerealsoseededontofreshlymixedCPCpaste,
incubated overnight, and stained. Nearly all of the
Onemilliliterofacell-alginatesolution(100,000cells/mL) cells observed with these approaches were dead with
was gelled in 0.25 mL of CaCl solution for 5 min. The only an occasional live cell ever visible. These exper-
2
cell-alginategelwaswashedandplacedinatroughformed iments were also very difficult to image so they were
by gluing together three glass slides (UV-sterilized) as de- simplified to that shown in Figure 1 where freshly
pictedinFigure8(A).CPCpaste(1gofCPCplus0.25mLof
mixed CPC paste was placed on monolayers of cells
water)wasplacedinthetroughagainstthecell-alginategel
adherent to TCPS [Fig. 1(C,D)].
such that it made direct contact with the gel across the
In this experiment, control monolayers (TCPS) that
lengthofthetrough.Afourthslidewasplacedontopofthe
werenotexposedtoCPC[Fig.1(A,B)]hadmostlylive
gel–CPCconstructtopreventmediumaccesstothegel–CPC
cells(green;panelA)andfewdeadcells(red;panelB).
interface(notethatbothendsofthetroughwerestillopen).
The construct was placed in a large Petri dish and covered Cells plated onto [Fig. 1(E,F)]21 or under [Fig. 1(G,H)]
with medium (50 mL). After an overnight incubation, the setCPCdiscswerealsonearlyalllivewithfewdead.
top slide was removed and the construct was Live-Dead Incontrast,themonolayersthathadCPCpasteplaced
634 SIMON, GUTHRIE, AND WANG
Figure5. Micrographsofcell-alginatebeadsafterseedingintoCPC.PanelsAandB(Control):aControlcell-alginatebead.
PanelsCandD(CPC-Exposed):acell-alginatebeadmixedintoCPCpastehavingpartofitssurfaceexposedtomedia.Panels
E and F (CPC-Covered): a cell-alginate bead mixed into CPC paste with none of its surface exposed to media (it was
completelycoveredwithCPC).PanelsGandH(Grease):acell-alginatebeadthatwascoatedinsiliconegrease.Allpanels
are from overnight incubations and were Live-Dead stained. Pairs of green (left) and red (right) panels are the same field
viewedwithagreen(live)orred(dead)filter.ThesizebarinPanelAappliestoallpanels.[Colorfigurecanbeviewedin
theonlineissue,whichisavailableatwww.interscience.wiley.com.]
on them [Fig. 1(C,D)] contained nearly all dead cells four bars of Figure 4. The “TCPS,” “CPC-Paste,”
(panel D) and very few live cells (panel C). The live “Cells on CPC Disc,” and “Cells under CPC Disc”
anddeadcellsinmicrographsfromtheseexperiments treatments had 98, 3, 97, and 98% live cells, respec-
were counted to determine percent live cells for each tively.TheseresultssuggestthatunreactedCPChasa
ofthetreatmentsandthesedataareshowninthefirst toxic effect on cells and that reacted CPC is nontoxic.
CELL SEEDING INTO CALCIUM PHOSPHATE CEMENT 635
5(A,B)].Theseobservationsdemonstratethatthealgi-
nate encapsulation procedure was not harmful to the
MC3T3-E1 cells. The CPC-Exposed beads also con-
tained primarily live cells [Fig. 5(C)] and only a few
deadcells[Fig.5(D)]showingthatthealginateencap-
sulation protected cells from the unreacted CPC.
Comparison of panels B and F of Figure 5 reveals
that the density of dead cells in beads that spent the
nightcoveredinCPC(withlimitedaccesstomedium)
was greater than that found in Control beads. Like-
wise, comparison of panels A and E (Fig. 5) reveals
fewerlivecellsinCPC-CoveredbeadsthaninControl.
Theseobservationssuggestthatsurvivaloftheencap-
sulated cells is better if the cell-alginate beads are not
Figure6. Percentlivecellsincell-alginatebeadsafterseed-
completely enclosed by CPC and have access to me-
ingintoCPCorsiliconegrease.Thenumberofliveanddead
dium.
cells was counted in micrographs from Control, CPC-Ex-
posed, CPC-Covered, and Grease beads. Bonferroni t tests Totestwhetheracellalginatebeadcontainsenough
showed that CPC-Covered was different from the other nutrientstosustaincellviabilityfor24h,cell-alginate
three treatments and that CPC-Exposed was different than beads were covered with silicone grease and incu-
Grease(p(cid:7)0.0083).
bated overnight (in the absence of any media). Beads
thatwerecoveredovernightingrease(“Grease”)con-
Cell culture insert and coverslip experiments were tained mostly live cells and few dead cells [Fig.
performed to determine whether prevention of direct 5(G,H)], similar to Control [Fig. 5(A,B)]. These results
contactbetweencellsandpastecouldprotectthecells showthatthereareenoughmediaandnutrientsinthe
fromunreactedCPC.PanelsA–DofFigure3arefrom beads to sustain cell viability for 24 h and they also
an experiment in which cells were seeded on the bot- suggest that a lack of media or nutrients is not the
tom of a six-well plate and CPC paste was placed cause of the decreased cell viability in the CPC-Cov-
abovethecellsinacellcultureinsert[seeFig.2(A)for ered beads [Fig. 5(E,F)].
diagram].CellsinbothControl[Fig.3(A,B)]andCPC The percent live cells present in cell-alginate beads
[Fig.3(C,D)]wellswerenearlyalllivewithfewdead. after the various treatments shown in Figure 5 was
Counting of live and dead cells in micrographs re- determined by counting live and dead cells in the
vealed that the “Control” and “CPC” treatments had
99and100%livecells,respectively[Fig.4(bars5and
6)]. When the experiment was reversed with cells in
the inserts and paste on the bottom of the wells [see
Fig.2(B)fordiagram],theoutcomewasthesame:cells
exposed to unreacted CPC paste were as viable as
control cells (data not shown) and 99% of the cells
were live in both treatments [Fig. 4 (bars 7 and 8)].
Next,coverslipscoveredbyamonolayerofcellswere
placed on CPC paste [see Fig. 2(C) for diagram].
Again, cells in both the Control [Fig. 3(E,F)] and CPC
[Fig. 3(G,H)] treatments were nearly all live with 98
and99%livecells,respectively[Fig.4(bars9and10)].
These coverslip and cell culture insert experiments
suggest that unreacted CPC is not toxic when it is
exposed to cells by an indirect means.
Our next objective was to encapsulate MC3T3-E1
cellsinagel(alginate),seedtheencapsulatedcellsinto
CPC, and determine whether the gel encapsulation
protected the cells from the unreacted CPC. Control
cell-alginate beads that had not been exposed to CPC
contained mostly live cells [Fig. 5(A)] and few dead Figure 7. Wst-1 assay of cell-alginate beads after seeding
intoCPC.TheviabilityofControl,CPC-Exposed,andCPC-
cells [Fig. 5(B)]. Comparison of panels A and B of
Covered cell-alginate beads was quantified with the Wst-1
Figure1topanelsAandBofFigure4revealsthatthe
assay (dehydrogenase activity). Bonferroni t tests showed
ratio of live to dead cells on TCPS controls [Fig. that CPC-Covered was different from Control and CPC-
1(A,B)] is similar to that in the alginate beads [Fig. Exposed(p(cid:7)0.017).
636 SIMON, GUTHRIE, AND WANG
Figure 8. Glass slide experiment. A cell-alginate gel was placed in contact with CPC paste in a trough formed by gluing
togetherglassslidesasdepictedinPanelA.TheconstructwasincubatedovernightandLive-Deadstained.PanelsBandC
aremicrographsoftheinterfacebetweentheCPCandthecell-alginategel.ThewhitedotsindicatetheinterfacebetweenCPC
andgel.PanelsDandEaremicrographsofanareaofthecell-alginategellocated2mmfromtheinterfacewiththeCPC.Pairs
ofgreen(left)andred(right)panelsarethesamefieldviewedwithagreen(live)orred(dead)filter.ThesizebarinPanel
Bappliestoallpanels.[Colorfigurecanbeviewedintheonlineissue,whichisavailableatwww.interscience.wiley.com.]
micrographs from these experiments. Control, CPC- unreacted CPC. When taking the micrographs for the
Exposed, CPC-Covered, and Grease beads contained experimentspresentedinFigure5(C,D)therewasnot
97,96,74,and99%livecells,respectively(Fig.6).Cell a “zone” of dead cells near the surface of the cell-
viability was also quantitated by assessing dehydro- alginate beads after an overnight incubation in unre-
genase activity using the Wst-1 assay. CPC-Exposed acted CPC. The few dead cells present in these beads
and CPC-Covered beads had 105 and 80% of the de- were evenly distributed throughout the bead and
hydrogenase activity in Control beads, respectively were not concentrated at the edges where the CPC
(Fig. 7). These quantitative Live-Dead (Fig. 6) and was in direct contact with the alginate. Even so, we
Wst-1(Fig.7)resultsagreewiththequalitativemicros- devisedthesystemdepictedinFigure8(A)inwhicha
copyresultspresentedinFigure5andtheyindicate:1. cell-alginate gel is placed against freshly mixed CPC
thatgelencapsulationcanprotectcellsfromunreacted paste to provide a clearer view of the interface be-
CPC, and 2. that isolation of the cell-alginate beads tween the CPC and the cell-alginate gel. Again, no
from the medium with CPC reduces cell survival. zone of dead cells was visible along the interface be-
To better characterize the ability of alginate to pro- tween the CPC and the alginate gel and the few dead
tect cells, we determined the physical depth to which cells present in the gel were uniformly dispersed
the alginate is able to provide protection from the throughout the alginate [Fig. 8(C)]. In addition, an
CELL SEEDING INTO CALCIUM PHOSPHATE CEMENT 637
examination of the cells located 2 mm from the CPC– al.25 when evaluated using a Live-Dead assay similar
gel interface [Fig. 8(D,E)] reveals that the density of to what we have used in the present study. These
live and dead cells in this section of the gel is equiv- effectsaresimilartothedecreaseinviabilityobserved
alent to that found in the section juxtaposed to the when we placed CPC paste on osteoblast monolayers
CPC [Fig. 8(B,C)]. These results show that cells are [Fig. 1(C,D)]. However, it is difficult to draw any
protectedfromunreactedCPCatanydepthinthegel conclusionsfromthisstudy25oranyoftheothers22–24
and that there is not a visible region at the CPC–gel inregardtoourownworkbecausedifferentmaterials
interface where cells are affected by the CPC. as well as different particle sizes, particle concentra-
tions, cell types, and assays were used in each study.
Although disparate observations were made in these
four studies, an obvious conclusion is that calcium
DISCUSSION
phosphatepowderscanaffectosteoblastfunctionand
we have clearly observed an effect of CPC paste on
The TCPS experiments presented in Figure 1 osteoblasts in the present experiments.
showed that unreacted CPC had toxic effects on cells Regardless of whether pH, ionic strength, or nutri-
but that reacted CPC was nontoxic. Unreacted CPC ent deprivation was responsible for the cell death
paste killed osteoblast-like cells (MC3T3-E1) within observedinourexperiments,CPCpastewasnottoxic
2hwhenitwasplacedontopofcellmonolayers[Fig. to cells when direct contact between the cells and the
1(C,D)].TheCPCsettingreactionmaybethecauseof CPC paste was prevented (Figs. 2–4). This was tested
the cell death because this reaction involves the dis- by exposing cells to CPC paste through porous mem-
solutionofcalciumphosphateparticlesthatmaycause branes or by placing coverslips covered by cells on
fluctuations in pH and ionic strength underneath the bedsofCPCpaste(Figs.2–4).CPCpastewasnottoxic
paste.3 These local fluctuations may contribute to the whenexposedtocellsviatheseindirectmeans,which
toxic effects of the unreacted CPC, especially because led to the supposition that gel encapsulation could
the calcium phosphate particles and the cells make protect cells from unreacted CPC during seeding.
close contact under these conditions. Nutrient depri- Many gel systems have been used for cell encapsu-
vation might also contribute to cell death because the lationbutwechosealginatebecauseofitsbiocompat-
cells were blanketed by a thin layer ((cid:7)1 mm) of CPC ibility, ease of use, and gentle encapsulation proce-
paste which may restrict diffusion. However, prelim- dure.16,17,26 Cells were encapsulated into alginate
inary work showed that cells seeded on top of CPC beads, mixed into CPC paste, and incubated for 24 h.
paste also died, suggesting that the toxic effect of the Cell viability in beads that were mixed into CPC was
paste results from more than just blockage of diffu- similartocontrolbeadsthatwerenotmixedwithCPC
sion. (Figs. 5–8) suggesting that gel encapsulation can pro-
Although previous studies have not examined the tect cells from unreacted CPC.
effects of placing CPC paste on cell monolayers, the In addition, cell survival in the beads whose sur-
effectsofplacingothercalciumphosphatepowderson faces were partially exposed to the medium was the
osteoblasts have been studied. Gre´goire et al.22 ob- same as in control beads and survival was slightly
served that HA particles ((cid:7)0.05 mm) or (cid:9)-tricalcium lessened in beads that were completely covered by
phosphate (TCP) particles ((cid:7)0.05 mm) caused an in- CPC(Figs.5–7).Theslightreductionincellviabilityin
crease in protein synthesis when added to MC3T3-E1 CPC-Covered beads may have been caused by CPC
cellsorprimaryhumanosteoblasts.Incontrast,Alliot- inhibiting diffusion of media or nutrients to the cells.
Licht et al.23 found that HA particles ((cid:7)0.01 mm) However, this possibility can be eliminated because
inhibitedthegrowthandalkalinephosphataseexpres- there was enough media and nutrients in a cell-alg-
sion of MC3T3-E1 cells, ROS 17/2.8 osteosarcoma inate bead to maintain cell viability during an over-
cells, and primary rat osteoblasts. Both investiga- night incubation of the bead in silicone grease in the
tions22,23 also reported that the osteoblasts phagocy- absence of any media [Fig. 5(G,H)]. Taken together,
tosed the calcium phosphate particles. these results suggest that the fluctuations in pH and
Sunetal.24alsoexposedcellstocalciumphosphate ionic strength during the CPC setting reaction may
particles and reported that HA particles (0.1 (cid:2)m) or become toxic to cells when washout of the ionic com-
(cid:9)-TCP particles (0.1 (cid:2)m) inhibited the growth of pri- ponents is restricted by the presence of CPC paste.
maryratosteoblastswhilecausinganincreaseintheir Thus,CPC-Exposedbeadsmaybeviablebecausethey
expression of alkaline phosphatase. In addition, Pio- have access to media and washout of the ionic com-
letti et al.25 observed a decrease in growth, viability, ponents of the setting reaction is not completely ob-
and synthesis of extracellular matrix in primary rat structedbypaste,whereasviabilityisslightlylessened
osteoblaststhatwereexposedto(cid:9)-TCPparticles(1–10 inCPC-Coveredbeadsbecausediffusionandwashout
(cid:2)m) or dicalcium dihydrate particles (1–10 (cid:2)m). A may be restricted by the covering of CPC.
90% decrease in viability was observed by Pioletti et The cell seeding system we have explored in the