92
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
Biocompa ibili y and osseoin eg a ion s udy o
new p os he ic ma e ials
Co espondence: Me cè Gine ([email p o ec ed])
Gine M1, San ana L2, Cos a AF3, Vázquez-Gámez MA4, Colmene o M3, Olmo FJ3, Chica di E2, To es Y2,
Mon oya-Ga cía MJ4
1 Depa men o No mal and Pa hological Cy ology and His ology. Se illa Uni e si y. Se illa (Spain)
2 Depa men o Enginee ing and Science o Ma e ials and T anspo a ion. Highe Poly echnic School o Se ille. Se illa (Spain)
3 In e nal Medicine. Vi gen Maca ena Uni e si y Hospi al. Se illa (Spain)
4 Depa men o Medicine. Se illa Uni e si y. Se illa (Spain)
Summa y
Obje i e: Bone implan s a e inc easingly used in clinical p ac ice and, among he ma e ials, Ti o i s alloys a e o e he
bes pe o mance gi en hei physicochemical p ope ies. Alloys such as TiNbTa ha e been shown o imp o e he bio‐
mechanical cha ac e is ics o comme cial pu e Ti (c.p.), howe e , i s osseoin eg a ion capaci y needs o be e alua ed.
The objec i e o he p esen s udy was o assess he cy o oxici y and he adhesion, p oli e a ion and di e en ia ion ca‐
paci y o os eoblas ic cells in cul u e, in luenced by discs o TiNbTa ma e ial e sus Ti c.p.
Ma e ial and me hods: A 4 and 7 days a e cul u e, we analyzed he MC3T3 cell line, cell iabili y (Alama Blue Cell
Viabili y Reagen . In i ogen, Spain), as well as cell p oli e a ion and di e en ia ion (alkaline phospha ase ac i i y (ALP)
and scanning elec on mic oscopy (Fixa ion o SEM) S uden 's es was pe o med o de e mine s a is ically signi ican
di e ences be ween he wo g oups o s udy discs.
Resul s: The esul s ob ained show e y good cell iabili y du ing he s udy pe iod, wi h no signi ican di e ences o bo h
ma e ials. Likewise, we de ec ed a d op in ALP le els ha was signi ican o bo h componen s be ween days 4 and 7 o
he s udy (p <0.05). Elec on mic oscopy images e ealed good adhesion capaci y o he ma e ial, as well as cell di e en‐
ia ion agains bo h ypes o discs.
Conclusions: The TiNbTa alloy as a ma e ial o bone implan s o e s good osseoin eg a i e capaci y, in addi ion o
sol ing biomechanical p oblems ha pu e i anium p esen s as a componen .
Key wo ds: TiNbTa, cy o oxici y, biocompa ibili y, os eoblas cells, cell cul u e, cell adhesion, Young's modulus.
Da e o eceip : 10/07/2020 - Da e o accep ance: 28/09/2020
9
Pape awa ded a FEIOMM g an o T ansla ional Resea ch 2018
INTRODUCTION
The gene a ion o unc ional issue h ough issue engi‐
nee ing has a high impac in a ious a eas o egene a‐
i e medicine, among which is skele al issue. The i s
implan s we e used in he ield o medicine, in 1909,
when Ki schne wi es and S einman nails we e de elo‐
ped o he ixa ion o bone ac u es, whe e s ainless
s eel was used. O e he yea s, s eel has been imp o ed,
making i mo e esis an o co osion and no causing
ha m ul e ec s on he human body. In 1940, he s udy
o i anium (Ti) began as a bioma e ial o bone implan‐
a ion1.
The phase change de e mines he change in he
c ys alline s uc u e o he ma e ial when subjec ed o
empe a u e changes. Ti anium’s allo opic ans o ma‐
ion occu s a 882ºC and goes om an α phase, which
has a compac and hexagonal s uc u e (HCP), li le de‐
o mable and esis an a oom empe a u e, o a β
phase cha ac e ized by a cubic s uc u e cen e ed on he
body (BCC), which is easily de o mable and allows o
ca ying ou hea ea men s o op imize he ma e ial’s
p ope ies2.
Among he cha ac e is ics ha make i anium one o
he bes ma e ials o bone implan s, i s g ea e speci ic
esis ance ( esis ance/densi y), i s high duc ili y and
lowe Young's modulus3compa ed o o he elemen s
such as, o example, s ainless s eel a e no ewo hy. A
he same ime, i is a non‐ e omagne ic ma e ial, which
does no p esen incon eniences when he pa ien wi h
a Ti implan unde goes magne ic esonance imaging.
Ancho age o bone issue is possible because o he
oxide laye o med in he ma e ial when passi a ed4.
DOI: h p://dx.doi.o g/10.4321/S1889-836X2020000300004
93
Biocompa ibili y and osseoin eg a ion s udy o new p os he ic ma e ials
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
Pu e comme cial Ti (c.p.) and o he alloys such as Ti‐
6Al‐4V a e cu en ly used mainly o bone implan s5. Ho‐
we e , bo h elemen s p esen a high elas ic modulus (E:
100‐112 GPa) compa ed o he elas ic modulus o co ical
bone (18.6‐20.7 GPa) and abecula (10.4‐14.8 GPa)
(Table 1) wha p oduces he s ess‐shielding e ec o
s ess‐shielding6. This phenomenon is due o he s i ness
o he bone implan ma e ial being g ea e han he s i ‐
ness o he bone, which places he en i e load on he bone
implan . Bone emodeling is la gely egula ed by he me‐
chanical loads o which i is subjec ed, so ha he p esence
o loads s imula es i s bone o ma ion, and he absence o
hem inc eases eso p ion. As a consequence o he bone’s
educed load suppo ed, i dec eases i s densi y in he a ea
nea he implan and, he e o e, bo h p ema u e ac u e
o he implan and loosening o he implan may occu 5.
A p esen , o elimina e he ol age shielding pheno‐
menon, educing he elas ic modulus is sough , wi h se‐
e al a ailable solu ions. One o hem, educe he densi y
o he ma e ial used, h ough po osi y. Howe e , as he
po osi y inc eases, he mechanical esis ance dec eases.
Ano he app oach would be o sea ch o ace‐cen e ed
cubic s uc u e Ti alloys (BCC) o β‐Ti alloys, which a e
low in elas ic modulus and do no show a dec ease in me‐
chanical s eng h. Fo he s abiliza ion o he β phase, β‐
s abilizing elemen s a e equi ed: Mo, V, Ta, Nb and Z as
β‐isomo phic elemen s and C , Co, Cu, Fe and Ni as β‐eu‐
ec oid elemen s. The ad an age o β‐isomo phic ele‐
men s is hei high amoun o subs i u e solid solu ion
and hei inabili y o o m in e me allic compounds o Ti,
which ha e high E. The e o e, i has been shown ha ele‐
men s such as Nb and Ta ha e a high le el o biocompa i‐
bili y and abili y o p e en he inc ease o pa icles,
which would a oid a bad cohesion in e ace7.
Niobium (Nb) and an alum (Ta) a e wo ansi ion
me als widely used in alloys; in pa icula , Nb is used in
he o ma ion o s eel. Un il 1866, i was hough ha
bo h elemen s we e he same, since hey ha e e y si‐
mila physicochemical cha ac e is ics. The TiNbTa alloy
s ands ou o i s high s abiliza ion o beha io , due o
he absence o β phase, which allows a dec ease in he
elas ic modulus (49 + 3 GPa), i s excellen elas ic esis‐
ance (σy>1860 MPa) and i s high biocompa ibili y7,8.
The e o e, a ma e ial such as TiNbTa, wi h a good com‐
bina ion o high s eng h and low Young's modulus close
o ha o bone, could be used o p e en loosening o im‐
plan s o a oid e ision su ge y (Table 1).
In ecen yea s, a whole se ies o echniques ha e been
de eloped wi h he aim o ob aining po ous ma e ials ha
p esen a Young's modulus close o ha o co ical bone.
Speci ically, Chica di e al. manu ac u ed a TiNbTa alloy
wi h physicochemical p ope ies mo e simila o hose o
bone9. Howe e , he imp o emen in he design o ma e‐
ials, des ined o be used as bone g a s, mus conside
hei osseoin eg a ion capaci y, and in his sense, e alua‐
ing he cy o oxici y and he adhesion, p oli e a ion and
di e en ia ion capaci y o os eoblas ic cells in luenced by
he ma e ial is equi ed. Ou main objec i e was o e a‐
lua e he osseoin eg a i e cha ac e is ics o he TiNbTa
alloy, wi h a Young's modulus simila o ha o abecula
bone, and o compa e hem wi h pu e Ti.
MATERIAL AND METHODS
1. Cell cul u e
The MC3T3 cell line (subclone 4) om ATTC (Manassas,
Vi ginia, USA) was cul u ed in α‐MEM medium (Gibco,
The mo Fische Scien i ic, Spain) supplemen ed wi h 1%
L‐glu amine (200 mM) and 10 % e al bo ine se um. To
induce di e en ia ion, cells we e ea ed wi h os eogenic
cul u e medium supplemen ed wi h 50 g / ml asco bic
acid (Me ck, Ge many) and 10 nM β‐glyce ophospha e
(S emCell Technologies, Canada). The cells we e seeded in
discs, 3 mm hick and 7 mm, o i anium c.p. and TiNbTa
a a densi y o 5x103, unde condi ions o 5% CO2a 37°C.
Media changes we e made e e y 48 h. Cul u es we e done
in iplica e and eadings o cell iabili y and alkaline
phospha ase ac i i y we e done a e 4 and 7 days.
2. Cell iabili y
The Alama Blue assay (Alama Blue Cell Viabili y Rea‐
gen . In i ogen, Spain) was ca ied ou acco ding o he
manu ac u e 's ins uc ions. Alama Blue is a non‐ oxic,
cell‐pe meable compound, blue in colo and non‐ luo es‐
cen . Viable cells main ain a educing en i onmen wi‐
hin hei cy oplasm. Alama Blue eagen is an oxidized
o m o edox and is blue in colo . When incuba ed wi h
iable cells i changes om blue o ed and becomes luo‐
escen . This change can be de ec ed using abso bance
me hods.
A 4 and 7 days, he cells wi h cell g ow h a e ans‐
e ed o a new well and 80 µL o Alama Blue a e added
on he disc and subsequen ly 720 µL o cul u e medium
a e added; This medium is incuba ed o 2 hou s a 37
[deg.] C. and he abso bance is measu ed a he espec‐
i e exci a ion and emission wa eleng hs o 570 and 600
nm (TECAN, In ini y 200 P o). The esul s a e p esen ed
as he pe cen age o educ ion. The expe imen s we e
ca ied ou in iplica e on each cul u e.
3. Alkaline phospha ase
We analyzed he ac i i y o alkaline phospha ase (ALP)
acco ding o he manu ac u e 's p o ocol (Colo ime ic
Alkaline Phospha ase Assay Ki , Abcam ab83369, UK) in
he cul u e supe na an . The es was ca ied ou a 4
and 7 days, h ough he con e sion o a colo less p‐ni‐
ophenyl phospha e in o a colo ed p‐ni ophenol. The
abso bance a 405 nm o 4‐ni ophenol (TECAN, In ini y
200 P o) was measu ed and he ALP ac i i y calcula ed
om a s anda d cu e. The expe imen s we e ca ied
ou in iplica e on each cul u e.
4. Fixa ion o SEM
To isualize he cells in scanning elec on mic oscopy
(SEM), MC3T3 cells we e g own on i anium discs c.p.
and Ti NbTa o 4 and 7 days in iplica e. The samples
we e ixed wi h 10% o malin, ollowed by a dehyd a‐
ion s ep wi h e hanolic solu ions and coa ed by gold
pla ing using a spu um coa ing (Pelco 91000, Ted Pella,
Redding, Cali o nia, USA). All mic og aphs we e ob ai‐
ned using a Jeol JSM‐6330F scanning elec on mic os‐
cope and he accele a ion ol age was 10 kV o SEM
images.
5. S a is ical analysis
All in i o expe imen s we e pe o med in iplica e o
each condi ion s udied. The a iables we e analyzed o
he dis ibu ion o no mali y using he Kolmogo o ‐
Smi no es . S uden 's es was pe o med o de e ‐
mine s a is ically signi ican di e ences be ween he
wo g oups.
Fo he s a is ical handling o esul s, he SPSS e ‐
sion 22.0 package o Windows (IBM Co p., A monk,
94 Gine M, San ana L, Cos a AF, Vázquez-Gámez MA, Colmene o M, Olmo FJ, Chica di E, To es Y, Mon oya-Ga cía MJ
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
New Yo k, USA) was used. In all cases, he le el o signi‐
icance was conside ed o be p<0.05. Da a a e exp essed
as mean ± s anda d de ia ion (SD).
RESULTS
The cell cul u es on he Ti discs c.p. and in he alloy hey
had a simila cell g ow h. A 7 days o g ow h, he cells
showed columna and basophilic mo phology unde he
ligh mic oscope (O.M.) compa ible wi h he beginning
o di e en ia ion (Figu e 1). The su ace o he disc does
no le ligh h ough, making i di icul o see cells clea ly.
Howe e , in he highligh ed pa s, i could be obse ed
ha some cellula adhesion had occu ed in he ma e ial,
which would indica e ha he alloy has good quali ies o
be used in implan s.
Cell iabili y
Figu e 2 ep esen s he iabili y o he cell line as a unc‐
ion o he cell g ow h ime (4 and 7 days) on he Ti discs
c.p. and TiNbTa. A 4 days o cul u e, cell iabili y in he
TiNbTa discs was simila o ha o he Ti c.p. While a
7, we obse ed a sligh inc ease in iabili y in he TiNbTa
samples and a dec ease in he Ti samples c.p., al hough
wi hou signi ican di e ences. In all cases, he pe cen‐
age o iabili y was always highe han 150%. A oxic
e ec is conside ed when he iabili y is less han 75%,
he e o e, wi h he esul s ob ained, i can be deduced
ha he cell cul u e is iable h oughou he en i e du‐
a ion o he cul u e unde all condi ions.
Alkaline phospha ase ac i i y
We quan i ied he alkaline phospha ase (ALP) ac i i y a‐
lues in MC3T3 cell cul u es a 4 and 7 days. Figu e 3 e‐
p esen s he mean and s anda d de ia ion o he esul s
ob ained in bo h cul u e condi ions. In he Ti discs c.p.,
he esul s ob ained showed a dec ease in he enzyma ic
ac i i y, he di e ence ound be ween days 4 and 7 being
s a is ically signi ican (p=0.001). In he TiNbTa discs, a
dec ease in ac i i y was also obse ed wi h cul u e ime
and he e was a signi ican di e ence be ween days 4
and 7 o cul u e (p=0.006). In bo h condi ions, a 4 days,
he maximum enzyma ic ac i i y was ob ained, since
he e is a g ea e p oli e a ion and cell g ow h, as he ia‐
bili y alues co obo a ed, and om day 7, he cells p e‐
sen ed a mo e di e en ia ed pheno ype, as obse ed in
he SEM images, and ALP ac i i y dec eased.
Cell mo phology by scanning elec on mic oscopy
A p elimina y s udy was ca ied ou o s udy he mo ‐
phology and cell adhesion on he TiNbTa s udy ma e ial
compa ed o he g ow h in Ti c.p. To do his, we isuali‐
zed he samples o bo h ypes o disc a 4 and 7 days o
cul u e. A 4 days, small cell clus e s we e obse ed on
bo h ypes o su ace and o simila densi y. A 7 days, he
images showed a monolaye g ow h o e he en i e su ‐
ace o he disk, being simila in bo h ma e ials. Cell‐cell
and cell‐bioma e ial junc ions we e also obse ed. The
cells adhe ed h ough ilopodia ( hin cell p ojec ions, ye‐
llow a ow) and lamelopodia (b oade ex ensions, ye‐
llow as e isk), hus demons a ing he connec ion o he
cells wi h he bioma e ial. We began o obse e he p e‐
sence o small esicles wi h a hexagonal s uc u e, sug‐
ges ing a possible nuclea ion o hyd oxyapa i e, on he
cell su ace, sugges ing he beginning o he mine aliza‐
ion p ocess de elopmen (Figu e 4).
DISCUSSION
In lamma o y and degene a i e p oblems o he bones and
join s a ec millions o people a ound he wo ld. In ac ,
hey ep esen hal o he ch onic diseases in people o e
50 yea s o age in de eloped coun ies10. These diseases
o en equi e su ge y, including eplacemen o he en i e
join in cases o de e io a ion. This ac , accompanied by
he inc ease in li e expec ancy and he aging o he popula‐
ion, en ails a g ea demand o heal hca e de i a i es,
among which he de elopmen o su gical implan s and
ma e ials wi h a longe use ul li e pe iod s and ou .
Bioma e ials cons i u e one o he mos impo an ad‐
ances in cu en medicine, imp o ing he pa ien ’s qua‐
li y o li e and educing he healing and con alescence
ime. In 2009, Bju s en highligh ed he impo ance o in‐
c easing implan ‐bone bonding, gi en ha he hal ‐li e o
an implan is be ween 10 and 15 yea s7,8, which implies
an inc ease in he numbe o e ision su ge ies.
Table 1. Physical cha ac e is ics o pu e i anium, he di e en phases and bone: elas ic modulus and elas ic esis ance
G oup Compound Young modulus (GPa) Yield limi (MPa)
Pu e i anium Ti c.p. 100 650
Phase α+β Ti‐6Al‐4V 112 1140
Phase β TiNbTa 46‐52 1860
Bone T abecula 0.5‐2.0 40‐60
Co ical 20‐25 150‐180
Figu e 1. In e ed op ical mic oscope pho og aph (Olym-
pus CKX53) wi h a 40X medium magni ica ion objec i e,
o cells g own a 7 days in he TiNbTa discs
95
Biocompa ibili y and osseoin eg a ion s udy o new p os he ic ma e ials
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
In ou s udy, we ha e ca ied ou es s o assess a new
ma e ial, TiNbTa alloy, wi h a Young's modulus simila
o ha o abecula bone. Nume ous s udies ha e
shown he dec ease in elas ic modulus in alloys wi h Nb
and Ta, such as Ti35Nb5Ta7Z , which shows an elas ic
modulus o 55GPa11. The ad an age compa ed o he Ti
c.p. o he Ti‐6Al‐4V alloy would be he lowe Young's
modulus o he alloy s udied, which would conside ably
educe he ol age shielding.
The pe cen age o iabili y is la gely ela ed o he bio‐
compa ible and cy o oxic p ope ies o he ma e ial; a‐
ious au ho s12,13 ha e shown how high pe cen ages o
Figu e 2. Cell iabili y o cells cul u ed in Ti discs c.p.
o he TiNbTn alloy a 4 and 7 days o cell g ow h. Re-
sul s p esen ed as mean ± s anda d de ia ion
Figu e 4. Scanning elec on mic oscope mic og aphs a 4 and 7 days o os eoblas cul u es on he su ace o Ti c.p. o TiNbTa.
Cell mo phology and p oli e a ion is shown on he wo su aces es ed. Cell-cell in e ac ions h ough ilopodia (yellow a ows)
and cell-su ace in e ac ions h ough lamellipodia (yellow as e isk)
Figu e 3. Ac i i y o alkaline phospha ase in cell g ow h
on Ti discs c.p. o TiNbTa o 4 and 7 days. *Ti c.p. 4 days
s. 7 days; ** TiNbTa 4 days s. 7 days. P<0.05
350
300
250
200
150
100
50
0
% educ ion
Pu e Ti
Cell iabili y
TiNbTa
Day 4 Day 7
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
U/mL
Pu e Ti
Alkaline phospha ase ac i i y
TiNbTa
Day 4 Day 7
4 days
Ti c.p.TiNbTa
7 days
96 Gine M, San ana L, Cos a AF, Vázquez-Gámez MA, Colmene o M, Olmo FJ, Chica di E, To es Y, Mon oya-Ga cía MJ
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
iabili y a e op imal o conside ing a candida e ma e ial
o be used as a bone implan in humans. Ou cul u es, a
di e en s udy imes, p esen ed a high deg ee o biocom‐
pa ibili y, as well as an absence o oxici y. Posi i e esul s
we e ob ained be ween he cell line and he ma e ial. I
could be sugges ed ha i his ma e ial we e implan ed,
i would end o syn hesize he bone ma ix, adhe e well
o he bone, and be biocompa ible14‐15.
The iabili y alues indica e ha he e a e no di e‐
ences in cell g ow h be ween he TiNbTa and Ti c.p
alloy, and in all condi ions he alues a e abo e 75%.
The e o e, bo h ma e ials would be non‐cy o oxic and
he e o e iable. On he o he hand, he adequa e ac i‐
i y o cellula me abolism con i ms ha he alloy does
no elease oxic esidues o ou cells15‐18. P e ious s u‐
dies o animal implan a ion o ma e ials such as Nb and
Ta in so and ha d issues o a s ha e shown he high
biocompa ibili y o me als and os eogenesis19,20.
We also quan i y cellula ac i i y by measu ing ALP
ac i i y. Inc eased ALP ac i i y is di ec ly ela ed o cell
p oli e a ion and is a ma ke o di e en ia ion o he os‐
eoblas ic pheno ype. In issues such as bone and ca i‐
lage, ALP is exp essed ea ly in he calci ica ion p ocess
and la e in de elopmen , ALP exp ession is dec eased.
I has been shown ha when ALP ac i i y dec eases, cell
di e en ia ion inc eases14.
Ou alues con i m ha he cul u e on he discs wi h
he new ma e ial p esen s ALP ac i i y ha a ies acco ‐
ding o he s udy ime. A he beginning o he cul u e, a
4 days, he MC3T3 cells showed a highe cell p oli e a‐
ion in Ti c.p. han hose g own on he alloy ma e ial, in‐
dica ing a slowe g ow h in he TiNbTa discs du ing he
i s days o cul u e. A 7 days, he ALP alues we e simi‐
la in bo h samples and lowe han he ini ial ones, which
shows ha he cul u e is beha ing wi h simila cha ac‐
e is ics in e ms o p oli e a ion and di e en ia ion. In
he images ob ained by SEM, we obse ed ha a 7 days
he cells co e ed he en i e su ace o he discs, cell
g ow h occu ed equally in bo h samples and hey ea‐
ched cell con luence, and he lowes ac i i y was consis en
wi h he images cells ha a e s a ing he mine aliza ion
p ocess. In addi ion, we began o obse e he sec e ion o
ma e ial ha will o m he ex acellula ma ix by he os‐
eoblas s, and an inc ease in molecules wi h he appea‐
ance o hyd oxyapa i e was obse ed on he su ace o
he cells21. On he o he hand, os eoblas s p esen ed ilo‐
podia and lamelopodia, essen ial cellula s uc u es du‐
ing he cell adhesion p ocess, which indica es ha he
union o o he cells and o he ma e ial is di ec ; o he
au ho s desc ibe hese junc ions in MC3T3 cells and Ti
discs c.p.22,23.
In conclusion, ou in i o s udy allows us o conclude
ha he no el alloy ha combines elemen s such as Nb
and Ta wi h Ti, in addi ion o imp o ing he mechanical
p ope ies o he ma e ial, is, in he sho e m, biocom‐
pa ible wi h os eoblas ic cells, beha ing wi h cha ac e‐
is ics o iabili y, p oli e a ion, di e en ia ion and
adhesion capaci y o os eoblas ic cell lines, in a e y si‐
mila way o ha o pu e Ti.
Funding: This wo k was unded by he Spanish Foun-
da ion o Bone Resea ch and Mine al Me abolism
(FEIOMM), by he FEIOMM T ansla ional g an in
2018.
Con lic o in e es s: The au ho s decla e no con lic o in e es .
9

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Biocompa ibili y and osseoin eg a ion s udy o new p os he ic ma e ials
Re Os eopo os Me ab Mine . 2020;12(3):92-97
ORIGINALS
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Gogia A‐K. Ti based bioma e ials, he
ul ima e choice o o hopaedic im‐
plan s. P og Ma e Sci. 2009;54:397‐
425.
2. Lü je ing G, Williams JC. Ti anium. Ed.
Sp inge Science & Business Media,
2007.
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Bibliog aphy