8Ac a Chimica Slo aca, Vol. 6, No. 1, 2013, pp. 8—14, DOI: 10.2478/acs-2013-0002
P epa a ion, cha ac e iza ion and in i o bioac i i y
o poly inyl alcohol-hyd oxyapa i e biphasique memb anes
Zuzana Balgo áa, Ma in Paloua, Ja omí Wasse baue a,
Gab iela Lu išano áb, Jana Kozánko áb
aFacul y o Chemis y, B no Uni e si y o Technology, Pu kyňo a 464/118, B no 612 00
bFacul y o Chemical and Food Technology, Slo ak Uni e si y o Technology in B a isla a,
Radlinského 9, 812 37 B a isla a, Slo akia
[email p o ec ed].cz
Abs ac : Six memb anes o poly inyl alcohol (PVA) wi h a ious weigh pe cen — 0 %, 10 %, 20 %, 30 %,
40 % and 50 % o hyd oxyapa i e (HA) we e p epa ed. Fou ie T ans o m In a ed (FTIR) spec oscopy
was used o iden i y he di e en unc ional g oups in memb ane composi es. The su ace mo phology
was examined h ough scanning elec on mic oscope. The in i o bioac i i y es s in Simula ed Blood Fluid
(SBF) ha e been pe o med up o 28 days, especially o memb ane con aining 50 w . % HA. SEM was
used o cha ac e ize su ace mic os uc u e o biocomposi e memb anes be o e and a e imme sion in
SBF. I was obse ed he o ma ion o clus e s wi hin memb anes wi h inc easing amoun o HA pa icles
due o hyd ogen bond and also he agglome a ion and c ys al g ow h o HA pa icles du ing d ying o
memb anes. The bioac i i y was ound inc easing wi h ime imme sion o biocomposi e ma e ials in SBF
solu ion.
Keywo ds: poly inylalcohol, nanohyd oxyapa i e, bioac i i y, simula ed body fl uid
In oduc ion
The de elopmen o composi e bioma e ials o
an app op ia e ha d and so issue eplacemen
implan s is one o he mos impo an opics in
issue enginee ing. Valle -Regí (2009) has defi ned
bioma e ials as implan able ma e ials ha pe o m
hei unc ion in con ac wi h li ing issues. De-
pending on he unc ion o pe o m, hey can be
manu ac u ed om e y di e en me al, polyme ,
ce amic ma e ials o hei composi es (Valle -Regí,
2009).
Biocomposi e ma e ials based on poly inylalcohol
ma ix ein o ced wi h nanohyd oxyapa i e gel
ha e been de eloped by Pan (2007) as an al e na i e
bioma e ial o i anium alloy in he eplacemen o
diseased o damaged ca ilage.
The applica ions o PVA as bioma e ials o eplace
o o epai some ha d and so issue like a icu-
la ca ilage ha e inc eased in he las yea s. The
main eason o he applica ion o PVA, namely as
injec able gel, is i s excellen biocompa ibili y and
bio ibological p ope ies. (Kobayashi e al., 2005;
Noguchi e al., 1991; Co e e al., 2003; Pan e al.,
2007, Zheng e al., 1998).
Howe e , he d awback o bioma e ials based
on PVA is hei adhesion o he biological issue.
Indeed, PVA i sel does no adhe e biologically o
chemically o issue due o i s bioine ness. Long-
e m fi xa ion o PVA implan on he su ace o
li ing issue by su u e o any way is also di fi cul
o gua an ee. O he sho ages o PVA implan s a e
hei low mechanical p ope ies incompa able wi h
na u al ha d issues.
Applica ion o implan s based on sin e ed cal-
cium phospha e ce amics like hyd oxyapa i e
(Ca10(PO4)6(OH)2) o bone and den al econs uc-
ion is wo ldwide known because o hei excellen
biocompa ibili y (Wozney e al. 1998, Suchanek e
al., 1998, Vi ko ič e al., 2009), high os eoconduc-
i i y (Ducheyne e al. , 1999) and ela i e good
mechanical p ope ies. I can be used as solid o
as injec able gel (Hua e al., 2010). Un o una ely,
HA-based implan s ha e limi ed applica ions
because o hei b i leness and poo pe o m-
ance o long e m mechanical s abili y suscep ible
o he egene a ion o non-load-bea ing bone
de ec s. Na u al HA and syn he ic HA can di e
in hei chemical composi ion and beha io . I is
known ha mos syn he ic HA a e s oichiome ic,
wi h a chemical composi ion o Ca10(PO4)6(OH)2.
By con as , human bones do no ha e pu e o
s oichiome ic HA. Human bones con ain o he
ions, mainly CO3
2– and aces o Na+, Mg2+, Fe2+,
Cl–, F–. Tha is why he Ca/P mola a io in bone
is lowe han 1.67, compa ed o a mola a io o
Ca/P in syn he ic HA (Lu išano á e al., 2012). I
was epo ed (E is e al., 2006) ha his a io can
be an impo an ac o in cell adhesion, p oli e a-
ion and in bone emodeling and o ma ion.
To o e come he sho ages o PVA and HA alone
as implan s, composi e polyme bioce amics we e
in ensi ely de eloped in he las decade (P amanik
e al, 2008, Ragel e al. 2002, Williams, 2009). The
9
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…
syn hesized biocomposi e ma e ials mus be sui -
ably ailo ed in o de o pe o m some specifi c
unc ions, o example o imp o e he mechanical
p ope ies o polyme o he oughness o HA. The
emendous amoun o esea ches on biocomposi e
polyme s-ce amics is jus ifi ed by he ac ha bone
is an aniso opic composi e consis ing mainly o
o ganic polyme s (collagen and fi b in p o ein)
and ino ganic ce amic mine als (poo ly c ys al-
line, nons oichiome ic, ca bona ed o subs i u ed
HA). Among a a ie y o biocomposi e ma e ials,
hyd oxyapa i e-polyamide (Wang e al., 2007),
hyd oxyapa i e-polye hylene (Wang e al., 2001),
hyd oxyapa i e-collagen-hyalu onic acid (Bakoš
e al., 1999) sca olds o issue enginee ing we e
p epa ed.
In ein o ced polyme ma ix, he p incipal ole
is played by he in e acial bond be ween polyme
molecules and fi lle s. Wang e al. (2001) has epo -
ed ha his bond depends on he hyd ophobici y o
pola i y o bo h fi lle s and polyme s.
The mine al o hyd oxyapa i e p esen in polyme
ma ixes p o ide ac i e si es o biomine aliza ion
and also o cellula a achmen . I is well es ab-
lished ha bioac i i y o ma e ials is conside ed as
i s abili y o o m a s ong bioin eg a ion wi h hos
issue h ough in e ace bone-bioma e ials due o
he o ma ion o bone like hyd oxyapa i e in i o
o in i o.
The de elopmen o biocomposi e ma e ials was
mo i a ed by he ac ha pu e ma e ial canno
alone mee all equi emen s o biomedical im-
plan s. Gene ally, composi e ma e ial consis s a
leas o wo chemically dis inc phases which coexis
as ma ix-fi lle en i y.
Many ac o s such as ype and con en o fi lle in
polyme ma ix and ma ix p ope ies (e.g. mo-
lecula weigh ) ha e a signifi can infl uence upon
he p ope ies o composi e ma e ials (Wang e al.,
2003). In he case o biocomposi es, o he ac o s
such as biocompa ibili y o he fi lle o ma ix, he
deg ada ion a e o ma ix and non- oxici y mus
be conside ed.
Expe imen al
Ma e ials
Poly inylalcohol Mowiol 10-98 wi h Mw 61 000 was
pu chased om Cla ian . Diammonium hyd ogen
phospha e (NH4)2HPO4, calcium ni a e e ahy-
d a e Ca(NO3)2·4H2O and ammonium hyd oxide
NH4OH we e om Sigma-Ald ich. Dis illed wa e
o all he p ocedu es was used.
Samples p epa a ion
Me hod o hyd oxyapa i e p epa a ion
Diammonium hyd ogen phospha e (NH4)2HPO4
and calcium ni a e e ahyd a e Ca(NO3)2·4H2O
we e mixed in aqueous solu ion in equi ed a io
Ca/P 1.67. The eac ion (Equa ion 1) was kep in
alkali en i onmen a pH alue 9 by ammonium
hyd oxide solu ion.
These condi ions and in ensi e s i ing lead o ge-
la ion o solu ion. Then, he gel was washed many
ime wi h dis illed wa e and pH measu emen s
we e aken un il he gel pH was neu al, so all he
ammonium ions we e washed ou .
Via his eac ion fi ne hyd oxyapa i e pa icles was
syn he ized:
6(NH4)2HPO4 + 10 Ca(NO3)2·4H2O + 8NH4OH J
J Ca10(PO4)6(OH)2 + 20NH4NO3 + 20H2O
P epa a ion o memb anes
Cla ian poly inylalcohol was dissol ed in dis illed
wa e unde cons an s i ing a he empe a u e
85 °C o 4 hou s un il all solid polyme was dis-
sol ed. The amoun o polyme used was 21 g dis-
sol ed in 400 ml o dis illed wa e . Homogenized
solu ions wi h a ious PVA: HA a ios we e pou ed
in o a mold. They we e d ied du ing 7 days a em-
pe a u e 30 °C in labo a o y own o ob ain 1mm
hin memb anes
Bioac i i y es ing
The assessmen o in i o bioac i i y o samples
was ca ied ou by soaking he memb anes in 40 ml
SBF, as p oposed by Kokubo e al. (1991). Value o
pH was 7.32 and empe a u e 37.50 ± 1 °C. The ion
composi ion o simula ed body fl uid (Table 1.) is
nea ly equal o ino ganic ion concen a ions o hu-
man blood plasma and i was p epa ed acco ding o
he li e a u e (Kokubo, 2006).
Cleaned samples o size 1 × 1 cm we e imme sed
and s o ed in he incuba ion appa a us (Binde
BD 115) o 2 hou s, 7 and 28 days a he empe a-
u e o 37.0 °C. The mic os uc u e o memb anes
Tab. 1. The ion concen a ions (mol/l) o SBF in compa ison wi h ions in human blood.
Na+K+Mg2+ Ca2+ Cl–HCO3
–HPO4
2– SO4
2–
Blood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
SBF 142.0 5.0 1.5 2.5 148.8 4.2 1.0 0.5
10
Fig. 1. Mic os uc u e o PVA/HA composi e
wi h 10 % HA in PVA ma ix.
Fig. 2. Mic os uc u e o PVA/HA composi e
wi h 20 % HA in PVA ma ix.
Fig. 3. Mic os uc u e o PVA/HA composi e
wi h 30 % HA in PVA ma ix.
Fig. 4. Mic os uc u e o PVA/HA composi e
wi h 40 % HA in PVA ma ix.
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…
su ace was es ed be o e and a e soaking in SBF
by SEM.
SEM
The su ace mo phology was examined using scan-
ning elec on mic oscope JEOL JSM-7600F. The
su ace o biocomposi e memb anes a e soaking
in SBF was analyzed by SEM (TESLA BS 300) o
de ec he s uc u e and homogenei y o he com-
posi es.
In a ed spec oscopy
In a ed spec a o composi e ma e ials we e eco d-
ed by Spec ome e The mo Nicole iS10 (The mo
Scien ifi c) using KB pelle s echnique. Pelle s we e
p epa ed by p essing he mix u e o p ecu so and
d y KB wi h he mass a io 1:100. All spec a we e
eco ded in he wa enumbe ange om 4000 o
500 cm–1.
Resul s and discussion
SEM
The SEM images o PVA-HA composi es a
500 magnifi ca ion a e displayed in Fig. 1—5. The
pu e PVA memb ane was clea . The PVA ma ix
was uni o m wi hou bubbles (no ep esen ed in
he manusc ip ). Hyd oxyapa i e in composi e is
p esen in od-like pa icles, which c ea e a ious
s uc u es – agglome a es. The PVA memb ane wi h
10 % o HA shows needles o HA dispe sed wi hin
he memb ane. Sample PVA wi h 20 % HA con ains
di e en ly shaped agglome a es om 10 % HA
sample. Sho agglome a ed clus e s o HA needles
and plen y o HA needles dis ibu ed i sel in he
PVA ma ix we e ound in his samples. The 30 %
PVA-HA sample con ains HA o simila s uc u es
as in sample wi h 10 % o HA, bu dispe sed wi hin
needles ones. The 40 % HA sample shows ha he
11
FTIR Cha ac e is ic
The FTIR o di e en biocomposi e memb anes a e
depic ed in Fig. 8. The FTIR spec a a e aligned om
pu e PVA o PVA wi h 50 w . % HA. The spec um
shows se e al bands cha ac e is ic o s e ching and
bending ib a ions o di e en unc ional g oups
like O—H, C—H, C
=
C and C—O g oups.
The esul s show an inc easing in ensi y o abso b-
ance a ying wi h he amoun o HA in he samples in
he adso p ion ange om 3000 cm–1 o 3500 cm–1.
B oad and s ong band a 3360 cm–1 which cha ac-
e izes O—H s e ching equency is well de ec ed
Fig. 5. Mic os uc u e o PVA/HA composi e
wi h 50 % HA in PVA ma ix.
Fig. 6. Shows de ailed iew o HA nano pa icles
a 5000 magnifi ca ion.
ma ix is sa u a ed wi h he HA fi lle . The HA is
dispe sed all o e he es ing a ea and ob iously he
HA g ows in o he su ace o he memb ane and
causes i s oughness. The 50 % HA shows huge
agglome a es, which g ow h ough he su ace and
made he specimen e y ough. Hyd oxyapa i e
pa icles we e o nanome e size, which is shown in
Fig. 6. The change in mo phology and size o HA
pa icle due he c ys al g ow h o nanopa icles is
ela ed no only o he concen a ion, bu o he ac-
ion o empe a u e (37 °C) and ime o p epa a ion
(7 days).
Fig. 7. FTIR spec a o pu e PVA and PVA-HA composi es.
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…
12
due he inc easing p esence o HA in composi e
ma e ials.
The PVA spec um indica es a wide and in ense band
due o he p esence o hyd oxyl g oups (O—H) a
3441 cm–1. The bands co esponding o he (—CH2—)
asymme ic and he symme ic s e ching a a ound
2800 cm–1. The band a 1400 cm–1 can be a ibu ed
o O—H and C—H bending. The abso p ion peaks
a 1110 cm–1 a e ela ed o C—O s e ching.
The band a 900 cm–1 esul s om an angula
de o ma ion ou side he plan o O—H bond. The
abso p ion bands a 1625 cm–1 is due o symme ic
s e ching o ca boxyla e anion (—COO—) (Wang
e al., 2004).
The in ensi y o spec um a a ound 1330 cm–1 in-
c eases wi h inc easing con en o HA in samples
and is assigned o he phospha e g oup PO43–. I
seems ha he in ensi y o he main peaks dec eases
while ha o appea ing one inc eases due o he
dec easing amoun o PVA in composi e ma e ials.
These bands ha e been a ibu ed o he s e ching
mode o C—O and C—C g oups.
Fig. 7 shows spec um o pu e HA powde . Bands
a 630 and 3570 cm–1 indica e s uc u al O—H
Fig. 8. FTIR spec um o pu e HA powde .
Fig. 9. Su ace mo phology o composi e
(50 % HA in PVA ma ix) coa ings
be o e soaking in SBF.
Fig. 10. Su ace mo phology o composi e
(50 % HA in PVA ma ix) coa ings
a e soaking in SBF o 2 hou s.
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…

13
g oups in he n-HAp c ys als. Bands loca ed a
abou 1000—1100 and 560—570 cm–1 a e a ibu ed
espec i ely o he ν3 and ν4 P—O ib a ion modes
o egula e ahed al PO4
3– g oups.
The obse ed bands a 604 cm–1 co esponds o
O—P—O bending and ν1 symme ic P s e ching
modes. The ν1 symme ic s e ching mode o phos-
pha e g oup is obse ed a 957 cm–1. The obse ed
bands a 1391 cm–1 is due o he s e ching mode o
ca bona e, which may be o he acquisi ion o ai
du ing mine al p ecipi a ion (Rajkuma e al. 2010).
Band a 1641 cm–1 indica es he p esence o H2O in
HAp c ys als (P amanik e al. 2008)
Bioac i i y Tes ing
The pic u es display o ma ion o HA on he su -
ace o 50 % HA composi e du ing es ing pe iods.
The in i o es ing p o ed he bioac i e p ope ies
o he PVA-HA composi e.
A e y small amoun o HA c ys als we e ound
on pu e PVA memb ane a e soaking in SBF o
28 days. Howe e , he c ys als we e obse ed on all
composi e memb anes jus a e 2 hou s o soaking
in SBF. Mos o he memb anes su ace was en i ely
co e ed wi h c ys als a e 7 days o soaking in SBF.
The o ma ion o he mine al laye was simila in
he case o o he composi e ma e ials.
Conclusion
Poly inyl alcohol-hyd oxyapa i e memb anes we e
p epa ed wi h a ious amoun s o hyd oxyapa i e.
These composi es we e examined by SEM, FTIR.
Fig. 11. Su ace mo phology o composi e
(50 % HA in PVA ma ix) coa ings
a e soaking in SBF o 28 days.
Fig. 12. De ail o new o med hyd oxyapa i e
on he su ace.
Bioac i i y was es ed by soaking in SBF. Wi h inc eas-
ing con en o HA in samples, memb anes become
mo e inhomogeneous and la ge c ys al o HA we e
obse ed due o he c ys alliza ion p ocess du ing
d ying. The bioac i i y o memb ane con aining
50 % o HA inc eased wi h inc easing soaking ime.
Coa se pa icles o new apa i e a e obse ed on he
su ace o composi e ma e ials. The o ma ion o
apa i e laye on he su ace o he composi es a e
soaking in SBF demons a es high in i o bioac i i y
o es ed samples, wha makes he composi e sui able
candida e o applica ions as a ifi cial ca ilage.
Acknowledgemen s
This wo k was suppo ed by P ojec Cen e s o ma e ials
esea ch a FCH BUT, eg. no. CZ.1.05/2.1.00/01.0012.
This wo k was also suppo ed by he VEGA g an No.
1/0934/11.
Re e ences
Bakoš D, Soldan M, He nandez-Fuen es I (1999) Bio-
ma e ials 20: 191—195.
Co e RJ, O RD, Ku DN (2003) Wea 255, 1064—1068.
Chen F, Wang ZCH, Lin CHJ (2002) Ma e ials Le e s 57:
858—861.
Ducheyne P, Qiu Q (1999) Bioma e ials 20: 2287—303.
E is Z, Sa o M, Webs e TJ (2006) Jou nal o he
Biomedical Ma e ials Resea ch 78(3): 500—507.
Hua L, Xia FCH, Chang RZ (2010) Key Enginee ing
Ma e ials, 434-435: 590—593.
Kobayashi M, Chang YS, Oka M (2005) Bioma e ials 26:
3243—3248.
Kokubo T (1991) Bioma e ials 12: 155—63.
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…
14
Kokubo T, Takadama H (2006) Bioma e ials 27(15):
2907—2915.
Lu išano á G, Palou MT, Kozánko á J (2011)
Ce amics — Siliká y 55(3): 199—207. Noguchi T,
Yamamu o T, Oka M (1991) Jou nal o Applied
Bioma e ials 2(2): 101—107.
Pan YS, Xiong DS, Ma RY (2007) Wea 262: 1021—1025.
P amanik N, Mohapa a S, Alam S (2008) Polyme
Composi es 29: 429—436.
Ragel CV, Valle -Regi M, Rod iguez-Lo enzo LM
Bioma e ials 23: 1865—1872.
Rajkuma M, Sunda am MN, Rajend an V (2010)
In e na ional Jou nal o Enginee ing Science and
Technology 2(6): 2437—2444.
Suchanek W, Yoshimu a M (1998) Jou nal o Ma e ials
Resea ch 13 , 94—117.
Valle -Regi M (2010) Comp es Rendus Chimie 13:
174—185.
Vi ko ič M, Maha SMN, Palou M, Jan o á S (2009) Cen al
Eu opean Jou nal o Chemis y 7(2): 246—251.
Wang RZ, Cui FZ, Lu HB, Wen HB, Ma CL, Li HD (1995)
Jou nal o Ma e ials Science Le e s 14: 490—492.
Wang M, Deb S, Bonfi eld W (2001) Bioma e ials 11:
1311—1320.
Wang M (2003) Bioma e ials 24: 2133—2151.
Wang T, Tu han M, Gunaseka an S (2004) Polyme
In e na ional 53: 911—918.
Wang H, Li Y, Zuo Y, Li J, Ma S, Cheng L (2007)
Bioma e ials 28: 3338—3348.
Williams DF (2009) Bioma e ials 30: 5897—5909.
Wozney JM, Rosen V (1998) Clinical O hopaedics and
Rela ed Resea ch 346: 26—37.
Zheng QG, Jiu MX, Xiang HZ (1998) Bio-Medical
Ma e ials and Enginee ing 8: 75—81.
Balgo á Z. e al., P epa a ion, cha ac e iza ion and in i o bioac i i y o PVA-hyd oxyapa i e…