Biocompatibility of Chitosan Collagen PVA Nanocomposite Containing Calcium Apatite | Wirjosentono | Proceedings of The Annual International Conference, Syiah Kuala University - Life Sciences & Engineering Chapter 5989 12366 1 SM
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
Biocom pa t ibilit y of Chit osa n/ Colla ge n/ PVA N a nocom posit e
Cont a ining Ca lciu m Apa t it e
* 1 Basuki Wirj osent ono, 1 M. Dim as Ekananda Dawandono, and 1 Tam rin
1
Depart m ent of Chem ist ry, Facult y of Mat hem at ics and Sciences Universit y of Sum at ra
Ut ara, Kam pus USU, Medan 20155
* Corresponding Aut hor: wirj [email protected]
Abst ra ct
Biopoly m er- based nanocom posit es cont aining bone m inerals hav e been used int ensiv ely as bonegraft in v arious brok en bone accident s. Main obj ect iv e of t his work is t o prepare and charact erise
biocom pat ible chit osan/ collagen/ PVA nanocom posit es con t aining calciu m apat it e suit able for bonegraft engineering. The calcium apat it e nanofillers were pr epared by calcinat ion of cow bones at
830 o C for 3 hours, followed by ball- m illing and ult rason icat ion processes and charact erizat ion using
part icle size analy sis ( PSA) and elect ron scanning m icroscopy ( SEM) . I t was found t hat part icle size
of t he filler ranging bet ween 100- 8000 nm due part ial aglom ersat ion. The bone- graft
nanocom posit es specim ens were prepared wit h v ariou s calcium apet it e loading ( 0- 10% ) in con st ant
rat io ( 1: 1: 1) of chit osan/ collagen/ PVA biopoly m ers. Com pr ession st rengt h of t he nanocom posit es
cont aining opt im u m filler of 6% was found 2.01 MPa, it s densit y was 1.19 g/ cm 3 and it s wat er
absorpt ion capacit y 58.3% and biodegradat ion rat e 2.67% / day . Degree of biocom pat ibillit y of t he
bone- graft was rev ealed aft er it s im plem ent at ion in m ice t issue which did not show any
hist opat hological effect aft er 14 day s.
Ke yw o rds: nanocalsiu m - apat it e, bone- graft , biocom pat ibilit y
I nt r oduct ion
Nowadays, various bone subst it ute m at erials in t erm s of st ruct ure and com posit ion sim ilar approach
t o nat ural bone have been developed. As it is well known, t he bone is a com posite consist ing of
organic and anorgnic phases. The organic phase consists of collagen and sm all am ount of ground
substance including glycosam inoglycans, proteoglycans, and glycoproteins, while t he inorganic phase
is com posed of calcium phosphate such as hydroxyapat it e and - t ricalcium phosphate ( Zhao et al.,
2002) . I n this works, bioceram ic of bovine bone, chit osan, collagen and poly( vinyl alcohol) were
chosen to prepare t he organic- inorganic com posites.
Bovine bone as biological wast e is very m any found in I ndonesia. I t s ut ilizat ion as biom aterial in bone
t issue engineering is st ill less. Bovine bone is m ore widely used as supplem entary food for livest ock.
The ut ilizat ion of bovine bone as raw m aterial of m anufact ure of bone graft subst it ut es or scaffolds
for bone t issue engineering has som e advant ages that are t he porosit y sim ilar t o nat ural bone and
containing essent ial m inerals such as hydroxyapat ite, - t ricalcium phosphate and calcium carbonate
t hat support the regenerat ion of bone ( Laurencin et al., 2006; Ooi et al., 2007). Chitosan is a
polysaccharide Obt ained from the N-deacet ylat ion of chit in, consist ing of polym eric ( 14) - linked 2am ino- 2-deoxy-- D-glucopyranose units. Chit osan can accelerate the growt h of new bone because it
st ruct ure is sim ilar t o glycosam ino glycans and hyaluronic acid found in cart ilage ( Risbud et al., 2005).
Collagen is a fibrous protein hollow and t here are lot s of j oint t issues, skin, m uscles, and tendons. This
nat ural fibrous connect s and support s ot her bodily t issues, such as bone and tendon (Hossein et al.,
2007) . I n t he field of derm at ology collagen is used as a growth enhancer net work. Polyvinyl alcohol is
a sem i- cryt alline synthet ic polym er t hat wat er-soluble, non- toxic, non-carcinogenic, biocom pat ible and
elast ic physical propert ies. I n addit ion PVA also has a st rong adhesive power so it is good as a
com posit e m at rix because it can im prove t he com pact ness and m echanical propert ies of t he
com posit e. These charact erist ics m ake t his polym er part icularly suitable for biom edical applicat ions.
I n t his st udy, bioceram ic was prepared by calcining bovine bones and crushed by ball m illing and
ult rasonicat ion in aquabidest . Chit osan- collagen-poly( vinyl alcohol) / bioceram ic com posit es were
prepared by m echanical m ixing and dried using freeze dryer. The m orphology and physicalchem ical
propert ies of t he com posites such as t he porosity, degradat ion and com pressive st rengt h were
invest igated. I n addit ion, also t he biocom pat ibilit y of t he com posites was prelim inary evaluated by in
vivo im plant at ion int o t he fem oral m uscle of whit e m ouse.
M a t e r ia ls a nd M e t hods
Preparat ion of Bioceram ic and Chitosan-Collagen-Poly( Vinyl Alcohol) / Bioceram ic Com posites
251
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
Bioceram ic was t ypically prepared by calcining bovine bone as follows. First ly, bovine leg bones were
cleaned and dried under sun, t hen boiled in boiling water for 5 hours, followed by boiling in a pot of
com m ercial pressure for 2 hours, t hen washed and t he process repeat ed t hree t im es. Furt herm ore,
bones were dried in oven at 105 oC for 3 hours, and t hen pounded by m ortar up int o sm all parts,
followed by im m ersion in n- hexane for 48 hours. Aft er t hat , t he part icles were filt ered, t hen t he
residue was dried in oven at t em perat ure of 200 oC t ill dried and sm okeless, and t hen proceed wit h
t he sm oothing process using blender. After t hat , t he bone powder was calcined at 830 oC for 3 hours
in furnace, t hen crushed by m ortar unt il sm oot h and t reat ed by ball m illing for 120 m inutes ( Retsch
PM 100), and t hen sieved by 140 m esh sieve. Aft er that, t he bone powder was t reated by
ult rasonicat ion ( 40 kHz, 250 W) for 180 m inutes ( Kudos). Then, t he bovine bone bioceram ic part icles
obtained were charact erized by FTI R, PSA and SEM.
The com posit es were prepared as follows. 3% chit osan solut ion was prepared in 1% acet ic acid. 3%
collagen solut ion and 10% PVA were prepared in dist illed wat er. Then, all of t he solut ions wit h a
weight rat io 1: 1: 1 were m ixed and st irred hom ogeneously for 1 hour at room tem perature. After t hat ,
added 4 g bioceram ics. Then t he m ixt ure was st irred t o form a hom ogeneous paste. After t hat , t he
past e was put into a cylindrical m old with a diam et er of ± 2 cm and height of ± 1.5 cm . Furt herm ore,
t he sam ples were frozen and t hen dried by freeze dryer. Do t he sam e t hing for t he m ass variat ion of
bioceram ics 5, 6 and 7 g which the com posit es were nam ed as C4, C5, C6, and C7 .
Biocom pat ibilit y
The biocom pat ibilit y was test ed for t he opt im al com posit e by im plant ing t he com posite in form of
granules int o t high m uscles of eight een 8- weeks-old m ale Sprague Dawley rat s which were obt ained
from Physiology and Anatom y of Anim al Laborat ory, Departem ent of Biology, Universit y of Sum atera
Ut ara. Before im plant at ion, t he granules were sterilized by im m ersion int o 70% et hanol solut ion,
followed by drying in UV- radit ion box. Besides, 12 experim ent anim als were adapted in a cage for 7
days. After t hat , the experim ent anim als were t reat ed by hair rem oval ,and then anest het ized by
ket am ine at dose of 1 m g/ kg.
Aft er im plant at ion at 7 and 14 days, t he experim ent anim als were t reated by dislocat ion and t hen
dissect ed t heir t high m uscles, next t he granules and surrounding t issue were t aken. The granule
edhering in t he t issue were fixed in bouin solut ion and t reat ed by em bedding in paraffin. I t was
sect ioned int o 5 m t hick slices perpendicular t o it s surface. The hist ological sect ions were stained
wit h hem at oxylin and eosin (HE) reagen and observed under an opt ical m icroscope.
Results a nd D iscussion
Preparat ion of Bioceram ic and Chitosan-Collagen-Poly( Vinyl Alcohol) / Bioceram ic Com posites
I n t he bioceram ic preparat ion, 121alcinat ions process on bovine bone aim s t o obt ain pure calcium apat it e m inerals wit hout t here are organic com pounds. The process caused t he bovine bone
undergoing physical changes, which t he bovine bone color before heat ing process at 200 oC and
121alcinat ions was whit e- yellow wit h 1000 g of weight . Aft er undergoing the processes , t he color of
bovine bone changed becom ing pure white wit h 516,8 g of weight or 51,68% of yield. The color and
weight changes of t he bovine bone were caused by releasing of organic com pounds cont ained in t he
bone ( Ooi et al., 2007) .
Milling process using ball m ill and ult rasonicat ion process produced finer part icles of bioceram ic t han
before being processed. The com posites are obtained seem hom ogeneous and also have pores.
Charact erizat ions
Part icle Size Analyzer
PSA analysis result shows that t he bioceram ics are obtained have part icle size dist ribut ion around
100- 8000 nm . Size take effect t o bioact ive propert ies of a biom aterial, which t he sm aller part icle or
nano m aterial has larger surface area so t hat has bet t er bioact ive propert ies t han coarse part icle
( Dorozhkin, 2010) .
Degradabilit y
Degradat ion of a scaffold is a very im portant param eter for bone graft subst itute applicat ion. I t should
degrade slowly t o allow bone regenerat ion ( Vacanti and Langer, 1999; Hollinger et al., 2005) . As
shown in Figure 1, t he all com posites can biodegrade slowly. The degradat ion of t he com posit es m ay
be due t o t he solubilit y of collagen and bet a-elim inat ion or t he prot onat ion of am ino/ im ine groups and
t he m echanical relaxat ion of coiled chitosan chains in SBF. The ions in SBF can accelerat e t he
hydrolysis of chitosan ( Zhou et al., 2012). Thus chit osan-collagen-poly( vinyl alcohol) / bioceram ic
252
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
com posit e possesses cont rolled biodegradat ion, which is suit able for a biom at erial for bone graft
subst it ut e.
Figure 1. Biodegradat ion of chit osan- collagen-poly ( vinyl alcohol) / bioceram ic com posites.
Mechanical Propert ies
Mechanical propert ies, especially com pressive st rength, are im port ant and usually im perat ive crit erion
in select ing t he bone graft subst it ut e m aterials ( Yin et al., 2003; Panzavolta et al., 2009) . Hence, t he
com pressive st rength of t hese m aterials were shown in Figure 2. From the records, it could be found
t hat t he com pressive st rengt h of t he com posites had a relat ion t o t he bioceram ic content in
com posit es which t he m echanical propert ies increased by rise of m ass of bioceram ics from 4 unt il 6 g
in t he com posites. However, t he m echanical propert ies decreased by adding t he bioceram ics m ore
t han 6 g or when t he m ass of the bioceram ics exceeded 7 g. it m eans t hat presence of sm all am ount
of t he bioceram ics could im prove the m echanical propert ies of t he com posites but t he large am ount of
t hose could reduce t he degree of t he m echanical propert ies. Zhou et al.. ( 2012) reported t hat
com pressive st rengt h of gelat in/ CM-chit osan/ - TCP declined wit h t he addit ion of fract ion of - TCP
m ore t han 5% . I n t his study, t he com posit e wit h 6 g m ass of bioceram ics is t he com posit e t hat has
t he com pressive st rengt h in range of t he degree of cancellous’s com pressive st rengt h which is 2- 12
MPa (Ficai, et al., 2011) . I t was pot ent ial used as bone graft subst it ute m at erials.
C4
C5
C6
C7
Figure 2. St ress- St rain curve of chit osan- collagen-poly ( vinyl alcohol) / bioceram ic com posites.
Fourier Transform I nfrared Spect roscopy
FTI R spect ra were recorde t o a bet ter underst anding of t he int eract ions bet ween t he com ponents in
t he com posit es. The FTI R spect ra of chit osan, collagen, poly( vinyl alcohol) , bioceram ic, and t he
com posit es before and after degraded were shown in Figure 3.
253
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
140
130
120
a
110
100
90
b
80
70
60
c
50
40
d
30
20
10
4000
e
f
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Figure 3. FTI R spect ra of ( a) chit osan, (b) collagen, (c) poly( vinyl alcohol) , (d) bioceram ic, (e)
chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite with 6 g bioceram ic cont ent before and ( f)
aft er degraded.
The FTI R spect ra of chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite wit h 6 g bioceram ic
content showed the absorpt ion at 3425.58cm - 1 was assigned t o –OH st retching vibrat ion from
hydroxyapat ite, chit osan, and poly( vinyl alcohol) . The peak of v 3 m ode of PO4 3- , v 4 m ode of PO4 3- , and
v 2 m ode of PO43- are also present at 1049.28 cm - 1 , 570.93 cm - 1 , and 470.63 cm - 1 , respect ively. I t
seem s t he m agnit ude of t hese bands becom es weaker. The absorpt ion bands of chit osan and collagen
such as am ide A, am ide B, am ide I , am ide I I , am ide I I I and COO-, and also v 1 m ode of PO4 3- and v 5
m ode of PO43- are absent in t he com posit es ( Figure 3e ) . t hese spect ral changes and absent s indicate
t here have been interact ions bet ween t he com ponents.
The com posit e after degraded for 7 days has sim ilar FTI R spect ra ( Figure 3f) wit h FTI R spect ra of the
bioceram ic ( Figure 3d) . The peak of t he all vibrat ion m ode of PO4 3- becom e st ronger and t he
absorpt ion of v 1 and v 5 m ode of PO4 3- , and also carbonate are present again in t he com posite. I t
indicat e t hat m ay be due to t he solubilit y of collagen, hydrolysis, beta-elim inat ion and the m echanical
relaxat ion of coiled chit osan chains in SBF.
Scaning Elect ron Microscope
Morphology of t he bioceram ic and chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posit e with 6 g
bioceram ic cont ent are shown in Figure 4. The m orphology of t he bioceram ic ( Figure 4a ) indicates
serious aggregat ion of it s. I t m ay be coused by ult rasonic t reat m ent which can prom ote t he
dist ribut ion of t he bioceram ic part icles. However, ext ensive ult rasonic t reatm ent will lead t o t he
aggregat ion of t he bioceram ic ( Zhou et al., 2012) . The m orphology of t he com posite significant ly
displays hom ogenous dist ribut ion of t he bioceram ic part icles wit hin t he chitosan- collagen-poly( vinyl
alcohol) m at rix and open pore st ructure wit h bot h m icropores ( 3.65 m ) and m acropores ( 81.5 µm ) .
an open pore st ruct ure can prom ot e t he form at ion of int ernal m ineralized bone and serve for nut rient
delivery. I n addit ion, it is also good for blood supply and cell at t achm ent ( Laurenchin et al., 2008;
Zhou et al., 2012) .
Alt hough, t he diam eter pores of t he com posit e are not in range of 100- 800 m t hat will benefit bone
regenerat ion ( Karageorgiou and Kaplan, 2005) , but there is no definite pore size for each bone t issue
in a body , because every bone part has different charact erist ics t hat are suit able t o perform the cell
m igrat ion, proliferat ion, adhesion, and different iat ion.
As shown in Figures 5 healing process of t he m ice wound were not affect ed by im plantat ion of the
nanocom posit e after day- 7 ( 1) and day- 14 ( 2) . I m plant at on of t he bone graft as biopolym ers and it s
com ponent s did not show any t oxicit y effects. The hydroxyapat ite, filler which was on nanosized eased
t he dissolut ion process int o t he m ice t issue. Chit osan and collagen m at rices were also polyionic
biopolym ers which m ay induced form at ion of new cell and t herefore revealed biocom pat ibilit y of t he
nanocom posit e into t he live m ice t issue.
254
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
b
a
Figure 4. SEM im ages of (a) bioceram ic and (b) chit osan-collagen- poly( vinyl alcohol) / bioceram ic
com posit e with 6 g bioceram ic cont ent .
Biocom pat ibilit y of chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite
Biocom pat ibilit y t est s of t he nanocom posit es as bone graft were carried out using live m ice wounded
t issue after t reatm ent for 7 and 14 days.
( 1)
( 2)
Figure 5. Mice t issue wound healing on day- 7 ( 1) and day- 14 ( 2) aft er im plantat ion of t he
nanocom posit e as bone graft
Conclusions
Mineral of calcium apet ite has been successfully isolat ed from bovine bone waste by calcinat ion at
830 oC and after ballm illing and charact erizat ion using part icle size analyser ( PSA) it was found t hat
t he part icle size was ranging 100 - 8000 nm . Nanocom posites of t he calcium apet ite- filled chit osancollagen showed good m echanicl propert ies ( com pression st rengt h: 2.01 MPa and m ore im portant ly
were biocom pat ible wit h live m ice t issue without indict ion of t oxicit y .
Re fe re n ce s
Dorozk hin, S. V. 2010. Nanosized and Nanocry st alline Calciu m Ort hophosphat es. Act a Biom at er ialia. 715- 734.
Ficai, A., Andronescu, E. , Voicu, G. , Ficai, D. 2011 . Adv ances in Collagen Hy drox y apat it e Com posit e Mat erial. I n
t ech. Am . 84: 454- 4 64.
Hossain, M.S and I qbal, A. 20 14. Pr oduct ion and Charact er izat ion of Chit osan from Shrim p Wast e. J. Bangladesh
Agril. Univ . 12 ( 1) . P 153- 16 0.
Hossein, H., Hosseink hani, M., Tian, F., Kobay ashi, H., Tabat a, Y. 2007.Bone Regenerat ion On Collagen Sponge
Self Assem bled Pept ide Am phiphiline Nanofiber Hy brid Scaffold, Tissue Engineering. I nt ernat ional
Journal.Vol. 13. No.1.
Karageorgiou, V., and Kaplan, D. 2005. Porosit y of 3D Biom at erial Scaffolds and ost eogenesis. Biom at erials
26: 5474- 5491.
Laurenchin, Cat o, T., Nair, Lak sh m i, S. 2008. Nanot echn ology and Tissue Engineering. CRC Press Tay lor and
Francis Group. Boca Rat on. P: 32 9- 347.
Ooi, C.Y., Ham di, M., Ram esh , S. 2007. Propert ies of Hy drox y apat it e from Produced by Annealing of Bov ine Bone.
Ceram ics I n t ernat ional. 33, 1171 - 1177.
Risbud, M.V., Di., M.A., Sihiger, M. 200 5. Chit osan: A Versat ile Biopoly m er for Ort hopedic Tissue Engineering.
Biom at erials. 26. 5983- 5990.
Zhou, Y., Xu, L., Zhang, X., Zhao, Y., Wei, S., Zhai, M. 2012. Radiat ion Sy nt hesis of Gelat in/ CM- Chit osan/ Tricalcium Phosphat e Com posit e Scaffold for Bone Tissue Engineering. ELSEVI ER. Mat erial Science and
Engineering C 32. 994- 1000 .
255
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
Biocom pa t ibilit y of Chit osa n/ Colla ge n/ PVA N a nocom posit e
Cont a ining Ca lciu m Apa t it e
* 1 Basuki Wirj osent ono, 1 M. Dim as Ekananda Dawandono, and 1 Tam rin
1
Depart m ent of Chem ist ry, Facult y of Mat hem at ics and Sciences Universit y of Sum at ra
Ut ara, Kam pus USU, Medan 20155
* Corresponding Aut hor: wirj [email protected]
Abst ra ct
Biopoly m er- based nanocom posit es cont aining bone m inerals hav e been used int ensiv ely as bonegraft in v arious brok en bone accident s. Main obj ect iv e of t his work is t o prepare and charact erise
biocom pat ible chit osan/ collagen/ PVA nanocom posit es con t aining calciu m apat it e suit able for bonegraft engineering. The calcium apat it e nanofillers were pr epared by calcinat ion of cow bones at
830 o C for 3 hours, followed by ball- m illing and ult rason icat ion processes and charact erizat ion using
part icle size analy sis ( PSA) and elect ron scanning m icroscopy ( SEM) . I t was found t hat part icle size
of t he filler ranging bet ween 100- 8000 nm due part ial aglom ersat ion. The bone- graft
nanocom posit es specim ens were prepared wit h v ariou s calcium apet it e loading ( 0- 10% ) in con st ant
rat io ( 1: 1: 1) of chit osan/ collagen/ PVA biopoly m ers. Com pr ession st rengt h of t he nanocom posit es
cont aining opt im u m filler of 6% was found 2.01 MPa, it s densit y was 1.19 g/ cm 3 and it s wat er
absorpt ion capacit y 58.3% and biodegradat ion rat e 2.67% / day . Degree of biocom pat ibillit y of t he
bone- graft was rev ealed aft er it s im plem ent at ion in m ice t issue which did not show any
hist opat hological effect aft er 14 day s.
Ke yw o rds: nanocalsiu m - apat it e, bone- graft , biocom pat ibilit y
I nt r oduct ion
Nowadays, various bone subst it ute m at erials in t erm s of st ruct ure and com posit ion sim ilar approach
t o nat ural bone have been developed. As it is well known, t he bone is a com posite consist ing of
organic and anorgnic phases. The organic phase consists of collagen and sm all am ount of ground
substance including glycosam inoglycans, proteoglycans, and glycoproteins, while t he inorganic phase
is com posed of calcium phosphate such as hydroxyapat it e and - t ricalcium phosphate ( Zhao et al.,
2002) . I n this works, bioceram ic of bovine bone, chit osan, collagen and poly( vinyl alcohol) were
chosen to prepare t he organic- inorganic com posites.
Bovine bone as biological wast e is very m any found in I ndonesia. I t s ut ilizat ion as biom aterial in bone
t issue engineering is st ill less. Bovine bone is m ore widely used as supplem entary food for livest ock.
The ut ilizat ion of bovine bone as raw m aterial of m anufact ure of bone graft subst it ut es or scaffolds
for bone t issue engineering has som e advant ages that are t he porosit y sim ilar t o nat ural bone and
containing essent ial m inerals such as hydroxyapat ite, - t ricalcium phosphate and calcium carbonate
t hat support the regenerat ion of bone ( Laurencin et al., 2006; Ooi et al., 2007). Chitosan is a
polysaccharide Obt ained from the N-deacet ylat ion of chit in, consist ing of polym eric ( 14) - linked 2am ino- 2-deoxy-- D-glucopyranose units. Chit osan can accelerate the growt h of new bone because it
st ruct ure is sim ilar t o glycosam ino glycans and hyaluronic acid found in cart ilage ( Risbud et al., 2005).
Collagen is a fibrous protein hollow and t here are lot s of j oint t issues, skin, m uscles, and tendons. This
nat ural fibrous connect s and support s ot her bodily t issues, such as bone and tendon (Hossein et al.,
2007) . I n t he field of derm at ology collagen is used as a growth enhancer net work. Polyvinyl alcohol is
a sem i- cryt alline synthet ic polym er t hat wat er-soluble, non- toxic, non-carcinogenic, biocom pat ible and
elast ic physical propert ies. I n addit ion PVA also has a st rong adhesive power so it is good as a
com posit e m at rix because it can im prove t he com pact ness and m echanical propert ies of t he
com posit e. These charact erist ics m ake t his polym er part icularly suitable for biom edical applicat ions.
I n t his st udy, bioceram ic was prepared by calcining bovine bones and crushed by ball m illing and
ult rasonicat ion in aquabidest . Chit osan- collagen-poly( vinyl alcohol) / bioceram ic com posit es were
prepared by m echanical m ixing and dried using freeze dryer. The m orphology and physicalchem ical
propert ies of t he com posites such as t he porosity, degradat ion and com pressive st rengt h were
invest igated. I n addit ion, also t he biocom pat ibilit y of t he com posites was prelim inary evaluated by in
vivo im plant at ion int o t he fem oral m uscle of whit e m ouse.
M a t e r ia ls a nd M e t hods
Preparat ion of Bioceram ic and Chitosan-Collagen-Poly( Vinyl Alcohol) / Bioceram ic Com posites
251
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
Bioceram ic was t ypically prepared by calcining bovine bone as follows. First ly, bovine leg bones were
cleaned and dried under sun, t hen boiled in boiling water for 5 hours, followed by boiling in a pot of
com m ercial pressure for 2 hours, t hen washed and t he process repeat ed t hree t im es. Furt herm ore,
bones were dried in oven at 105 oC for 3 hours, and t hen pounded by m ortar up int o sm all parts,
followed by im m ersion in n- hexane for 48 hours. Aft er t hat , t he part icles were filt ered, t hen t he
residue was dried in oven at t em perat ure of 200 oC t ill dried and sm okeless, and t hen proceed wit h
t he sm oothing process using blender. After t hat , t he bone powder was calcined at 830 oC for 3 hours
in furnace, t hen crushed by m ortar unt il sm oot h and t reat ed by ball m illing for 120 m inutes ( Retsch
PM 100), and t hen sieved by 140 m esh sieve. Aft er that, t he bone powder was t reated by
ult rasonicat ion ( 40 kHz, 250 W) for 180 m inutes ( Kudos). Then, t he bovine bone bioceram ic part icles
obtained were charact erized by FTI R, PSA and SEM.
The com posit es were prepared as follows. 3% chit osan solut ion was prepared in 1% acet ic acid. 3%
collagen solut ion and 10% PVA were prepared in dist illed wat er. Then, all of t he solut ions wit h a
weight rat io 1: 1: 1 were m ixed and st irred hom ogeneously for 1 hour at room tem perature. After t hat ,
added 4 g bioceram ics. Then t he m ixt ure was st irred t o form a hom ogeneous paste. After t hat , t he
past e was put into a cylindrical m old with a diam et er of ± 2 cm and height of ± 1.5 cm . Furt herm ore,
t he sam ples were frozen and t hen dried by freeze dryer. Do t he sam e t hing for t he m ass variat ion of
bioceram ics 5, 6 and 7 g which the com posit es were nam ed as C4, C5, C6, and C7 .
Biocom pat ibilit y
The biocom pat ibilit y was test ed for t he opt im al com posit e by im plant ing t he com posite in form of
granules int o t high m uscles of eight een 8- weeks-old m ale Sprague Dawley rat s which were obt ained
from Physiology and Anatom y of Anim al Laborat ory, Departem ent of Biology, Universit y of Sum atera
Ut ara. Before im plant at ion, t he granules were sterilized by im m ersion int o 70% et hanol solut ion,
followed by drying in UV- radit ion box. Besides, 12 experim ent anim als were adapted in a cage for 7
days. After t hat , the experim ent anim als were t reat ed by hair rem oval ,and then anest het ized by
ket am ine at dose of 1 m g/ kg.
Aft er im plant at ion at 7 and 14 days, t he experim ent anim als were t reated by dislocat ion and t hen
dissect ed t heir t high m uscles, next t he granules and surrounding t issue were t aken. The granule
edhering in t he t issue were fixed in bouin solut ion and t reat ed by em bedding in paraffin. I t was
sect ioned int o 5 m t hick slices perpendicular t o it s surface. The hist ological sect ions were stained
wit h hem at oxylin and eosin (HE) reagen and observed under an opt ical m icroscope.
Results a nd D iscussion
Preparat ion of Bioceram ic and Chitosan-Collagen-Poly( Vinyl Alcohol) / Bioceram ic Com posites
I n t he bioceram ic preparat ion, 121alcinat ions process on bovine bone aim s t o obt ain pure calcium apat it e m inerals wit hout t here are organic com pounds. The process caused t he bovine bone
undergoing physical changes, which t he bovine bone color before heat ing process at 200 oC and
121alcinat ions was whit e- yellow wit h 1000 g of weight . Aft er undergoing the processes , t he color of
bovine bone changed becom ing pure white wit h 516,8 g of weight or 51,68% of yield. The color and
weight changes of t he bovine bone were caused by releasing of organic com pounds cont ained in t he
bone ( Ooi et al., 2007) .
Milling process using ball m ill and ult rasonicat ion process produced finer part icles of bioceram ic t han
before being processed. The com posites are obtained seem hom ogeneous and also have pores.
Charact erizat ions
Part icle Size Analyzer
PSA analysis result shows that t he bioceram ics are obtained have part icle size dist ribut ion around
100- 8000 nm . Size take effect t o bioact ive propert ies of a biom aterial, which t he sm aller part icle or
nano m aterial has larger surface area so t hat has bet t er bioact ive propert ies t han coarse part icle
( Dorozhkin, 2010) .
Degradabilit y
Degradat ion of a scaffold is a very im portant param eter for bone graft subst itute applicat ion. I t should
degrade slowly t o allow bone regenerat ion ( Vacanti and Langer, 1999; Hollinger et al., 2005) . As
shown in Figure 1, t he all com posites can biodegrade slowly. The degradat ion of t he com posit es m ay
be due t o t he solubilit y of collagen and bet a-elim inat ion or t he prot onat ion of am ino/ im ine groups and
t he m echanical relaxat ion of coiled chitosan chains in SBF. The ions in SBF can accelerat e t he
hydrolysis of chitosan ( Zhou et al., 2012). Thus chit osan-collagen-poly( vinyl alcohol) / bioceram ic
252
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
com posit e possesses cont rolled biodegradat ion, which is suit able for a biom at erial for bone graft
subst it ut e.
Figure 1. Biodegradat ion of chit osan- collagen-poly ( vinyl alcohol) / bioceram ic com posites.
Mechanical Propert ies
Mechanical propert ies, especially com pressive st rength, are im port ant and usually im perat ive crit erion
in select ing t he bone graft subst it ut e m aterials ( Yin et al., 2003; Panzavolta et al., 2009) . Hence, t he
com pressive st rength of t hese m aterials were shown in Figure 2. From the records, it could be found
t hat t he com pressive st rengt h of t he com posites had a relat ion t o t he bioceram ic content in
com posit es which t he m echanical propert ies increased by rise of m ass of bioceram ics from 4 unt il 6 g
in t he com posites. However, t he m echanical propert ies decreased by adding t he bioceram ics m ore
t han 6 g or when t he m ass of the bioceram ics exceeded 7 g. it m eans t hat presence of sm all am ount
of t he bioceram ics could im prove the m echanical propert ies of t he com posites but t he large am ount of
t hose could reduce t he degree of t he m echanical propert ies. Zhou et al.. ( 2012) reported t hat
com pressive st rengt h of gelat in/ CM-chit osan/ - TCP declined wit h t he addit ion of fract ion of - TCP
m ore t han 5% . I n t his study, t he com posit e wit h 6 g m ass of bioceram ics is t he com posit e t hat has
t he com pressive st rengt h in range of t he degree of cancellous’s com pressive st rengt h which is 2- 12
MPa (Ficai, et al., 2011) . I t was pot ent ial used as bone graft subst it ute m at erials.
C4
C5
C6
C7
Figure 2. St ress- St rain curve of chit osan- collagen-poly ( vinyl alcohol) / bioceram ic com posites.
Fourier Transform I nfrared Spect roscopy
FTI R spect ra were recorde t o a bet ter underst anding of t he int eract ions bet ween t he com ponents in
t he com posit es. The FTI R spect ra of chit osan, collagen, poly( vinyl alcohol) , bioceram ic, and t he
com posit es before and after degraded were shown in Figure 3.
253
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
140
130
120
a
110
100
90
b
80
70
60
c
50
40
d
30
20
10
4000
e
f
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Figure 3. FTI R spect ra of ( a) chit osan, (b) collagen, (c) poly( vinyl alcohol) , (d) bioceram ic, (e)
chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite with 6 g bioceram ic cont ent before and ( f)
aft er degraded.
The FTI R spect ra of chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite wit h 6 g bioceram ic
content showed the absorpt ion at 3425.58cm - 1 was assigned t o –OH st retching vibrat ion from
hydroxyapat ite, chit osan, and poly( vinyl alcohol) . The peak of v 3 m ode of PO4 3- , v 4 m ode of PO4 3- , and
v 2 m ode of PO43- are also present at 1049.28 cm - 1 , 570.93 cm - 1 , and 470.63 cm - 1 , respect ively. I t
seem s t he m agnit ude of t hese bands becom es weaker. The absorpt ion bands of chit osan and collagen
such as am ide A, am ide B, am ide I , am ide I I , am ide I I I and COO-, and also v 1 m ode of PO4 3- and v 5
m ode of PO43- are absent in t he com posit es ( Figure 3e ) . t hese spect ral changes and absent s indicate
t here have been interact ions bet ween t he com ponents.
The com posit e after degraded for 7 days has sim ilar FTI R spect ra ( Figure 3f) wit h FTI R spect ra of the
bioceram ic ( Figure 3d) . The peak of t he all vibrat ion m ode of PO4 3- becom e st ronger and t he
absorpt ion of v 1 and v 5 m ode of PO4 3- , and also carbonate are present again in t he com posite. I t
indicat e t hat m ay be due to t he solubilit y of collagen, hydrolysis, beta-elim inat ion and the m echanical
relaxat ion of coiled chit osan chains in SBF.
Scaning Elect ron Microscope
Morphology of t he bioceram ic and chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posit e with 6 g
bioceram ic cont ent are shown in Figure 4. The m orphology of t he bioceram ic ( Figure 4a ) indicates
serious aggregat ion of it s. I t m ay be coused by ult rasonic t reat m ent which can prom ote t he
dist ribut ion of t he bioceram ic part icles. However, ext ensive ult rasonic t reatm ent will lead t o t he
aggregat ion of t he bioceram ic ( Zhou et al., 2012) . The m orphology of t he com posite significant ly
displays hom ogenous dist ribut ion of t he bioceram ic part icles wit hin t he chitosan- collagen-poly( vinyl
alcohol) m at rix and open pore st ructure wit h bot h m icropores ( 3.65 m ) and m acropores ( 81.5 µm ) .
an open pore st ruct ure can prom ot e t he form at ion of int ernal m ineralized bone and serve for nut rient
delivery. I n addit ion, it is also good for blood supply and cell at t achm ent ( Laurenchin et al., 2008;
Zhou et al., 2012) .
Alt hough, t he diam eter pores of t he com posit e are not in range of 100- 800 m t hat will benefit bone
regenerat ion ( Karageorgiou and Kaplan, 2005) , but there is no definite pore size for each bone t issue
in a body , because every bone part has different charact erist ics t hat are suit able t o perform the cell
m igrat ion, proliferat ion, adhesion, and different iat ion.
As shown in Figures 5 healing process of t he m ice wound were not affect ed by im plantat ion of the
nanocom posit e after day- 7 ( 1) and day- 14 ( 2) . I m plant at on of t he bone graft as biopolym ers and it s
com ponent s did not show any t oxicit y effects. The hydroxyapat ite, filler which was on nanosized eased
t he dissolut ion process int o t he m ice t issue. Chit osan and collagen m at rices were also polyionic
biopolym ers which m ay induced form at ion of new cell and t herefore revealed biocom pat ibilit y of t he
nanocom posit e into t he live m ice t issue.
254
Proceedings of The 5 th Annual I nt er nat ional Conf er ence Syiah Kuala Universit y ( AI C Unsyiah) 2015
I n conj unct ion w it h The 8 th I nt ernat ional Conference of Chem ical Engineering on Science and Applicat ions ( ChESA) 2015
Sept em ber 9- 11, 2015, Banda Aceh, I ndonesia
b
a
Figure 4. SEM im ages of (a) bioceram ic and (b) chit osan-collagen- poly( vinyl alcohol) / bioceram ic
com posit e with 6 g bioceram ic cont ent .
Biocom pat ibilit y of chit osan-collagen-poly( vinyl alcohol) / bioceram ic com posite
Biocom pat ibilit y t est s of t he nanocom posit es as bone graft were carried out using live m ice wounded
t issue after t reatm ent for 7 and 14 days.
( 1)
( 2)
Figure 5. Mice t issue wound healing on day- 7 ( 1) and day- 14 ( 2) aft er im plantat ion of t he
nanocom posit e as bone graft
Conclusions
Mineral of calcium apet ite has been successfully isolat ed from bovine bone waste by calcinat ion at
830 oC and after ballm illing and charact erizat ion using part icle size analyser ( PSA) it was found t hat
t he part icle size was ranging 100 - 8000 nm . Nanocom posites of t he calcium apet ite- filled chit osancollagen showed good m echanicl propert ies ( com pression st rengt h: 2.01 MPa and m ore im portant ly
were biocom pat ible wit h live m ice t issue without indict ion of t oxicit y .
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