Carburos de silicio biomórficos como cerámicas portadoras de fármacos para su aplicación en implantes óseos

Depa amen o de Fa macia y Tecnología Fa macéu ica
Facul ad de Fa macia
Campus Vida
15782 San iago de Compos ela
Ca bu os de silicio biomó icos como
ce ámicas po ado as de á macos pa a su
aplicación en implan es óseos
Pa icia Díaz Rod íguez
San iago de Compos ela, 2014
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aguan an odas las cha las cien í icas que les doy y me animan en odo lo que me
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Manolo po consegui hace me desconec a los ines de semana y po p eocupa se
siemp e po mí.
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consecuencias y po se el copilo o ideal en es e iaje.
A mi amilia, especialmen e a mis íos Suso, Te esa, Glo ia, Manolo y mis p imos Ja i,
Rubén y Yessica po que de nada si e llega a la me a si e sien es solo. G acias po
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p esen es, po odos sus consejos y po habe me hecho la niña más eliz de es e
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alo a me al y como soy.
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A TODOS, MUCHÍSIMAS GRACIAS
3
Resumen/Summa y
Los hid ogeles o mados po algina o y poloxame ambién ue on capaces de inclui
ec o es i ales ecombinan es adeno-asociados ( AAV) p omo iendo su libe ación
sos enida y man eniendo su e icacia de ansducción. El sis ema polimé ico con
es uc u a más compac a ha ob enido un inc emen o de la exp esión angénica más
p olongado. La inco po ación de los hid ogeles ca gados en los bioSiCs no ha sido
capaz de ob ene una adecuada e icacia de ansducción siendo necesa ios es udios
adicionales.
En su conjun o, los esul ados ob enidos con i man el ele ado po encial de las
ce ámicas de ca bu o de silicio como sis emas bio uncionales con aplicación en
implan es óseos. La posibilidad de emplea nume osos p ecu so es na u ales y a ias
écnicas de ca ga pe mi i ía la selección del sis ema más adecuado en unción de la
necesidad eque ida.

5
Resumen/Summa y
Summa y
The inco po a ion o he apeu ic molecules in o implan able bioma e ials o in si u
d ug elease gi es a no el gene a ion o bio unc ional ma e ials. This esea ch wo k is
ocused on he syn hesis o ce amics de i ed om na u al p ecu so s by a wo-s ep
p ocess, which allows biomo phic silicon ca bides (bioSiCs) o be ob ained.
bioSiCs po en ial o egene a e bone issue was es ablished by e alua ing hei
mo phological p ope ies, hei cellula esponses p oduced and hei abili y o load
and elease he apeu ic molecules.
The bioSiCs syn hesis om di e en na u al p ecu so s (pine, oak and sapelli) has led
o sys ems wi h a ious mo phological p ope ies. Thei s uc u al and su ace
cha ac e iza ion has led o he conclusion ha highe po osi y ce amics a e hose
esul ing om pine wood; he highe po e size is ob ained using oak wood as
p ecu so and sapelli wood gene a es he denses and he oughes ce amic, also wi h
he highes po e in e connec i i y.
The e alua ion o he biocompa ibili y o he h ee ce amic s uc u es has been
pe o med by he cell cul u e wi h mesenchymal s em cells showing excellen esul s.
Resumen/Summa y
6
In addi ion, sys ems wi h la ge po e size induced cell di e en ia ion, leading o highe
concen a ions o os eoblas ic di e en ia ion ma ke s.
The s udy o he in e ac ions o silicon ca bide ce amics and hei ca bonaceous
p ecu so s wi h issue and blood componen s showed he mo phological and
s uc u al pa ame e s ha modula e hese in e ac ions.
The h ee ce amic sys ems we e loaded wi h ancomycin by unspeci ic adso p ion
showing po ous s uc u e dependen elease p o iles. The he apeu ic ac i i y o hese
sys ems was e alua ed by he abili y o ea a bac e ial bio ilm and he abili y o sapelli
sys ems o inhibi i s o ma ion. Sapelli sys ems signi ican ly inhibi bio ilm o ma ion
and all loaded sys ems we e able o dec ease he numbe o colony o ming uni s
(CFUs) p esen in he p e o med bio ilm.
The Vascula Endo helial G ow h Fac o (VEGF) was inco po a ed in o he bioSiCs by
ionic in e ac ions be ween he p o ein and he unc ional g oups o he ce amic
su aces. The loaded sys ems show a sus ained elease p o ile dependen on he
po ous s uc u e which was able o main ain he bioac i i y o he p o ein bo h in i o
and in i o. The addi ion o VEGF in o he ce amic has achie ed a as e os eoblas ic
di e en ia ion, indica ing a syne gis ic e ec be ween VEGF and bioSiCs opog aphy.
Hyd ogel-ce amic composi e sys ems we e ob ained by mixing algina e and poloxame .
The sys ems achie ed sus ained elease p o iles o poo ly soluble d ugs, also modula ed
by he mo phology o he ma e ials. The in i o e alua ion o indome hacin loaded
sys ems showed a sa is ac o y an i-in lamma o y ac i i y o all he samples and also a
dec ease o he ca abolic ac i i y o os eoa h i ic chond ocy es.
The hyd ogels o med by algina e and poloxame we e also capable o including
ecombinan adeno-associa ed i al ec o s ( AAV) p omo ing hei con olled elease
and main aining hei ansduc ion e iciency. The polyme ic sys em wi h he mos
7
Resumen/Summa y
compac s uc u e was able o ob ain a longe inc ease in ansgene exp ession. The
inco po a ion o loaded hyd ogels in o bioSiCs has no been able o ob ain adequa e
ansduc ion e iciencies, addi ional s udies being equi ed.
Taken oge he , he esul s o his wo k con i m he high po en ial o silicon ca bide
ce amics as bio unc ional sys ems wi h applica ions in bone implan s. The la ge numbe
o na u al p ecu so s and di e en loading echniques a ailable, should allow he
selec ion o he igh i in each case acco ding o he equi ed needs.

Capí ulo 1
In oducción gene al
1.1
Implan able ma e ials o local d ug deli e y in bone egene a ion
19
Implan able ma e ials o local d ug deli e y in bone egene a ion
ma ix (28, 34, 38-40) o a ce amic laye (35), adso bed di ec ly on o hese implan
coa ings (36, 41-44), nonspeci ically adso bed on o he implan su ace (45) o bound
o he me al su ace (30, 46, 47). Ti anium is he mos used me al o his pu pose.
1.1.3.2 Ce amics
Ce amics a e de ined as ino ganic non-me allic ma e ials. I is a complex g oup
including calcium phospha e based ce amics, silica based bioac i e glasses and ine
ce amics, such as zi conia and silicon ca bides. Thei use as biomedical ma e ials da es
back o la e 1960s, when hey we e in oduced o imp o e he applicabili y o he
me als. Ce amics p esen , as main ad an ages, hei lowe wea a es a he a icula ing
su ace and he elease o lowe concen a ions o ine wea pa icles (48, 49). They
a e ha d e ac o y, polyc ys alline compounds, di icul o shea plas ically wi h high
mel ing empe a u es, low elec ic conduc i i y and co osion esis ance (15, 15, 48).
Ce amics could be classi ied in o wo majo g oups: biodeg adable and non-abso bable
ce amics.
1.1.3.2.1 Biodeg adable ce amics
Thei abili y o in e ac wi h issue en i onmen acili a ing hei su ace mine aliza ion
and he e o e he bone g ow h makes hem he mos a ac i e g oup in issue
enginee ing. Biodeg adable ce amics a e eabso bed a e implan a ion by dissolu ion,
hus being eplaced by endogenous issues. The deg ada ion o he ce amic ma ix
leads o he o ma ion o pa icles which a e phagocy ed by mac ophages and gian
cells whe eas la ge ma e ial olumes a e eabso bed by os eoclas (33, 48, 50).
Calcium phospha e ce amics. As p e iously men ioned he main mine al componen o
bone is hyd oxyapa i e (HAp), a speci ic ype o calcium phospha es. This ac jus i ies
he in e es in calcium phospha e ce amics when ying o simula e he na u al bone

Capí ulo 1.1
20
composi ion (41, 43). I has been shown ha hese ce amics a e able o imp o e he
bone bonding s eng h wi hou inducing he o ma ion o ib ous issue showing
excellen biocompa ibili y (49, 51). Despi e he hyd oxyapa i e chemical composi ion
being he closes o he mine al bone, i s high s abili y makes he de elopmen o
chemical modi ied de i a i es wi h be e aqueous solu ion p ope ies o imp o e
eso p ion a e in i o necessa y (50, 52-54).
Calcium phospha e ce amics deg ada ion a e could be ailo ed by combining wo o
mo e calcium phospha es wi h di e en s abili y. T icalcium phospha e (TCP) wi h
Ca/P a io o 1.5 and ou polymo phs is widely used. The combina ion o β-TCP and
hyd oxyapa i e named biphasic calcium phospha e is a ma e ial whose eso p ion a e
can be modi ied acco ding o he bone issue egene a ion and wi h highe
os eoinduc ion o e pu e HAp, xenog a s and in some cases au ologous bone g a
(48, 55, 56). I has been shown ha hese ce amics ha e been eplaced in i o by new
and unc ional bone issue which ep esen s a signi ican ad an age compa ed wi h
o he biomedical ma e ials (56-58).
Calcium phospha e ce amics a e commonly used o local d ug deli e y as po ous
sca olds, coa ings and sel -ha dening cemen s and in he la e yea s as nanopa icles
(32).
As cemen s, one o mo e calcium phospha es can be mixed wi h an aqueous solu ion
o gi e injec able pas es. A e implan a ion and an in si u cu ing p ocess, a inal
ca bona e apa i e p oduc is o med which is cha ac e ized by an adequa e mechanical
s eng h ha a oids he mig a ion o he cemen o undesi able si es (53, 59). Thei
abili y o mold he de ec si e and hei os eoconduc ion makes hem a ac i e o
o hopedic eco e y, being app o ed by he Food and D ug Adminis a ion (FDA) in
1996 o he epai ing o c anio acial de ec s in humans (60). D ugs could also be
inco po a ed by mixing hem in o one o he wo phases o hei componen s (53).
21
Implan able ma e ials o local d ug deli e y in bone egene a ion
Phospha e ce amic nanopa icles a e high biocompa ible and easy o handle. They a e
biodeg adable gi ing no oxic deg ada ion p oduc s. Nanopa icles can be used as d ug
deli e y sys ems e en o ec o ing RNA and DNA. These nanopa icles could be
ob ained using a ious syn hesis me hods which lead o he e icien inco po a ion o
an i umo al agen s and p o eins (32). These nanopa icles could also be coa ed by a
d ug-polyme solu ion o ming nanocomposi e sys ems which a e able o elease
an ibio ics and pep ides (61-63).
A common combina ion is he use o ce amic coa ings in me als which could be
pe o med by di e en app oaches; plasma sp aying, sol-gel deposi ion, elec opho e ic
deposi ion, simul aneous apo deposi ion, pulsed lase o elec on beam deposi ion
and biomime ic p ecipi a ion (32, 41). The inal ma e ial is cha ac e ized by highe
os eoin eg a ion han he subs a e alone (42). Howe e , mos o hese coa ing
echniques use high p ocessing empe a u es making d ug loading di icul (32). This
makes biomime ic calcium phospha e-d ug cop ecipi a ion he mos used echnique in
o de o ob ain d ug loaded coa ing (64-66).
Calcium phospha e ce amic po ous sca olds could be loaded by speci ic and unspeci ic
d ug adso p ion (67-69). Howe e , he combina ion wi h polyme s gi ing a inal
composi e sys em is he mos common echnique (55, 70-72).
Bioac i e glasses and glass-ce amics. Silica-based bioac i e glasses a e nonc ys alline
compounds made om ine ce amic c ys alli es in a glassy ma ix, which p esen as he
main ad an ages high su ace eac i i y, good deg ada ion a e, os eoconduc ion and
os eoinduc ion, c ea ing an apa i e laye p ecipi a e in he p esence o simula ed body
luid (48, 49, 73-76). SiO2 is he mayo cons i uen o he glass p o iding s abili y o he
ma e ial by he o ma ion o a co alen ly bonded ne wo k (73). The inco po a ion o
new echniques in he ob aining p ocess o bioac i e glasses has lead o he p oduc ion
o mesopo ous bioac i e glasses which a e cha ac e ized by well s uc u ed po e
Capí ulo 1.1
22
channels wi h an a e age po e size o 5-20 nm and high su ace a ea. Those p ope ies
make he loading o se e al he apeu ic molecules o he su ace modi ica ion o
enhance d ug a ini y possible, and jus i y he high numbe o published s udies on his
subjec (49, 73, 77-81).
1.1.3.2.2 Non abso bable ce amics
Non abso bable ce amics a e ma e ials ha main ain hei physical and mechanical
p ope ies once implan ed in o he pa ien . Alumina, zi conia and py oli ic ca bon a e
examples o his g oup. Thei excellen biocompa ibili y, s eng h and oughness a e
hei main ad an ages. Howe e , hey ha e a lack o long e m s abili y and poo
os eoin eg a ion p ope ies (33, 48, 82). Non-oxide ce amics, such as silicon ca bide o
silicon ni ide a e conside ed mo e s able and no as sensi i e o he cha ac e is ic
slow c ack g ow h o ce amics, which should lead o a be e eliabili y. The ob aining
o po ous silicon ca bide using he wood s uc u e as empla e has lead o biomo phic
silicon ca bide ce amics (bioSiCs) cha ac e ized by consis en biomime ic
mic os uc u e, high deg ee o in e connec i i y, high s eng h and he mal
conduc i i y, good esis ance o oxida ion, biocompa ibili y and he mal conduc i i y
whose inal p ope ies a e con olled by he densi y and mic os uc u e o he wood
p ecu so (83-88).
1.1.3.3 Polyme s
Polyme s a e he p oduc o co alen bonding o small molecules called unime s
o ming long chain molecules widely used in biomedical de ices (27). Acco ding o hei
o igin hey could be di ided in wo g oups: na u al and syn he ic polyme s.
23
Implan able ma e ials o local d ug deli e y in bone egene a ion
1.1.3.3.1 Na u al polyme s
The mos common na u al polyme s used in biomedical ield a e collagen, algina e,
aga ose, ib in, chi osan and hyalu onic acid (5, 57). They a e biodeg adable,
bio eso bable and e sa ile (24) and could be used as gels, po ous sca olds, ilms and
nano ibe s (24, 47).
Collagen is a s uc u al p o ein o bones widely used in bone egene a ion, he
c osslinking wi h di e en agen s o educe i s deg ada ion a e is a commonly used
echnique o inc ease i s s abili y (5, 17). A good al e na i e o collagen is he use o i s
dena u ed s a e, gela in. The p esence o sequences a ginine-glycine aspa ic acids in i s
s uc u e imp o es os eoin eg a ion and s imula es he adhesion o os eoblas s (89).
Algina e is a polysaccha ide p esen in b own algae, o med by D-mannu onic acid and
L-glucu onic acid, his composi ion allows he binding o di alen ca ions (Ca2+, Cd2+,
Cu2+, Mn2+) p oducing he gela ion o he polyme ic solu ion. Mo eo e , calcium is he
mos equen ly used ion o p omo e gela ion o algina e sys ems o biomedical
applica ions due o i s p esence in he human body especially in bone issue (89, 90).
The use o his polyme has been success ul in he con olled elease o g ow h ac o s
(24).
Chi osan is a hyd ophilic polysaccha ide wi h a simila s uc u e o na u ally o med
glycosaminoglycans ha could be deg aded by human enzymes (3, 5). I s ca ionic na u e
acili a es he in e ac ion wi h anionic glycosaminoglycans and p o eoglycans which
ha e g ea a ini y o cy okines and g ow h ac o s. On he o he hand due o i s N-
ace ylglucosamine moie y, chi osan is able o in e ac di ec ly wi h g ow h ac o s,
ecep o s and adhesion p o eins (91). This ac , oge he wi h i s an ibac e ial ac i i y
and i s app op ia e physicochemical and biological p ope ies, make i an excellen
Capí ulo 1.1
24
ma e ial o he p epa a ion o biomedical ma e ials and d ug deli e y sys ems (34, 92,
93).
Aga ose is used o many issue enginee ing applica ions such as d ug deli e y o cell
cul u e beads (57) whe eas ib in has been used as issue sealan in su ge y. Fo his
applica ion ib in gels a e p epa ed by he combina ion o ib inogen and h ombin
solu ions con aining calcium ions o ming an adhesi e glue adequa e o he
adminis a ion o endogenous plasma ich in g ow h ac o s (24, 90, 94).
Hyalu onic acid is a linea polysaccha ide, essen ial componen in ex acellula ma ix.
I is cha ac e ized by high biocompa ibili y and may media e in cellula signaling, wound
epai and ma ix o ganiza ion. The use o his polyme in ex acellula ma ix
biomime ic hyd ogels has shown good esul s as g ow h ac o s deli e y sys ems (95).
O he na u al polyme s ha e been use ul as implan able bone d ug deli e y sys ems
such as silk (96), chond oi in sul a e (25) and bac e ial cellulose (97). Na u al polyme ic
sys ems a e able o load d ugs in o he polyme ic ma ix ob aining di usion/e osion
con olled d ug elease p o iles (90, 98) ha can be ailo ed modeling he d ug load
cha ge, he polyme weigh and c osslinking a iables (99).
1.1.3.3.2 Syn he ic polyme s
Al hough na u al polyme s p esen adequa e biological p ope ies hei
immunogenici y, he di icul y in p ocessing and he po en ial isk o ansmi ing
animal-o igina ed pa hogens make i necessa y o ob ain syn he ic al e na i es (47).
Poly(α-es e s) such as poly(cap olac one) (PCL), poly(p opylene uma a e) (PPF),
poly(lac ic acid) (PLA), poly(glycolic acid) (PGA) and hei copolyme poly(lac ic-
glycolic acid) (PLGA) and poly(e hylene glycol) (PEG) a e b oadly used in he medical
ield wi h minimal o eign body eac ion (2, 3). These polyme s ha e been app o ed by
he FDA o hei use in di e se clinical applica ions such as su u es, spinal usion cages,

25
Implan able ma e ials o local d ug deli e y in bone egene a ion
co ona y s en s, sys emic d ug deli e y sys ems, ne e condui s and ixa ion sc ews (2,
43). PLGA is he mos used as implan able bone ma e ial. I s deg ada ion a e and
mechanical p ope ies could be ailo ed by selec ing i s molecula weigh and
copolyme a io which makes his polyme a ac i e o con olled d ug deli e y
sys ems applica ions (3, 24, 100, 101). G ea e o s ha e been ca ied ou in his ield
in o de o ob ain he pe ec ma e ial wi h he adequa e elease p o ile; he use o
PLGA mic osphe es alone o included in o di e en ma ices has been one o he mos
common echnological app oaches used o achie e he desi able bone local elease. In
his sense, good esul s ha e been ob ained in he elease o di e en he apeu ic
molecules as BMP-2 (102, 103), BMP-7, IGF (insulin-like g ow h ac o ), dexame hasone
(104, 105), alend ona e (106) o gen amicin (107). The main disad an age o hese
molecules (PLA, PGA and PLGA) is hei acidic deg ada ion p oduc s as a consequence
o hei hyd oly ic p ocess ha can cause he deg ada ion o he apeu ic molecules
(91).
PCL is a biocompa ible, biologically ine polyme whose mechanical p ope ies and
deg ada ion a e could be also modi ied in o de o ob ain he desi able cha ac e is ics.
I shows minimal oxici y and immune esponse (18, 25).
PPF is a linea polyes e , highly biocompa ible which has been shown o suppo and
guide bone o ma ion once c osslinked (108).
The abili y o PEG o bind p o eins o pep ides gi ing s able nanosized complexes o
hei con olled elease is used in he de elopmen o p o ein comme cial d ug
deli e y sys ems (57, 109). This polyme has also been use ul o bone local deli e y o
BMPs (24).
The syn hesis o new biodeg adable polyme s such as poly(lac ic acid)-p-dioxane-
poly(e hylene glycol) (PLA-DX-PEG) has been use ul o he con olled elease o BMP-
Capí ulo 1.1
26
2. This polyme is able o unde go a sol-gel ansi ion by he change o he empe a u e
and i s deg ada ion a e could be ailo ed in o de o achie e he desi able d ug elease
p o ile (24, 110-113).
Al hough, he abo e men ioned polyme s wi h ele a e biocompa ibili y and
biodeg adabili y ha e high in e es in he o hopedic ield, he gold s anda d in
polyme s o o hopedic p os heses a e poly(me hyl me hac yla e) (PMMA) cemen s
and beads (114). They a e cha ac e ized by hei easy ab ica ion and excellen physical
p ope ies including ensile modulus, ensile s eng h, lexu al igidi y and esis ance o
c eep (15, 33). Today, he s a egy app o ed o local an ibio ic deli e y in ch onic
os eomyeli is a e su gical deb idemen o dead bone is he use o an ibio ic loaded
PMMA implan s. Howe e , he poo os eoin eg a ion and non-biodeg adabili y o he
polyme makes i necessa y o pe o m addi ional su ge y o emo e he polyme
de ice once he pa hology is epai ed (115, 116). Fu he mo e, he high d ug doses
loaded in o he cemen s a e no comple ely eleased due o he absence o
deg ada ion. Fo o e coming his limi a ion, di e en app oaches ha e been made wi h
be e esul s as he inco po a ion o loaded PLGA mic osphe es in o he cemen s
(117) o he combina ion wi h o he polyme s (118).
1.1.3.4 Composi es
As i can be deduced, each ma e ial has ad an ages and disad an ages. No s ange hen
ha s udies aimed a he collec ion and e alua ion o composi es a e a ac ing
inc easingly a en ion. In he las ew yea s new complex bioma e ials ha e been
de eloped in o de o ha e he ad an ages o a ious ma e ials simul aneously.
A composi e could be de ined as a con inuous phase ma e ial, made om wo o mo e
ing edien s wi h signi ican ly di e en physical and/o chemical p ope ies, whose
cha ac e is ics a e a om hose o he aw ma e ials (33). Se e al possible
27
Implan able ma e ials o local d ug deli e y in bone egene a ion
combina ions poin ed ou by Hab aken and cowo ke s and Bose and cowo ke s (32,
119) can be used as implan ma e ial and also o local d ug deli e y. The mos used
combina ion is he inclusion o pa icles o ibe s o calcium phospha e ce amics and
bioac i e glasses in o biodeg adable polyme ic ma ices (120). Composi es o chi osan
and PLA whe e PLA p o ides mechanical s eng h and s i ness whe eas he ca ionic
na u e o chi osan minimizes he educ ion o pH caused by he PLA deg ada ion (91)
a e also employed o , wi h he same objec i e, mix u es o componen s able o elease
calcium and silicon ions, as bioac i e glasses o phospha e ce amics wi h polyme ic
sys ems as PLGA (121) ha e been de eloped.
The combina ion o wo ypes o ce amics in o de o couple bo h mechanical
p ope ies and elec ical conduc ion could be also used; an example is he case o
ca bon nano ubes and silica o ob aining a inal ma e ial wi h good in i o esul s
s imula ing cell p oli e a ion o human os eoblas s a e elec ical s imuli (122).
Mo e complex sys ems can also be syn hesized including me als, ce amics and
polyme s. In many o hem he s abili y o he polyme ic componen , mos ly PLGA, is
inc eased by he addi ion o he ce amic phase, calcium phospha es o bioac i e glasses
ha neu alize he su ounding acid en i onmen and educe i s au oca aly ic e ec
(43). These e na y sys ems ha e been shown o be use ul in con olled elease o
an ibio ics (34).
1.1.4 The apeu ic molecules wi h in e es in bone egene a ion
Going back o Figu e 1.1.1, i can be seen ha he bone healing is an ex emely
complex p ocess depending on di e en ac o s. Despi e he g ea de elopmen o new
ma e ials in he ew las yea s, he ideal implan ma e ial has no ye been de eloped.
Some po en ial candida es, despi e possessing app op ia e po osi y and mechanical
p ope ies, ha e a lack o os eoinduc ion. In his si ua ion, hei loading wi h bone
Capí ulo 1.1
28
mo phogene ic p o eins and/o o he g ow h ac o s should enhance hei alue by
imp o ing cellula a achmen and bone o ma ion (23, 123).
Al hough bone induc ion is impo an in he p os hesis os eoin eg a ion, implan
ixa ion is also c ucial in o de o a oid i s ailu e. I has been shown ha he local
adminis a ion o bisphosphona es, an i eso p i e d ugs, imp o e implan ixa ion in i o
(124). PMMA cemen s a e cha ac e ized by low po osi y and in e connec i i y making
he g ow h o bone cells in o hei inne s uc u e di icul . This complica ion could be
o e come by he addi ion o N-ace yl cys eine, a s ong an ioxidan ha ac s as a
sca enging agen du ing he polyme iza ion, inc easing po osi y and consequen ly
os eoconduc i i y (125).
As can be seen he combina ion o bioma e ials and d ugs has lead o he enhancemen
o he ma e ial p ope ies making he he apeu ic success easie . Ma e ial-d ug
combina ions a e no only adequa e o he imp o emen o implan ma e ials bu also
allow local d ug deli e y. Local he apeu ic ea men s inc ease he ime o
pe manence o he d ug a he a ge si e, main ain high local d ug concen a ion and
educe he occu ence o side e ec s (23, 117).
In addi ion, he inco po a ion o labile he apeu ic molecules as g ow h ac o s o
bioma e ial sys ems could enhance hei s abili y p o ec ing hem om enzyma ic o
chemical deg ada ion bo h in polyme ic sys ems (126-128) and in ce amic ma ices
(13). The use o solid implan ma e ials loaded wi h BMP-2 able o suppo enough
space o cell g ow h has been ound o be mo e adequa e o bone egene a ion han
hyd ogels. These sys ems ha e achie ed highe le els o bone o ma ion due o he
s imula ion o isula comp ession, he key in bone induc ion (67, 129). Mo eo e , high
implan olume o ma e ials has p oduced a high bone o ma ion (110). This could be
explained by he in luence o mechanical signals in he bone healing modula ing
35
Implan able ma e ials o local d ug deli e y in bone egene a ion
combine hype he mia wi h local d ug deli e capaci y. The high empe a u e achie ed
a e applying magne ic s imula ion o he implan causes he umo cells dea h (185).
1.1.4.7 O he s
Sim as a in, a common hypolipidemic d ug is also o in e es because i has been
epo ed o s imula e bone o ma ion by inducing he exp ession o BMP-2 in di e en
animal models, and he e o e enhances os eogenesis and also induces os eoblas ic
di e en ia ion o mesenchymal s em cells (186-189). Jeon and cowo ke s (190) ound
ha i s in e mi en elease om de ices p omo es os eoblas ic s imula ion while
con inuous s imula ion educes cell iabili y.
The p oposal o he use o lac o e in, a p o ein p esen in he g anules o neu ophils
and in b eas milk wi h os eogenic pu poses, is based on i s abili y o modi y
p oli e a ion and di e en ia ion o os eoblas s, inc easing he calci ica ion o
ex acellula ma ix (191).
Lidocaine, is a local anes he ic ha could be used o dec easing pain a e he su ge y.
I s sho hal -li e in blood se um (1.5-2 h) makes he design o con olled elease
sys ems necessa y when using his d ug (192).
Di e en he apeu ic ions ha e also been s udied as possible candida es o local
con olled elease om implan s. Con olled elease o sil e ions (Ag) om icalcium
phospha e pa icles has shown an adequa e p o ec ion agains bac e ial e-in ec ion
(193). Selenium (Se) eleased om coa ed i anium has an icance ac i i y in i o
agains a ious cance cell lines (182). Whe eas s on ium (S ) p omo es bone
egene a ion and inhibi s bone eso p ion enhancing bone olume and mic os uc u e
(194, 195).

Capí ulo 1.1
36
Some polyme s also ha e he apeu ic in e es such as poloxamines. These iblock
syn he ic polyme s o med by poly(e hylene oxide)-poly(p opylene oxide) wi h an
e hylenediamine co e no only a e adequa e o d ug solubiliza ion bu also (196) ha e
he abili y o p omo e os eoblas ic di e en ia ion o mesenchymal s em cells in i o
(197, 198). O he polyme s a e able o elease p oline when hey a e deg aded,
p omo ing an an i-in lamma o y e ec (199).
In he de elopmen o new complex sys ems i is also possible o combine wo o
mo e d ugs om di e en g oups. Combina ions o an ibio ics and g ow h ac o s ha e
been ound use ul o he os eoin eg a ion p ocess by bo h enhancing os eoblas s
unc ion and inc easing an ibac e ial ac i i y (29) while he combina ion o
dexame hasone and alend ona e has been shown o be use ul o he enhancemen o
os eoblas ic di e en ia ion bo h in i o and in i o (176).
1.1.5 Mechanism o loading d ugs in o implan ma e ials and elease
kine ics
The apeu ic molecules could be inco po a ed in o he ma e ial ma ix o adso bed on
he ex e nal bioma e ial su ace. Figu e 1.1.2 shows he di e en mechanisms o
loading implan ma e ials wi h d ugs. D ugs could be included du ing he ma e ial
syn hesis p ocess (physical en apmen loading) o on he su ace, a e he inal
bioma e ial is ob ained. In he la e case, he d ug can be nonspeci ically adso bed on
he ma e ial su ace, co alen ly linked o he su ace o a ached by physical
in e ac ions o he in e nal o ex e nal su ace o he ma ices o polyme ic gel
ne wo ks.
Me allic implan s a e usually d ug loaded h ough he o ma ion o a polyme ic o
ce amic coa ing he e o e d ug loading s a egy depends mainly on he coa ing
echnique selec ed. Howe e , some o i anium su ace chemical modi ica ions enhance
37
Implan able ma e ials o local d ug deli e y in bone egene a ion
he p esence o unc ional g oups use ul in he co alen bonding o BMP-2 showing a
con olled elease (30, 46).
Unspeci ic adso p ion is no mally used in ce amic sys ems when he e is no a ini y
be ween he d ug and he ce amic. Ce amic sys ems can also be loaded by di ec ly
mixing hem wi h d ug powde s (32) o by co-p ecipi a ion (64, 65) (e.g. physical
en apmen loading o he d ug in calcium cemen s).
Figu e 1.1.2 D ug loading echniques on bioma e ials, c i ical ac o s and associa ed
elease mechanisms and p o iles.
The unc ional g oups o biodeg adable ce amics con e g ea a ini y o ce ain d ugs
such as bisphosphona es, ob aining he loading by physical in e ac ions.
Capí ulo 1.1
38
Polyme ic implan s a e he mos s udied g oup. Those sys ems can be loaded wi h
d ugs using all echniques. Gene ally, hose made wi h na u al polyme s make use o
he physical in e ac ions, while he syn he ic polyme s implan s load d ugs by physical
en apmen (200).
1.1.5.1 Unspeci ic adso p ion
The imme sion o po ous ma e ials in o high concen a ion d ug solu ions allows he
d ug loading by unspeci ic adso p ion which a e mainly dependen on s uc u al
pa ame e s o he bioma e ials like speci ic su ace, o al po osi y and po e size
dis ibu ion (201).
Once in con ac wi h he dissolu ion medium, hese loaded sys ems unde go he d ug
deso p ion. Release p o iles a e cha ac e ized by an ini ial bu s e ec caused by he
as dissolu ion o d ug molecules adso bed on he ex e nal s uc u e ollowed by a
elease highly dependen on d ug solubili y and ma e ial po e s uc u e (68, 139, 202).
Fo simila o al po osi ies, he p esence o small size po es p omo es d ug sus ained
elease due o he enhancemen o he su ace a ailable o d ug adso p ion (77). On
he con a y, big po es imp o e he wa e up ake and quick d ug solubiliza ion and
elease (203). The modula ion o po e size dis ibu ion has been used as an app oach
o con ol and ex end zolend ona e d ug elease om mesopo ous bioce amics (204).
An al e na i e me hod could be applying acuum du ing he loading p ocess o
ob aining slow d ug elease p o iles. This p ocedu e makes i possible o he loaded
solu ion o each he small size po es o he s uc u e, hampe ing he we ing and he
d ug elease (205). The inco po a ion o a polyme ic coa ing as a complemen o
unspeci ic adso p ion me hod has been used as a way o educe he ini ial bu s in
an ibio ic elease p o iles by c ea ing a local d ug di usion ba ie (206, 207).
39
Implan able ma e ials o local d ug deli e y in bone egene a ion
When g ow h ac o s a e loaded on polyme ic elec ospining ibe s and sca olds using
unspeci ic adso p ion me hods as and uncon olled elease p o iles a e achie ed (99,
208). This makes he imp o emen o he loading echniques necessa y in o de o
ob ain a mo e con olled d ug elease.
1.1.5.2 Physical in e ac ions
Physical in e ac ions including hyd ogen bonding, Van de Waals o ces, elec os a ic o
hyd ophobic in e ac ions a e widely used o load highly hyd ophobic d ugs in
bioma e ials and achie e desi able con olled elease p o iles (183, 209, 210).
Wi h he excep ion o he hyd ophobic in e ac ions, all a e highly dependen on pH
condi ioning o bo h d ug loading and elease p ocesses. The modi ica ion o he pH
medium is c i ical in p o ein loading and deli e y and can be used as an app oach o
modula e p o ein elease. A a pH lowe han he p o ein isoelec ic poin , he
molecule is posi i ely cha ged whe eas a pH highe han isoelec ic poin a e
nega i ely cha ged. This modi ica ion can inc ease o dec ease he a ini y o p o eins
o he bioma e ial (211). I he p o eins a e loaded unde hyd ophobic condi ions
di e en pH alues do no cause modi ica ion in p o ein elease. The s abiliza ion o
he p o ein molecule unde hese condi ions is unable o ionize a any pH (212).
The s ong ionic in e ac ion be ween bisphosphona es and di alen me al ions can be
used as an app oach o loading bioma e ials. In he p esence o hose ions,
bisphosphona es gi e a low soluble calcic sal p ecipi a e ha allows bioma e ial su ace
loading by he o ma ion o a d ug coa ing and he consequen con olled elease (42,
45). The elec os a ic in e ac ions be ween calcium phospha es and bisphosphona es
a e hough o be be ween wo phospha e anions o calcium phospha e and wo
phosphona e g oups o he bisphosphona e (183). These d ugs ha e been shown o
ha e high a ini y o calcium phospha e and bioac i e glasses ob aining good con olled
Capí ulo 1.1
40
elease sys ems (69, 176, 213, 214). The binding a ini ies o bisphosphona es o
hyd oxyapa i e a e di e en acco ding o hei molecula s uc u e ( om highes o
lowes zolend ona e > alend ona e > iband ona e > isend ona e > e id ona e >
clod ona e) (215).
Also he a io be ween calcium and phospha e, he su ace cha ge o he ce amics and
he su ace hyd ophobici y ha e an impo an in luence on d ug-ce amic a ini y, being
s onge o ce amics wi h he highes concen a ion o calcium (183, 215) and making
he loading o hyd ophobic ca bon nano ubes by hyd ophobic in e ac ions possible
(122).
I has been demons a ed ha he he apeu ic esul s o bisphosphona es a e be e
when deli e ed locally, loaded on ce amic coa ings, han a e hei sys emic
adminis a ion, inding an imp o ed implan -bone con ac (42, 216).
Acidic su aces show g ea e capaci y o bisphosphona e inco po a ion han he neu al
su aces, which allows he load wi h one highe dose o bisphosphona e in ma ices
ha con ain phospho us due o in e ac ions wi h he unc ional g oups o he
bisphosphona e (175).
The loading o g ow h ac o s as BMP-2 o VEGF in implan s can also be pe o med
h ough elec os a ic in e ac ions using hepa in as media o (217, 218). Hepa in is a
highly sul a ed glycosaminoglycan able o bind many g ow h ac o s by elec os a ic
in e ac ions be ween i s nega i ely cha ged sul a e g oups and he posi i ely cha ged
amino acid g oups o p o eins (219). The immobiliza ion o he hepa in molecule on
bioma e ial su ace can be pe o med by di e en echniques like i s binding o
polyme s by ionic in e ac ions (219) o i s chemical conjuga ion o polyme ic sys ems
(220) and demine alized bone ma ices (221). The in e ac ion o hepa in wi h p o eins
no only enhances he d ug con en in he inal ma e ial (17) wi h a con olled elease

41
Implan able ma e ials o local d ug deli e y in bone egene a ion
(217) bu also s abilizes p o eins, p o ec ing hem agains p o eoly ic deg ada ion and
inc easing hei bioac i i y (219).
Hepa in can be also use ul o loading o he he apeu ic molecules con aining amine
g oups in hei s uc u e such as gen amicin. I has been shown ha he combina ion o
gen amicin and BMP-2 loaded using hepa in immobilized on i anium su ace, p esen s a
sui able an ibac e ial ac i i y and p omo es p e-os eoblas s di e en ia ion (29).
The mechanism o hepa in immobiliza ion has an impo an e ec on d ug elease.
When hepa in is bound h ough ionic in e ac ions, he bioma e ial-hepa in a ini y plays
a key ole in he con olled elease o he d ug (219). On he con a y, when hepa in is
co alen ly linked o he bioma e ial su ace, i is he d ug-hepa in a ini y, he d ug
di usion and he sys em deg ada ion which a e he impo an a iables in d ug deli e y
(221). The d ug concen a ion should no exceed he loading abili y o he hepa in,
o he wise an ine icien loading o he implan able sys em is p oduced (37).
I is possible o syn hesize he apeu ic pep ides including a mine al binding domain in
hei s uc u e ha allows hei ionic in e ac ion wi h calcium phospha es. P o ein
elease om hose loaded ce amics is highly in luenced by he ce amic solubili y ha
could be modi ied by changing he ca bona e phase con en (153). I has been shown
ha calcium phospha es ha e high a ini y o some p o eins (BMP-2, BSA) inc easing
he sca old load capaci y and being able o modula e hei elease (23, 55, 65). As an
example, he high a ini y o COO-, OH and NH2 g oups o BMP-2 and he nega i ely
cha ged hyd oxyapa i e gi es a s ong in e ac ion h ough wa e b idged hyd ogen
bonds ha p omo es he slow elease o he p o ein (222, 223). Highe su ace
a ailable o he in e ac ion wi h p o eins inc eases he loading abili y (224). Those
in e ac ions could be also used o loading an ibac e ial pep ides ha simul aneously
inhibi bac e ial g ow h and enhance bone o ma ion (225).
Capí ulo 1.1
42
Addi ionally, bioac i e glasses could in e ac wi h cha ged ampicillin (34), gen amicin
(202) o ancomycin (226) ob aining sys ems wi h slow an ibio ic elease p o iles. P-
OH and Si-OH g oups o bioac i e glasses a e able o gene a e hyd ogen bonding wi h
an ibio ic hyd oxil and amine g oups. Those combina ions would be sensi i e o a pH
ha can be modula ed o achie e he an ibio ic con olled elease (227, 228).
The inc ease o Si-OH g oups o bioac i e glasses aises he loading abili y o p o eins
due o he p esence o highe numbe o unc ional g oups. This e ec also depends on
he molecula weigh o he loaded p o ein. The highe molecula weigh o p o ein,
he lowe i s loading due o he need o la ge a ailable su ace o he in e ac ion wi h
he sca old (210). The inclusion o s on ium in o mesopo ous bioac i e glasses has
been shown o be adequa e in o de o achie e a con olled elease o dexame hasone
(229).
Polyme ic sys ems can also be loaded h ough physical in e ac ions. F equen ly,
chemical modi ica ions o na u al polyme s such as dex an ha e been ca ied ou o
inc easing hei a ini y o BMP-2 (174). Ano he app oach is o inco po a e an
addi i e o modula e he he apeu ic molecule-polyme a ini y. Fo example, he
inco po a ion o ke a ose o collagen hyd ogels inc eases g ow h ac o s a ini y
h ough ionic in e ac ions gi ing inal hyd ogels wi h good con olled elease p ope ies
du ing ou weeks (230). On he con a y, he addi ion o chond oi in sul a e o
collagen educes i s a ini y o p o eins by enhancing he pola g oups o he hyd ogels
and he su ace a ea, his modi ica ion allows a highe bu s e ec o be ob ained ha
inc eases he os eoinduc i e ac i i y in i o possibly by he enhancemen o chemo axis
(163).
The cond oi in sulpha e applied as a coa ing on PCL/β-TCP sca olds has been also
shown use ul o p omo ing con olled elease o BMP-2 o 15 days. Addi ionally, he
amoun o loaded p o ein can be con olled by he pH modi ica ion (161).
43
Implan able ma e ials o local d ug deli e y in bone egene a ion
Chemical in e ac ions also explain he di e en app oaches o de eloping d ug
con olled elease sys ems based on o he polyme s as gela in o chi osan. Gen amicin
in e ac s wi h gela in h ough a Schi eac ion gi ing slow d ug elease p o ile pa icles.
The d ug loaded amoun and he elease mechanism (e osion, di usion) can be
de e mined by he selec ion o app op ia ed basic o acid gela in (89, 191, 231).
The in e ac ions be ween g ow h ac o s (TGF-β1) and chi osan allow he p oduc ion
o su ace-loaded chi osan beads ha elease TGF-β1 a an adequa e a e o p omo e
os eoblas ic di e en ia ion (232).
Bone mo phogene ic p o eins could be inco po a ed in o polyelec oly e ilms by he
pH modi ica ion (25, 161). In hose sys ems d ug elease is de e mined by he cha ge,
he empe a u e o he deg ada ion o he polyelec oly e mul ilaye (233).
1.1.5.3 Physical en apmen
The apeu ic molecules (an ibio ics, g ow h ac o s and ions) can be physically
en apped in o ce amics o polyme ic sys ems du ing hei syn hesis o p oduc ion
p ocess (234, 235). Homogeneous d ug dis ibu ion is an impo an ac o o be
conside ed in he de elopmen o hose sys ems in o de o a oid an ini ial bu s
elease. They gene ally achie e mo e p olonged elease p o iles han d ug-adso bed
sys ems, he elease con olled being mainly by di usion and bioma e ial e osion
depending on he polyme deg ada ion (2, 213).
Fo inco po a ing d ugs in o o hopedic cemen s, hey can be physically mixed (181,
236) o dissol ed (53) in one o he componen s o he cemen sys em o in all o
hem. Bo h echniques ha e ad an ages and disad an ages. The addi ion o he d ug in
he solid phase allows mo e slow- elease sys ems o be ob ained, while inco po a ing
he d ug in he liquid phase gi es ise o mo e homogeneous sys ems (170). The
Capí ulo 1.1
44
inco po a ion o high amoun s o d ugs in o hese sys ems changes he p ope ies o
he inal cemen (237).
The ex eme ope a ion a iables du ing cemen s p oduc ion o hei inal
cha ac e is ics (e.g. pH) can limi he u ili y o his loading p ocedu e. As an example,
he high empe a u e necessa y o he polyme iza ion p ocess du ing PMMA cemen
p oduc ion may accele a e he mo sensi i e d ug deg ada ion (136). The pH o he
cemen is c ucial o main ain he ac i i y o he apeu ic molecules deg ading
ancomycin a pH 9.5 (238). Finally, PMMA cemen s ha e a low po osi y and an
ex emely slow deg ada ion a e ha limi he elease o d ugs physically en apped
in o hose ma e ials (136).
In o de o o e come hese limi a ions, se e al polyme s as ca boxime hycellulose
(117) o ce amics as silica nanopa icles (142) can be mixed wi h PMMA cemen s.
Those addi i es modi y he po osi y and/o he wa e up ake in o he PMMA ma ices
enabling he dissolu ion and he elease o he d ug.
D ug elease can be also imp o ed by inc easing he amoun o d ug in o PMMA
cemen s which p omo es ma e ial agili y and i s c acking (239) o h ough ex e nal
s imuli like ul asounds (240, 241).
An al e na i e o he PMMA is he use o calcium phospha e cemen s in which a
ha dening p ocess akes place a oom o body empe a u e. They ha e gene ally high
in insic po osi y ha can e en be imp o ed by adding soluble o ganic addi i es (60,
242). I has been shown ha calcium phospha e cemen s imp o e he he apeu ic
ac i i y o ce ain p o eins (53, 242) showing op imal elease p o iles o local elease
o an ibio ics and an i-in lamma o y d ugs (243).
Techniques o inco po a e he apeu ic molecules in o ce amics include i s biomime ic
cop ecipi a ion o ming a coa ing on me allic implan s (35), he co-p ecipi a ion o
51
Implan able ma e ials o local d ug deli e y in bone egene a ion
been desc ibed ha some o hese sys ems a e able o elease g ow h ac o s h ough
cell-media ed polyme deg ada ion mechanism (280).
D ug elease p o iles om polyme ic o ce amic coa ings (281) can be modula ed by
he hickness (282, 283) and he deg ada ion p ope ies o he coa ing (284, 285).
Na u al ilmogen polyme s such as zein o chi osan a e an a ac i e al e na i e o he
con olled elease o d ugs om implan s, being adequa e o an i umo als, an ibio ics
and g ow h ac o s wi h kine ics dependen on d ug solubili y (40, 254, 286).
Addi ionally, some d ug depending ac o s can be poin ed ou . The selec ion o high
soluble (e.g. sal s) o low soluble de i a i es can be used as a echnological app oach o
modula e he elease p o iles o some d ugs, educing he bu s elease o imp o ing
he dissolu ion p ocess (192, 252, 287).
The inclusion o high concen a ions o d ugs could acili a e bioma e ial deg ada ion by
sys em ins abili y o due o he cellula chemo axis a ec ing d ug elease p o iles (111).
When calcium phospha e cemen s a e loaded, high amoun s o d ugs can modi y hei
heological p ope ies and se ing kine ics inc easing he po osi y o he inal ma e ial
and p omo ing as elease kine ics. The e ec is s ong o molecules ha in e ac wi h
calcium and phospha e ions (53, 243).
The inclusion o wo p o eins simul aneously in o a hyd ogel could a ec he elease o
bo h p o eins due o a compe i i e mechanism. As an example, he combina ion o
SDF-1 (s omal cell-de i ed ac o 1) and BMP-2 in o a gela in hyd ogel inc eases he
bu s e ec o SDF-1 (156).

Capí ulo 1.1
52
1.1.6.1 D ug elease kine ic analysis
Ma hema ical modeling o d ug deli e y and p edic abili y o d ug elease om
pha maceu ical o mula ions is a ield o eno mous impo ance ha his o ically has
a ac ed g ea a en ion (288).
Despi e he condi ions o he in i o s udies a e eally he e ogeneous and no allowing
easy compa isons be ween hem, di e en au ho s ha e modeled he d ug elease
beha io om implan s, o make p edic ions abou he he apeu ic e ec o he de ice
(35), o compa ing o mula ions (64) o o imp o ing he unde s anding o he d ug
elease mechanism (274).
The kine ics mos commonly used o e alua e he elease o d ugs om implan s a e as
ollows (289, 290):
Ze o o de kine ics: F = ko , whe e F is he ac ion o eleased d ug a ime , and ko is
an appa en elease a e. The elease a e is independen on he d ug concen a ion
(64, 274). BMP-2 and insulin–like g ow h ac o ha e been deli e ed a ze o o de
kine ics o pseudo-ze o o de kine ics om c osslinked gela in coa ing sys ems (168).
Simila ly, he elease o ibup o en om asymme ic coa ing i anium allows dependence
on osmo ic p essu e (89). The elease o o he an ibio ics and co icoids
elec odeposi ed on o pu e Ti dependen on-demand elec ical s imula ion can also
ollow ze o o de kine ic p o iles (291).
Fi s o de kine ics: ln(1-F) = – k1 , whe e F ep esen s he ac ion o d ug eleased a
ime , and k1 is he i s -o de elease a e cons an . The elease a e is dependen on
d ug concen a ion, d ug solubili y and di usi i y (35, 274).
Higuchi model: F = kH ½, whe e F ep esen s he ac ion o d ug eleased a ime , and
kH is he Higuchi dissolu ion cons an . This equa ion pe ec ly desc ibes elease
53
Implan able ma e ials o local d ug deli e y in bone egene a ion
p ocesses whe e d ugs a e dispe sed in monoli hic sys ems wi h no changes du ing he
elease p ocess (cons an po osi y, no swelling…) and elease is pu ely di usion
con olled wi h cons an di usion coe icien s. This model has been shown o be
adequa e especially o he ini ial s ages o d ug deli e y om calcium phospha e
ce amics and calcium phospha e cemen s. A e ha , d ug elease is also modula ed by
ma ix deg ada ion (53, 64, 236, 243, 281).
Hixon-C owell model: Mo1/3 – M 1/3 = ks , whe e Mo is he ini ial amoun o d ug in he
bioma e ial, M is he emaining amoun o he d ug in he composi e a ime , and kS
is he cons an inco po a ing he su ace olume ela ion. Di iding he abo e equa ion
by Mo1/3 and simpli ying gi es (1-F)1/3 = 1 – kE , F = 1 – (M /Mo), whe e F ep esen s he
d ug dissol ed ac ion a ime , and kE is he elease a e cons an . This model
desc ibes sys ems whe e he d ug elease is dependen on he sys em e osion (64).
Ko smeye -Peppas model: F = kP n, whe e F ep esen s he d ug ac ion eleased a ime
, kP is he elease a e cons an and n is he di usional exponen which indica es he
d ug elease mechanism n = 0.45 Fickian di usion, 0.45 < n < 0.89 anomalous di usion,
n = 0.89 case II anspo , n > 0.89 supe case II anspo o ypical ze o-o de elease
(57, 64). This model has been used o explain an ibio ics elease mechanisms om
i anium coa ings o calcium algina e and gela in (51). The use o his model is also
adequa e o sys ems whose elease p o iles a e con olled by a ious ac o s especially
hyd ophilic polyme ic sys ems in whe e di usion, dissolu ion and swelling a e key
pa ame e s (57, 98, 250).
Weibull model: F= Mo [1-e-( -T)b/a] whe e F ep esen s he amoun o d ug dissol ed as a
unc ion o ime . Mo is he o al amoun o d ug being eleased, he elease p o iles
a e cha ac e ized by he ela ionship wi h he pa ame e shape ha de ine he
anspo mechanism (61). This model, ypically used o ac al sys em
cha ac e iza ion, has been success ully applied o cha ac e ize d ug dissolu ion om
Capí ulo 1.1
54
mesopo ous composi e ibe s (202). This model has also been ound use ul in he
analysis o elease kine ics om calcium phospha e bone cemen s wi h in e ac ion
be ween d ug and cemen componen . In hese cases di e en pa ame e s modula e
d ug elease as d ug solubili y, in e ac ion be ween d ug and cemen composi ion and
he p ecipi a ion o bone cemen componen s (150).
The sui abili y o he he apeu ic molecule elease p o ile may ex emely condi ion he
he apeu ic success o he implan . Di e en au ho s ha e analyzed hese e ec s in
o de o ob ain he adequa e elease p o ile o he success o g ow h ac o
ea men s in s imula ing an os eogenic esponse (168).
This ield can be expec ed o become an in eg al pa o implan de ices de elopmen .
The wide a ie y o ma e ials a ailable makes i unlikely ha he e will be one gene al
heo y applicable o any ype o implan . I is much mo e likely ha he e will be a
b oad spec um o di e se ma hema ical models, applicable o speci ic ypes o de ices
di e ing in composi ion, geome y and d ug load. In silico p edic ion and op imiza ion
should help in accu acy and easiness o applica ion o d ug loaded bone implan s.
1.1.7 T ansla ion o he human si ua ion
In i o s udies allow d ug elease p o iles wi h good ma hema ical i ing o be
ob ained. Howe e , esul s a e pa icula ly di icul o be ex apola ed o he in i o
si ua ion (286). I in i o-in i o co ela ions a e always di icul o any adminis a ion
ou e, hese cases a e e en mo e p oblema ic as he physiological en i onmen is
a iable and dependen on mul iple ac o s. Di e en au ho s ha e desc ibed high le els
o an ibio ic in o he bone issue a e implan a ion o loaded ma e ials despi e he
elease kine ics in i o we e ma kedly slowe han he one ob ained in i o du ing he
elease s udies in PBS (149).
55
Implan able ma e ials o local d ug deli e y in bone egene a ion
Se e al animal models ( a s, mice, sheeps, abbi s, dogs, pigs…) ha e been used o he
in i o e alua ion o d ug loaded implan ed ma e ials (50, 70, 130, 149, 155, 279),
showing ha hei he apeu ic e ec is highly dependen on he hema oma size,
loca ion and size o inju y (292). All hese pa ame e s modi y he physiological
en i onmen and he e o e, he d ug elease (292).
A key ac o in he elucida ion o he in i o esul s when s udies a e ca ied ou using
implan s loaded wi h bone egene a ion ac o s is o dis inguish be ween he e ec o
he he apeu ic molecule and he in insic bone egene a ion cha ac e is ics o he
animal. Adequa e con ols a e needed. In some s udies, no he BMP ac i i y bu he
egene a ion abili y o he bone i sel is esponsible o he good he apeu ic esul s
(292).
1.1.8 Fu u e pe spec i es
Success ul bone egene a ion equi es he combina ion o many e en s, cells ha
unde go di e en ia ion o o m os eoblas s, biological ac o s ha con ol g ow h and
cell di e en ia ion and cellula a achmen , mig a ion and p oli e a ion (47). To help in
his na u al p ocess and inc easing he apeu ic success, bioma e ials play an impo an
ole. They mus be p ope ly ailo ed o ob ain he co ec p ope ies and hey can be
loaded wi h g ow h ac o s o p omo e bone healing, an ibio ics o educe in ec ions,
analgesics and an i-in lamma o ies o educe pain and eco e y ime o an i umo al
d ugs o a oid me as asis. They can also be used o gene- he apy pu poses by
inco po a ing DNA plasmids and small-in e e ing RNA. The sus ained deli e y o
pDNA and siRNA om mesopo ous silica nanopa icles (MSNPs) has been shown o
inc ease he ans ec ion le els o hese molecules. All hese applica ions need
op imiza ion and well o ganized s a egies o he e icien deli e y o d ugs a a ge
si es.
Capí ulo 1.1
56
The deli e y o cells wi h he apeu ic ac i i y is he nex s ep in he de elopmen o
bio unc ional ma e ials. I has been shown ha he use o au ologous bone ma ow
mononuclea cells and bone ma ow mesenchymal s em cells inc ease angiogenesis and
bone egene a ion by he sus ained elease o bFGF (293, 294). Addi ionally, he
combina ion o os eoblas s and BMP-2 has been ound o s imula e alkaline
phospha ase ac i i y (295) and human bone ma ow mesenchymal s em cells oge he
wi h VEGF ha e been use ul in he enhancemen o he bone egene a i e mechanism
(296).
The bone d ug deli e y sys ems a e conside ed combined (o hopedic ma e ials +
d ug) sys ems by he FDA which equi es a longe egis a ion p ocess han o
adi ional o hopedic implan s hampe ing hei clinical use (297).
The de elopmen o new local deli e y sys ems o bone egene a ion and/o he
op imiza ion o he al eady de eloped ones need expe ise om bo h, bioma e ial
enginee ing and pha maceu ical echnology ields which ha e been adi ionally a a
dis ance (41). Mul idisciplina y wo ks linking bone subs i u e p oduc ion and he “know
how” o pha maceu ical companies a e c ucial o acili a e he clinical use o hese new
ma e ials.
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Capí ulo 1.1
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292. Kempen DHR, Yaszemski MJ, Heijink A, He e an TE, C eeme s LB, B i son J, e
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1.2
Ce ámicas biomó icas de ca bu o de silicio como nue os ma e iales
pa a egene ación ósea
99
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
p ocesos de deposición química po apo . Sin emba go, es as elabo aciones suelen
eque i empe a u as supe io es a 2000 ºC y esul an ca as, lo que es a u ilidad a los
ma e iales pa a una amplia gama de aplicaciones (36).
En los úl imos años se ha abo dado el desa ollo de nue os mé odos de p oducción
pa a ob ene ce ámicas biomó icas de ca bu o de silicio con meno consumo
ene gé ico y po lo an o meno cos e (25, 37). Su po osidad abie a, lige eza y
excelen es p opiedades mecánicas en lexión y comp esión son algunas de las en ajas
del ca bu o de silicio biomó ico (bioSiC) en e al ca bu o de silicio con encional (38-
41).
La Figu a 1.2.1 sin e iza las di e en es opciones disponibles pa a la ob ención de
ce ámicas biomó icas de ca bu o de silicio; la in il ación con silicio en ase apo , la
educción ca bo é mica y la in il ación con silicio líquido (20). Es os mé odos
equie en el empleo de al as empe a u as pa a la con e sión de la made a en
ce ámicas (9, 14) y en odos ellos, la po osidad e in e conexión de los po os del
p ecu so de e minan el p oceso de in il ación (11, 24).
Figu a 1.2.1 Mé odos de sín esis del ca bu o de silicio biomó ico (bioSiC) a pa i de
made a y p ecu so es p ocesados.

Capí ulo 1.2
100
1.2.3.1 In il ación con silicio en ase apo
Es e p ocedimien o de sín esis es e sá il. Las piezas de made a desecadas y moldeadas
de la o ma ap opiada se pi olizan e in il an con apo de silicio (Si7, SiO8 o CH3SiCl3)
a ele ada empe a u a (27, 42). La elección del agen e in il an e pe mi e la ob ención
de di e en es p oduc os de silicio, como SiC-Si3N4, lo que pe mi e además modula la
esis encia a la oxidación del ma e ial (43, 44). Sin emba go, su p incipal incon enien e
es la uen e de ob ención del silicio, la descomposición de p ecu so es me alo gánicos
(45), lo que implica el empleo de al as empe a u as y la gos p ocesos (26, 46).
1.2.3.2 Reducción ca bo é mica
Cuando se emplea es e p ocedimien o, las piezas de made a se in il an con dióxido de
silicio (SiO2) y pos e io men e se pi olizan. El SiO2 puede se ob enido di ec amen e
po una in il ación con silica gel o po el empleo de o os eac i os. Pa a la ob ención
del ma e ial deseado es necesa io lle a a cabo a ios ciclos de in il ación-pi ólisis. El
p oceso se inaliza median e una educción ca bo é mica a empe a u as supe io es a
1000 ºC de los p ecu so es in il ados y pi olizados. Es e p ocedimien o da luga a
ma e iales p incipalmen e o mados po ase β-SiC (47) aunque la ob ención de
ma e iales pu os SiC ecuen emen e equie e a amien os é micos adicionales a
empe a u as muy supe io es debido a la o mación de ases in e medias (48). La
o mación del ca bu o de silicio es á de e minada p incipalmen e po las eacciones en
ase de apo , dando luga a ma e iales de meno esis encia que los ob enidos po
in il ación de silicio líquido (15).
Con el in de mejo a los esul ados de es e p ocedimien o se han ealizado di e en es
es udios cen ados en el con ol del acío y/o de la p esión du an e la imp egnación
sol-gel con SiO2 o en el uso de luidos supe c í icos (12, 45).
101
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
La in il ación sol-gel puede ealiza se ambién u ilizando o os eac i os, como
e ae ilo osilica o (TEOS), poli(me il enil inilsilsesquioxano) (PMPVS),
poli(me ilhid osiloxano) (PMHS) o poli(ca bome ilsilano) (PCMS) (27, 49, 50).
Es e mé odo es sencillo y de bajo cos e, lo que cons i uye sus p incipales en ajas.
1.2.3.3 In il ación con silicio líquido (LSI)
Según es e p ocedimien o, las ce ámicas biomó icas de ca bu o de silicio (bioSiCs) se
ab ican median e un p oceso en dos e apas; una pi ólisis con olada de la made a en
a mós e a ine e, no malmen e a gón, seguida po una in il ación eac i a ápida y
ambién con olada con silicio undido a una empe a u a supe io al pun o de usión
del silicio (1.410 ºC) (36, 51-53).
C(s)+ Si(l)→ β-SiC
Ecuación 1.2.1 Reacción química du an e la sín esis de ca bu o de silicio median e la
in il ación con silicio líquido (14).
El ma e ial inal es á o mado p incipalmen e po β-SiC y silicio lib e en la supe icie de
los po os, pe o ambién puede con ene algunos elemen os aza como Al, S, B, Na (9,
14). El con enido medio de silicio se si úa en e el 20 y el 30% (14).
La mayo pa e de la pé dida de peso de la made a du an e el p oceso de pi ólisis se
lle a a cabo po encima de 500 ºC. El uso de empe a u as supe io es du an e es a
e apa p omue e cambios es uc u ales, disminuyendo el amaño de po o de la
p e o ma de ca bón y di icul ando la pos e io in il ación (54).
Las ca ac e ís icas de las made as p ecu so as seleccionadas modulan la
mic oes uc u a de la p e o ma de ca bón y és a a su ez condiciona el p oceso de
in il ación de silicio, ya que de e mina las ue zas capila es y su ciné ica. Además, las
Capí ulo 1.2
102
p opiedades de la supe icie del ca bón in luyen en el ángulo de con ac o y la
humec ación con el silicio líquido. Po o a pa e, la in il ación del ca bón en la
di ección axial a o ece la con e sión a ca bu o de silicio debido al anspo e del
undido a a és de los canales na u ales del molde (20). Así, se ob iene una buena
ansmisibilidad de la mic oes uc u a en e la o iginal de la made a, la del ca bón
ege al y la de la ce ámica de SiC inal (55).
El silicio undido in e acciona con la p e o ma de ca bón dando luga a una eacción
exo é mica espon ánea que disuel e el ca bón gene ando g upos Si-C que c is alizan. Si
no hay su icien e silicio pa a disol e el ca bón, la capa inicial de ca bu o de silicio
o mada inhibe las eacciones adicionales en e Si y C, y la o mación de ca bu o de
silicio pos e io depende de un p oceso de di usión, cuya impo ancia es al a a
empe a u as supe io es al pun o de usión del silicio. Es e mecanismo explica las
ca ac e ís icas mic oes uc u ales obse adas en las mues as de ca bu o de silicio
ob enidas (36, 56). Du an e el p oceso de in il ación, los po os de meno amaño son
eliminados debido a la expansión de olumen del 58% que se gene a as la
c is alización del ca bu o de silicio (54).
Las condiciones en las que se p oduce el p oceso de in il ación, además de la
na u aleza del p ecu so , de e minan la p esencia de ca bón esidual que a ec a á
nega i amen e a las p opiedades de la ce ámica (16, 52, 57). Po ello se han explo ado
di e en es opciones, como el uso de la adiación ul asónica, con el in de mejo a el
p oceso de in il ación y, po lo an o, las p opiedades inales de los ma e iales
ob enidos (49).
La amplia a iedad de made as exis en e o ece la posibilidad de p oduci ce ámicas de
ca bu o de silicio biomó ico con di e en es mic oes uc u as idimensional y
p opiedades a medida (densidad, po osidad abie a, in e conec i idad, esis encia a la
ac u a...) pa a la aplicación eque ida (36, 37).
103
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
El mé odo de in il ación con silicio líquido p esen a di e en es en ajas pa a la
p oducción de ce ámicas biomó icas de ca bu o de silicio con espec o a los demás
p ocedimien os (8, 52, 56-58):
 Resul a espe uoso con el medio ambien e ya que usa ma e iales eno ables y
el p oceso es poco con aminan e.
 Sus eque imien os ene gé icos y é micos ( eacción exo é mica) son
educidos.
 No p ecisa adi i os adicionales.
 El p ocedimien o es ápido y de bajo cos e.
El ca bu o de silicio es químicamen e ine e, no eabso bible y ex emadamen e
esis en e a la co osión y a la e osión. Los ca bu os de silicio son ma e iales
semiconduc o es an o é mica como eléc icamen e (2.36 y 3.05 eV pa a β- y α-SiC,
espec i amen e). En gene al, las ce ámicas de ca bu o de silicio son du as y
esis en es, an o a empe a u a ambien e como a empe a u as supe io es, y
p esen an buenas p opiedades ibológicas (19, 59, 60). Su e osión se p oduce
median e la o mación y p opagación de g ie as la e ales y adiales.
La a iabilidad obse ada en las p opiedades de los ca bu os de silicio biomó icos se
explica en unción del ma e ial p ecu so y el p oceso de ab icación empleado
( empe a u a de p ocesado, iempo de eacción, elación C/Si empleada), ya que es os
de e minan su composición inal, su mic oes uc u a (po osidad, mo ología y
dis ibución de amaño de po o), su aniso opía y su densidad (8, 41).
Los e ec os de cada a iable implicada en su p oceso de elabo ación sob e sus
p opiedades se de allan a con inuación:
Capí ulo 1.2
104
1.2.3.3.1 Ma e ia p ima
La disposición de las células ege ales en la made a, con o mando áqueas o
aqueidas, de e mina la mic oes uc u a del ca bu o de silicio biomó ico (17, 61). La
densidad inal de los bioSiCs que oscila en e 1,1 y 2,6 g/cm3 (62) es ambién
ex emadamen e dependien e de la made a p ecu so a, habiéndose encon ado una
elación lineal en e ambos pa áme os (58):
ρSiC=(2,39∓0,01)ρWood R2= 0,99
Ecuación 1.2.2 Relación en e la densidad de la made a p ecu so a y la densidad de
bioSiC.
El empleo de made as y p ocesados de ele ada densidad (made a de ced o p ensada
de al a densidad o ablones de al a densidad) conduce a mues as con una esis encia a
la comp esión dos eces supe io a las ob enidas a pa i de moldes de baja densidad
(paulonia) (25).
El ca bu o de silicio biomó ico, p esen a la aniso opía del ma e ial de pa ida, es
deci , sus p opiedades son ambién di e en es en di ección axial espec o a las
di ecciones adial o angencial, aunque no de o ma an ma cada como en la made a
o iginal. Es as di e encias son pa icula men e impo an es en lo que se e ie e a las
ca ac e ís icas mecánicas y eléc icas (37, 63).
En las ce ámicas biomó icas, a di e encia de sus made as p ecu so as, la de o mación
en di ección axial no sólo depende de la comp esión axial de las pa edes celula es de la
made a y en la comp esión angencial, la de o mación no es sólo debida a la lexión
plás ica de las pa edes celula es. Los sis emas ce ámicos inales son complejos y su
compo amien o no solamen e es á condicionado po las es uc u as celula es
implicadas (63).

105
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
La esis i idad eléc ica del bioSiC aumen a con el con enido de silicio esidual en
di ección axial, mien as que en la di ección angencial, es e enómeno no se obse a
(64).
El ca ác e aniso ópico mecánico de las mues as de ca bu o de silicio se puede
modula median e la in il ación con mezclas undidas de Al3-Si9-Mg, ob eniendo
ca bu os de silicio compues os, e o zados de aluminio, que p esen an una mejo
esis encia ans e sal a la comp esión y una mayo igidez (16).
1.2.3.3.2 Relación Si/C empleada
La elación inicial en e Si y C es un pa áme o c í ico du an e el p oceso de
p oducción. Cuan o mayo es la can idad de silicio espec o a la de ca bón, más
ele ada es la p opo ción de SiC/C en el p oduc o inal, y po lo an o, la densidad, la
du eza y la esis encia mecánica del ma e ial ob enido (65).
Índices Si/C po debajo de 2,33 dan luga a ce ámicas con po os acíos. La o mación
de c is ales de ca bu o de silicio ob u an los po os de la p e o ma de ca bón y
educen la po osidad. Así, p opo ciones supe io es a 2,33, dan luga a ma e iales con
po os pa cial o comple amen e ob u ados con silicio esidual (11, 66). Cuando la
p opo ción de Si/C es mayo que es, las ce ámicas biomó icas ob enidas es án
o madas po SiC y Si esidual y no p esen an ca bón sin eacciona (26).
La can idad de silicio esidual condiciona, en e o as, las p opiedades eléc icas del
bioSiC, especialmen e a empe a u as en e -268 y 228 ºC. En es e ango, el Si esidual
cons i uye una ed in e conec ada, esponsable de su compo amien o me álico. Es e
e ec o no se obse a a empe a u as supe io es a 228 ºC, en las que el bioSiC se
con ie e en un ma e ial semiconduc o como consecuencia de la mayo con ibución
del ca bu o de silicio (37).
Capí ulo 1.2
106
El exceso de silicio esidual puede se eliminado median e su a amien o con ácidos,
usando una solución de ácido luo híd ico (HF) y ácido ní ico (HNO3) en una elación
mola de 1,66 en agua. Con olada median e un mecanismo de di usión, se lle a a cabo
la eacción es equiomé ica siguien e (67):
3Si + 12HF + 4HNO3 3SiF4 + 4NO + 8H2O
Ecuación 1.2.3 Reacción es equiomé ica de eliminación de silicio median e a amien o
ácido.
El iempo de a amien o ácido de e mina el silicio esidual y la apa ición de po os que
ac úan como núcleos de o mación de g ie as. Tiempos de a amien o ex ensos,
mejo an la in e conec i idad de las mues as (68). Es a eacción ambién depende de la
aniso opía y la po osidad de la mues a, siendo más ápida en la di ección axial y
ob eniéndose di e en es coe icien es de di usión e icaz en unción de la po osidad.
1.2.3.3.3 Tempe a u a de p ocesado
La empe a u a de p ocesado condiciona el p oceso de in il ación de silicio y el ipo
de p oduc o inal ob enido. A empe a u as al ededo de 1.550 ºC se ob iene la o ma
β-SiC, mien as que en e 2.200 y 2.500 ºC se p oducen los poli ipos hexagonales (α-
SiC) (11, 66). Se ha demos ado que los bioSiCs p oducidos a al as empe a u as, son
muy densos y ienen una ele ada esis encia a la de o mación (9).
1.2.3.3.4 Tiempo de eacción
El iempo de eacción con ibuye, al igual que la p opo ción Si/C u ilizada, a la
modulación de la elación en e el SiC y C en los ma e iales inales. A medida que el
iempo se inc emen a, el po cen aje de ca bón esidual disminuye, y pa alelamen e, la
po osidad del ma e ial y su esis encia a la ac u a (26). El iempo de eacción op imo
107
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
depende de la es uc u a po osa de la p e o ma de ca bón, así como del amaño del
ma e ial in il ado y de la empe a u a empleada.
1.2.4 Aplicaciones del ca bu o de silicio biomó ico
Las ce ámicas de ca bu o de silicio biomó ico poseen excelen es p opiedades
mecánicas y de esis encia a la oxidación, la co osión, la empe a u a y el desgas e que
las hacen adecuadas pa a su empleo en aplicaciones de ingenie ía a anzada, así como
pa a el desa ollo de nue os ma e iales con ines biomédicos (56).
1.2.4.1 Sopo es ca alí icos
Debido a su es uc u a po osa, los bioSiCs se han mos ado ú iles como sopo es
ca alí icos de níquel pa a la oxidación pa cial de me ano en la p oducción de gas de
sín esis y ambién pa a la oxidación selec i a de H2S, el a amien o de gases de escape
pa a mo o es de abajo pesado y la deshid ogenación de n-bu ano. Además, su
ecub imien o con zeoli a pe mi e inc emen a la u ilidad pa a es a aplicación, ya que
és a p esen a una ele ada supe icie especí ica que con ibuye posi i amen e a los
p ocesos de adso ción, sepa ación y ca álisis (69).
1.2.4.2 Re ue zo ce ámico en ho migones
El e ue zo de ho migones con pa ículas en o ma de aguja de ca bu o de silicio es una
o ma e ec i a pa a mejo a sus p opiedades mecánicas mos ando, desde un pun o de
is a mecánico, esul ados simila es al e ue zo con ilamen os me álicos pe o con una
mayo es abilidad química as el p oceso de cu ado (56, 70).
1.2.4.3 Aplicaciones de al a empe a u a
Los bioSiCs se pueden emplea en el desa ollo de mo o es a anzados y como
componen es es uc u ales de in e cambiado es de calo (44, 49). Con el in de
Capí ulo 1.2
108
mejo a su conduc i idad é mica se puede ob ene compues os de cob e-ca bu o
silicio, de o ma que se combine, en un único ma e ial, la ele ada conduc i idad é mica
del cob e y el bajo coe icien e de expansión é mica del ca bu o de silicio, ab iendo
nue as posibilidades pa a las aplicaciones de ges ión é mica (54).
La capacidad de las ce ámicas de silicio co alen es (SiC, Si3N4...) pa a o ma una capa
de óxido supe icial (SiO2) con una baja pe meabilidad al oxígeno hace posible la
ob ención de ma e iales cuya esis encia a la oxidación es la más ele ada de en e las
ce ámicas no oxídicas (41, 44, 49).
1.2.4.4 Sis emas de il ación
La mic oes uc u a de los ca bu os de silicio biomó ico los hace adecuados como
ma e iales de il ación pa a la limpieza de gases en calien e en la indus ia de
gene ación de ene gía eléc ica, donde se necesi an como p incipales ca ac e ís icas,
una baja emisión y una ele ada e iciencia. Pa a es as aplicaciones, se han p opues o
como los más adecuados los ob enidos a pa i de ablones de densidad media, con
iempos co os de eacción (71).
1.2.4.5 Aplicaciones biomédicas
El ca bu o de silicio se ha p opues o como ma e ial de ecub imien o de disposi i os
biomédicos (s en s co ona ios, ál ulas) ya que es capaz de inc emen a su
hemocompa ibilidad median e la educción de su ombogenicidad y de la espues a
in lama o ia (72-74). En los úl imos años, el ca bu o de silicio se ha p obado como
ma e ial ú il pa a el diseño y desa ollo de nue os disposi i os biomédicos como
memb anas, agen es de diagnós ico po imagen y biosenso es. Además, es
pa icula men e in e esan e su po encial aplicación como bioma e ial en implan es
o opédicos (59).
115
Ce ámicas bioSiCs como nue os ma e iales pa a egene ación ósea
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o biomo phic SiC ce amics coa ed wi h bioac i e glass. Bol Soc Esp Ce am Vid io.
2006;45(2):109-14.
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84. Bo ajo JP, Gonzalez P, Se a J, Lis e S, Chiussi S, Leon B, e al. Biomo phic silicon
ca bide ce amics coa ed wi h bioac i e glass o medical applica ions. Ma e Sci Fo um.
2006;514-516(P . 2, Ad anced Ma e ials Fo um III):970-4.
85. Bo ajo JP, Se a J, Lis e S, Gonzalez P, Chiussi S, Leon B, e al. Pulsed lase
deposi ion o hyd oxylapa i e hin ilms on biomo phic silicon ca bide ce amics. Appl
Su Sci. 2005;248(1-4):355-9.
86. Gonzalez P, Se a J, Lis e S, Chiussi S, Leon B, Pe ez-Amo M, e al. New
biomo phic SiC ce amics coa ed wi h bioac i e glass o biomedical applica ions.
Bioma e ials. 2003;24(26):4827-32.
87. Rial L, Rodal P, Lopez-Al a ez M, Bo ajo JP, Solla E, Se a J, e al. Bioce amic
coa ings on biomo phic SiC by elec opho e ic deposi ion. Ma e Sci Fo um. 2008;587-
588(Ad anced Ma e ials Fo um IV):86-90.
88. de Ca los A, Bo ajo JP, Se a J, Gonzalez P, Leon B. Beha iou o MG-63
os eoblas -like cells on wood-based biomo phic SiC ce amics coa ed wi h bioac i e
glass. J Ma e Sci Ma e Med. 2006;17(6):523-9.
89. Will J, Hoppe A, Muelle FA, Raya CT, Fe nandez JM, G eil P. Bioac i a ion o
biomo phous silicon ca bide bone implan s. Ac a Bioma e . 2010;6(12):4488-94.
90. Fila do G, Kon E, Tampie i A, Cabezas-Rod iguez R, Di Ma ino A, Fini M, e al.
New bio-ce amiza ion p ocesses applied o ege able hie a chical s uc u es o bone
egene a ion: An expe imen al model in sheep. Tissue Eng, Pa A. 2014;20(3-4):763-
73.
91. A cos D, Valle -Regi M. Bioce amics o d ug deli e y. Ac a Ma e 2013;61(3):890-
911.
Capí ulo 2
Obje i os
131
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA in ec ions
3.1 Abs ac
Implan - ela ed in ec ions a e a se ious complica ion in o hopaedic and den al su ge y
esul ing in p olonged hospi aliza ion, high medical cos s and pa ien mo ali y. The
de elopmen o po ous implan s loaded wi h an ibio ics may enable a local d ug
deli e y o p e en ing su ace coloniza ion and bio ilm o ma ion. A new gene a ion o
bio-de i ed po ous ce amic ma e ial ha mimics hie a chical s uc u es om na u e
was e alua ed. Silicon ca bide ce amics (bioSiCs) de i ed om sapelli wood we e
ob ained by py olysis o En and oph agma cylind icum wood ollowed by in il a ion wi h
mol en silicon. This p ocess ende s disks ha keep he bimodal po e size dis ibu ion
(3 and 85 µm) o he o iginal ma e ial and a e highly cy ocompa ible (BALB/3T3 cell
line). The abili y o he bioce amic o load he an imic obial agen ancomycin was
e alua ed by imme sion o disks in d ug solu ions co e ing a wide ange o
concen a ions. The disks eleased a pH 7.4 an impo an amoun o d ug du ing he
i s 2 h (up o 11 mg/g bioSiC) ollowed by a slowe elease, which is ela ed o he
p esence o mac o and mesopo es. Finally, he an ibio ilm e ec agains me hicillin
esis an S aphylococcus au eus was assessed and a conside able educ ion (92%) o he
bac e ial ilm was obse ed. Resul s highligh he bioSiC po en ial as componen o
medica ed medical de ices.

Capí ulo 3
132
3.2 In oduc ion
A e oughly 100 yea s o clinical use o ce amics in den is y and o hopaedics, he e
is s ill a need o no el bioma e ials. Che alie and G emilla d (1), in hei ecen and
ex ensi e e iew on ce amics o medical applica ions, s a ed he impe a i e need o
ob aining no el, ough and s able ma e ials (wi h special men ion o non oxide ce amics
as silicon ca bide o silicon ni ide) as o hopedic ma e ial candida es. Addi ionally,
hese au ho s poin ed ou he in e es o de eloping hose new ma e ials h ough a
biomime ic app oach. Huebsch and Mooney (2) emphasize ha he e is a conside able
body o esea ch on he impo ance o physical a iables, including opological and
mechanical p ope ies o bioma e ials, in guiding a biological esponse. Some ma e ials
o igina ed om li ing o ganisms, as bones, ha e ou s anding p ope ies due o hei
ino ganic na u e bu also o hei complex s uc u al o ganiza ion. To achie e a
syn he ic ma e ial ha ma ches o bone, one has o ake ca e o bo h hese aspec s.
Bones a e made up o a collec ion o ma e ials buil ou o a common basic building
block, he mine alized collagen ib il, ha can be a anged in di e en pa e ns. All
o ms o bone possess mechanical s eng h and oughness ou o each om i s
cons i uen ma e ials (3). Nume ous a emp s ha e been made o mimic he s uc u e
o bone (4–6). Highly po ous calcium phospha e ce amic sca olds o o ganic–ino ganic
composi es a e well known examples ha ha e been used success ully (7), bu none o
hem simul aneously combined he mic oscopic and mac oscopic s uc u e o he
bone. Mimicking bone s uc u e con inues o be a challenging ask.
Bio-de i ed silicon ca bide based ce amics (bioSiC), ob ained by Si-mel in il a ion o
ca bonaceous sca olds de i ed om wood empla es ha e been p oposed as new
enginee ing ce amic ma e ials wi h po en ial use in biomedical applica ions (8, 9). As a
esul o he e olu ion, wood combines a good balance be ween esis ance/weigh a io
and luids ci cula ion. BioSiC a e non oxide ce amics ha keep he complex na u al
133
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA in ec ions
s uc u al o ganiza ion o wood, esembling o a ce ain ex en ha o bones.
Biocompa ible bioSiC can be p oduced wi h low cos , nea ne - shape and adequa e
mechanical p ope ies (8–10). The possibili y o a ying he wood cellulosic p e o ms
and/o he p ocess a iables makes he bioSiC app oach ex emely e sa ile and allows
o ailo mic os uc u es esul ing in ma e ials wi h di e en densi ies, mo phologies,
po e size dis ibu ions, le els o aniso opy, mechanical s eng h, e c. (8) and, hus,
po en ially use ul as candida es o he de elopmen o o hopedic and den al implan s.
Bone in ec ions a e ypically caused by bac e ia in oduced om auma, su ge y,
implan use, o by di ec coloniza ion om a p oximal in ec ion o ia sys emic
ci cula ion. Pos ope a i e os eomyeli is is s ill an impo an p oblem in o hopedic and
den al su ge y (11). The bac e ial bio ilm, ex emely esis an o bo h he immune
sys em and an ibio ics, is conside ed he p ima y cause o implan -associa ed in ec ion.
Du ing he i s 6 h a e su ge y, an implan is pa icula ly suscep ible o su ace
coloniza ion and bio ilm o ma ion (11). E iciency o he sys emic ea men o
os eomyeli is is limi ed by he di icul access o he an ibio ic o he in ec ion si e (12,
13). The use o implan s loaded wi h an imic obial agen s is a p omising app oach o
p e en pos -ope a i e in ec ions (14). The e ec i eness o he an ibio ic/de ice
combina ions is s ongly dependen on he mechanism and he a e o d ug elease
(15). Sub-inhibi o y d ug concen a ions (i.e., hose below he minimal inhibi o y
concen a ion) does no p e en he o ma ion o a mic obial bio ilm and may e en
exace ba e complica ions o induce esis ance in wound-si e bac e ia. An ini ial ‘‘bu s ’’
elease om he de ice may su icien ly educe he likelihood o he p ima y bio ilm
in ec ion and, as a consequence, imp o e he p ophylaxis agains in ec ion and speed-up
he pa ien s eco e y. Recen s udies ha e demons a ed ha mic opo ous ma e ials
may be pa icula ly use ul as d ug-elu ing implan s. Po ous hyd oxyapa i e enables
mo e abso p ion and longe an ibac e ial ac i i y (up o 2 days) in i o han dense
Capí ulo 3
134
hyd oxyapa i e (only 12 h) (16). On he o he hand, mesopo ous silica wi h a highly
egula nano-po ous s uc u e and a as su ace a ea p o ided con olled elease and
an excellen p o ec ion o he loaded gues molecules (17).
The aim o he p esen wo k was o e alua e he po en ial o a bioSiC om sapelli
wood o he loading and he elease o ancomycin in o de o p e en local bac e ial
in ec ions, including bio ilms o med by me hicillin esis an S aphylococcus au eus
(MRSA). To he bes o ou knowledge he sui abili y o bioSiC as d ug deli e y sys em
has no been e alua ed ye . Vancomycin is a highly soluble (>100 mg/mL) an imic obial
agen , which is usually adminis e ed sys emically a e bone su ge y o p e en bac e ial
in ec ion because o i s b oad spec um and pa icula e iciency agains s aphylococci
(18, 19). Fi s , he po osi y and opog aphy o he bioSiC was cha ac e ized in de ail in
o de o con i m he mimicking o he sma hie a chical s uc u e o he ee
empla e. Cy ocompa ibili y and cell conduc i e p ope ies we e hen es ed. Finally,
he abili y o bioSiC o load ancomycin and o p e en he bio ilm o ma ion was
e alua ed and ela ed o he pa icula hie a chical s uc u e o he disks.
3.3 Ma e ials and me hods
3.3.1 Bio-inspi ed silicon ca bide
Pieces o bioSiC we e ob ained om ans e sal cu s o sapelli wood (En and oph agma
cylind icum). The bioce amiza ion p ocess consis ed o d ying he wood a 60 ºC o 24
h, a e which pieces we e subjec ed o a py olysis s ep up o 800 ºC in an ine
a mosphe e wi h well con olled hea ing and cooling amps. Finally, he ca bon
pe o m ob ained was in il a ed wi h mol en silicon a 1,550 ºC in acuum o 30 min
(9). The inal ma e ial was cu o ob ain disks o Ø 6 mm x 2 mm.
135
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA in ec ions
3.3.2 BioSiC cha ac e iza ion
The mic os uc u e and opog aphy o bioSiC disks we e e alua ed by Scanning
Elec on Mic oscopy (SEM Philips XL 30), In e e ome ic P o ilome y (WYKO NT-
1100) and Con ocal Lase Scanning Mic oscopy (CLSM Bio-Rad MRC 1024). The
ma e ial densi y was de e mined, by iplica e, using a helium-ai pycnome e
(Quan ac ome Mod. PY2, USA). The po e size dis ibu ion was e alua ed by me cu y
in usion po osime y using a Mic ome i ics Au opo e IV 9500 (No c oss, GA, USA)
i ed wi h a 3 mL pene ome e o solids. The wo king p essu es co e ed he ange
0.6–2.5 x 104 psi. The speci ic su ace a ea was e alua ed by he B unaue –Emme –
Telle (BET) me hod (20) which in ol ed he de e mina ion o he amoun o he
adso p i e gas (N2 in his case) equi ed o co e he ex e nal and he accessible
in e nal po e su ace o he ma e ial wi h a comple e monolaye . The disks we e
degassed by hea ing a 60 ºC and 10-3 mmHg. Then, samples we e exposed o N2 gas a
77 K and 0.01–0.98 ela i e p essu e on an au oma ic su ace a ea analyze
(Mic ome i ics ASAP 2000, USA). The BET su ace a ea (SBET) was calcula ed om he
iso he ms acco ding o he BET equa ion: SBET (m2/g) = 4.37 Vm (cm3/g), whe e Vm is
he olume o ni ogen necessa y o o m he monolaye .
3.3.3 Cell iabili y es
The in i o cy ocompa ibili y o bioSiC disks was es ed, in iplica e, by using a
BALB/3T3 cell line (CCL 163, ATCC, USA), acco ding o he 10993-5 p o ocol o he
In e na ional S anda diza ion O ganiza ion (ISO). BioSiC disks we e placed in 24-well
pla es. Then a cell suspension o 200,000 cells/well in 2 mL o DMEM (GIBCO®),
supplemen ed wi h 10% e al bo ine se um (FBS) and 1% gen amicin, was added in o
he wells and he pla e was incuba ed a 37 ºC o 24 h in 5% o CO2 and 90% o
ela i e humidi y en i onmen . A con ol (cells wi hou bioSiC disk) was ea ed in he
same way. The bioSiC samples we e collec ed and dyed in o de o assess li e/ dead
Capí ulo 3
136
popula ions by means o a calcein/p opidium iodide s aining using con ocal mic oscopy
(Con ocal Spec al Mic oscopy Leica TCS-SP2 LEICA, We zla , Ge many). To calcula e
hei a io ( iabili y), li e and dead popula ions we e coun ed using a ligh mic oscope
(Op ipho 2, Nikon, Japan) wi h g een and ed il e s and an Image Analysis so wa e
(So Imaging Sys em GmbH, Ve sion 3.2 Build 0.607). Cells emaining adhe ed o he
issue cul u e polys y ene (TCP) well we e ypsinized and cen i uged. The pelle was
esuspended wi h cell cul u e medium, cy ospinned on o a glass slide, and dyed wi h
calcein and p opidium iodide.
3.3.4 Vancomycin loading
The high solubili y in wa e o ancomycin enabled he loading o his an imic obial
agen in o bioSiC disks by a simple imme sion in 3 mL o d ug aqueous solu ions wi h
concen a ions anging om 0.05 o 42.5 mg/mL. The disks we e le in he d ug
solu ion o 24 h wi h mechanical shaking. Vacuum (75 mmHg) was applied o he i s
2 h o elimina e he ai om he po es o he pieces and o p omo e he lux o d ug
solu ion in o he disks. The amoun o ancomycin loaded in each disk was calcula ed
as he di e ence be ween he ini ial and he inal concen a ions in he su ounding
solu ion, de e mined by UV spec opho ome y a 280 nm (Agilen 8453, Böblingen,
Ge many). All he expe imen s we e ca ied ou in duplica e. D ug-loaded disks we e
desicca ed a 40 ºC un il cons an weigh . BioSiC ma e ial loaded wi h ancomycin was
e alua ed as i was indica ed abo e.
3.3.5 Vancomycin elease
D ied d ug-loaded disks we e ans e ed o ials con aining 3 mL o phospha e bu e
(PBS) pH 7.4 a 37 ºC and kep unde mechanical shaking. Samples o he elease
medium we e wi hd awn a egula in e als and e u ned o he ial immedia ely a e
hei d ug concen a ion was measu ed spec opho ome ically a 280 nm. Wa e

137
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA in ec ions
up ake by he disks was moni o ed in pa allel o he elease es s by placing o d ied
disks in a Gay-Lussac pycnome e (A o a, Spain) and weighing he pycnome e a
di e en imes a e illing wi h wa e .
3.3.6 MRSA bio ilm o ma ion
BioSiC ma e ial and ancomycin-loaded bioSiC disks, p epa ed by imme sion in 42.5
mg/mL o d ug solu ion du ing 24 h, we e subjec ed o his mic obiological s udy by
using a clinical MRSA isola e. This bac e ial isola e ( eco e ed om a pa ien a he
Ghen Uni e si y Hospi al, Ghen , Belgium) was g own on T yp ic Soy Aga (TSA)
(Oxoid, D ongen, Belgium) a 37 ºC. MRSA bio ilms we e o med in wo di e en
model sys ems. Fi s , MRSA bio ilms we e o med in he Modi ied Robbins De ices
(MRD), as desc ibed p e iously (21). In his sys em, g ow h medium is con inuously
eplaced. Secondly, bio ilms we e o med on d ug loaded-bioSiC disks using 24-well
mic o i e pla es (MTP, T asadingen, Swi ze land). To his end, disks we e placed in 1
mL MRSA suspensions wi h a densi y o app . 106 CFU/mL (in 1:5 dilu ed T yp ic Soy
B o h, TSB) o 1 h. Subsequen ly, disks we e gen ly insed wi h 0.9% (w/ ) NaCl o
emo e non-adhe en cells and we e placed in 1 mL dilu ed TSB o an addi ional 24 h
a 37 ºC. To quan i y he bio ilm o ma ion in bo h me hods he MRD and MTP, each
disk was ans e ed o es ubes wi h 10 mL 0.9% ( /w) NaCl and he ubes we e
subjec ed h ee imes o 30 s o sonica ion (B anson 3510, 42 kHz, 100 W, B anson
Ul asonics Co p., Danbu y, USA) and 30 s o o ex mixing o de ach he bio ilm om
he disks. Using his p ocedu e all cells we e emo ed om he disks and clumps o
cells we e b oken apa . Sessile S. au eus cells we e pla ed on TSA, and incuba ed a
37 ºC o 48 h. Finally, he numbe o colony o ming uni s (CFU) pe disk was
calcula ed by coun ing colonies on he pla es. All expe imen s we e ca ied ou on a
leas 3 disks o each composi ion. The S uden es was used o e alua e he e icacy
in educing cell colonies and he di e ences be ween MRD and MTP me hods.
Capí ulo 3
138
3.4 Resul s
P ocessing a iables du ing bioSiC p oduc ion may a ec he in e connec ed
mic os uc u e o he wood empla e. Mo phological cha ac e iza ion o bioSiC was
ca ied ou by SEM mic og aphs (Figu e 3.1) ha e idenced he pa icula po ous
mic os uc u e o he sapelli ee. Mac opo es (≈ 80 µm) in g oups o wo o h ee,
cha ac e is ic o he sapelli ee (A), can be seen a he su ace o he c oss sec ion o
bioSiC. Mo e in de ail (B) a second popula ion o mesopo es (less han 10 µm) a he
su ace o he ma e ial can be no iced, wi h he silicon ca bide c ys als making he
s uc u e up (C). The walls o he essels we e main ained a e in il a ion gi ing a
ce amic ma e ial wi h po es unidi ec ional connec ed, as i can be no ed a he
longi udinal sec ion (Figu e 3.2) cha ac e ized by in e e ome ic p o ilome y (A) and
con ocal lase scanning mic oscopy (B).
Figu e 3.1 SEM mic og aphs (c oss-sec ion) o bioSiC ce amics p oduced om Sapelli
wood. Th ee magni ica ions a e shown: (A; X100), (B; X500) and (C; X4,000).
Mic os uc u e was also con i med om me cu y in usion po osime y measu emen s
(Figu e 3.3). Resul s e idence a o al in usion olume o 0.243 ± 0.005 mL/g and a o al
po osi y o 41.49 ± 0.05% wi h a bimodal po e size dis ibu ion (Figu e 3.3) including
mac opo es (mean diame e ≈ 85 µm) and mesopo es (mean diame e ≈ 3 µm).
Quan i a i e di e ences in he mic os uc u al p ope ies be ween ancomycin loaded
139
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA in ec ions
and unloaded bioSiC disks could no be es ablished using me cu y in usion
po osime y.
Figu e 3.2 Topog aphic cha ac e is ics o a bioSiC ce amic (longi udinal sec ion) by
in e e ome ic p o ilome y (A) and con ocal lase scanning mic oscopy (B).
The ni ogen adso p ion analysis con i med he absence o mic opo osi y and allowed
he es ima ion o speci ic su ace (1.198 ± 0.005 m2/g).
The abili y o bioSiC disks o sus ain cell a achmen and g ow h was assessed by an in
i o biocompa ibili y es ollowing ISO 10993-5 p ocedu e. The assays we e
conduc ed using Balb cell line. A e 24 h in con ac , a homogeneous well dis ibu ed
laye o li ing cells and a signi ican ly smalle amoun o dead cells on he op o he
bioSiC disks can be obse ed (Figu e 3.4). The a io be ween li ing and dead cells was
used o calcula e he iabili y o cells on he disk. The ob ained alues we e 79.9% (sd
13.1) on bioSiC disks and 98.0% (sd 1.9) on he TCP om he wells. Vancomycin
loaded bioSiC we e also es ed o assu e in i o biocompa ibili y achie ing a pe cen age
o cell iabili y o 95.8% (sd 6.0).
Capí ulo 3
140
Figu e 3.3 Po e size dis ibu ion and pe cen age o po osi y o bioSiC de i ed om
sapelli wood.
Figu e 3.5 shows he adso p ion cu e o ancomycin on bioSiC ma e ial which ela es
he concen a ion o he solu e on he adso ben (mg ancomycin pe g am o d ied
ma e ial) o he concen a ion o he solu e in he su ounding solu ion a he
equilib ium. The shape o he adso p ion cu e can be classi ied as Class S acco ding o
Giles and cowo ke classi ica ion (22). A he highes loading concen a ion s udied (6
mg/mL) a signi ican amoun o ancomycin (nea ly 50 mg/g o bioSiC) was loaded.
Figu e 3.4 Calcein-p opidium iodide s aining obse ed by con ocal mic oscopy 24 h
a e seeding on bioSiC samples. Li ing cells in g een and dead cells in ed.
Capí ulo 7
Algina e-poloxame -silicon ca bide composi es o he con olled
elease o indome hacin dec ease in i o in lamma ion

245
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
7.1 Abs ac
Composi es o biomo phic silicon ca bides (bioSiCs) and hyd ogels a e p oposed in
o de o ob ain ma e ials able o load and elease poo soluble d ugs wi h applica ion
in bone pa hologies he apy. Hyd ogels composed by algina e and poloxame we e
loaded wi h indome hacin, inco po a ed in o he ce amics and c osslinked. The
indome hacin elease p o ile is dependen on he mic os uc u e o he bioSiC
selec ed. The loaded oak and sapelli bioSiCs composi es ha e adequa e elease p o iles
o p omo e he dec easing o he sec e ion o p o-in lamma o y cy okines in LPS
s imula ed mac ophages, showing s onge an i-in lamma o y e ec s han pine bioSiC
composi es. The eleased indome hacin is able o modula e he deg ada ion o
chond ocy es ex acellula ma ix and p omo e he o ma ion o new collagen.
Pa icles de i ed om mechanical wea o biomo phic silicon ca bides do no show
high oxici y, being simila o he zi conia pa icles.
Capí ulo 7
246
7.2 In oduc ion
Bone is a complex hie a chically o ganized issue o med by an o ganic ma ix, mainly
collagen which is sequen ial mine alized wi h an ino ganic componen , hyd oxyapa i e
(HAp), by he ac ion o speci ic cells (1, 2). Due o his complex s uc u e he
de elopmen o new bone biomime ic ma e ials has led o he p oduc ion o new
po ous 3D composi e sys ems o med by he combina ion o one o mo e han one
o ganic componen (na u al o syn he ic polyme s) wi h an ino ganic componen .
Composi e sys ems should be able o mee all he physical and biological equi emen s
o bone egene a ion, combining he ad an ages o all componen s (3).
These composi e sys ems ha e been ound o be adequa e also o he elease o
g ow h ac o s as BMP-2 (4-6), VEGF (7), pla ele de i ed g ow h ac o s (8, 9) o
d ugs such as dexame hasone (10, 11), ancomycin (12, 13) and gen amicin (14). In he
las ew yea s, hey ha e been also p oposed o eleasing a combina ion o he apeu ic
molecules such as BMP-2 and ancomycin (15), amikacin and gen amicin (16) o BMP-2
and VEGF (17). This new app oach makes hem an a ac i e al e na i e o he
ea men o se e al bone pa hologies.
Biomo phic silicon ca bide ce amics (bioSiCs) ob ained om na u al esou ces (18)
ha e been shown o main ain he o iginal s uc u e o hei p ecu so s being highly
po ous and biocompa ible ma e ials (19) sui able o he egene a ion and
e ascula iza ion o issues. The inco po a ion o a polyme componen in o hei
s uc u e mus inc ease hei he apeu ic alue. The use o ionic c osslinking polyme s,
such as algina e makes i possible o easily ob ain a h ee dimensional ne wo k polyme
h ough he addi ion o di alen ions (20). Algina e hyd ogels can exe he unc ion o
an o ganic ma ix sui able o cellula g ow h and encapsula ion wi hin he ce amic
sys em and also acili a e he inco po a ion o d ugs and g ow h ac o s in o i s h ee
dimensional s uc u e while modula ing hei elease. The e o e, he combina ion o
247
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
algina e hyd ogels and bioSiCs can be p esen ed as a p omising s a egy in he
de elopmen o complex sys ems o issue egene a ion and con olled d ug elease.
The addi ion o syn he ic block copolyme s such as poloxame s o poloxamines o he
hyd ogel componen should be able o inc ease he solubili y o low aqueous solubili y
d ugs. The abili y o hese polyme s o o m micelles in an aqueous solu ion, whe e
hyd ophobic d ugs can be inco po a ed, leads o an inc ease on hei solubili y and
he e o e makes hei adminis a ion possible (21, 22). Fu he mo e, i was obse ed
han poloxamines we e able o s imula e he os eoblas ic di e en ia ion o adipose
de i ed mesenchymal s em cells by hemsel es (23). The use o poloxame s has also
been ound o inhibi P-glycop o ein unc ion dec easing he esis ance o mul id ug
esis an (MDR) cell lines (24, 25).
Cu en ly, he long- e m s abili y o he p os hesis con inues o be a challenge in he
de elopmen o bone subs i u es. Ma e ial wea deb is is one o he main d awbacks
associa ed o he use o a i icial ma e ials as implan able sys ems. The p oduc ion o
ma e ial pa icles may cause asep ic loosening and he ac i a ion o he su ounding
mac ophages leading o bone des uc ion and pe ip os he ic os eolysis (26-28). Se e al
pa ame e s modula e he in lamma o y eac ion caused by ma e ial pa icles such as
hei size (29) o hei chemical composi ion (30, 31). Despi e he ac ha biomo phic
silicon ca bide has been p e iously desc ibed as a highly biocompa ible ma e ial he
in lamma o y eac ions caused by i s po en ial deb is ha e no been documen ed.
The aim o he p esen wo k is o de elop composi es able o load by en apmen and
elease an an i-in lamma o y d ug, indome hacin. The de elopmen o composi e
sys ems o med by a na u al (algina e) and a syn he ic polyme (poloxame ) oge he
wi h he biomo phic ce amic (silicon ca bide) should allow us o ob ain an implan
ma e ial sui able o load and elease, in a con olled way, he indome hacin wi h an
Capí ulo 7
248
amoun enough o show an adequa e an i-in lamma o y e ec on os eoa h i ic
chond ocy es.
Addi ionally, he po en ial oxic e ec s o pa icles ob ained by mechanical wea o
biomo phic silicon ca bides a e e alua ed.
7.3 Ma e ials and me hods
7.3.1 P epa a ion o composi e sys ems
Th ee di e en ypes o biomo phic silicon ca bide (bioSiC) samples we e ob ained as
desc ibed elsewhe e om oak (Que cus obu ), pine (Pinus pinnas e ) and sapelli
(En hand oph agma cylind icum). Disks o 6 mm in diame e we e s e ilized by
au ocla ing a 121 ºC o 20 min (32).
Two polyme ic componen s we e used; Poloxame 407 (Plu onic F127® (PF127)) ha
was kindly dona ed om BASF (Ludwigsha en, Ge many) and sodium algina e
(GRINDSTED® Algina e PH 155) was pu chased om Danisco (Copenhagen,
Denma k).
Plu onic was dissol ed in phospha e bu e (PBS) o achie e a inal concen a ion o
2.5%. A e i s comple e dissolu ion, indome hacin (2.38 mg/mL) and algina e (2%) we e
sequen ially added. The inal solu ion was au ocla ed a 121 ºC o 20 min.
30 µL o he polyme ic solu ion was added on he bioSiC samples and c osslinked by
he imme sion o he loaded sample in o a s e ile solu ion o calcium chlo ide (Pan eac;
Ba celona, Spain) a a concen a ion o 20% o 10 seconds. A e he c osslinking,
sys ems we e washed wice wi h 2 mL o PBS o en seconds.
C osslinked algina e-poloxame beads p epa ed by d opping he polyme ic solu ion
in o he calcium chlo ide solu ion we e used as con ol.

249
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
7.3.2 Cha ac e iza ion o he polyme ic componen
The mechanical s abili y o he c osslinked and unc osslinked polyme ic sys ems was
analyzed be o e and a e he au ocla ing p ocess using a con olled s ess heome e
(Rheolys AR-1000N TA ins umen s, Su ey, UK). Ramps o empe a u e om 15 °C
o 60 °C a 2 °C/min wi h an oscilla o y s ess o 0.1 Pa a 5 ad/s we e ca ied ou o
all he samples.
The di e en ial scanning calo ime ic (DSC Q200, TA ins umen s, Su ey, UK) was
used o e alua e he po en ial deg ada ion o he polyme ic chains du ing he
au ocla ing p ocedu e. Ramps o empe a u e we e ca ied ou i s om oom
empe a u e o -30 °C a 10 °C/min and hen om his empe a u e o 50 °C a 10
°C/min.
7.3.3 Isola ion o human os eoa h i ic chond ocy es
Human os eoa h i ic ca ilages we e p o ided by he Ins i u o de O opedia y Banco
de Tejidos Musculoesquelé icos o he Uni e si y o San iago de Compos ela. Pieces o
he issue we e cu and placed in o s e ile ubes wi h ypsin and cell cul u e medium.
The issue was main ained in he solu ion a 37 ºC o 30 min in o de o kill he
ib oblas s p esen in he ex ac s. Then, issue samples we e imme sed in collagenase
1.5% in cul u e medium and kep o e nigh a 37 ºC unde mechanical s i ing.
The solu ions we e cen i uged a 1,056 g o 4 min. Cells we e esuspended in DMEM
supplemen ed wi h 10% o e al bo ine se um and 1% penicillin/s ep omycin and
cul u ed a 37 ºC wi h 5% o CO2 and 90% o ela i e humidi y.
Capí ulo 7
250
7.3.4 In i o elease o indome hacin
Loaded composi e sys ems we e imme sed in 3 mL o phospha e bu e a 37 ºC. A
p ese imes he concen a ion o indome hacin was quan i ied by UV- isible
spec opho ome y a 320 nm. All he expe imen s we e ca ied ou in iplica e.
7.3.5 An i-in lamma o y e ec o indome hacin loaded composi es
The an i-in lamma o y e ec s o loaded indome hacin composi es we e e alua ed in
wo cell ypes, ex ac ed human os eoa h i ic chond ocy es and a mu ine mac ophage
cell line (Raw 264.7).
The mac ophage cell line was cul u ed in DMEM-F12 HAM supplemen ed wi h 10%
FBS and 1% penicillin/s ep omycin and main ained a 37 ºC wi h 5% o CO2 and 90%
ela i e humidi y. Loaded composi e sys ems we e placed in 24-well pla es, cul u ed
wi h 100,000 cells pe well and s imula ed wi h lipopolysaccha ide (LPS) a a
concen a ion o 100 ng/mL.
The composi es an i-in lamma o y e ec was e alua ed a e 24 and 72 hou s o cul u e
by he quan i ica ion o p os aglandin E2 (PGE2) (A bo ), TNF-α (eBioScience), ni ic
oxide (Cayman) and IL-1α (eBioScience).
The composi es cy o oxici y was analyzed by he quan i ica ion o lac a e
dehyd ogenase (LDH) (Roche). Indome hacin a 100 µM, unloaded silicon ca bide
samples, and he polyme ic solu ion we e used as con ols.
Composi es we e also cul u ed wi h os eoa h i ic chond ocy es in 24-well pla es a a
densi y o 60,000 cells pe well wi h 2 mL o supplemen ed DMEM. The e ec on
ex acellula ma ix syn hesis o os eoa h i ic chond ocy es was e alua ed by he
quan i ica ion o glycosaminoglycans (GAGs) and collagen (I-V) p oduc ion a e 15
days o cul u e.
251
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
Cell cul u e supe na an s we e cen i uged a 10,000 g o 10 min. Two colo ime ic
assays we e used, Blyscan (Biocolo ) o GAGs and Si col Collagen Assay ki (Biocolo )
o collagen. Single cell cul u e medium was used as nega i e con ol and cells ea ed
wi h indome hacin a a concen a ion o 100 µM equi alen o 100% o d ug elease
we e used as a posi i e con ol. Unloaded bioSiCs and c osslinked loaded and
unloaded polyme ic componen s we e also used as con ols.
Addi ionally, he concen a ion o in lamma o y cy okine (IL-1β) and he sec e ed PGE2
we e measu ed a 1, 2, 5 and 15 days h ough an ELISA assay (Bende MedSys em) and
a comme cial colo ime ic assay (A bo ) espec i ely.
7.3.6 E alua ion o in lamma ion caused by silicon ca bide eleased pa icles
Pa icles we e ob ained by di ec mechanical ic ion o wo oak silicon ca bide
samples. One o he samples was immobilized on he su ace o a s e ile plas ic
con aine whe eas he o he was ixed on an impelle a ached o a o o (Ika
RW20DZM) o a ing a 150 pm o one week. Ul apu e wa e (milliQ) was added o
he con aine in o de o acili a e he eco e y o he pa icles. Pa icle size was
analyzed by Scanning Elec on Mic oscopy (ZEISS EVO LS 15, Ge many) and hei
composi ion by EDX using he same equipmen . Pa icles size dis ibu ion was analyzed
using a ze asize (Ze asize Nano ZSP, Mal e n, Uni ed Kingdom).
The in lamma ion caused by he pa icles was e alua ed using a mac ophage cell line
(Raw 264.7) and cul u ed as p e iously desc ibed. Cells we e seeded in 24-well pla es
wi h 1 mL o cell cul u e medium and pa icles we e 10- old dilu ed and added o he
wells. Cells s imula ed wi h 1 µg/mL o LPS we e used as a posi i e con ol o
in lamma ion and comme cial Zi conium (IV) oxide pa icles as e e ence ma e ial
(Sigma, USA).
Capí ulo 7
252
In lamma o y cy okines we e quan i ied a e 24 hou s o cul u e, IL-1α and TNF-α
(Bende MedSys ems).
7.3.7 S a is ical Analysis
Expe imen al esul s we e exp essed as means and s anda d de ia ions. The s a is ical
signi ican di e ences be ween ea men s we e es ablished by he analysis o a iance
(ANOVA). When he F- a io sugges s signi ican di e ences be ween g oups, he leas
signi ican di e ence (LSD) es was used o compa e hem in pai s. ANOVA and LSD
we e pe o med by S a g aphics Cen u ion®X64 so wa e (USA).
7.4 Resul s and discussion
7.4.1 Cha ac e iza ion o he polyme ic componen
A s ong in e ac ion could be obse ed be ween algina e and poloxame , his
in e ac ion is modi ied by he au ocla ing p ocess. Figu e 7.1 shows he dynamic and
elas ic moduli o bo h, Alg and Alg-PF127 o mula ions be o e (Figu e 7.1A) and a e
(Figu e 7.1B) he au ocla ing p ocess. The addi ion o poloxame o algina e solu ion
caused an inc ease in bo h moduli and consequen ly in he complex iscosi y a 37 ºC
( om 1.94 o 3.12 Pa.s) (Figu e 7.1A). The s e iliza ion p ocess led o a ma kedly
dec ease in he complex iscosi y alues a 37 ºC, being 0.06 Pa.s and 0.04 Pa.s o he
Alg and Alg-PF127 o mula ions espec i ely. A e his p ocess he elas ic modulus
could no be de ec ed. In e es ingly, whe eas in uns e ilized sys ems he inco po a ion
o poloxame inc eased he complex iscosi y, a e au ocla ing hese seem o ha e he
opposi e e ec . Complex in e ac ions we e es ablished be ween algina e chains and
poloxame micelles in he uns e ilized sys ems. Polysaccha ide chains could be placed
a ound he micelles hampe ing he in e ac ion be ween poloxame and wa e and
inc easing micelle-micelle in e ac ions and he e o e, he iscosi y o he sys em (33). In
he same way, Lin and cowo ke s ound ha he addi ion o algina e o poloxame
259
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
The con olled elease o indome hacin by he composi e sys ems, pa icula ly hose
p oduced using oak and sapelli bioSiC, p omo e he highes an i-in lamma o y e ec
acco ding o all he analyzed cy okines.
Figu e 7.5 Le els o NO and PGE2 sec e ed by LPS s imula ed mac ophages a e 24
and 72 o cell cul u e. (C-) is mac ophages no s imula ed and (C+) is mac ophages
s imula ed wi h LPS wi hou any ea men . The homogeneous g oups a e indica ed by
an equal numbe o as e isks (*) abo e he columns (α < 0.05).
Os eoa h i is (OA) is a non-in lamma o y join disease and he mos common o m o
a h i is. I is cha ac e ized by he loss o ca ilage om he a icula ing su aces,
os ophy e o ma ion, changes in he syno ial memb ane, subchond al bone scle osis
and an inc eased olume o syno ial luid wi h less iscosi y and poo lub ica ion
p ope ies (40, 41). I s cu en ea men is symp oma ic, including in a-a icula
injec ions o glucoco icoids and hyalu onic acid o mula ions o he adminis a ion o
non-s e oidal an i-in lamma o y d ugs (NSAIDs) (40, 42). I is also well known ha an
imbalance be ween i s me abolism and deg ada i e signals is p esen in os eoa h i ic
ca ilage. Os eoa h i ic chond ocy es inc ease he sec e ion o in lamma o y cy okines
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e+ind Ind C-
C+
0
50
100
150
200
250
300
350
400
**
*
*
***
PGE2 (pg/mL)
24h
72h
*
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e
PF127+algina e+ind Ind C- C+
0
10
20
30
40
50
60
70
80
90
100
110
120
NO (mM)
24h
72h
*
A
A
B

Capí ulo 7
260
and dec ease he collagen syn hesis (43). I has been epo ed ha he sec e ion o
p os aglandin E2 is enhanced in os eoa h i ic ca ilage, syno ium and syno ial luid (38).
Cell iabili y and he sec e ion o p oin lamma o y cy okines a e 15 days o cell
cul u e ha e been analyzed o os eoa h i ic chond ocy es ea ed wi h he di e en
o mula ions.
Cell iabili y esul s, all highe han 60%, (Figu e 7.6), show no s a is ically signi ican
di e ences be ween indome hacin alone and d ug loaded sys ems. The addi ion o he
d ug o he cell cul u e medium, shows a sligh oxici y and p omo es a dec ease in cell
iabili y.
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
Ind
0
20
40
60
80
100
120
cell iabili y (%)
15 days
Figu e 7.6 Cell iabili y alues ob ained by MTT assay a e 15 days o cell cul u e
co ec ed by he nega i e con ol (cul u e pla es). No s a is ical signi ican di e ences
we e obse ed (α < 0.05).
The le els o NO and PGE2 sec e ed by os eoa h i ic chond ocy es a e 48 hou o
ea men wi h he di e en o mula ions (Figu e 7.7) poin ou ha loaded composi es
p omo e a signi ican dec ease in cy okines p oduc ion in compa ison o un ea ed
chond ocy es (C-).
261
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
Figu e 7.7 Le els o NO and PGE2 sec e ed by os eoa h i ic chond ocy es a e 48
hou s o cell cul u e. The nega i e con ol (C-) is os eoa h i ic chond ocy es wi hou
ea men . The homogeneous g oups a e indica ed by an equal numbe o as e isks (*)
abo e he columns (α < 0.05).
The sec e ion o IL-1β induces he ca abolic cascade o os eoa h i ic chond ocy es
including he ac i i y o cyclooxygenase enzymes and, he e o e, he p oduc ion o
PGE2, he main p o-in lamma o y ac o (38). The Le els o IL-1β we e unde ec able
o all he ea men s s udied.
In o de o e alua e he ex acellula ma ix ca abolism and me abolism, he
concen a ion o he main s uc u al mac omolecules, glycosaminoglycans (GAGs) and
collagen I-V, in he cell cul u e medium a e i een days o assay ha e been quan i ied.
Figu e 7.8 shows he no s a is ically signi ican di e ences be ween ma e ials wi h
ega d o he amoun o eleased GAGs and syn he ized collagen o bo h loaded and
unloaded composi es. The elease o indome hacin om loaded composi es p omo es
a signi ican dec ease in GAGs oge he wi h a signi ican inc ease in collagen
p oduc ion om he os eoa h i ic chond ocy es which can be explained by he
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e+ind C-
Ind
0
50
100
150
200
250
300
350
400
48h
PGE2 (pg/mL)
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e+ind C-
Ind
0
10
20
30
40
50
60 *
*
NO (mM)
48h
*
A
B
Capí ulo 7
262
dec easing o hei anabolic ac i i y, leading o he syn hesis o new collagen molecules
and o he educ ion o he diges ion o he ex acellula ma ix.
Figu e 7.8 Le els o GAGs (A) and collagen I-V (B) sec e ed by os eoa h i ic
chond ocy es a e 15 days o cell cul u e. The nega i e con ol (C-) is os eoa h i ic
chond ocy es wi hou any ea men .
7.4.4 E alua ion o in lamma ion caused by silicon ca bide eleased pa icles
Despi e he excellen ibological p ope ies o silicon ca bides and hei high
co osion esis ance unde no mal biological condi ions (26), i is ele an o e alua e
he in lamma ion and mac ophage ac i a ion caused by hei mechanical wea deb is
a e hei hypo he ical in i o implan a ion. The simula ion o mechanical wea be ween
wo silicon ca bide samples allowed he ob aining o small pa icles cha ac e ized by a
mean diame e o 481.33 nm o (Figu e 7.9). The analysis o pa icle composi ion show
ha hey we e mainly composed by silicon and ca bon a oms acco ding o EDX
measu emen s.
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e+Ind
c-
c+
0.00
0.05
0.10
0.15
0.20
0.25
0.30
GAGs (mg/mL)
15 days
loaded pine SiC
loaded sapelli SiC
loaded oak SiC
pine SiC
sapelli SiC
oak SiC
PF127+algina e+Ind
c-
Ind
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Collagen I-V (mg/mL)
15 days
A
B
263
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
100 1000 10000
0
2
4
6
8
10
12
14
Mean In ensi y (%)
Size (nm)
Figu e 7.9 Pa icle size dis ibu ion ob ained using a pa icle size analyze a oom
empe a u e.
Mac ophages we e able o up ake wea deb is om he medium as can be seen in
Figu e 7.10 whe e he pa icles a e p esen inside he cy oplasm o he cells (black
poin s) while he nuclei emain anspa en .
Figu e 7.10 Mac ophage cellula up ake o biomo phic silicon ca bide pa icles a e 24
hou s o cell cul u e (B) compa ed o nega i e con ol (A) a 20X.
The ac i a ion o mac ophages causes he sec e ion o nume ous p o-in lamma o y
cy okines ha cause he in lamma o y cell ec ui men as IL-1β, TNF-α, IL-6, and IL-8
A
B
Capí ulo 7
264
(44). Two cy okines we e selec ed o e alua e he ac i a ion o mac ophages; umo
nec osis ac o alpha (TNF-α) ha had been ound o be c ucial media ing in lamma o y
cell ec ui men de i ed om pa icula e sys ems, (45) and in e leukin-1 (IL-1) ha
s imula es bone eso p ion and he consequen pe ip os he ic os eolysis (30). The
le els o TNF-α and IL-1 we e compa ed wi h he cy okines sec e ed a e a simila
ea men wi h zi conia pa icles.
Figu e 7.11 Le els o umo nec osis alpha (TNF-α) and in e leukin 1 be a (IL-1β)
sec e ed by he mu ine mac ophage cell line compa ed o posi i e con ol (cells
s imula ed wi h lipopolisaccha ide). The homogeneous g oups a e indica ed by an equal
numbe o as e isks (*) abo e he columns (α < 0.05).
A e he addi ion o pa icles ob ained by mechanical wea simila le els o TNF-α
sec e ion han o zi conia pa icles (46) we e ound. Bo h le els we e signi ican ly
lowe han hose p omo ed by he posi i e con ol (Figu e 7.11). On he o he hand
he sec e ion o IL-1β was quan i a i ely simila o he one p omo ed by he posi i e
con ol and he zi conia pa icles. I was ound ha he addi ion o lipopolisaccha ide
(con ol+) was no able o s imula e he p oduc ion o IL-1β. I has been shown ha
zi conia pa icles show simila le els o in lamma ion o alumina wi h be e esul s
SiC pa icles Zi c pa icles C+
0
1000
2000
3000
4000
5000
24h
TNF-(pg/mL)
*
SiC pa icles Zi c pa icles C+
0
10
20
30
40
50
60
70
IL-1 (pg/mL)
24h
A
B

265
Algina e-poloxame -SiC composi es o indome hacin con olled elease dec ease in lamma ion
han polyme s like high-densi y polye hylene (46). Al hough he p esence o zi conia
pa icles may p oduce some ad e se eac ions, hey a e less in ense han hose
p omo ed by i anium pa icles, he e o e zi conia is conside ed he mos sui able
bioma e ial in e ms o in lamma ion p omo ed by ma e ial deb is (47). Simila esul s
we e ob ained o silicon ca bide and zi conia pa icles showing he accep able
biocompa ibili y o ou biomo phic silicon ca bide pa icles.
7.5 Conclusions
New composi e sys ems including biphasic algina e-poloxame hyd ogels and bioSiCs
om di e en p ecu so s ha e been de eloped and analyzed wi h ega d o hei
capabili y o loading and con ol indome hacin elease. All he ce amic-hyd ogel
composi es ha e shown high biocompa ibili y. The loaded oak and sapelli bioSiCs
composi es had adequa e elease p o iles able o p omo e he dec easing o he p o-
in lamma o y cy okines sec e ion in LPS s imula ed mac ophages, showing s onge
an i-in lamma o y e ec s han pine bioSiC composi es. Those di e ences could no be
obse ed when ea ing os eoa h i ic chond ocy es. In hose cases he indome hacin
eleased om he composi es was also able o modula e he deg ada ion o
chond ocy e ex acellula ma ix and p omo e he o ma ion o new collagen.
Pa icles de i ed om wea deb is o biomo phic silicon ca bide did no show high
oxici y, being simila o he zi conia pa icles.
These de eloped composi es p esen g ea po en ial o he local ea men o bone
pa hologies.
7.6 Re e ences
1. Ragha an RN, Mu hukuma T, Somana han N, Sas y TP. Biomime ic mine aliza ion
o no el silane c osslinked collagen. Ma e Sci Eng, C. 2013;33(4):1983-8.
Capí ulo 7
266
2. Sil e man LD, Lukasho a L, He man OT, Lane JM, Boskey AL. Release o gen amicin
om a icalcium phospha e bone implan . J O hop Res. 2007;25(1):23-9.
3. Dessi M, Bo zacchiello A, Mohamed THA, Abdel-Fa ah WI, Amb osio L. No el
biomime ic he mosensi i e β- icalcium phospha e/chi osan-based hyd ogels o bone
issue enginee ing. J Biomed Ma e Res, Pa A. 2013;101A(10):2984-93.
4. He nandez A, Sanchez E, So iano I, Reyes R, Delgado A, E o a C. Ma e ial- ela ed
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in eg a ion o g ow h ac o -elu ing laye -by-laye polyelec oly e mul ilaye coa ed
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10. Son JS, Choi Y, Pa k E, Kwon T, Kim K, Lee K. D ug deli e y om hyd oxyapa i e-
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B. 2013;101B(2):247-57.
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hyd oxyapa i e sca old wi h h ee-dimensional localized d ug deli e y sys em using
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15. Do y HA, Leedy MR, Cou ney HS, Hagga d WO, Bumga dne JD. Composi e
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(Ams e dam, Ne h ). 2013;454(1):285-95.
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18. Gonzalez P, Bo ajo JP, Se a J, Chiussi S, Leon B, Ma inez-Fe nandez J, e al. A
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Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA
in ec ions. J Ma e Sci: Ma e Med. 2011;22(2):339-47.
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sus ained deli e y o ancomycin. Eu J Pha m Biopha m. 2012;80(1):103-12.
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lapachone hyd ogels by a i icial neu al ne wo ks. In J Pha m (Ams e dam, Ne h).
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23. Rey-Rico A, Sil a M, Coucei o J, Conchei o A, Al a ez-Lo enzo C. Os eogenic
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Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
8.1 Abs ac
Vi al ec o s a e commonly used o deli e a gene in o a speci ic cell ype in o de o
es o e i s no mal p o ein exp ession. The inclusion o i al ec o s in o implan able
sys ems is a p omising al e na i e o he con en ional he apy. Howe e , li le is known
abou he equi emen s o elease sys ems o success ully include i al ec o s and
achie e a sui able ansgenic exp ession. This wo k is ocused on he de elopmen o
di e en hyd ogel s uc u es, able o include ecombinan adeno-associa ed i al
ec o s ( AAV) and p omo e hei con olled elease in o de o elucida e he bes
condi ions o achie e he highes ansduc ion e iciency. The inclusion o AAV loaded
hyd ogels in o silicon ca bide ce amics allow he ob aining o implan able sys ems o
he elease o i al ec o s o local adminis a ion. The ansduc ion e iciencies o he
loaded hyd ogels sys ems we e in ag eemen wi h hei in i o elease p o iles. The
addi ion o poloxame o he sys ems was able o enhance i al ec o s ansduc ion
e iciencies a e one day o s udy. The inco po a ion o loaded hyd ogels in o bioSiCs
has no been able o ob ain adequa e ansduc ion e iciencies, being equi ed addi ional
s udies.

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8.2 In oduc ion
Gene he apy is an a ac i e app oach o he ea men o nume ous diseases due o
i s abili y o s ably in oduce a unc ional gene in o a a ge cell, allowing o he
sus ained p oduc ion o a he apeu ic candida e molecule. This can be pe o med
ei he ia di ec injec ion o DNA, by encapsula ion o DNA in ca ionic lipids o
polyme s, o h ough i al gene deli e y sys ems (1, 2). Se e al non-in eg a ing and
in eg a ing i al ec o s a e a ailable o achie e his goal. Non-in eg a ing ec o s such
as hose de i ed om adeno i uses o he he pes simplex i us (1, 3) a e o ela i ely
limi ed clinical in e es due o he ini ia ion o dele e ious, i us-speci ic immune and/o
oxic eac ions by he hos . The de elopmen o adequa e deli e y sys ems is hus
necessa y in o de o ensu e ha sa e gene exp ession may occu a app op ia e le els
o e ex ended pe iods o ime using such ec o s (3, 4).
The mos commonly used i al ec o s a e based on e o i al and ecombinan
adeno-associa ed i al ( AAV) ec o s (1). Adminis a ion o AAV ec o s ha de i e
om he human nonpa hogenic AAV i us is he goal o human gene he apy. They
ha e been ound o be mos adequa e as hey can ansduce bo h di iding and non-
di iding cells in ma ked con as wi h he e o i al ec o s ha can only modi y
di iding cells, ca ying he addi ional isk o inse ional mu agenesis. Fu he mo e,
AAV allow o he di ec , e ec i e ansduc ion in animal models wi h low
immunogenici y, being sui able o he ea men o human pa hologies, especially hose
ha a ec he a icula ca ilage ( auma ic de ec s, os eoa h i is) (5, 6). T ansduc ion
e iciencies up o 80% ha e been epo ed in a icula chond ocy es in i o, in si u, and
in i o using epo e bu also he apeu ic candida e genes including he insulin-like
g ow h ac o I (IGF-I), ans o ming g ow h ac o be a (TGF-β), and ib oblas g ow h
ac o 2 (FGF-2), es o ing a close o no mal me abolic balance in os eoa h i ic
ca ilage (7, 8). AAV ha e been also success ul o ac i a e he p ocesses o
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Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
chond ogenic di e en ia ion in human bone ma ow de i ed mesenchymal s em cells
(hMSCs) in i o upon gene ans e o he ca ilage-speci ic ansc ip ion ac o SOX9
(9) and o enhance he healing o os eochond al de ec s ollowing di ec adminis a ion
o he same ec o cons uc (10). O e all, hese indings sugges ha his ec o ype
migh be he mos adap ed gene ehicle o elabo a e u u e, e ec i e ea men s
agains ca ilage inju ies in pa ien s. While sys emic injec ion o AAV ec o s can be
pe o med in human subjec s, he high amoun s o ec o s gene ally needed o achie e
a he apeu ic e ec (~ 5 x 1013 g/kg) se e ely es ic hei use in he clinics ia his
ou e o adminis a ion (11). Se e al app oaches ha e been explo ed o imp o e he
deli e y o such ec o s by including hem in o polyme ic sys ems h ough di e en
echnological app oaches as a means o inc ease hei s abili y while dec easing
po en ial immune esponses ha may be aised agains he i al capsids (3, 12, 13). The
ideal sys em would inc ease he le els and du a ion o ansgene exp ession and
imp o e he sa e y o he gene ans e sys em h ough con olled elease o he
ec o s, ensu ing longe esidence ime by educ ion o he i al clea ance (3, 14). To
achie e his goal, se e al na u al and syn he ic polyme ic sys ems like ib in, gela in,
collagen, poly(e hylene glycol) (PEG), and aga ose ha e been es ed (15, 16). Pa en e al
adminis a ion o co-adminis a ion o ca ionic polyme s such as poly-L-lysine o poly-
a ginine ha e been also desc ibed, allowing o enhance he e icacy o AAV gene
deli e y. Such polyme s may a ec he pe missi i y o he a ge s o he ec o pa icle
ei he by modi ying he cha ge o he capsid o by inc easing he in e ac ions be ween
he i al pa icle and i s cell memb ane ecep o , a hepa an sul a e p o eoglycan (11).
Despi e ecen ad ances, he de elopmen o con olled i al ec o elease sys ems
emains challenging. The high size o i al pa icles gene ally impai s hei elease om
he mos con en ional pha maceu ical echnology app oaches. Some s udies showed
he abili y o poloxame s like Plu onic F68® o inc ease AAV-media ed ansgene
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exp ession in di e en issues like adipose issue (17). Biomo phic silicon ca bide
(bioSiC) ce amics ha e been epo ed o hei excellen mechanical p ope ies, wi h
an adequa e po osi y and po e in e connec i i y o p omo e bone egene a ion and a
good biocompa ibili y o induce he os eoblas ic di e en ia ion o hMSCs (18). To ou
bes knowledge, such compounds ha e ne e been examined o hei abili y o elease
AAV ec o s as a means o imp o e he ec o deli e y p ocess o ca ilage epai .
The goal o he p esen wo k was he e o e o p oduce po ous algina e-poloxame
sys ems in a sui able ne wo k s uc u e ha is capable o eleasing such p omising,
clinically ele an gene deli e y ec o s o op imal ansduc ion e icacy o epo e
genes in hMSCs. The inclusion o he op imized hyd ogel in o a po ous bioSiC ce amic
may p o ide adequa e biphasic sys ems o u u e applica ions o ea a icula
ca ilage de ec s in pa ien s.
8.3 Ma e ials and me hods
8.3.1 Reagen s
Sodium algina e (GRINDSTED® AlgPH155) was pu chased a Danisco (Copenhagen,
Denma k). Poloxame 407 (Plu onic F127® (PF127)) was kindly p o ided by BASF
(Ludwigsha en, Ge many). Biomo phic silicon ca bide (bioSiC) samples we e ob ained
om sapelli wood (En hand oph agma cylind icum) as p e iously epo ed (19). The Cell
P oli e a ion Reagen WST-1 and β-gal S aining Se we e om Roche Applied Science
(Mannheim, Ge many). The Be a-Glo® Assay Sys em was om P omega (Mannheim,
Ge many).
8.3.2 Cells
Human bone ma ow de i ed mesenchymal s em cells (hMSCs) we e p epa ed om
bone ma ow aspi a es ob ained om he dis al emu s o pa ien s unde going o al
knee a h oplas y (n = 5). The s udy was app o ed by he E hics Commi ee o he
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Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
Saa land Physicians Council. All pa ien s p o ided in o med consen be o e inclusion in
he s udy. All p ocedu es we e pe o med in acco dance wi h he Helsinky
Decla a ion. Cells we e isola ed, expanded in DMEM, 100 U/mL penicillin and 100
µL/mL s ep omycin, 10% FBS (g ow h medium), and cha ac e ized o cell su ace
ma ke s and mul ilineage po en ial as p e iously desc ibed (9, 20). Cul u e medium was
eplaced e e y 2-3 days. Cells a passage 1-2 we e used o he expe imen s.
8.3.3 AAV plasmids and ec o s
The cons uc s we e de i ed om pSSV9, an AAV-2 genomic clone (21, 22). AAV-
lacZ ca ies he lacZ gene o E. coli β-galac osidase and AAV-RFP a Discosoma sp. ed
luo escen p o ein (RFP) cDNA agmen , bo h unde he con ol o he
cy omegalo i us immedia e-ea ly (CMV-IE) p omo e (5, 8-10, 23). The ec o s we e
packaged as con en ional (no sel -complemen a y) ec o s using a helpe - ee, wo-
plasmid ans ec ion sys em in he 293 cell line (an adeno i us- ans o med human
emb yonic kidney cell line) wi h he packaging plasmid pXX2 and he Adeno i us
helpe plasmid pXX6 as p e iously desc ibed (8). The ec o p epa a ions we e
pu i ied by dialysis and i e ed by eal- ime PCR (5, 8-10, 23), a e ing 1010 ansgene
copies/mL.
8.3.4 Capsule p epa a ion and cha ac e iza ion
In o de o achie e adequa e ec o s abili y and elease p o iles, h ee di e en
condi ions we e selec ed om he ini ial condi ions s udied. Capsules we e p epa ed
wi h 0.3% algina e (AlgPH155) o wi h algina e con aining 9% PF127 (AlgPH155 +
PF127) in a solu ion o 10% suc ose. In he la e case, he e ec s o empe a u e upon
iscosi y and inal s uc u e o he combina ion con aining a he mosensi i e polyme
we e examined by c osslinking he sys ems a oom empe a u e (AlgPH155 + PF127
[C]) o a 50 °C (AlgPH155 + PF127 [H]). Unloaded and AAV ec o -loaded capsules
Capí ulo 8
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we e p epa ed by d opping he dispe sion o polyme s alone o con aining AAV o a
calcium chlo ide solu ion (102 mM) in a 2 mL ube using a sy inge wi h a needle o 18
½ G. The c osslinking p ocedu e was pe o med a oom empe a u e o a 50 °C o
a b ie pe iod o ime (30 sec) o a oid i e e sible c osslinking o all he polyme ic
bead. Capsules we e hen kep in cul u e in 96-well pla es. Polyme ic dispe sions o
AlgPH155 and AlgPH155 + PF127 in suc ose 10% we e p epa ed by di ec dissolu ion
and used as con ols.
The heological p ope ies o he polyme ic dispe sions we e e alua ed using a
heome e (Rheolys AR-1000N TA ins umen s, UK) equipped wi h a Pel ie pla e o
empe a u e con ol and a cone-pla e geome y (60-mm diame e wi h an angle o
1.58°, gap 59 µm). Ramps o empe a u e om 15 °C o 60 °C a 2 °C/min wi h an
oscilla o y s ess o 0.1 Pa a 5 ad/sec we e ca ied ou . Gel empe a u e (Tgel) was
es ima ed om he c oss poin be ween he s o age moduli (G´) and loss moduli (G´´).
8.3.5 AAV ec o encapsula ion e iciency
The abili y o he capsules o en ap AAV du ing c osslinking was e alua ed by
measu ing he numbe o i al pa icles in each bead. The capsules we e placed in 50
µL o bead dissolu ion medium (55 mM sodium ci a e, 0.15 M sodium chlo ide, 30 mM
EDTA) (24), o exed o 2 min ollowed by he addi ion o 50 µL i al dilu ion bu e
(10% SDS, 1 M T is pH 7.5, 0.5 M EDTA) and incuba ed o 10 min a 56 °C. A e a
quick spin, he ec o concen a ions we e measu ed spec opho ome ically a 260
nm (VPcapsule). The ini ial amoun o i al pa icles in he loading solu ion was also
e alua ed (VPloading) and used as 100% o AAV ec o encapsula ion e iciency (EE)
calcula ed as:
EE (%) = [VPcapsule/VPloading] x 100

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Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
8.3.6 Capsule s abili y and deg ada ion
The s abili y o he ec o -loaded and unloaded capsules was e alua ed by es ing hei
weigh and size upon imme sion in cell cul u e medium in he p esence o absence o
hMSCs. Each ype o bead was placed in a well o 96-well pla es con aining 150 µL o
g ow h medium. A p ese imes (0, 1, 3, 5, 10 and 21 days), each capsule was weigh ed
and he mean diame e s and o al a eas we e measu ed unde ligh mic oscopy
(Olympus BX45, Hambu g, Ge many) using he analySIS® p og am (Olympus). Simila ly,
s abili y agains dilu ion was analyzed by modi ying he medium olume whe e he
capsules we e imme sed. The esul s we e exp essed as he pe cen age o a ea loss o
weigh loss o he ini ial a ea and weigh , espec i ely.
8.3.7 AAV ec o elease om he capsules
The ec o elease p o iles we e ob ained by placing he capsules in 96-well pla es
con aining 150 µL o g ow h medium. A p ese imes (1, 3, 5, 10 and 21 days), he
elease medium was emo ed and eplaced by esh medium. The numbe o i al
pa icles in he emo ed medium a he selec ed ime poin s was es ima ed by
spec opho ome y as desc ibed abo e. Unloaded capsules we e used as nega i e
con ols and loading solu ions as posi i e con ols. A e 21 days, he capsules we e
placed in 50 µL o bead dissolu ion medium and he numbe o emaining i al pa icles
was es ima ed in a simila way.
8.3.8 Cell iabili y
hMSC iabili y was es ima ed by placing AAV-lacZ-loaded o unloaded capsules in
con ac wi h cells in monolaye cul u e (3,500 cells/well in 96-well pla es) o 1, 3, 5, 10
o 21 days using he Cell P oli e a ion Reagen WST-1, wi h OD p opo ional o he
cell numbe s as p e iously desc ibed (9). Equi alen ec o solu ions wi h o wi hou
algina e and poloxame we e used as con ols.
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8.3.9 T ansduc ion e iciencies om eleased AAV ec o s
In o de o analyze he po en ial e ec s o algina e and poloxame on he AAV
ansduc ion e iciencies, hMSCs in monolaye cul u e (7,500 cells/well in 96-well
pla es) we e ea ed wi h AAV-lacZ solu ions wi h and wi hou equi alen
concen a ions o polyme s used o p epa e he beads. A e 24 h, he e iciencies we e
measu ed om he luminescence p oduced a e 30 min o incuba ion wi h he Be a-
Glo® assay acco ding o he manu ac u e ’s ecommenda ions. The alues ob ained
we e exp essed as Rela i e Luminescence Uni s (RLUs) no malized o he cell numbe s
as de e mined by using he Cell P oli e a ion Reagen WST-1 (9).
The ansduc ion e iciencies o eleased ec o s we e quan i a i ely and quali a i ely
using hMSCs in monolaye cul u e (109,400 cells/cm2). Fo he i s app oach based on
AAV-lacZ gene ans e , X-Gal s aining was pe o med ollowing ixa ion o he cells
and u he p ocessing acco ding o he manu ac u e ’s ecommenda ions o examine
posi i e s aining unde ligh mic oscopy (Olympus BX45). Quan i a i e es ima ion o
he ansduc ion e iciencies o AAV-lacZ-loaded and unloaded capsules was ca ied
ou measu ing he luminescence p oduced a e 30 min o incuba ion wi h he Be a-
Glo® assay as desc ibed abo e wi h RLUs no malized o he cell numbe . The
ansduc ion e iciencies we e quali a i ely es ima ed by de ec ion o li e luo escence
in ansduced cells as wi h polyme s alone a e 1, 3, 5 and 10 days o con ac wi h
polyme ic AAV-RFP-loaded capsules unde a luo escen mic oscope wi h a 568 nm
il e (Olympus CKX41).
8.3.10 Hyd ogel-ce amic composi es
In o de o e alua e he possibili y o include he hyd ogel sys ems in a ce amic ma ix,
known amoun s o each op imized polyme ic solu ion we e added o bioSiC samples.
The composi e sys ems ob ained we e hen c osslinked wi h calcium chlo ide by
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Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
imme sion in calcium solu ion (102 mM) o 30 sec. The ansduc ion e iciencies o he
composi e sys ems using AAV-lacZ we e e alua ed by X-Gal s aining a e 1, 5 and 10
days o monolaye cul u e wi h hMSCs (12,500 cells/well in 24-well pla es). The e ec s
o he p esence o bioSiC on he ansduc ion e iciencies we e e alua ed simila ly.
8.3.11 S a is ical analysis
All expe imen s we e pe o med in quin uplica e o each condi ion and ime poin .
Resul s a e exp essed as mean ± s anda d de ia ion. S a is ical signi ican di e ences
be ween ea men s we e e alua ed by analysis o a iance (ANOVA) using he
S a g aphics Cen u ion® X64 so wa e (S a poin Technologies, USA). P alues o less
han 0.05 we e conside ed s a is ically signi ican .
8.4 Resul s
8.4.1 Hyd ogel and bead cha ac e iza ion
The po en ial in e ac ions be ween AlgPH155 and PF127 we e s udied by analyzing he
heological p ope ies o he polyme ic dispe sions. The dispe sion o AlgPH155 in
wa e p oduced a iscous solu ion cha ac e ized by a complex iscosi y ha dec eases
as he empe a u e inc eases wi h no gela ion phenomena. The low concen a ion o
AlgPH155 selec ed p oduced solu ions wi h nega i e alues o elas ic modulus a all he
empe a u es s udied (da a no shown). PF127 dispe sion showed a gela ion
empe a u e o 55.50 °C (Figu e 8.1A). The combina ion AlgPH155 + PF127 p omo ed
a educ ion in he gel empe a u e o a lowe alue (44.55 °C) (Figu e 8.1B), p obably
due o enhanced in e ac ions o poloxame chains ia AlgPH155 (Figu e 8.1C).
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Figu e 8.1 Hyd ogel and bead cha ac e iza ion. The heological p ope ies o he
polyme ic dispe sions we e analyzed by using amps o empe a u e om 15 °C o 60
°C a 2 °C/min wi h an oscilla o y s ess o 0.1 Pa a 5 ad/sec. Values o s o age
moduli (G´) and loss moduli (G´´) a e shown o PF127 (A) and AlgPH155 + PF127 (B).
The gel empe a u es we e ob ained by he c oss poin be ween bo h moduli. A
schema ic dis ibu ion o polyme ic chains a e c osslinking wi h calcium ions is
depic ed in (C).
The s abili y pa ame e s as e alua ed by measu ing he loss o capsule a ea and weigh
a e p esen ed in Figu e 8.2. The di e ences in po ous s uc u es esul ed in a ia ions
15 20 25 30 35 40 45 50 55 60 65
1
10
100
G', G'' (Pa)
empe a u e (°C)
G'
G''
20 25 30 35 40 45 50 55 60 65
1
10
100
G', G'' (Pa)
empe a u e (°C)
G'
G''
A
B
C
291
Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
Figu e 8.6 Li e luo escence emission o ansduced hMSCs in monolaye cul u es a e
1, 3, 5 and 10 days o ea men wi h AAV-RFP-loaded capsules.
Quali a i e analysis was also ca ied ou by X-Gal s aining o hMSCs ea ed wi h
unloaded and AAV-lacZ-loaded capsules. Figu e 8.7 shows lacZ exp ession a e 1, 3, 5
and 10 days. Capsules con aining algina e and poloxame c osslinked a oom
empe a u e and a 50 °C showed highe s aining a e one day o ea men han
capsules o med by algina e alone. On he o he hand, capsules con aining single
AlgPH155 o AlgPH155 + PF127 [H] showed highe ansduc ion e iciencies a e one
day o assay. Di e en cell mo phology was obse ed in hese wo ypes o capsules,
cells ea ed wi h algina e capsules show ib ous shape whe eas cells ea ed wi h
AlgPH155 + PF127 capsules show ound shape. I was clea ly obse ed ha while
posi i e con ol su e ed a dec ease in lacZ exp ession a e 3 days o cul u e,

Capí ulo 8
292
AlgPH155 and AlgPH155 + PF127 [H] main ained s aining in ensi y o e he ime.
Con olled elease o AAV was able o p omo e a sui able ansduc ion e iciency
o e ime in such sys ems.
Figu e 8.7 X-Gal s aining o hMSCs in monolaye cul u e a e 1, 3, 5 and 10 days o
ea men wi h capsules including AAV-lacZ.
Es ima ion o he ansduc ion e iciencies was ca ied ou using AAV-lacZ-loaded and
unloaded capsules as con ols. Figu e 8.8 shows RLUs/cell numbe o each one o he
s udied condi ions a di e en imes. Acco ding o he expe imen al esul s, all sys ems
we e able o achie e signi ican highe alues o ansgene exp ession han nega i e
con ol (Con ol -). Capsules including PF127 we e ound o be able o inc easing cell
ansduc ion in compa ison wi h bo h, he posi i e con ol and single algina e capsules
o med a e 24 h o s udy. This e ec may be a ibu ed o he ini ial bu s elease
293
Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
and/o he polyme i sel in ag eemen wi h ou p e ious esul s (Figu es 8.3 and 8.4B).
The addi ion o poloxame is able o inc ease he ansduc ion e iciencies o he
ec o s.
AlgPH155
AlgPH155 + PF127 [C]
AlgPH155 + PF127 [H]
Con ol -
Con ol +
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
*
*
**
RLU/cell nume co ec ed
1 day
3 days
5 days
10 days
21 days
**
Figu e 8.8 T ansduc ion e iciencies o eleased AAV-lacZ om capsules con aining
AlgPH155 wi h o wi hou PF127. The homogeneous g oups a e indica ed by an equal
numbe o as e isks (*) abo e he columns (P < 0.05).
A e he i s ime poin o cell cul u e, poloxame -algina e capsules shown simila
le els o gene ansduc ion e iciencies han algina e capsules bu signi ican ly highe
han nega i e and posi i e con ols. Fu he mo e, single algina e capsules o med by
AlgPH155 we e able o achie e s able cell ansduc ion wi h simila RLU alues a all
ime poin s e alua ed whe eas o he o he capsules, he ansduc ion e iciencies
dec eased wi h ime. In spi e o he e ec o algina e dec easing ansduc ion e iciency
ound o his polyme alone (Figu e 8.4B), he con olled elease achie ed in hese
samples was able o p omo e an adequa e in i o ansduc ion e iciency wi h
s a is ically signi ican highe le els o nega i e con ol and AlgPH155 + PF127 [C] e en
Capí ulo 8
294
a e 21 days o cell cul u e. In e es ingly, capsules o med by he wo polyme s
c osslinked a oom empe a u e we e no able o achie e highe le els o
ansduc ion e iciencies han nega i e con ol a e 10 and 21 days o cell cul u e.
Acco ding o he expe imen al da a capsules o med by algina e alone ha e been
shown he bes elease p o iles and a mo e p olonged inc ease in angene exp ession.
Poloxame -algina e capsules c osslinked a 50 °C ha e also shown good expe imen al
esul s. These wo hyd ogel s uc u es we e selec ed o hei inco po a ion in o
bioSiC ce amics.
8.5.6 Hyd ogel-ce amic composi es
Selec ed hyd ogels including AAV ec o we e used o be loaded in o ce amic
ma ices in o de o ob ain a composi e sys em able o p omo ing a local con olled
elease o AAV use ul o bone epai implan s. Two composi ions we e selec ed o
his pu pose in ligh o hei be e in i o esul s: AlgPH155 and AlgPH155 + PF127
[H]. As con ols, equi alen concen a ions o AAV we e di ec ly loaded in he bioSiC
samples, ec o s added o cell cul u e medium we e used as posi i e con ol and cells
cul u ed wi hou any ea men as nega i e con ols. Figu e 8.9 shows he esul s o X-
Gal s aining o he s udied condi ions. Bo h posi i e con ol and silicon ca bide
con aining AAV-lacZ showed simila s aining in ensi y a di e en imes s udied. The
p esence o his ce amic hus did no a ec he ansduc ion e iciency. On he o he
hand, low s aining was obse ed on bo h composi e sys ems. Agina e-bioSiC showed
ew ansduced cells while no s ained cells we e obse ed using AlgPH155 + PF127-
bioSiC. This may be due o he lowe hyd ogel-cell cul u e in e acial su ace ha
dec eases he elease a e o he ec o s, hus dec easing ansduc ion. Op imiza ion
o he composi e p ocess need o be nex pe o med o ob ain he desi able
he apeu ic e ec o hese composi e sys ems.
295
Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
Figu e 8.9 X-Gal s aining o hMSCs in monolaye cul u es a e 1, 3, 5 and 10 days o
ea men wi h he hyd ogel-bioSiC composi es including AAV-lacZ.
8.5 Discussion
The combina ion o wo polyme s allowed o gene a e polyme ic sys ems capable o
combining he ad an ages o each polyme . Algina e is a sa e na u al polyme ex ac ed
om b own algae commonly used as able binding agen and as a di usion ba ie in
con olled elease o mula ions, dec easing d ug molecules mig a ion (25). The
selec ion o algina e as pa o he polyme ic composi ion allows ob aining c osslinked
hyd ogels by simple addi ion o di alen ions (26). Syn he ic block polyme s such as
poloxame s a e capable o o ming micelles in aqueous solu ions wi h a hyd ophilic
shell and a hyd ophobic co e adequa e o hyd ophobic d ug inclusion, inc easing hei
solubili y. Mo eo e , poloxame s can unde go a sol- o-gel ansi ion a a empe a u e
highe han he gel empe a u e (27).
Capí ulo 8
296
The addi ion o algina e o poloxame dispe sions was able o dec ease he gel
empe a u e o he sys ems. This e ec could be caused by complex in e ac ions
be ween he chains o bo h polyme s whe e wa e could ac as c osslinking agen
o ming hyd ogen bonds be ween he polyme s (28).
The iono opic c osslinking o he polyme ic dispe sions wi h calcium chlo ide a wo
di e en empe a u es was ca ied ou being he ob ained capsules o di e en
cha ac e is ics. The selec ion o a c osslinking empe a u e highe han he gel
empe a u e o he mix u e algina e-poloxame should make possible o ob ain
hyd ogel sys ems wi h di e en po ous s uc u es. The agg ega ion o micelles a e
he gel empe a u e (50 °C) could be used as empla es in o de o ob ain sys ems
wi h highe po ous size. The complex in e ac ions be ween he wo polyme s whe e
algina e chains should be placed a ound he polyme ic micelles could inc ease he
hyd ophobici y o micelles and enhance micelle-micelle in e ac ions and he consequen
gela ion as is shown in Figu e 8.1B.
The signi ican di e ences in capsule s abili y pa ame e s sugges ha he p esence o
block polyme ic micelles in algina e medium modi ies he in e ac ion be ween algina e
chains and calcium ions du ing he c osslinking p ocess. Micelles could impai hese
in e ac ions gi ing inal highly po ous hyd ogels he e o e wi h lowe s abili y. When
he c osslinking p ocedu e was ca ied ou a 50 °C, a empe a u e o e he sol-gel
ansi ion (Tgel 44.50 °C), he plu onic micelles can unde go agg ega ion. The inc ease
in iscosi y which s ongly may hinde he ionic in e ac ions be ween algina e and
calcium oge he wi h p esence o micelle agg ega es, which ac as po ogen agen s, may
cause he syn hesis o an e en mo e po ous hyd ogel wi h la ge po e size (Figu e 8.2).
Good encapsula ion e iciencies we e ob ained o all he sys ems s udied. Howe e ,
despi e he highe iscosi y o he polyme ic composi ion, he lowes encapsula ion
e iciency was ob ained o high empe a u e c osslinked sys ems. This migh be esul

297
Con olled elease o AAV ec o s om algina e-poloxame -SiC composi e sys ems
o he highe po ous s uc u e on hei ex e nal hyd ogel su ace laye , leading o a
loss o AAV om he pa icles when is compa ed o sys ems c osslinked a oom
empe a u e.
The con olled AAV elease was go e ned by a di usion mechanism h ough he
hyd ogel ma ix. The h ee po ous s uc u e ob ained we e able o achie e di e en
elease p o iles as a unc ion o he a ia ions in hyd ogel s uc u e ha a e in
ag eemen wi h he s abili y esul s.
The e alua ion o he e ec o he polyme p esence on ansduc ion e iciencies has
poin ed ou an inc ease o angene exp ession o PF127 acco ding o a simila
phenomenon ha has been p e iously desc ibed o a simila block copolyme , PF68
(29). On he con a y he addi ion o algina e dec eases he ansduc ion e iciency. I
has been epo ed ha his polyme could dec ease he a e o i us anspo , hus
dec easing he in i o ans ec ion o cance cells, bu no a ec ing he bioac i i y o he
ec o s (30).
Quali a i e e alua ion o angene exp ession o capsule eleased ec o s h ough li e
luo escence emission and X-Gal s aining showed an enhancemen on he ansduc ion
e iciency o he con olled elease ec o s when hey a e compa ed o posi i e
con ol a long pe iods o ime. I was p e iously epo ed ha an adequa e AAV
elease p o ile is able o inc ease ansgene exp ession o cells and educe mac ophage
ac i a ion, inc easing he he apeu ic u ili y o he sys ems (13).
Acco ding o he quan i a i e analysis o ansduc ion e iciencies da a i is an e idence
ha he p esence o plu onic in he capsule composi ion enhance he ansduc ion
e iciency o he elease ec o s a e one day o s udy. This ac could be a ibu ed o
a change in he cellula memb ane pe meabili y (27) and was p e iously epo ed o
len i i al and adeno i al ec o s (31).
Capí ulo 8
298
Biomo phic silicon ca bide ce amics ha e been shown o ha e highly po ous s uc u es
in which he op imized hyd ogels could be included. The addi ion o i al ec o s o he
ce amic s uc u es did no a ec he ac i i y o he ec o s.
8.6 Conclusions
In he p esen s udy, we we e able o de elop h ee hyd ogel ne wo ks due o he
he mosensi i e p ope ies o PF127 and hei complex in e ac ions wi h algina e
chains du ing calcium c osslinking. These sys ems we e s able agains dilu ion and we e
able o p oduce h ee di e en elease p o iles o AAV. The p esence o PF127 was
able o inc ease he ansduc ion e iciency o AAV. C osslinked capsules showed
excellen biocompa ibili y esul s and hei ansduc ion e iciency e alua ion had lead
o be e esul s o sys ems con aining algina e alone because o he long ime
con olled elease obse ed o such sys ems. Despi e he e ec o PF127 on he
ansduc ion e iciency he as e elease on hese sys ems was no able o achie e
highe long- e m ansduc ion e iciencies han nega i e con ol a e i e days o assay
o oom empe a u e c osslinked sys ems. The inclusion o he hyd ogels in a po ous
ce amic was no able o achie e an adequa e ansduc ion e iciency due o he lowe
hyd ogel su ace and he consequen lowe ec o elease. Fu he s udies should be
done in o de o achie e adequa e ce amic-hyd ogel combina ions o he ea men o
os eochond al de ec s.
8.7 Re e ences
1. Koo s a NA, Ve ma IM. Gene he apy wi h i al ec o s. Annu Re Pha macol
Toxicol. 2003;43:413-39.
2. Ib aheem D, Elaissa i A, Fessi H. Gene he apy and DNA deli e y sys ems. In J
Pha m. 2014;459(1-2):70-83.
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3. Wang C, Pham P. Polyme s o i al gene deli e y. Expe Opin D ug Deli .
2008;5(4):385-401.
4. Xu X, Yang J, Cheng Y. Pha macokine ic s udy o i al ec o s o gene he apy:
P og ess and challenges. Vi al Gene The . 2011:435-50.
5. Weime A, Mad y H, Venka esan JK, Schmi G, F isch J, Wezel A, e al. Bene i s o
ecombinan adeno-associa ed i us ( AAV)-media ed insulin-like g ow h ac o I (IGF-
I) o e exp ession o he long- e m econs uc ion o human os eoa h i ic ca ilage by
modula ion o he IGF-I axis. Mol Med. 2012;18(3):346-58.
6. Huang S, Kamihi a M. De elopmen o hyb id i al ec o s o gene he apy.
Bio echnol Ad . 2013;31(2):208-23.
7. Cucchia ini M, Thu n T, Weime A, Kohn D, Te willige EF, Mad y H. Res o a ion
o he ex acellula ma ix in human os eoa h i ic a icula ca ilage by o e exp ession
o he ansc ip ion ac o SOX9. A h i is Rheum. 2007;56(1):158-67.
8. Venka esan JK, Rey-Rico A, Schmi G, Wezel A, Mad y H, Cucchia ini M. AAV-
media ed o e exp ession o TGF-be a s ably es uc u es human os eoa h i ic
a icula ca ilage in si u. J T ansl Med. 2013;11:211-24.
9. Venka esan JK, Ekici M, Mad y H, Schmi G, Kohn D, Cucchia ini M. SOX9 gene
ans e ia sa e, s able, eplica ion-de ec i e ecombinan adeno-associa ed i us
ec o s as a no el, powe ul ool o enhance he chond ogenic po en ial o human
mesenchymal s em cells. S em Cell Res The . 2012;3(3):22-36.
10. Cucchia ini M, O h P, Mad y H. Di ec AAV SOX9 adminis a ion o du able
a icula ca ilage epai wi h delayed e minal di e en ia ion and hype ophy in i o. J
Mol Med. 2013;91(5):625-36.
11. Moulay G, Bou in S, Masu ie C, Sche man D, Kichle A. Polyme s o imp o ing
he in i o ansduc ion e iciency o AAV2 ec o s. PLoS One. 2010;5(12): e15576-83.
Capí ulo 8
300
12. Zeng Y, Tseng S-, Kempson IM, Peng S, Wu W, Liu J. Con olled deli e y o
ecombinan adeno-associa ed i us se o ype 2 using pH-sensi i e poly(e hylene
glycol)-poly-l-his idine hyd ogels. Bioma e ials. 2012 12;33(36):9239-45.
13. Lee S, Kim J, Chu HS, Kim G, Won J, Jang J. Elec ospun nano ib ous sca olds o
con olled elease o adeno-associa ed i al ec o s. Ac a Bioma e ialia. 2011
11;7(11):3868-76.
14. Kilpa ick LA, Li Q, Yang J, Godda d JC, Feke e DM, Lang H. Adeno-associa ed
i us-media ed gene deli e y in o he scala media o he no mal and dea ened adul
mouse ea . Gene The . 2011;18(6):569-78.
15. Kidd ME, Shin S, Shea LD. Fib in hyd ogels o len i i al gene deli e y in i o and in
i o. J Con ol Release. 2012;157(1):80-5.
16. Tseng S-, Kempson IM, Peng S, Ke B, Chen H, Chen P, e al. En i onmen acidi y
igge s elease o ecombinan adeno-associa ed i us se o ype 2 om a unable
ma ix. J Con ol Release. 2013;170(2):252-8.
17. Zhang F-, Jia S-, Zheng S-, Ding W. Celas ol enhances AAV1-media ed gene
exp ession in mice adipose issues. Gene The . 2011;18(2):128-34.
18. Díaz-Rod íguez P, Pé ez-Es é ez A, Seoane R, González P, Se a J, Landin M.
Sui abili y o biomo phic silicon ca bide ce amics as d ug deli e y sys ems agains
bac e ial bio ilms. ISRN Pha m. 2013:104529-36.
19. Díaz-Rod íguez P, Landin M, Rey-Rico A, Coucei o J, Coenye T, González P, e al.
Bio-inspi ed po ous SiC ce amics loaded wi h ancomycin o p e en ing MRSA
in ec ions. J Ma e Sci Ma e Med. 2011;22(2):339-47.
20. Elsle S, Sche ing S, Schmi G, Kohn D, Mad y H, Cucchia ini M. E ec i e, sa e
non i al gene ans e o p ese e he chond ogenic di e en ia ion po en ial o human
mesenchymal s em cells. J Gene Med. 2012;14(7):501-11.
307
Discusión gene al
Figu a 9.1 Relación en e la po osidad de los bioSiCs y sus p e o mas de ca bón
ob enida median e po osime ía de in usión de me cu io.
La igu a 9.2 ep esen a g á icamen e algunos de los pa áme os ca ac e ís icos de las
ce ámicas biomó icas p oducidas y sus p ecu so es de ca bón. Mien as que el pino
gene a la ce ámica de mayo po osidad o al (46.97%) y meno supe icie especí ica,
con mac opo os de pequeño amaño (50 ± 20 µm), el oble da luga al bioSiC con
meno po osidad o al (27.85%) pe o con la mayo supe icie po osa, una densidad
in e media y mac opo os de mayo amaño 250 ± 20 µm (Capí ulo 6).
Los bioSiCs ob enidos de la made a de sapelli se ca ac e izan po p esen a : una
dis ibución de amaños de po o bimodal, una po osidad o al simila a la del pino
(40.72%), una supe icie especí ica in e media y la mayo densidad de los es sis emas
es udiados, con mac opo os de un amaño medio de 140 ± 30 µm.
0,01 0,1 110 100
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20 bioSiC de Sapelli
Ca bón de Sapelli
Volumen de po o inc emen al (ml/g)
Diáme o medio (m)
0,01 0,1 1 10 100
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20
Volumen de po o inc emen al (ml/g)
Diáme o medio (m)
bioSiC de oble
ca bón de oble
0,01 0,1 1 10 100
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
0,18
0,20
Volumen de po o inc emen al(ml/g)
Diáme o medio (m)
bioSiC de pino
ca bón de pino

Capí ulo 9
308
0,60,81,01,2 1,4 1,6 1,8 2,0
0
1
2
3
20
30
40
50
60
bioSiC de oble
bioSiC de sapelli
bioSiC de pino
Ca bón de oble
Ca bón de sapelli
densidad eal (g/cm3)
po osidad (%)
a ea supe icial (m2/g)
Ca bón de pino
Figu a 9.2 Relación en e po osidad, á ea supe icial (ob enida po adso ción de
ni ógeno) y densidad eal (ob enida median e picnome ía de helio) de los es
sis emas ce ámicos bioSiCs.
Los es bioSiCs, a di e encia de sus co espondien es p e o mas de ca bón, se
ca ac e izan po se ma e iales hid o ílicos, con un ángulo de con ac o medio de 38 ±
7º (Capí ulo 5) sin di e encias signi ica i as en e ellos. Sin emba go, el empleo de
luidos de di e en e na u aleza pe mi e la es imación de alo es de ene gía supe icial
a iables. Así, los bioSiCs de pino p esen a on una ene gía supe icial posi i a, mien as
que los de oble y sapelli p esen a on alo es nega i os (Capí ulo 6).
La ugosidad supe icial, e aluada median e pe ilome ía in e e omé ica (Capí ulo 5),
ambién pone de mani ies o a iaciones impo an es. El bioSiC de sapelli p esen a
mayo ugosidad supe icial con un Rq de 11.05 µm, muy supe io al del pino y el
oble, con alo es de 6.91 µm y 5.91 µm espec i amen e. El análisis de la supe icie de
los bioSiCs median e FT-IR mos ó en odas ellas, la p esencia de g upos uncionales
OH y SiO (Capí ulo 6).
309
Discusión gene al
La modelización de la es uc u a po osa de los sis emas ce ámicos a pa i de las
cu as de po osime ía de in usión de me cu io median e un so wa e especializado
(Po eXpe ®) pe mi e alo a la in e conec i idad de sus po os, su o uosidad y
ambién ealiza p edicciones sob e el po encial p oceso de pene ación de un luido
en la es uc u a po osa cuando la ce ámica en e en con ac o con un medio líquido
(Capí ulo 6). La Figu a 9.3 p esen a la ciné ica de cap ación de agua a 0 MPa simulada
pa a los di e en es sis emas. Como puede obse a se, es la mic oes uc u a del bioSiC
de sapelli la que p esumiblemen e es capaz de cap a más agua y más ápidamen e. Es e
sis ema p esen a la mayo conec i idad y la meno o uosidad de los es es udiados.
A pesa de que la capacidad de cap ación de agua es simila en los bioSiCs de pino y de
oble a 110 ms, la modelización pone de mani ies o que la conec i idad del bioSiC de
oble es supe io a la del pino.
Figu a 9.3 Simulación de la cap ación de agua po pa e de los es sis emas po osos
bioSiCs ob enida as la modelización de la es uc u a po osa con el so wa e
especializado Po eXpe ®.
Capí ulo 9
310
9.2 E aluación de la espues a celula e inmunológica a los bioSiCs
La cap ación de luidos po pa e de las es uc u as po osa modi ica no sólo la
po encial capacidad de ca ga de á macos, sino ambién la adhesión celula y la
espues a in lama o ia. Po ello, las di e en es es uc u as de ca bu o de silicio
debe ían da luga a un compo amien o celula di e en e y pe iles de ca ga y cesión
a iables. De es a mane a la e aluación de la espues a celula y isula a los sis emas
bioSiCs esul a c ucial pa a analiza su po encial aplicación en egene ación ósea.
9.2.1 E aluación de la biocompa ibilidad de los bioSiCs
La in oducción de nue os ma e iales como sus i u os óseos ha de es a p esidida po
la capacidad del bioma e ial pa a eje ce su unción sin p oduci e ec os ad e sos. El
bioma e ial ideal ú il en egene ación ósea debe se económico, iable y segu o, a la ez
que biocompa ible, os eoinduc i o, os eoconduc i o y p e e en emen e biodeg adable
(3, 4). Si el de ec o óseo es g ande y el implan e ha de ab ica se con ma e ial no
biodeg adable, es necesa io que és e sea capaz de es imula la o mación de ejido
óseo y/o p omo e su os eoin eg ación. En es os casos las ca ac e ís icas
mic oes uc u ales y supe iciales del ma e ial son c í icas. Di e sos au o es han
pues o de mani ies o que el amaño de los po os del ma e ial condiciona la o mación
de nue os asos en el implan e y, po an o, el c ecimien o del ejido óseo (5-8).
El es udio p elimina de la biocompa ibilidad de los bioSiCs ue ealizado con una línea
celula de ib oblas os (BALB/3T3) usando como ma e ial ce ámico el bioSiC de sapelli
(Capí ulo 3). Los esul ados expe imen ales demos a on la excelen e
biocompa ibilidad de los sis emas obse ándose la o mación de una monocapa de
células as 15 días de es udio (Figu a 9.4).
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Discusión gene al
Figu a 9.4 Fo og a ía de mic oscopía elec ónica de ba ido (SEM) de sapelli as 15
días de cul i o con la línea celula ib oblás ica BALB/3T3.
La biocompa ibilidad de los es sis emas bioSiCs (pino, oble y sapelli) ue
pos e io men e e aluada usando células mad e mesenquimales ob enidas de médula
ósea humana (Capí ulo 6). Los esul ados de iabilidad celula así como las imágenes de
mic oscopía con ocal, la cuan i icación de ci oquinas p oin lama o ias (IL-1β) y de
ma cado es de apop osis (caspasa-3), mues an que odos los sis emas son al amen e
biocompa ibles con ni eles no de ec ables de IL-1β y concen aciones de caspasa-3
equi alen es al con ol nega i o a odos los iempos es udiados. De es a mane a, odos
los ca bu os de silicio cumplen el equisi o de biocompa ibilidad.
9.2.2 E ec o de las es uc u as po osas de los bioSiCs sob e la in e acción
con los componen es sanguíneos
Las ca ac e ís icas supe icies de los ma e iales ambién de e minan su in e acción con
los componen es sanguíneos en el momen o de su implan ación. La o mación de
coágulo, la ac i ación de plaque as y la in lamación condicionan el p oceso no mal de
egene ación ósea y modulan el c ecimien o óseo al ededo del implan e y la
os eoconducción (3, 9-12). También ha sido desc i o que la ene gía supe icial, el
Capí ulo 9
312
ángulo de con ac o, la libe ación de iones, la esis i idad y la ugosidad supe icial
modi ican es as in e acciones, y po an o, la egene ación ósea (13-16).
La in e acción de las supe icies de los ca bu os de silicio y los componen es
sanguíneos ue e aluada en lo que espec a a la hemólisis p oducida, la adso ción de
p o eínas, la coagulación, la adhesión de plaque as y la ac i ación del sis ema de
complemen o con el in de ob ene los pa áme os mic oes uc u ales cla e que
condicionan dichas in e acciones (Capí ulo 5). Así, se ha obse ado que el amaño de
po o es undamen al a la ho a de p edeci la hemólisis gene ada. La u ilización de
sis emas con meno amaño de mac opo o (pino y sapelli) da luga a alo es de
hemolisis más educidos.
La elación en e la adso ción de p o eínas hid o ílicas (se oalbúmina bo ina) y
p o eínas hid o óbicas ( ib inógeno) condicionan la adhesión celula y como
consecuencia la pos e io os eoin eg ación. Todos los bioSiCs es udiados se
ca ac e izan po posee una mayo adso ción de albúmina que de ib inógeno lo que
debe acili a el econocimien o celula de las supe icies. Ello además, se co elaciona
con una baja o mación de ombo y con una mayo supe icie especí ica de los
ma e iales. De la misma mane a, se obse ó que la ac i ación del sis ema de
complemen o, di ec amen e elacionada con la espues a inmune, es á condicionada
po la adso ción de p o eínas. La adhesión de plaque as po pa e de los sis emas
ce ámicos mos ó que és as man ienen una mo ología edondeada, lo que indica una
adecuada adhesión celula , sin causa su ac i ación.
La e aluación de o mación de coágulo pa a los es sis emas ensayados mos ó
mayo es alo es pa a el bioSiC de sapelli lo que es á di ec amen e co elacionado con
la mayo ugosidad supe icial. Se ha obse ado que la o mación de coágulo es
dependien e de la supe icie ex e na mien as que la adso ción de p o eínas se e
modulada an o po la supe icie ex e na como po la in e na. Los esul ados

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Discusión gene al
expe imen ales ob enidos pe mi en conclui que las ce ámicas de ca bu o de silicio
biomó ico p esen an una hemocompa ibilidad adecuada pa a su aplicación como
sis emas implan ables.
Además, la o mación de coágulo obse ada debe ía da luga a una adecuada
in eg ación de los implan es. La educida espues a in lama o ia pod ía indica la
ausencia de o mación de ejido ib oso al ededo del implan e a o eciendo su
in eg ación (17). De acue do con los esul ados expe imen ales de ugosidad
supe icial y la o mación de coágulo obse ada los ca bu os de silicio de sapelli se
p esen an como los más p ome edo es pa a una posible aplicación clínica.
9.2.3 E ec o de la opog a ía de los bioSiCs sob e la di e enciación celula
La ugosidad supe icial, el ángulo de con ac o, la c is alinidad y la composición química
modulan la adhesión, la mo ología, la uncionalidad y la mig ación celula en el in e io
del ma e ial lo que condiciona la ijación del implan e y su in eg ación (5, 18-27).
El e ec o de las ca ac e ís icas opog á icas de los ca bu os de silicio biomó icos así
como de su es uc u a po osa sob e la di e enciación os eoblás ica de células mad e
mesenquimales, se e aluó en unción de la sec eción de indicado es de di e enciación
(os eocalcina, os eopon ina y os a asa alcalina) (Capí ulo 6). De acue do con da os
bibliog á icos p e ios, se ha obse ado que las p opiedades de los bioSiCs modi ican el
compo amien o celula . Así, los ca bu os de silicio que p esen an mayo amaño de
po o ( oble y sapelli) son capaces de es imula la di e enciación os eoblás ica de las
células mad e mesenquimales as quince días de cul i o ob eniéndose alo es de
os eocalcina y os eopon ina simila es a los de las células cul i adas en p esencia de
medio de di e enciación os eoblás ica. Es posible que la di e enciación obse ada no
sólo es é condicionada po la mo ología de los sis emas, sino ambién po la p esencia
de silicio, ya que se ha desc i o que es e compues o es capaz de p omo e la
Capí ulo 9
314
di e enciación os eoblás ica, e inc emen a la p oli e ación de los os eoblas os y su
p oducción de os eocalcina (28). Los esul ados expe imen ales ob enidos pe mi en
conclui que seleccionando un p ecu so con una es uc u a po osa adecuada se
pod ían ob ene ce ámicas de ca bu o de silicio biomó ico con capacidad
os eoinduc o a.
9.2.4 Ac i idad in lama o ia de las pa ículas de bioSiC ob enidas po
desgas e mecánico
O o aspec o impo an e de los bioma e iales es su po encial pa a gene a p oduc os
de deg adación óxicos as su implan e. Po ejemplo, el uso de políme os de i ados
del ácido lác ico p omue e la p oducción de sus ancias de ca ác e ácido como
p oduc o de deg adación que gene an in lamación en los ejidos adyacen es (29).
El desgas e mecánico de los ma e iales implan ados puede da luga a la o mación de
pa ículas, que causen la ac i ación de los mac ó agos ci cundan es, el
desencadenamien o de una espues a in lama o ia aguda y la consiguien e des ucción
de ejido óseo y allo del implan e (30-32).
Los ca bu os de silicio biomó icos son ma e iales sólidos, poco iables, cuya elocidad
de deg adación es ex emadamen e len a, meno de 30 nm po año en condiciones
isiológicas no males (30). Se es udió la capacidad de las pa ículas de ca bu o de silicio
biomó ico pa a es imula mac ó agos y p o oca una espues a in lama o ia con is as
a p edeci su po encial oxicidad a la go plazo as su implan ación (Capí ulo 7). Las
pa ículas de ca bu o de silicio nanomé icas ob enidas po desgas e mecánico a pa i
de dos piezas de ca bu o de silicio some idas a ozamien o ue on in e nalizadas po
los mac ó agos, como se mues a en la Figu a 9.5. A pesa de ello, la sec eción de
ci oquinas p oin lama o ias po los mac ó agos (TNF-α e IL-1β) mos ó alo es
simila es a los ob enidos con las pa ículas de zi conio usadas como e e encia. De
315
Discusión gene al
acue do con los esul ados expe imen ales se puede a i ma que el ca bu o de silicio
causa unos ni eles acep ables de in lamación, simila es a un ma e ial, el zi conio, que
es á econocido como el más adecuado desde el pun o de is a in lamación de
pa ículas (33).
Figu a 9.5 Cap ación de las pa ículas de ca bu o de silicio po pa e de los mac ó agos
en compa ación con el con ol nega i o (A).
9.3 Desa ollo de sis emas bio uncionales de ca bu o de silicio
La inco po ación de moléculas e apéu icas en sis emas implan ables pe mi e do a de
alo añadido a los ma e iales. De acue do con lo desc i o en el Capí ulo 1.1 exis en
di e en es mecanismos de ca ga que pueden se empleados pa a la ob ención de
ma e iales bio uncionales y que condicionan los pe iles de libe ación de á maco
ob enidos. En es e abajo se han u ilizado es mecanismos di e en es pa a la
inco po ación de moléculas e apéu icas: la adso ción inespecí ica, las in e acciones
iónicas y la inclusión en una ma iz.
9.3.1 Adso ción inespecí ica de an ibió icos
A pesa de los a amien os p e en i os con an ibió icos a ni el sis émico, la
os eomieli is pos qui ú gica con inúa siendo una se ia complicación en la ci ugía
A
B
Capí ulo 9
316
o opédica y den al (34). Los ma e iales implan ados en el o ganismo son a menudo
obje o de colonización mic obiana, lo que e i a la adhesión celula y da luga al allo de
la p ó esis (35). Cuando la con aminación es se e a, la os eomieli is se c oni ica y la
adminis ación sis émica de an ibió icos no es capaz de e adica la debido a las bajas
concen aciones locales de á maco que se alcanzan. El p o ocolo pa a el a amien o
de es a p oblemá ica supone la e i ada de la p ó esis y la implan ación p o isional de
sis emas polimé icos de polime il-me ac ila o (PMMA) ca gados con an ibió icos
capaces de alcanza un adecuado pe il de libe ación. El ca ác e no biodeg adable del
PMMA hace necesa ia su e i ada median e una nue a ci ugía (36, 37).
La posibilidad de desa olla sis emas pa a la egene ación ósea, ca gados con
an ibió icos que p e engan o a en la os eomieli is ep esen a una p ome edo a
al e na i a en egene ación ósea.
En es e es udio se han desa ollados sis emas de ca bu o de silicio biomó ico ca gados
con ancomicina median e un mecanismo de adso ción inespecí ica. La necesidad de
alcanza concen aciones ele adas de á maco a iempos co os que uncionen como
dosis de a aque a los mic oo ganismos p esen es en el medio y e i en la colonización
del implan e po pa e de las bac e ias hacen de es e mecanismo el más adecuado a
p io i pa a el desa ollo de sis emas bioSiC ca gados con ancomicina.
Como e aluación p elimina de la capacidad de ca ga y cesión de los bioSiCs se
seleccionó el ca bu o de silicio ob enido a pa i de made a de sapelli (Capí ulo 3). La
iso e ma de adso ción del á maco mues a la o mación inicial de una monocapa de
moléculas de ancomicina pa a luego da luga a consecu i as capas de á maco a
medida que la concen ación de ca ga se e inc emen ada de acue do con un pe il
ca ac e ís ico de adso ción inespecí ica.
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Discusión gene al
( AAV) han mos ado u ilidad pa a la ansducción de cond oci os (60, 61). Sin
emba go, el ele ado amaño y la educida es abilidad de es os i us di icul a su
aplicación median e sis emas de libe ación con encionales, lo que ab e in e esan es
pe spec i as pa a el desa ollo de nue as al e na i as pa a su adminis ación.
Como ase inal de es e abajo hemos desa ollado combinaciones de es uc u as
po osas de ca bu o de silicio biomó ico con políme os, algina o y poloxame , con el
obje i o de ca ga y libe a de o ma con olada es os ec o es i ales pa a su
po encial aplicación en la egene ación de de ec os os eocond ales.
La p esencia de algina o y poloxame modi ica la e icacia de ansducción de los
ec o es i ales, de o ma simila a lo obse ado po o os au o es (62-64). Así, el
poloxame inc emen a la e icacia de ansducción, posiblemen e debido a la
modi icación de la pe meabilidad de la memb ana (65), mien as que la adición de
algina o al medio de cul i o educe la e icacia de ansducción de los ec o es en
solución.
La inco po ación de los ec o es i ales en los sis emas bina ios algina o-poloxame
e iculados con calcio da luga a cápsulas con pe iles de libe ación condicionados po
su composición y condiciones de p ocesado. De es a mane a, las cápsulas o madas po
algina o solo poseen una es uc u a más compac a en su pa e ex e na. Los sis emas
o mados po algina o y poloxame e iculados a empe a u a ambien e y a 50 ºC
poseen una es uc u a menos compac a. En es os úl imos, el ealiza la e iculación a
una empe a u a supe io a la de geli icación (44.55 ºC) pe mi i ía que los ag egados
micela es de poloxame ac ua an como moldes pa a ob ene sis emas de mayo
amaño de po o.
Los pe iles de libe ación ob enidos de los es hid ogeles con es uc u a a iable se
co elacionan con la e icacia de ansducción obse ada. El pe il de libe ación más

Capí ulo 9
324
con olado, ob enido po los sis emas que con ienen solo algina o, ue el que log ó una
e icacia de ansducción más p olongada. Los sis emas que con ienen poloxame
mos a on un inc emen o de la capacidad de ansducción de los ec o es i ales as
el p ime día de cul i o. Sin emba go, es e e ec o no se obse ó a iempos más
p olongados.
La inco po ación de los dos sis emas con mejo libe ación en el seno de ma ices
polimé icas de bioSiC no log ó un pe il de libe ación adecuado capaz de alcanza unos
ni eles deseados de ansducción (Figu a 9.8). La adición de los ec o es i ales en las
ce ámicas de ca bu o de silicio biomó ico no modi icó la e icacia de ansducción de
los ec o es. Las condiciones de sín esis de los sis emas complejos debe ían de se
modi icadas con el in de alcanza el ni el óp imo de ansducción.
Figu a 9.8 Tinción X-Gal co espondien es a células mad e mesenquimales cul i adas
en monocapa as 1, 5 y 10 días de a amien o con los sis emas compues os hid ogel-
bioSiC con eniendo el ec o i al AAV-lacZ.
325
Discusión gene al
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339
Conclusiones/Conclusions
10. Conclusions
The wo k p esen ed and discussed he ein led o he ollowing conclusions:
1. The bioce amiza ion p ocess employed o he syn hesis o bioSiCs in wo
s eps, py olyza ion and in il a ion wi h liquid silicon o na u al p ecu so s
(pine, oak and sapelli wood) allows h ee ce amic sys ems wi h di e en
p ope ies o po osi y, po e size dis ibu ion, su ace p ope ies and densi y o
be ob ained, which de e mine i s use ulness as bone subs i u es able o load
and elease he apeu ic molecules.
2. All ma e ials showed excellen biocompa ibili y alues when es ed on an
ib oblas cell line (BALB/3T3) and mesenchymal s em cells de i ed om
human bone ma ow. Fu he mo e, he mic os uc u e o ma e ials p omo es
he os eoblas ic di e en ia ion, pa icula ly hose silicon ca bides wi h la ge
po e size (oak and sapelli) in which, a e i een days o cell cul u e, os eocalcin
and os eopon in le els we e simila o hose o cells cul u ed in he p esence
o he os eoblas ic di e en ia ion medium.
Silicon ca bide pa icles p oduced by mechanical wea showed no oxic e ec
and an accep able cellula esponse.

Capí ulo 4
340
Signi ican di e ences in he in e ac ions o bioSiCs and hei espec i e ca bon
p ecu so s in con ac wi h blood componen s we e obse ed acco ding o
hei mic os uc u e and su ace p ope ies. Thus, he hemoly ic esponse is
p ima ily de e mined by he po e size, while clo o ma ion depends on he
su ace oughness and he p o ein adso p ion and complemen ac i a ion on
he speci ic su ace.
3. The an ibio ic loading o he sys ems h ough unspeci ic in e ac ions lead o
mul i laye s o d ug molecules on he su ace o he ma e ial. These
dis ibu ion esul s in a wo phase elease p o ile wi h an ini ial bu s ollowed
by a slowe elease, dependen on he mic os uc u e o he ma e ial selec ed.
The lowe po osi y o oak bioSiC leads o mo e sus ained elease p o iles.
Loaded sys ems a e able o inhibi bac e ial bio ilm o ma ion and e en o ea
an al eady o med bio ilm.
4. The inclusion o VEGF in o he ce amics by ionic in e ac ions leads o
bio unc ional sys ems which main ain he bioac i i y o he p o ein, inc easing
he essel o ma ion in an in i o animal model. The addi ion o he g ow h
ac o achie es an ini ial s imula ion o he di e en ia ion o mesenchymal s em
cells, showing a syne gis ic e ec be ween he p o ein and he ma e ial
opog aphy.
5. The combina ion o bioSiCs wi h wo polyme s, algina e and poloxame , allows
low aqueous solubili y d ugs o be loaded by he d ug inclusion in o he
syn he ic polyme micelles and he hyd ogel di usion con olled elease.
Fu he mo e, he bioSiC mic os uc u e de e mines he medium access and
d ug elease, so ha signi ican di e ences in he an i-in lamma o y e ec o
indome hacin loaded bioSiCs composi es we e obse ed.
341
Conclusiones/Conclusions
6. The mix u e o hese polyme s, algina e and poloxame , has also allowed he
loading and elease o i al ec o s main aining hei ansduc ion e iciency
a e 21 days o assay, especially o hose sys ems wi h close s uc u es. The
inco po a ion o hese hyd ogels in o he ce amic ma ix has no p oduced
sys ems ha achie e adequa e gene ansduc ion, hus equi es u he
op imiza ion wo k.
Taken all oge he , he esul s o his s udy con i m he high po en ial o silicon ca bide
ce amics as bio unc ional sys ems, wi h an applica ion as implan s o he ea men o
a ious bone pa hologies. The la ge numbe o na u al p ecu so s and di e en loading
echniques a ailable, should allow he selec ion o he igh i in each case acco ding o
he equi ed needs.