2021
37
Ja ie A agón Fe nández
Nano ib ous memb anes ob ained
by elec ospinning o bone issue
enginee ing and wound d essing
applica ions
EXTRACTO
Depa amen o
Di ec o /es
Ingenie ía Química y Tecnologías del Medio
Ambien e
I us a Alde e e, Sil ia
Rica do, Ana Aguia
De Ba olo, Lo edana
© Uni e sidad de Za agoza
Se icio de Publicaciones
ISSN 2254-7606
En cumplimien o del a ículo 14.6 del Real Dec e o 99/2011, de 28 de ene o,
po el que se egulan las enseñanzas o iciales de doc o ado, los au o es que
puedan e se a ec ados po alguna de las excepciones con empladas en la
no ma i a ci ada debe án solici a explíci amen e la no publicación del con enido
ín eg o de su esis doc o al en el eposi o io de la Uni e sidad de Za agoza.
Las si uaciones excepcionales con empladas son:
•Que la esis se haya desa ollado en los é minos de un con enio de
con idencialidad con una o más emp esas o ins i uciones.
• Que la esis ecoja esul ados suscep ibles de se pa en ados.
•Alguna o a ci cuns ancia legal que impida su di usión comple a en abie o.
El p esen e documen o es un ex ac o de la esis o iginal deposi ada en el
A chi o Uni e si a io.
Ja ie A agón Fe nández
NANOFIBROUS MEMBRANES OBTAINED BY
ELECTROSPINNING FOR BONE TISSUE
ENGINEERING AND WOUND DRESSING
APPLICATIONS
Di ec o /es
Ingenie ía Química y Tecnologías del Medio Ambien e
I us a Alde e e, Sil ia
Rica do, Ana Aguia
De Ba olo, Lo edana
Tesis Doc o al
Au o
2019
Reposi o io de la Uni e sidad de Za agoza – Zaguan h p://zaguan.uniza .es
UNIVERSIDAD DE ZARAGOZA
[Ex ac o]
Nano ib ous memb anes ob ained by elec ospinning
o bone issue enginee ing and wound d essing
applica ions.
A hesis submi ed o ob ain he deg ee o doc o , p esen ed by
Ja ie A agón Fe nández
Za agoza, 2018

Nano ib ous memb anes ob ained by elec ospinning o bone
issue enginee ing and wound d essing applica ions.
A hesis
P epa ed in he amewo k o
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) o ob ain mul iple
Doc o al deg ees issue by
Uni e sidad de Za agoza, Depa amen o de Ingenie ía Química y Tecnología del Medio Ambien e
Uni e si à della Calab ia, Is i u o pe la Tecnologia delle Memb ane (ITM)
Uni e sidade No a de Lisboa, Facul ade de Ciência e Tecnologia
Supe iso s:
D . Sil ia I us a Alde e e, Associa e Resea ch P o esso , Depa amen o de Ingenie ía Química y
Tecnología del Medio Ambien e, Uni e sidad de Za agoza, Spain
D . Lo edana De Ba olo, Senio Resea che , Is i u o pe la Tecnologia delle Memb ane, I aly
D . Ana Isabel Aguia -Rica do, Full P o esso , Faculdade de Ciências e Tecnologia o Uni e sidade
No a de Lisboa, Po ugal
INDEX
SUMMARY AND OBJECTIVES 1
CHAPTER I 21
In oduc ion 21
I.1 Bioma e ials 23
I.1.1 Bioma e ial classi ica ion 25
I.1.1.1 Me allic bioma e ials 25
I.1.1.2 Ce amic bioma e ials 27
I1.1.3 Polyme ic bioma e ials 29
I.1.1.4 Composi e bioma e ials 31
I.2 D ug deli e y sys em 33
I.3 Wound d essing ma e ials 37
I.3.1 Wound d essing classi ica ion 40
I.4 Tissue enginee ing 47
I.4.1 Bone issue enginee ing 50
I.4.1.1 Classi ica ions o sca olds in bone issue enginee ing 54
I.4.1.1.1 Me allic sca olds in bone issue enginee ing. 55
I.4.1.1.2 Ce amic sca olds in bone issue enginee ing. 55
I.4.1.1.3 Polyme ic sca olds in bone issue enginee ing. 56
I.5 Techniques o p oduce ma e ials o biomedical applica ions 58
Re e ences 63
CHAPTER II 79
Composi e sca old ob ained by elec o-hyd odynamic echnique o in ec ion
p e en ion and ea men in bone epai 79
II.1 In oduc ion 82
Objec i e 84
II.2 P epa a ion o polyme ic pa icles and memb ane 84
II.2.1 PLGA pa icles p oduc ion 84
II.2.2 Elec ospun sca olds p oduc ion 85
II.3 Resul s and discussion 85
II.3.1 Cha ac e iza ion o composi e sca olds 85
II.3.2 MIC and MBC de e mina ion 94
II.3.3 In i o cell s udies 95
II.4 Conclusions 98
Re e ences 99
CHAPTER III 103
Lase - ea ed elec ospun ibe s loaded wi h nano-hyd oxyapa i e o bone issue
enginee ing. 103
III.1 In oduc ion 106
Objec i e 108
III.2 Syn hesis o ino ganic nanopa icles and elec ospun sca olds 108
III.2.1 Syn hesis o hyd oxyapa i e nanopa icles 108
III.2.2 P epa a ion o elec ospun sca olds 108
III.3 Resul s and discussion 109
III.3.1 Hyd oxyapa i e cha ac e iza ion 109
III.3.2 Sca olds cha ac e iza ion 111
III.3.2.1 As spun sca olds 111
III.3.2.1 Lase ea ed sca olds 114
III.3.3 In i o bioac i i y 116
III.3.4 In i o cell mo phology and iabili y 117
III.4 Conclusions 125
Re e ences 126
CHAPTER IV 130
Polyme ic elec ospun sca olds o bone mo phogene ic p o ein 2
deli e y in bone issue enginee ing. 130
IV.1 In oduc ion 133
Objec i e 134
IV.2 Sca old ab ica ion 135
IV.3 Resul s and discussion 137
IV.3.1 Sca old cha ac e iza ion 137
IV.3.2 In i o p o ein elease 141
IV.3.3 In i o enzyma ic deg ada ion 144
IV.3.4 Cell iabili y and mo phology 148
IV.3.5 Os eogenic, os eoinduc i e, and os eoconduc i e ac i i ies o
sca olds 150
IV.4 Conclusions 155
Re e ences 156
CHAPTER V 161
Elec ospun asymme ic memb anes o wound d essing applica ions 161
V.1 In oduc ion 164
Objec i e 166
V.2 Memb ane p epa a ion 167
V.3 Resul s and discussion 167
V.3.1 Memb anes cha ac e iza ion by SEM and FTIR 167
V.3.2 Mechanical p ope ies 171
V.3.3 Fluids handling p ope ies 172
V.3.4 Ca ac ol elease 175
V.3.5 An imic obial p ope ies 177
V.3.6 Cy ocompa ibili y 178
V.3.7 Cell sc a ch assay 181
V.4 Conclusions 183
Re e ences 185
CHAPTER VI 190
GENERAL CONCLUSIONS 190
APPENDIX 1 202
Ma e ials and me hods 202
A.1.1 Ma e ials 205
A.1.2 Physical-chemical cha ac e iza ion 206
A.1.3 In i o s udies in simula ed body luid (SBF) on “Lase - ea ed
Elec ospun ibe s loaded wi h nano-hyd oxyapa i e” (Chap e III) 207
A.1.4 Mechanical p ope ies207
A.1.4.1 Mechanical p ope ies o “Composi e sca old loaded wi h RFP”
(Chap e II) 207
A.1.4.2 Mechanical p ope ies on “Elec ospun asymme ic memb anes”
(Chap e V) 207
A.1.5 Memb anes pe meabili y in “Polyme ic elec ospun sca olds
o bone mo phogene ic p o ein” (Chap e IV) 208
A.1.6 Encapsula ion e iciency 208
A.1.6.1 Encapsula ion e iciency in “Composi e sca old loaded wi h RFP”
(Chap e II) 208
A.1.6.2 Encapsula ion e iciency in “Polyme ic elec ospun sca olds o bone
mo phogene ic p o ein” (Chap e IV) 209
A.1.6.3 Encapsula ion e iciency in “Elec ospun asymme ic memb anes”
(Chap e V) 209
A.1.7 In i o elease s udy and kine ic modeling 209
A.1.7.1 In i o elease s udy and kine ic modeling o “Composi e sca old
loaded wi h RFP” (Chap e II) 209
A.1.7.2 In i o elease s udy and kine ic modeling o “Polyme ic
elec ospun sca olds o bone mo phogene ic p o ein” (Chap e IV) 210
A.1.7.3 In i o elease s udy and kine ic modeling o “Elec ospun
asymme ic memb anes” (Chap e V) 210
A.1.8 D ug elease kine ics 210
A.1.9 Swelling s udies o “Elec ospun asymme ic memb anes” (Chap e V) 212
A.1.10 Wa e apo ansmission o “Elec ospun asymme ic memb anes”
(Chap e V) 212
A.1.11 Biodeg ada ion s udies 213
A.1.11.1 Enzyma ic deg ada ion in “Polyme ic elec ospun sca olds o bone
mo phogene ic p o ein” (Chap e IV) 213
A.1.11.2 Biodeg adabili y in a mimic eal wound en i onmen o “Elec ospun
asymme ic memb anes” (Chap e V) 213
6
RESUMEN Y OBJETIVOS | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
RESUMEN Y OBJETIVOS
Es a esis doc o al se ha ealizado den o del ma co de un acue do de co-
u ela en e la Uni e sidad de Za agoza (Uni e sidad de o igen), la Uni e sidad de
Calab ia (Uni e sidad an i iona) y la Facul ad de Ciencias y Tecnología de la
Uni e sidad NOVA de Lisboa (FCT NOVA) (Uni e sidad an i iona). El abajo de
in es igación se ha lle ado a cabo den o del p og ama de Doc o ado en Ingenie ía
de Memb anas E asmus Mundus (EUDIME), (FPA 2011-0014), inanciado po la
Unión Eu opea.
La esis se cen ó p incipalmen e en el uso de la écnica de elec ohilado pa a
p oduci di e en es ipos de memb anas que puedan se u ilizadas en dis in as
aplicaciones biomédicas. Se sin e iza on y p oduje on nanopa ículas o gánicas e
ino gánicas pa a se u ilizadas como ellenos o como po ado es (sis ema de
adminis ación de á macos), así como memb anas nano ib osas elec ohiladas.
Es e abajo se lle ó a cabo en el Ins i u o de Nanociencia de A agón (INA),
especí icamen e en el g upo de Nanos uc u ed Films and Pa icles (NFP) bajo la
supe isión de la p o eso a Sil ia I us a y la D a. G acia Mendoza. Una pa e
impo an e de la ca ac e ización ísico-química se ealizó en el INA.
En la Uni e sidad de Calab ia se abajó bajo la supe isión de la D a.
Lo edana de Ba olo en el Ins i u o de Tecnología de Memb anas (ITM). Allí se
u iliza on écnicas especí icas an o pa a la ca ac e ización como pa a es udia
di e en es señales biológicas p oducidas po las memb anas sin e izadas, bajo la
supe isión. Po o o lado, la mo ilidad lle ada a cabo en la Facul ad de Ciencias y
Tecnología (FCT NOVA) de la Uni e sidade NOVA (FCT NOVA) bajo la supe isión
de la p o eso a Ana Isabel Aguia -Rica do, pe mi ió ealiza una ca ac e ización
comple a de dos memb anas asimé icas siguiendo di e en es No mas
In e nacionales que es ablecen di e en es ensayos a ealiza en apósi os p ima ios
u ilizados en he idas.
El desa ollo de nue os sca olds ca gados con p o eínas mo ogené icas o
an ibió icos es de g an in e és en el campo de la ingenie ía de ejidos óseos.
Sca olds elec ohilados con una mic opo osidad mejo ada puede se bene icioso

E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | RESUMEN Y OBJETIVOS
7
pa a mejo a la iabilidad celula debido a que una al a po osidad jun o a la
p esencia de mic opo os puede p opo ciona un en o no idimensional (3D) que
no solamen e acili a la siemb a y di usión celula sino ambién p opo ciona una
mejo di usión de los nu ien es y esiduos a a és del sca olds. La adición de
ce ámica de os a o de calcio ha sido ampliamen e in es igada pa a ab ica
sca olds al amen e po osos pa a la ingenie ía de ejidos óseos debido a que
p esen an una composición muy simila al hueso, incluyendo excelen es
p opiedades de biocompa ibilidad, os eoinduc i as y os eoconduc o as. Pa ículas
ca gadas con p o eínas mo ogené icas de hueso dis ibuidas homogéneamen e en
el sca olds pod ían asegu a una libe ación con inua del ac o de c ecimien o
p opo cionando de es a o ma las señales bioquímicas necesa ias pa a la
epa ación y egene ación ósea.
Los sca olds ca gados con an ibió icos pueden p opo ciona una libe ación
sos enida del á maco en el si io de in e és, así como el man enimien o de
p opiedades os eogénicas mejo adas pa a la egene ación exi osa del hueso.
E i ando de es a o ma que se alcancen ni eles de oxicidad o ni eles ine icaces en
la zona de in e és, así como la apa ición de e ec os secunda ios indeseados en los
pacien es que p o ocan un echazo a los a amien os p olongados de á macos
po ía sis emá ica ( ía o al e in a enosa).
O a aplicación biomédica in e esan e de las memb anas elec ohiladas es la
ab icación de apósi os in eligen es e icien es pa a el a amien o de he idas. Pa a
log a una cu ación ápida de la he ida es necesa io desa olla memb anas
ap opiadas con po os in e conec ados capaces de p e eni la deshid a ación
ápida y la pene ación de bac e ias. Pa a man ene un ambien e húmedo en el
lecho de la he ida se necesi a una al a capacidad de abso ción y una adecuada
ansmisión de apo de agua. Además, si la memb ana elec ohilada p esen a
p opiedades bac e icidas acili a á el p oceso de cu ación.
El obje i o p incipal de es a esis ue el desa ollo median e elec ohilado de
memb anas ib osas con las ca ac e ís icas ap opiadas pa a se u ilizadas en la
ingenie ía de ejidos óseos o como apósi o pa a he idas. En los Capí ulos II al V se
plan ean una se ie de obje i os especí icos con el in de cumpli el obje i o
p incipal.
8
RESUMEN Y OBJETIVOS | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Es e documen o de esis se di idió en las siguien es secciones:
 CAPÍTULO I, co esponde a la in oducción gene al donde se
desc iben los concep os de bioma e iales, sca olds, ingenie ía de ejidos y el
obje i o p incipal de los sis emas de libe ación de á macos. Así como, la
clasi icación de los bioma e iales y la ingenie ía de ejidos según el o igen de los
ma e iales. Además se ponen de mani ies o odos los ac o es que deben ene se
en cuen a pa a desa olla y aplica adecuadamen e los apósi os pa a he idas. Se
menciona on las di e en es écnicas u ilizadas en la li e a u a haciendo én asis en
el uso de elec ohilado y elec opul e ización pa a p oduci sca olds o memb anas
pa a su uso en la ingenie ía del ejido óseo y como apósi os pa a he idas.
 CAPÍTULO II, se en oca en el desa ollo y mejo a de andamios 3D
capaces de p omo e una e icien e egene ación ósea jun o con la libe ación de
an ibió icos di igidos pa a p e eni la colonización de bac e ias. El obje i o de es e
abajo ue sin e iza y ca ac e iza un sis ema de libe ación de á macos que
consis e en nano ib as elec ohiladas de policap olac ona (PCL) deco adas con
pa ículas de poli (ácido lác ico-coglicólico) (PLGA) ca gadas con i ampicina
(RFP). Es e ma e ial debe p omo e la epa ación ósea e i ando el de e io o del
sca olds p o ocado po una in ección. Se ealizó la e aluación in i o de la
capacidad bac e icida del ma e ial elec ohilado sin e izado con a bac e ias G am
posi i as (S aphylococcus au eus) y G am nega i as (Esche ichia coli), así como su
ci ocompa ibilidad en cul i os 3D con os eoblas os humanos. Es os esul ados se
en ia on a la Re is a de a macia “In e na ional Jou nal o Pha maceui ics” pa a su
publicación en o ma o de a ículo y es á bajo e isión.
 CAPÍTULO III, se desc ibe la sín esis y ca ac e ización de
memb anas con es uc u a de núcleo-en ol u a de PCL y ace a o de poli inilo
(PVAc) ob enidas po elec ohilado. Las ib as se ca ga on con nanopa ículas de
hid oxiapa i a sin é ica (HAn) pa a aumen a la bioac i idad de los ma e iales. Los
sca olds desa ollados se a a on con ablación láse pa a c ea ca ac e ís icas
opog á icas deseadas a ni el mic omé ico con el obje i o de a o ece la
adhesión y c ecimien o celula .
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | RESUMEN Y OBJETIVOS
9
Todas las memb anas ob enidas p esen a on una es uc u a de po os
idimensionalmen e in e conec ados y el a amien o con láse p o ocó un
aumen o en la iabilidad y densidad celula . Además, el aumen o en la
biocompa ibilidad de los sca olds sugie e que los mic opo os pequeños a o ecen
la adhesión y p oli e ación celula . Es os esul ados ue on publicados en el
a ículo i ulado “Lase - ea ed elec ospun ibe s loaded wi h nano-
hyd oxyapa i e o bone issue enginee ing”. Ja ie A agon, Nu ia Na ascues,
G acia Mendoza, Sil ia I us a. In e na ional Jou nal o Pha maceu ics 525,112–122,
2017. DOI:10.1016/j.ijpha m.2017.04.022.
 CAPÍTULO IV, se e ie e al desa ollo de un sca old elec ohilado
compues o po ib as con es uc u a de núcleo-cubie a de PCL o PCL/PVAc
ca gado con HAn sin é ica. Es as ib as se deco a on con pa ículas de PLGA
ca gadas con p o eína mo ogené ica ósea 2 (BMP2) median e el uso simul aneo
de elec ohilado coaxial y elec opul e ización. El obje i o de es e abajo ue
e alua las p opiedades es uc u ales y ísico-químicas así como el p oceso de
biodeg adación de los nue os sca olds desa ollados y su capacidad pa a abo da
las ca ac e ís icas a qui ec ónicas, bioquímicas y uncionales del ejido óseo. Pa a
es o, se p obó la bioac i idad del sca old median e el cul i o de os eoblas os
humanos sob e ellos y se moni o eo de la iabilidad celula du an e 4 semanas. Se
e aluó la ac i idad os eogénica in i o de las células semb adas sob e los sca olds
de e minando la ac i idad de la os a asa alcalina (ALP) y la exp esión de
os eocalcina (OCN) y os eopon ina (OPN) como p o eínas os eogénicas. Es os
esul ados ue on publicados en el a ículo i ulado “Polyme ic elec ospun
sca olds o bone mo phogene ic p o ein 2 deli e y in bone issue
enginee ing”. Ja ie A agón, Simona Sale no, Lo edana De Ba olo, Sil ia I us a
and G acia Mendoza. Jou nal o Colloid and In e ace Science, 531 (2018) 126–137.
DOI:10.1016/j.jcis.2018.07.029.
 El CAPÍTULO V, desc ibe la sín esis de un apósi o an imic obiano
pa a he idas, con una esis encia mecánica adecuada que es capaz de abso be
exudados y e i a la deshid a ación ápida de una he ida. Se p epa a on
memb anas asimé icas de PCL/PVAc ca gadas con ca ac ol (CRV) median e el
10
RESUMEN Y OBJETIVOS | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
uso simul áneo de elec ohilado y elec opul e ización. Las memb anas cons an de
dos capas; la p ime a es una capa de PCL elec ohilado; la segunda, una lámina de
PVAc que es a ía en con ac o con la piel libe ando a su ez el compues o
an imic obiano. Se demos ó que el uso de di e en es disol en es pueden da luga
a la ob ención de di e en es mo ologías de la capa PVAc-CRV. Los alo es
ob enidos de elongación máxima de las memb anas an es de ompe se son
adecuados pa a se u ilizados como apósi os pa a he idas ya que es án en el
mismo ango epo ado de elongaciones en la piel humana. Las memb anas
p esen an una asa óp ima de T ansmisión de apo de agua (WVTR) con alo es
que se encuen an en el ango eque ido pa a man ene un buen balance en e
humedad y pé dida de agua en la he ida. En la p ime a semana, se libe ó más del
60 % del CRV ca gado, mien as que después de es semanas, las memb anas
libe a on en e el 85 y el 100 % del CRV ca gado median e la con ibución de un
p oceso de di usión de ipo Fickiano y la elajación delas cadenas polimé icas. Las
memb anas sin e izadas son candida as po enciales pa a se u ilizadas como
apósi os pa a he idas. El manusc i o que esume es os esul ados se en ió a la
e is a “Ma e ials Science and Enginee ing C” y es á bajo e isión
(MSEC_2018_3013).
 CAPÍTULO VI, esume las conclusiones gene ales del abajo de
esis.
 APÉNDICE 1, desc ibe las p incipales écnicas de ca ac e ización y
los mé odos pa a e alua di e en es p opiedades en unción de las posibles
aplicaciones.
 APÉNDICE 2, esume los a ículos publicados y la pa icipación en
o os cien í icos du an e el pe íodo de esis.
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | SOMMARIO E OBIETTIVI
11
SOMMARIO E OBIETTIVI
La p esen e esi di do o a o è s a a s ol a in base ad un acco do di co-
supe isione a l'Uni e si à di Sa agozza (Uni e si à di appa enenza),
l'Uni e si à della Calab ia (Uni e si à ospi an e) e la Facol à di Scienze e
Tecnologia dell'Uni e si à NOVA di Lisbona (FCT NOVA) (Uni e si à ospi an e) .
Ques a ice ca è s a a condo a all'in e no del p og amma E asmus Mundus
Doc o a e in Memb ane Enginee ing (EUDIME), (FPA 2011-0014), inanzia o
dall'Unione Eu opea.
Ques a esi si è concen a a p incipalmen e sull'uso della ecnica di
ele o ila u a pe p odu e di e si ipi di memb ane pe applicazioni biomediche.
Sin esi e p oduzione di nanopa icelle ino ganiche e o ganiche da u ilizza e come
ille s o e o i (sis ema di somminis azione di a maci) e p oduzione di
memb ane nano ib ose ele o ila e. Ques o la o o è s a o condo o all'in e no
dell'Ins i u e o Nanoscience o A agon (INA), in pa icola e nel g uppo
Nanos uc u ed Films and Pa icles (NFP) so o la supe isione della P o esso essa
Sil ia I us a e della do .ssa G acia Mendoza. All’in e no dello s esso is i u o INA si
è s ol a una pa e impo an e della ca a e izzazione isico-chimica.
Lo s udio di di e si segnali biologici e l'uso di ecniche speci iche pe la
ca a e izzazione delle memb ane è s a o e e ua o p esso l'Uni e si à della
Calab ia so o la supe isione della do .ssa Lo edana de Ba olo p esso l’Is i u o
pe la Tecnologia delle Memb ane (ITM). In ine, la mobili à s ol a p esso la Facol à
di Scienze e Tecnologia (FCT NOVA) dell'Uni e sidade NOVA (FCT NOVA) so o la
supe isione della p o .ssa Ana Isabel Aguia -Rica do, ha pe messo una
ca a e izzazione comple a di due memb ane asimme iche in acco do a di e si
s anda d in e nazionali sull’esecuzione dei es pe il a amen o p ima io di una
e i a.
Lo s iluppo di nuo i sca old ca ica i con p o eine mo ogene iche o
an ibio ici sono di g ande in e esse nel campo dell'ingegne ia del essu o osseo. Gli

12
SOMMARIO E OBIETTIVI | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
sca old ele o ila i con una maggio e po osi à o po i mic oscopici po ebbe o
esse e u ili pe p omuo e e la i ali à cellula e e la p oduzione di ma ice
ex acellula e. In a i un inc emen o della po osi à e della dimensione dei po i
po ebbe o ni e un ambien e idimensionale age olando la semina/di usione
cellula e e miglio ando la di usione di nu ien i e i iu i a a e so gli sca old.
L’addizione di ce amiche di os a o di calcio è s a a ampiamen e s udia a pe la
abb icazione di sca olds al amen e po osi u ili pe l’ingegne ia del essu o osseo
alla composizione chimica simile al essu o osseo, l’eccellen e biocompa ibili à, le
p op ie à os eoindu i e e os eocondu i e. Una dis ibuzione omogenea delle
pa icelle ca ica e con la p o eina mo ogene ica ossea lungo l'in e o sca old
po ebbe ga an i e un ilascio con inuo del a o e di c esci a, s imolo biochimico
necessa io pe la ipa azione e la igene azione ossea.
Gli sca old ca ica i con an ibio ici possono o ni e un ilascio mi a o e
p olunga o di a maci, e i ando la somminis azione mul i a maco o ale e
endo enosa a lungo e mine, che implica e e i colla e ali ossici, bassa e ogazione
al si o a ge e bassa ade enza al a amen o da pa e del pazien e, ol e a
sos ene e un inc emen o dell’os eogenici à u ile pe la igene azione dell'osso.
Un'al a in e essan e applicazione biomedica delle memb ane ele o ila e è
la ealizzazione di medicazioni in elligen i pe il a amen o delle e i e. Una
gua igione apida delle e i e ichiede lo s iluppo di memb ane app op ia e con
po i in e connessi pe p e eni e la apida disid a azione e la pene azione dei
ba e i. Un'ele a a capaci à di asso bimen o e una asmissione adegua a del
apo e acqueo sa anno necessa i pe man ene e un ambien e umido nel le o della
e i a. Inol e, p op ie à ba e icide miglio e anno il p ocesso di gua igione.
L'obie i o p incipale di ques a esi è lo s iluppo di memb ane ib ose
median e ele o ila u a pe applicazioni nell'ingegne ia del essu o osseo o pe la
medicazione delle e i e. Pe aggiunge e ques i obie i i, sono s a i de ini i di e si
obie i i speci ici, desc i i nei Capi oli da II a V.
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | SOMMARIO E OBIETTIVI
13
Ques o documen o è s a o di iso nelle seguen i sezioni:
 CAPITOLO I, è un'in oduzione in cui engono desc i i i conce i di
bioma e iali, sca old e ingegne ia issu ale e l'obie i o p incipale dei sis emi di
somminis azione di a maci. Inol e, iene desc i a anche la classi icazione dei
bioma e iali e dell'ingegne ia issu ale in base all'o igine dei ma e iali. Vengono
inol e elenca i u i i a o i pe lo s iluppo e l’applicazione di una medicazione.
Sono s a i menziona i di e si ipi di ecniche u ilizza e in le e a u a pe p odu e
sca old o memb ane pe l'ingegne ia del essu o osseo e le medicazioni delle
e i e, concen andosi sull'uso di elec ospinning ed elec osp ay.
 CAPITOLO II, si concen a sullo s iluppo di impalca u e 3D a anza e
in g ado di p omuo e e una igene azione ossea e icien e insieme a ilascio di
an ibio ici mi a i pe p e eni e la colonizzazione dei ba e i. Lo scopo di ques o
la o o è sin e izza e e ca a e izza e un sis ema di somminis azione di a maci
cos i ui o da nano ib e ele o ila e di policap ola one (PCL) deco a e con
i ampicina (RFP) ca ica e in pa icelle di acido poli(la ico-co-glicolico) (PLGA).
Ques o ma e iale p omuo e ebbe la ipa azione ossea e i ando il danneggiamen o
dello sca old a causa dell'in ezione. La capaci à ba e icida del ma e iale
ele o ila o sin e izza o è s a a alu a a in i o con o i ba e i G am posi i i
(S aphylococcus au eus) e G am nega i i (Esche ichia coli), nonché la
ci ocompa ibili à nelle col u e umane di os eoblas i 3D. Un manosc i o con ques i
isul a i è s a o p esen a o all'In e na ional Jou nal o Pha maceu ics ed è in ase di
e isione (Ms. Re . No.: IJP-D-18-01794).
 CAPITOLO III, desc i e la sin esi e la ca a e izzazione delle
memb ane co e-shell di PCL e poli inilace a o (PVAc) o enu e pe ele ospinning.
Le ib e sono s a e ca ica e con nanopa icelle di id ossiapa i e sin e ica (HAn) pe
aumen a e la bioa i i à dei ma e iali. Gli sca old p epa a i sono s a i quindi
a a i median e ablazione lase pe c ea e le ca a e is iche opog a iche su
mic oscala deside a a al ine di a o i e l'adesione e la c esci a delle cellule.
Tu e le memb ane o enu e hanno mos a o una s u u a e icola e dei po i
in e connessa idimensionalmen e e il a amen o lase ha causa o un aumen o
della i ali à e della densi à cellula e. Inol e, un aumen o della biocompa ibili à
14
SOMMARIO E OBIETTIVI | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
degli sca old sugge isce che dimensioni di mic opo i più piccole a o iscono
l'adesione e la p oli e azione cellula e. Ques i isul a i sono pubblica i nell'a icolo
"Lase - ea ed elec ospun ibe s loaded wi h nano-hyd oxyapa i e o bone
issue enginee ing". Ja ie A agon, Nu ia Na ascues, G acia Mendoza, Sil ia
I us a. In e na ional Jou nal o Pha maceu ics 525, 12-122, 2017. DOI: 10.1016 /
j.ijpha m.2017.04.022.
 CAPITOLO IV, si i e isce allo s iluppo di un'impalca u a
ele o ila a composi a di ib e co e-shell PCL o PCL / PVAc ca ica e con HAn
sin e ico. Ques e ib e sono s a e deco a e con la p o eina mo ogene ica ossea 2
(BMP2) ca ica a in pa icelle di PLGA median e ele o il azione simul anea ed
ele o ila u a coassiale. Lo scopo di ques o la o o è di alu a e le p op ie à
s u u ali e isico-chimiche e i p ocessi di biodeg adazione degli sca old
ecen emen e s iluppa i e la lo o capaci à di a on a e le ca a e is iche
a chi e oniche, biochimiche e unzionali del essu o osseo. A ale scopo, la
bioa i i à dello sca old è s a a es a a col i ando gli os eoblas i umani sugli
sca old e moni o ando la i ali à cellula e ino a 4 se imane. L'a i i à os eogenica
in i o delle cellule semina e sugli sca old è s a a esamina a alu ando l'a i i à
della os a asi alcalina (ALP) e l'esp essione delle p o eine os eogeniche
os eocalcina (OCN) e os eopon ina (OPN). Ques i isul a i sono pubblica i
nell'a icolo "Polyme ic elec ospun sca olds o bone mo phogene ic p o ein 2
deli e y in bone issue enginee ing". Ja ie A agón, Simona Sale no, Lo edana De
Ba olo, Sil ia I us a e G acia Mendoza. Jou nal o Colloid and In e ace Science, 531
(2018) 126-137. DOI: 10.1016 / j.jcis.2018.07.029.
 CAPITOLO V, desc i e la sin esi di un ma e iale pe la medicazione
delle e i e an imic obiche, con un'adegua a esis enza meccanica che e i a la
apida disid a azione e l'asso bimen o degli essuda i. Le memb ane asimme iche
PCL / PVAc ca ica e con ca ac olo (CRV) sono s a e p epa a e median e
ele ospinning e ele osp ay simul aneamen e. Le memb ane sono cos i ui e da
due s a i; il p imo è un oglio ele o ila o di PCL; il secondo, un oglio di PVAc che
du an e il con a o con il essu o epide mico do ebbe ilascia e il compos o
an imic obico. L'uso di di e si sol en i de e mina mo ologie di e en i pe lo
s a o PVAc-CRV. I alo i di de o mazione ino a o u a sono nella gamma della
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | SOMMARIO E OBIETTIVI
15
pelle umana, essendo adegua i pe esse e deposi a i su una supe icie della e i a. I
campioni p esen ano alo i di asmissione del apo e acqueo (WVTR)
nell'in e allo ichies o pe man ene e un buon equilib io id ico con la pe di a di
acqua dalla e i a alla equenza o imale. Nella p ima se imana, ol e il 60 % del
CRV ca ica o è s a o ilascia o men e dopo e se imane le memb ane sono s a e
ilascia e a l'85 e il 100 % del CRV ca ica o a a e so una di usione di Fick e una
di usione do u a al ilassamen o del polime o. Le memb ane sin e izza e sono
po enzialmen e candidabili pe le applicazioni di medicazione. Il manosc i o che
iassume ques i isul a i è s a o p esen a o alla Ma e ials Science and Enginee ing
C ed è in ase di e isione (MSEC_2018_3013).
 CAPITOLO VI, iassume le conclusioni gene ali del la o o di esi.
 APPENDICE 1, desc i e le p incipali ecniche di ca a e izzazione e i
me odi pe alu a e le di e se p op ie à in base alle possibili applicazioni.
 APPENDICE 2, iassume gli a icoli pubblica i e la pa ecipazione a
o um scien i ici du an e il pe iodo della esi.
CHAPTER I .................................................................................................................................................................... 21
In oduc ion ............................................................................................................................................................ 21
I.1 Bioma e ials ...................................................................................................................................................... 23
I.1.1 Bioma e ial classi ica ion .................................................................................................................... 25
I.1.1.1 Me allic bioma e ials .................................................................................................................... 25
I.1.1.2 Ce amic bioma e ials ................................................................................................................... 27
I1.1.3 Polyme ic bioma e ials ................................................................................................................ 29
I.1.1.4 Composi e bioma e ials .............................................................................................................. 31
I.2 D ug deli e y sys em .................................................................................................................................... 33
I.3 Wound d essing ma e ials .......................................................................................................................... 37
I.3.1 Wound d essing classi ica ion .......................................................................................................... 40
I.4 Tissue enginee ing ......................................................................................................................................... 47
I.4.1 Bone issue enginee ing ...................................................................................................................... 50
I.4.1.1 Classi ica ions o sca olds in bone issue enginee ing .................................................. 54
I.4.1.1.1 Me allic sca olds in bone issue enginee ing. ........................................................... 55
I.4.1.1.2 Ce amic sca olds in bone issue enginee ing. .......................................................... 55
I.4.1.1.3 Polyme ic sca olds in bone issue enginee ing. ...................................................... 56
I.5 Techniques o p oduce ma e ials o biomedical applica ions ................................................... 58
Re e ences ................................................................................................................................................................ 63

E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
23
I.1 Bioma e ials
The loss o a body pa o an o gan gene a es, besides he loss o unc ion, social
and psychological diso de s. This has de e mined no only he in e es o inding new
ma e ials, bu cons an ly new echnologies a e de eloped o imp o e hem and
p o ide ma e ials ha mee he mos mode n equi emen s in his ield. Fo mo e
han a cen u y, e o s ha e been de o ed o inding ma e ials wi h he app op ia e
cha ac e is ics o sol e he p oblems ela ed o he econs uc ion o issues and
o gans inju ed due o aging diseases o due o auma om acciden s o alls.
The widesp ead de ini ion o bioma e ials was sugges ed by he Ame ican
Na ional Ins i u e o Heal h, "bioma e ial is any subs ance o combina ion o
subs ances, o he han d ugs, syn he ic o na u al in o igin, which can be used o any
pe iod o ime, which augmen s o eplaces pa ially o o ally any issue, o gan o
unc ion, in o de o main ain o imp o e he quali y o li e o he indi idual" (1).
Gold and i o y we e he i s ma e ials used by Egyp ians and Romans as
bioma e ials o eplacemen s o c anial de ec s (1). Amnio ic memb ane (inne mos
laye o he placen a) was he i s biological ma e ial documen ed o i s use as a
su gical ma e ial in skin ansplan a ion wi h be e esul s when compa ed o
xenog a o cada e ic co e ings (2). Polyme hyl me hac yla e (PMMA) is a non-
deg adable polyac yla e and was one o he i s polyme s accep ed o be used in
o hopedics applica ions in he mid-1950s (3). The issue biocompa ibili y o his
polyme became u he appa en when Plexiglas agmen s we e acciden ally
implan ed in eyes and o he body issues o Wo ld Wa II igh e pilo s du ing ai c a
c ashes (3).
The ad ance in mode n medicine has implied he inc ease in human li e and,
hus, a g ea e use o bioma e ials. Nowadays, bioma e ials play an essen ial ole in
medicine acili a ing healing and ees ablishing he main unc ions o issues a e
inju y o disease. The mode n ield o bioma e ials combines no only chemis y,
physics, biology and medicine bu also ecen in luences om ma e ials science,
egene a i e medicine and issue enginee ing (Figu e I.1) (4).
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CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Figu e I.1. Main disciplines ha in eg a e he bioma e ials sciences and enginee ing.
Modi ied om on Recum and LaBe ge (4).
Bioma e ials science and enginee ing is an in e disciplina y esea ch ield ha
equi es knowledge om di e en a eas, which a e men ioned in Table I.1.
Table I.1. Disciplines equi ed o he de elopmen o bioma e ials.
Discipline
Essen ial Knowledge
Basic Science
Physics, chemis y and Biology.
Ma e ials Science and Enginee ing
S uc u e, p ope y, ela ionship o
syn he ic and biological ma e ials including
me als, ce amics, polyme s, composi es, body
issues, design and p o o ype de elopmen ,
applied mechanics, he modynamics, e c.
Medical Science
Composi ion o human body (cells,
issues, o gans and sys ems), ana omy,
physiology, pha macology, immunology and
pa hology wi h some biochemis y,
mic obiology, molecula biology, gene ics, e c.
Clinical Science
Clinical special ies, among which a e
includes: su ge y, o hopedics, maxillo acial,
plas ic and econs uc i e su ge y, den is y,
oph halmology, neu osu ge y, obs e ics and
gynecology, , ca dio ascula su ge y,
e e ina y medicine, e c
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
25
The global ma ke o bioma e ials was a ound 70 billion o USD in 2016 and is
expec ed o each abou 150 Billion o USD by 2021 wi h a compound annual g ow h
a e (CAGR) o 16.0% (5). Acco ding o hei applica ions, he bioma e ials ma ke is
sec ioned in o ca dio ascula , o hopedic, oph halmology, den al, plas ic su ge y,
wound healing, issue enginee ing, neu ological/cen al ne ous sys em, and o he
applica ions (5). The plas ic su ge y and wound healing segmen s and he polyme ic
bioma e ials a e expec ed o g ow a he highes CAGRs du ing he o ecas pe iod. As
well as, polyme hylme hac yla e (PMMA) is expec ed o lead he polyme bioma e ials
ma ke . Among he main companies ha comme cialize bioma e ials we can ind:
Royal DSM (Ne he lands), BASF SE (Ge many), Co bion N.V. (Ne he lands), Co es o
(Ge many), In ibio L d. (U.K.), Ca pen e Technology Co po a ion (U.S.), E onik
Indus ies AG (Ge many), Be keley Ad anced Bioma e ials, Inc. (U.S.), CAM
Bioce amics BV (Ne he lands), and Celanese Co po a ion (U.S.) (5).
I.1.1 Bioma e ial classi ica ion
Bioma e ials can be classi ied acco ding o he na u e o he ma e ial, being he
mos impo an g oups: ce amics, me als, polyme s and composi es. They can be
de ined ei he as passi e bioma e ials (gene ally emain neu al in hei biological
en i onmen and ha e no inhe en powe o ac ion) o ac i e bioma e ials (able o
in e ac wi h hei en i onmen and may e en become an in eg al pa o he body)
wi h di e en bioac i i y (6).
I.1.1.1 Me allic bioma e ials
Me allu gic indus y is able o p oduce a la ge numbe o me als and alloys, bu
only a ew a e biocompa ible and long- e m success ul as an implan ma e ial. The
combina ion o high mechanical s eng h and ac u e oughness makes me als mo e
sui able o load-bea ing applica ions compa ed wi h ce amics o polyme ic
ma e ials. A he beginning, me allic implan s we e de eloped o be used mainly in
bone epai (in e nal ac u e ixa ion o long bones), playing a majo ole in mos
o hopedic de ices, including empo al and pe manen de ices (e.g. bone pla es, pins,
26
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
sc ews and o al join eplacemen s) (7). The main me hods o ob ain hem a e by
cas ing, o ging, p essing, olling and machining. A e wa ds, he me allic implan s
de eloped we e used no only in o hopedic su ge y, bu also in den al and
o hodon ic p ac ice, including oo h illings and oo s. Cu en ly, se e al esea ches
poin o he use o me allic bioma e ials in applica ion o noncon en ional
econs uc i e su ge y o ha d issues/o gans (NiTi shape memo y alloys as ascula
s en s) and o de elop new magnesium-based alloys o bone issue enginee ing and
egene a ion (7).
Chen and Thouas summa ized he cu en s a us and clinical applica ions o he
ou classes o me allic bioma e ial (7): S ainless s eels, Co-based and Ti-based
alloys a e ou inely applied in: empo a y de ices ( ac u e pla es, sc ews, hip nails,
e c.); s em and cup o o al hip eplacemen s wi h cobal –ch omium-molybdenum o
ce amic emo al heads; o al join eplacemen s (w ough alloys); den is y cas ings
and o he pe manen de ices (nails, pacemake s). The las ca ego y co esponds o
"Miscellaneous" and wi hin he e a e di e en alloys such as an alum alloys, used
as a adiog aphic ma ke and wi e su u es o plas ic su ge y and neu osu ge y, bo h
app o ed by US Food and D ug Adminis a ion (FDA); magnesium alloys, ha ha e
been epo ed in di e en pape s as a biodeg adable o hopedic implan s and nickel-
i anium alloys (known as Ni inol), which ha e been FDA app o ed o di e en
applica ions: o hodon ic den al a chwi es, ascula s en s, ena ca a il e ,
in ac anial aneu ysm clips, ca he e guide wi es, o hopedic s aples and con ac ile
a i icial muscles o an a i icial hea .
Despi e all i s ad an ages o bone epai cu en me allic bioma e ials p esen
limi a ions o disad an ages, among which can be highligh ed (8):
 possible elease o oxic me allic ions and/o pa icles h ough
co osion o wea p ocesses, educing biocompa ibili y, leading o he beginning o
he in lamma o y cascades and igge ing issue loss
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
27
 he elas ic moduli a e no well ma ched wi h ha o na u al bone issue,
p oducing s ess shielding e ec s educing he emodeling and s imula ions o new
bone g ow h, comp omising he implan s abili y
 a e essen ially neu al in i o, emaining as pe manen ix u es, causing
a second su gical p ocedu e a e he issue has healed su icien ly o emo e hem
sa ely, such a e he cases o pla es, sc ews and pins used o secu e se ious ac u es.
I.1.1.2 Ce amic bioma e ials
Ce amic ma e ials a e composed o ino ganic, non-me allic subs ances
c ys alline, semi-c ys alline o non-c ys alline (amo phous compounds), glasses and
glass-ce amics (pa ially c ys allized glasses).
Ce amic bioma e ials (Table I.2) can be ca ego ized acco ding wi h he
in e ac ions o a achmen be ween ma e ial and hos issue (bioine o bioac i e).
Fu he mo e, he bioac i e ce amics may be di ided as eso bable o non- eso bable
and hey may be manu ac u ed ei he in g anula es, as coa ings, po ous o dense in
bulk o m (9).
Ce amic bioma e ials ha e been widely used o epai he skele al sys em,
comp ising bone, join s and ee h and o augmen bo h ha d and so issue (10).
Addi ionally, hese ma e ials ha e been used as ca ie s o enzymes, an ibodies,
an igens and as mic oinjec able deli e y sys em o adioac i e iso opes o in si u
ea men o umo s. They a e also used o eyeglasses, diagnos ic ins umen s,
chemical wa e, he mome e s, issue cul u e lasks and ibe op ics o endoscopy (9).
In den is y, ce amics a e used as es o a i e ma e ials (including inlays and onlays),
in gold-po celain c owns, glass- illed ionome cemen s, mul i-uni b idges and
den u es (1,9).

28
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table I.2. Types o Ce amic bioma e ials (9).
In e ac ion wi h
hos issue
P ope ies
Implan –Tissue
Response
Ce amic
Bioine
Non- oxic ma e ial dense
o non-po ous and
biologically inac i e (nea ly
ine )
A ib ous issue o
a iable hickness is
o med and adhe ed
by g ow h in o
su ace i egula i ies
by cemen ing he
de ice in o he
issues o by p ess-
i ing in o a de ec
Aluminum oxide
(single c ys al and
polyc ys alline)
Zi conia (10)
Non- oxic ma e ial po ous
(po e diame e be ween
50-150µm) and biologically
inac i e (nea ly ine )
An ing own occu s
ha mechanically
a aches he issue o
he ma e ial
Aluminum oxide
(polyc ys alline)
Hyd oxyapa i e-
coa ed po ous
me als
Bioac i e
Non- oxic ma e ial
biologically ac i e, dense o
non-po ous, glasses and
glass-ce amics wi h a
eac i e su ace
They connec
di ec ly by chemical
bonding wi h he
issue o ming an
in e acial bond
Bioac i e glasses
Bioac i e glass-
ce amics
Hyd oxyapa i e
(HA)
Non- oxic eso bable
ma e ial biologically ac i e,
dense, non-po ous o
po ous able o dissol e in
biological en i onmen
The su ounding
issue eplaces i
slowly
Calcium sul a e
T icalcium
phospha e
Calcium–
phospha e sal s
The main me hods o manu ac u ing ce amic bioma e ials a e plasma sp aying,
liquid phase sin e ing ( i i ied), hyd o he mal syn hesis and solid s a e sin e ing (9).
In addi ion, he main ad an ages o he use o ce amic bioma e ials a e hei
high biocompa ibili y, high esis ance o comp ession, lowe wea a es, esis ance o
co osion, chemical ine ness, elease o e y low concen a ions o ‘ine ’ wea
pa icles, low he mal and elec ical conduc i i y, easy ob aining and low p oduc ion
cos s (10,11). I s main disad an ages a e associa ed wi h he low impac esis ance
and he di icul ies in p ocessing speci ic geome ic o ms (11).
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
29
I1.1.3 Polyme ic bioma e ials
Polyme s a e he bioma e ials mos used in medicine because hey show se e al
unique p ope ies which make hem use ul in a wide ange o applica ions, such as
ha d and so issue eplacemen s, o hopedics, den al o ca dio ascula de ices (12).
The pola i y, s i ness and o ganiza ion o he polyme ic chains in o e y in e es ing
a chi ec u es a e he main cha ac e is ics o polyme ic bioma e ials (12). These
ma e ials a e o en mechanically weake han o he classes o bioma e ials (me als
and ce amics), because hey a e linked by seconda y in e ac ions, such as hyd ogen
bonding, dipole-dipole in e ac ions and London o ces. None heless, hey can exhibi
physical beha io mo e simila o na i e issue (12).
Polyme s bioma e ials can be classi ied ega ding i s o igin in na u al o
syn he ic. Biodeg adabili y is conside ed he main cha ac e is ic o na u al polyme s,
bu syn he ic polyme s a e mo e a ailable and usually ha e a mo e cos -e ec i e
ab ica ion compa ed wi h he ob aining o na u al polyme s (13). Many esea che s
conside ha na u al polyme s a e inding inc easing applica ions in he a ea o bone
eplacemen and ha d issue augmen a ion bu i s a ailabili y and cos o ob aining is
i s main disad an age (13). Among he mos used na u al polyme s can be ound:
collagen, chi osan, algina e, s a ch, cellulose, hyalu onic acid, elas in, ke a in, silk, e c.
(14,15) . Some o hem ha e a highly desi able o induce apid bone coloniza ion since
hey can p o ide a empla e o biomime ic apa i e o ma ion (15).
The main me hods o p ocess syn he ic polyme s a e he mally induced phase
sepa a ion, po ogen leaching/sol en cas ing, elec ospinning, gas oaming, apid
p o o yping o 3D p in ing a e some o he me hods used o p ocess syn he ic
polyme s (16). Syn he ic polyme s such as, poly( ε -cap olac one) (PCL), poly lac ic
acid (PLA), poly glycolic acid (PGA) and hei copolyme s, e.g. poly(lac ide-co-glycolic
acid) (PLGA) ha e been widely used o c ea e 3D cons uc s o bone egene a ion
due o i s biocompa ibili y and hei ailo able biodeg ada ion (16,17).
30
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
In 2016, Teo and collabo a o s epo ed a lis o syn he ic polyme s app o ed by
he FDA (Table I.3), commonly used as medical implan s (13).
Table I.3. Syn he ic polyme s app o ed by FDA o biomedical applica ions.
Syn he ic Polyme
Biomedical Applica ions
Polye hylene
Anes hesiology, Ca dio ascula , O ola yngology,
Gas oen e ology, U ology, Hema ology, Pa hology,
Neu ology, Oph halmic and O hopedic
Poly e a luo e hylene
Anes hesiology, Ca dio ascula , Gas oen e ology, U ology,
Gene al Su ge y, Plas ic Su ge y, Hema ology, Pa hology,
Neu ology and Oph halmic
Polyamide
Anes hesiology, Ca dio ascula , Gas oen e ology, U ology,
Hema ology, Pa hology, Neu ology Gene al Su ge y, Plas ic
Su ge y and Oph halmic
Polyp opylene
Ca dio ascula , Gas oen e ology, U ology, Gene al Su ge y,
Plas ic Su ge y, Obs e ic and Gynecologic
Polye hylene e eph hala e
Ca dio ascula , Gene al Su ge y and Plas ic Su ge y
Polydime hylsiloxane
Ca dio ascula , O ola yngology, Gas oen e ology, U ology,
Gene al Su ge y, Plas ic Su ge y and Neu ology
Polyhyd oxyalkanoa es
Ca dio ascula , Gas oen e ology, U ology, Neu ology and
O hopedic
Polyme hylme hac yla e
Den al and Oph halmic
Liquid c ys al polyme
O ola yngology and Neu ology
Silicone
O ola yngology, Gas oen e ology, U ology, Gene al Su ge y,
Plas ic Su ge y, Obs e ic and Gynecologic
Pa ylene
O ola yngology, Neu ology
Polyimides
Neu ology
SU-8
Neu ology
Polye he E he Ke one
O hopedic
Polyu e hane
Obs e ic and Gynecologic
Despi e all i s ad an ages and applica ions, polyme ic bioma e ials p esen a low
mechanical esis ance and se e al o hem a e easily biodeg adable. These wo
p ope ies mus be aken in o accoun when de eloping a new bioma e ial (11).
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
31
I.1.1.4 Composi e bioma e ials
Composi e ma e ials we e de ined by William in he Dic iona y o Bioma e ials,
as a "s uc u al ma e ial made o wo o mo e dis inc ly di e en ma e ials, whe e
each componen con ibu es posi i ely o he inal p ope ies" (18). In ou body, many
issues a e conside ed "composi e" wi h an addi ional complexi y due o hei
hie a chical s uc u e, such as ex acellula ma ix (ECM), endons, ligamen s, skin,
and bone, among o he s (19). Gene ally, composi e bioma e ials a e associa ed o
combina ions o wo o mo e componen s om he basic bioma e ials classes, me als,
ce amic and polyme s, a he han a combina ion o ma e ials wi hin he same class
(18). Componen s p esen in a composi e should be dis inguishable a a mic oscopic
le el. Dis ibu ion, con en and in e ac ion o he cons i uen ma e ials oge he wi h
hei physical, chemical and mechanical p ope ies, ha e a s ong epe cussion o e
he inal p ope ies o he composi e bioma e ials (19).
Composi e bioma e ials usually consis o one o mo e discon inuous phases
(commonly known as ille o ein o cing ma e ial) embedded wi hin a con inuous
phase (known as ma ix). The p incipal unc ions o he ille a e o inc ease he
mechanical p ope ies and bioac i i y and change he physical and chemical
p ope ies. Ce amic and glass pa icles as well as ca bon, polyme and glass ibe s
ha e been he main ein o cing ma e ials used in composi e bioma e ials (Table I.4).
Mos composi e bioma e ials used in biomedical applica ions ha e a polyme ic
ma ix (Table I.5), ha can be bio abso bable o no . Ce amic and me allic ma ices
composi es a e used mainly in non-biomedical applica ions, only a ew composi e
bioma e ials ha e a ce amic ma ix, such as calcium phospha e bone cemen s (19).
38
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
No mal wound healing physiological p ocess equi es skin es o a ion ia e-
epi helializa ion and collagen o ma ion (30,31). Se e al ype o d essing a e widely
used o p o ec he wound, o inc ease he speed and quali y o wound healing.
The main cha ac e is ic o a wound d essing is o p o ide he op imum
condi ions o wound healing and, a he same ime, o e p o ec ion o he wound
om in asion by pa hogenic mic oo ganisms and u he auma (31). I is also
essen ial ha he d essing does no in eg a e in o he issue and can be de ached
wi hou causing any auma o he wound su ace du ing d essing changes (32).
The ob aining o an ideal d essing is e y complica ed because no only mus
ake in o accoun he cha ac e is ics o each ype o wound, bu also should mimic he
p ope ies and unc ions o human skin (33):
 P o ec ing he o ganism om i s en i onmen e en while main aining i
in unin e up ed communica ion wi h he en i onmen ;
 P e en ing body luid om escaping and ex e nal luids om pene a ing;
 The mo egula ion;
 Syn hesis ( i amin D and melanin)
 S o age (lipids, melanin and wa e );
 Resis ance o mechanical s ess;
 Abso p ion;
 Exc e ion; e c.)
To selec an app op ia e wound d essing, i is necessa y o ake in o accoun
mul iple ac o s, based on hei unc ional and pe o mance cha ac e is ics (24).
 Deb idemen (wound cleansing): Enhances he mig a ion o leukocy es
in o he wound bed and suppo s he accumula ion o enzymes. Nec o ic issue,
o eign bodies and pa icles p olong he in lamma o y phase and se e as a medium
o bac e ial g ow h.
 P o ide o main ain a mois wound en i onmen : P e en s desicca ion
and cell dea h, enhances epide mal mig a ion, p omo es angiogenesis and connec i e
issue syn hesis, and suppo s au olysis ia he ehyd a ion o desicca ed issue.

E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
39
 Abso p ion. Remo al o blood and excess exuda e: In ch onic wounds,
he e is an excess o exuda e, con aining issue-deg ading enzymes, which block he
p oli e a ion and ac i i y o cells, and b eak down ex acellula ma ix ma e ials and
g ow h ac o s, hus delaying wound healing. The excess o exuda e can also mace a e
he su ounding skin.
 Gaseous exchange (wa e apo and ai ): Pe meabili y o wa e apo
con ols he managemen o exuda e. Low issue oxygen le els s imula e angiogenesis.
Raised issue oxygen s imula es epi helializa ion and ib oblas s.
 P e en in ec ion: p o ec he wound om bac e ial in asion: In ec ion
p olongs he in lamma o y phase and delays collagen syn hesis, inhibi s epide mal
mig a ion and induces addi ional issue damage.
 P o ision o he mal insula ion: No mal issue empe a u e imp o es
he blood low o he wound bed and enhances epide mal mig a ion.
 Low adhe ence. P o ec s he wound om auma: Adhe en d essings
may be pain ul and di icul o emo e, and cause u he issue damage.
 Cos -e ec i e low equency o d essing change: D essing compa isons
based on ea men cos s a he han uni o pack cos s should be made (cos -bene i -
a io). Al hough many d essings a e mo e expensi e han adi ional ma e ials, he
mo e apid esponse o ea men may sa e conside ably on he o al cos .
Global wound d essings ma ke was es ima ed o be alued a 6.3 billion o USD
in 2016, and is expec ed o g ow a a CAGR o 6.0% om 2016 o 2021, o each
a ound 8.5 Billion o USD by 2021 (34). Ad anced wound d essings segmen
egis e ed he la ges ma ke sha e in 2015 and i is expec ed o g ow a a CAGR o
6.9%. Inc easing knowledge ega ding wound ca e managemen , he high g ow h
po en ial o eme ging economies, and he inc easing numbe o acquisi ions by key
companies ha e opened up a wide ange o oppo uni ies o he g ow h o his
ma ke in upcoming yea s. Among he main companies ha ma ke wound d essings
a e Smi h & Nephew plc (U.K.), Aceli y L.P., Inc. (U.S.), In eg a Li esciences
Co po a ion (U.S.), Coloplas Co po a ion (Denma k), 3M Company (U.S.),
40
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
O ganogenesis Inc. (U.S.), Con aTec Inc. (U.S.), Hollis e Wound Ca e (U.S.), Mölnlycke
Heal h Ca e AB (Sweden), and BSN Medical (Ge many) (34).
In he es ima ed global p e alence by wound ype acco ding o he epo om
MedMa ke Diligence, LLC (Figu e I.3), i is expec ed ha he mos equen inju ies in
he wo ld in 2020 will be su gical wounds (abou 450 million o people), ollowed by
diabe ic ulce s (mo e han 60 million o people), enous ulce s (abou 40 million o
people), p essu e ulce s and lace a ions (mo e han 20 million o people) and bu n
wounds (abou 10 million o people) (35). This indica es he impo ance o de eloping
new wound d essings o he e icien ea men o hese ypes o wounds.
Figu e I.3. Es ima e global p e alence by wound ype epo ed by
Ma ke sandMa ke s™ in 2016 (34).
I.3.1 Wound d essing classi ica ion
T adi ionally, wound d essings a e classi ied based on i s na u e o ac ion as:
passi e p oduc s (gauze and ulle d essings, which is he la ges ma ke segmen );
in e ac i e p oduc s (polyme ic ilms and oams ecommended o low exuding
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
41
wounds, which a e mos ly anspa en , pe meable o wa e apo and oxygen bu
impe meable o bac e ia); and bioac i e p oduc s (d essings cons uc ed om
ma e ial which has endogenous ac i i y o d essings able o ac as a d ug deli e y
sys em o bioac i e compounds in wound healing) (36).
In 1999, wound d essing ca ego ies we e eclassi ied by he FDA as ollow (36):
 Non eso bable Gauze/Sponge D essing o Ex e nal Use (s e ile o
nons e ile de ice, o be placed di ec ly on a pa ien ’s wound o abso b
exuda es).
 Hyd ophilic Wound D essing (non- eso bable ma e ials wi h
hyd ophilic p ope ies a ailable in s e ile o nons e ile o m, o co e a
wound and abso b exuda es).
 Occlusi e Wound D essing (non- eso bable ma e ial s e ile o
nons e ile wi h o wi hou an adhesi e backing, in ended o co e a
wound, o p o ide o suppo a mois wound en i onmen and o allow
he exchange o gases such as oxygen and wa e apo h ough he
de ice).
 Hyd ogel Wound and Bu n D essing (non- eso bable ma ix s e ile o
nons e ile made o hyd ophilic polyme s o o he ma e ial in
combina ion wi h a leas a 50 % o wa e , designed o co e a wound, o
abso b wound exuda es, o con ol bleeding o luid loss, and o p o ec
agains ab asion, ic ion, desicca ion and con amina ion).
 In e ac i e Wound and Bu n D essings ( educes he coloniza ion coun ,
exuda es le el, imp o es he wound bed mois u e e en ion, imp o es
he wound collagen ma ix, emo es cellula p oduc s and p o ides
p o ec ion o he epi helializing bed).
A p esen , wound d essing ma ke o e s mo e han 3,000 o d essing p oduc s
egis e ed o sui he needs acco ding o he condi ion o he wound (37) and
acco ding o he base o d essings hey can be di ided in o 8 main ca ego ies (Table
I.7).
42
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table I.7. Wound d essing ca ego ies and i s applica ions.
D essing ca ego ies
Main ea u es
Applica ions
Disad an ages
Gauze and
Imp egna ed Gauze
D essings
Highly pe meable and ela i ely
non-occlusi e, inexpensi e, and
used as a non- ime o sho -
e m use.
Seconda y d essings commonly used
o bo h in ec ed and non-in ec ed
wounds o any size, shape, dep h, o
e iology.
Requi e mo e o ce o emo e
and may lea e esidue o lin
in he wound bed.
Films
Flexible, pe meable o wa e
apo and gas, bu impe meable
o bac e ia and wa e .
Supe icial wounds such as lace a ions,
ab asions, pa ial hickness wounds,
su u ed wounds, and g a dono si es
wi h minimal d ainage. They may also
be used on g anula wounds and a eas
o ic ion.
No o be used on in ec ed
wounds, wounds wi h
mode a e o hea y d ainage
(li le abso p i e capabili ies),
o pa ien s wi h agile skin.
Hyd ogels
Pe meable o gas and wa e ,
p o ide mois u e o d y wounds
and abso bs a minimal amoun
o luid, e ec i e o so ening
escha s.
A ailable in shee s, gels, o
imp egna ed gauzes a e indica ed o
any hickness wounds wi h minimal o
mode a e d ainage. They can dec ease
pain and p o ide padding o dec ease
shea o ces.
Mos hyd ogels a e almos
non-adhesi e, hus equi ing a
seconda y d essing. Should
no be used on in ec ed
wounds and on hea ily
d aining wounds.
Foams
Po ous 3 laye ed polyu e hane
oams able o quickly and
e ec i ely d aw he exuda es
deeply in o he abso ben
ma e ial and eliably hold i
he e. Hyd ophobic ou e laye
p e en s bac e ia om
pene a ing. Middle abso p ion
laye is de ised o e ain
abso bed wound exuda es. The
inne con ac laye is
hyd ophilic wi h speci ically
po es size.
Uni e sally used on wounds wi h
minimal o hea y exuda es, because
hey a e easy o place and p o ide
he mal insula ion. Foams d essing
should be able o quickly and e ec i ely
d aw he exuda es deeply in o he
abso ben ma e ial and eliably hold i
he e. I should also p o ide so co e
o he wound si e, in o de o manage
he wound en i onmen .
Semipe meable oams a e no
indica ed o d y o escha
co e ed wounds because i
can adhe e o he wound
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
43
Table I.7. Con inued
Hyd ocolloids
Hyd ophilic colloidal pa icles wi h a
s ong ilm o a oam adhesi e
backing shee p o ide he mal
insula ion. Impe meable o wa e , gas,
and bac e ia and he e o e can be
e ec i e ba ie s agains u ine, s ool,
and pa hogenic mic oo ganisms.
Indica ed o any hickness
wounds, may be sa ely used on
bo h g anula and nec o ic
wounds. They should only be
used wi h skin sealan s on
pa ien s wi h good skin in eg i y.
O en lea e esidues a e
emo al. No app op ia e o
bleeding o hea ily d aining
wounds. They a e
con aindica ed o in ec ed
wounds and mus be used
wi h cau ion on immuno-
comp omised pa ien s.
Algina es and
Hyd o ibe s
Reac wi h se um and wound
exuda es o o m a hyd ophilic gel o
p o ide a mois wound en i onmen .
They a e highly pe meable and non-
occlusi e.
Ideal d essings o mode a e o
highly d aining wounds ( hey can
abso b up o 20 imes hei
weigh o exuda es). Indica ed o
pa ial and ull hickness d aining
wounds and can also be used o
in ec ed wounds.
They equi e a seconda y
d essing and a e no indica ed
o d y o minimally d aining
wounds and on wounds wi h
exposed endon, capsule, o
bone
Biological
D essings
De i ed om na u al issues, such as
skin o amnion. Gene ally, hey
p o ide and main ain mois wound
en i onmen ha is conduci e o
egene a ion and mig a ion o
ib oblas s and epi helial cells and ac
as a bac e ial ba ie o p o ec he
wound om in ec ion and luid loss.
Dec ease he pain associa ed wi h
open wounds. Some ypes may adhe e
i mly o he wound by ascula
connec ions.
Tempo a y co e age o open and
la ge wounds. Ideal skin
subs i u e o use in acu e and
ch onic wounds (38).
Low a ailabili y and high
p oduce cos . (38) The mos
se ious po en ial liabili y o
biologic wound d essings is
ansmission o in ec ion;
howe e , he ac ual incidence
o such ansmission is
ex emely low.

44
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table I.7. Con inued
Composi e
D essings and
Adjunc i es
Mul iple laye s and each laye is
physiologically dis inc . They
usually ha e 3 laye s: inne
con ac laye (is non adhe en ,
p e en ing auma o he wound
bed), middle laye (abso bs
mois u e and wicks i away o
p e en mace a ion ye
main aining a mois wound bed)
and ou e laye (commonly a
semipe meable ilm, se es as a
bac e ial ba ie ).
Con enien o bo h pa ial and ull
hickness wounds. I can be used as
p ima y o seconda y d essings.
Less lexibili y and mo e
expensi e (30).
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
45
As desc ibed abo e, each d essing a ie y has unique p ope ies, which makes i
sui able o di e en wound condi ions. The disad an age o a d essing o a speci ic
ype o wound may be bene icial o he ea men o ano he wound. Some o he
comme cial p oduc s a e desc ibed in Table I.8 (36). Howe e , he e is s ill no
supe io p oduc ha heals ch onic wounds like enous leg ulce s, diabe ic wound
and p essu e ulce s which o en ail o achie e comple e healing. Hence, de eloping a
d essing ma e ial ha add esses he majo in e e ing ac o s o no mal healing
p ocess will help pa ien s and wound ca e p ac i ione s (30).
Table I.8. Some comme cial d essing ma e ials (36).
D essing
Type
Company
Ma e ial
Bioclusi e®
Film
Johnson & Johnson
PU
Cica-Ca e
Film
Smi h & Nephew
Silicone
Mepi o m®
Film
Mölnlycke Heal h Ca e
Silicone
Mepilex®
Film/ o m
Mölnlycke Heal h Ca e
Silicone/PU
Me ilm®
Film
Mölnlycke Heal h Ca e
PU
Mi a lex®
Film
Mölnlycke Heal h Ca e
PU
Omide m®
Film
Ia o Medical
PU
Opsi e®
Film
Smi h & Nephew
PU
Spy oso b®
Film
B i Cai
PU
Tegade m®
Film/pad/ oam
3M Heal h Ca e
Hyd ocolloid/ac ylic
Bia ain®
Foam/ ilm
Coloplas
PU
PolyMem®
Foam/ ilm
Fe is M g. Co p.
PU
Con ee ®
Foam/ ilm
Coloplas
PU con aining sil e
Ca i-ca eTM
Foam
Smi h & Nephew
Silicone
Lyo oam®
Foam
Se on Heal hca e
PU
Alle yn®
Foam
Smi h & Nephew
PU
Tielle®
Foam
Johnson & Johnson
PU
Ac iso b plus®
Deodo izing
Johnson & Johnson
Ac i a ed cha coal clo h
wi h sil e
Ca bone ®
Deodo izing
Smi h & Nephew
Ac i a ed cha coal clo h
Alione
Bioac i e
Coloplas
Hyd ocolloid
Bio ilm
Bioac i e
Bio ol
Hyd ocolloid
CombiDERM®
Bioac i e
Con aTec
Hyd ocolloid
Com eel Con ou ®
Bioac i e
Coloplas AS
Hyd ocolloid
Cu ino a Hyd o
Bioac i e
Smi h & Nephew
Hyd ocolloid
46
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table I.8. Con inued
Kal oca b®
Deodo izing
Con aTec
Ac i a ed cha coal clo h
wi h algina e
G anuGel® Pas e
Bioac i e
Con aTec
Hyd ocolloid pas e
G anu lex®
Bioac i e
Con aTec
Hyd ocolloid
Tegaso b®
Bioac i e
3M Heal h Ca e
Hyd ocolloid
Duode m®
Bioac i e
Con aTec
Hyd ocolloid
Mesi an®
Bioac i e
Theo Manu ac u ing BV
Hyd ocolloid wi h honey
Aquacel®
Bioac i e
Con aTec
Hyd o ibe
Ve si a®
Bioac i e
Con aTec
Ca boxyme hyl cellulose
Aqua o m®
Bioac i e
Aspen Medical
Hyd ogel
Gelipe m®
Bioac i e
Geis lich Sons L d.
Hyd ogel
G anugel®
Bioac i e
Con aTec
Hyd ogel
In asi e Gel®
Bioac i e
Smi h & Nephew
Hyd ogel
Nu-Gel®
Bioac i e
Johnson & Johnson
Hyd ogel
Pu ilon Gel®
Bioac i e
Coloplas L d.
Hyd ogel
S e igel®
Bioac i e
Se on
Hyd ogel
Vigilon
Bioac i e
Ba d
Hyd ogel
Algisi e®
Bioac i e
Smi h & Nephew
Algina es
Algos e il®
Bioac i e
B o hie
Algina es
Kal os a ®
Bioac i e
Con aTec
Algina es
Melgiso b
Bioac i e
Mölnlycke Heal h Ca e
Algina es
SeaSo b®
Bioac i e
Coloplas AS
Algina es
So bsan®
Bioac i e
Pha ma-Plas L d.
Algina es
Tegagel®
Bioac i e
3M Heal h Ca e
Algina es
Tegagen
Bioac i e
3M Heal h Ca e
Algina es
Mepo e®
T adi ional
Mölnlycke Heal h Ca e
Nonwo en polyes e
ab ic
Deb isan®
Beads
Pha macia and Upjohn
L d.
Polysaccha ide
Iodoso b
Beads
Smi h & Nephew
Polysaccha ide
Iodo lex
Beads
Smi h & Nephew
Polysaccha ide
Ac icoa
Sil e
Smi h & Nephew
High-densi y polye hylene
mesh and nonwo en
ab ic o ayon and
polyes e
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
47
Table I.8. Con inued
P omog an
Fo m
Johnson & Johnson
Collagen/ egene a ed
cellulose
Ky oCel®
Fo m
Aspen Medical
Chi osan ibe s
CeloxTM
Fo m/pad/g anules
Med ade P oduc s L d.
Chi osan
HemCon®
Fo m
HemCon Medical Tech.,
Inc.
Chi osan
Aquano aTM
Pad
MedT ade P oduc s L d.
Chi osan
Beschi in®
Sponge/ ilm
Uni ika Medical P oduc s
Chi in
I.4 Tissue enginee ing
One o he mos de as a ing, cos ly and equen p oblem in human heal h ca e
is he loss o ailu e o an o gan o issue (39). E e y yea , millions o people su e
issue loss o end s age o gan ailu e. Tissue enginee ing is conside ed a new ield
da ing om la e 80's, ha bene i s/ akes ad an age o he p inciples o enginee ing
and biology o he de elopmen o unc ional subs i u es o damaged issue.
In 1988, issue enginee ing was de ined by Y.C. Fung as "an in e disciplina y
ield ha applies he p inciples o enginee ing and he li e sciences owa d he
de elopmen o biological subs i u es ha es o e, main ain, o imp o e issue
unc ion" (17).
In 1993, h ee gene al s a egies we e p oposed o he c ea ion o new issue
(39):
 Isola ed cells o cell subs i u es. I allows he eplacemen o only he
a ec ed cells and pe mi s manipula ion o cells be o e using hem. I s limi a ions
include ailu e o he cells used o main ain hei unc ion in he ecipien and
immunological ejec ion.
 Tissue-inducing subs ances. This app oach no only depends on he
la ge-scale p oduc ion and pu i ica ion o app op ia e signal molecules, bu also on
he de elopmen o me hods o deli e hese molecules (g ow h ac o s, d ugs o any
bioac i e ing edien ) o hei a ge s.
 Cells placed on o wi hin ma ices. The ma ices a e p oduced om
na u al o syn he ic ma e ials (commonly known as sca olds). Immunological
54
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table I.9. Con inua ion
Ac i use
Silica e subs i u ed calcium phospha e.
Bax e
In e na ional
Inc., Dee ield,
IL, USA
Vi oss
Bioac i e glass and calcium phospha e.
O ho i a Inc.
Mal e n, PA, USA
OP-1® Pu y
hBMP-7, Type I bo ine collagen ma ix and
pu y addi i e ca boxyme hylcellulose
sodium.
Olympus
Bio ech,
Hopkin on, MA,
USA
OP-1 Implan
Os eoinduc i e and os eoconduc i e bone
g a ma e ial consis ing o collagen ma ix,
1 g o Type I bo ine collagen, 3.3 mg o
hBMP-7 and 2–3 cc o saline.
I.4.1.1 Classi ica ions o sca olds in bone issue enginee ing
Sca olds used in bone issue enginee ing mus p o ide a s able mechanical
en i onmen and adequa e deg ada ion a e o e a ce ain pe iod o ime in o de o
achie e op imal bone issue ing ow h and egene a ion. In 2013, Henkel and
Hu mache p oposed a no el app oach o design sca olds-based bone issue
enginee ing (Figu e I.6) (69).
Sca olds designed o bone issue enginee ing should p o ide a sui able
mic oen i onmen o he issue egene a ion capaci y, p omo e he c ucial s eps in
he o gan ma u a ion p ocess and issue emodeling (69). Sca olds can be classi ied
acco ding o he na u e o he ma e ial (me als, ce amic and polyme sca olds wi h o
wi hou modi ica ions). Polyme ic sca olds wi h modi ica ions (addi ion o ce amics)
a e he mos used in his ield.

E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
55
Figu e I.6. Ta ge ed design and applica ion o sca olds o bone issue enginee ing.
Images om Se ie Medical A , © 2013 SERVIER. All Righ s Rese ed (55,69).
I.4.1.1.1 Me allic sca olds in bone issue enginee ing.
Me allic sca olds ac mo e like pe manen implan s han sca olding. Pu e
me als like i on, magnesium, i anium, an alum, zi conium and niobium, and di e en
alloys such as, ch ome-cobal , magnesium-calcium, i on-magnesium, i anium-
aluminum- anadium, nickel- i anium and s ainless s eel, ha e been used. Clinical
applica ions as well as hei ad an ages and disad an ages a e discussed in I.1.1.1
sec ion
I.4.1.1.2 Ce amic sca olds in bone issue enginee ing.
Bio-ce amics p ac ically mimic he bone issue and p o ide a highe os eoblas s
adhe ence and p oli e a ion because solubili y and su ace opog aphy ha e a
signi ican in luence in cell beha io compa ed o o he ma e ials. Calcium phospha e
ce amics such as hyd oxyapa i e, icalcium phospha e, biphasic and amo phous
calcium phospha es ha e been g ea ly s udied o bone issue epai in he o m o
sca olding o in combina ion wi h polyme s o me als.
56
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
I.4.1.1.3 Polyme ic sca olds in bone issue enginee ing.
Se e al physiochemical cha ac e is ics (solubili y, po osi y, po e size, enzyma ic
eac ions, biocompa ibili y and alle gic esponse) a e usually mo e con ollable in
polyme ic ma e ials used o design sca olds. Na u al polyme sca olds could be
issue o cell de i ed ha show os eoinduc i e p ope ies. They a e composed mainly
by p o eins (collagen, elas in, gela in, ke a in, ib inogen and silk), polysaccha ides
(cellulose, glycosaminoglycans, amylose, chi in and dex an), polynucleo ides (DNA,
RNA) and ex acellula ma ix .
Table I.10. Mos used polyme ic sca olds in bone epai . Modi ied om Ghassemi e
al (70).
Name
Mechanical
P ope ies
Modi ica ions
Ad an ages
Syn he ic polyme s
PLLA*
+++
HA inco po a ion o enhance cell
g ow h.
- biocompa ible
- biodeg adable
- suppo cell adhesion
PGA*
+++
Alkaline hyd olysis o inc easing
cell eplacemen and cells
bioma e ials in e ac ion
imp o emen .
- biocompa ible
- biodeg adable
- suppo cell adhesion
PCL*
++
- High RGD concen a ion o
inc easing os eoblas a achmen .
- CNT addi ion o mechanical
p ope ies, BMSCs p oli e a ion and
di e en ia ion enhancemen .
- biodeg adable
PLGA*
+
- HA inco po a ion o enhancing
comp essi e s eng h.
- Diamond nanopa icles
inco po a ion o highe mechanical
esis ance.
Inco po a ion o CNTs o highe
a e o cell a achmen ,
p oli e a ion, and di e en ia ion.
- biodeg adable
- suppo cell adhesion
PET*
+++
-
- highly biocompa ible
- biodeg adable
- impac esis ance
PAG
+
-
- biocompa ible
PAA*
+
-
- non-biodeg adable
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
57
Table I.10. Con inued
PBT*
++
-
- highly biocompa ible
- biodeg adable
- impac esis ance
PEG*
+
RGD pep ides o
acili a ing cell adhesion
and sp eading.
- biocompa ible
- s ee ing cells in o sca olds
- osmo ic e ec s in body
PVA
+++
CNT and CNF inco po a ion
o highe concen a ion o
ALP and mine alised
ma ix.
- Non-biodeg adable
- g ea esis ance agains
o ganic sol en s
PPF*
++
linked RGD pep ides o
os eoblas mig a ion
egula ion.
- biocompa ible
- sui able physical p ope ies
and decomposi ion a e
PU
+
-
- a iable deg adabili y
- injec able
Na u al polyme s
Collagen
( ype I, II and
III)
+
- mixing wi h calcium o
mechanical in eg i y
inc ease.
- blending wi h PCL o
mechanical imp o emen .
- biocompa ible
- deg adable
Chi osan
-
Nanoc ys alline
hyd oxyapa i e and SWCNT
inco po a ion o
mechanically and
cy ocompa ibili y
enhancemen .
-
Chi in
+
-
- biocompa ible
- biodeg adable
Algina e
+
Addi ion o HA, calcium
phospha e cemen s,
bioglass and o he na u al
and syn he ic polyme s o
upg ading cell adhesion
and mechanical p ope ies.
- biocompa ible
- deg adable
- minimally in asi e manne
(gel- o ming)
- ease o chemical
modi ica ion wi h adhesion
ligands and con olled
elease o issue induc ion
ac o s (e.g., BMP, TGF-β)
Mechanical p ope ies: +++ good, ++ a e age, + poo
FDA app o ed: *
58
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
On he o he hand, biocompa ible and biodeg adable syn he ic polyme s such as
PCL, PLA and PGA ha e been he mos u ilized in bone issue enginee ing o hei
excellen mechanical p ope ies and i s low cos o ob aining. Bu some polyme s lose
hei comp essi e s eng h due o apid deg ada ion in i o (poly(p opylene
uma a e), gene a ing a local acidic en i onmen which can cause ad e se issue
esponses. Table I.10 shows some o he mos used polyme ic sca olds modi ied and
hei ad an ages.
I.5 Techniques o p oduce ma e ials o biomedical applica ions
Bioma e ials may be designed and ab ica ed using all kind o ma e ials:
polyme s, ce amics and me als, as well as hei combina ions (71). In he li e a u e,
he e a e di e se me hods o p oduce bioma e ials o medical applica ions,
speci ically o wound d essing applica ions and bone issue enginee ing (71–73).
Sale no and Ne i summa ized some o hem (F eeze d ying, Pa icles sin e ing, Phase
in e sion, Solid ee o m ab ica ion, Re e se empla ing, Sp aying, Tex iles,
Emulsion, Gas oaming, Biop in ing, Mic o luidic, Sel -assembly) (71).
The design o an app op ia e bioma e ial depends no only on i s speci ic
medical applica ion bu also on he cha ac e is ics o he pa icula pa ien . I is e y
di icul o desc ibe speci ic cha ac e is ics o ake in o accoun in such de elopmen s,
bu any de ice used should be able o main ain i s unc ionali y du ing all he accu a e
ime wi hou ejec ion and sa ely.
On he o he hand, o p epa e 3D po ous sca olds, he mos used me hods a e
ibe bonding, comp ession molding, ex usion, high in e nal phase emulsion
empla ing, emulsion eeze d ying, sol en cas ing/pa icula e leaching, high
p essu e p ocessing, supe s uc u e enginee ing, supe c i ical luid p ocessing, gas
oaming/pa icula e leaching, he mally induced phase sepa a ion, elec ospinning
and apid p o o yping (46,74).
Elec ospinning is a e sa ile, simple, scalable and cos -e ec i e spinning
echnique o p oduce sca olds o memb anes in o m o ibe s wi h a andom
o ien a ion o wi h a ce ain deg ee o alignmen (Figu e I.7). Cu en ly, se e al
na u al (hyalu onic acid, collagen, algina e, cellulose, chi osan, gela in, pullulan, zein,
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
59
silk ib oin) o syn he ic (polyamide, polycap olac one, polylac ic acid, poly (lac ic-co-
glycolic acid), poly inyl alcohol, polyu e hane, poly (e hylene-co- inil alcohol),
polys y enee c, e c.) (75,76) polyme s a e elec ospun and he ibe s ob ained by
hese echnique ha e been used in a ious applica ions such as issue enginee ing
sca olds, wound d essing and in nume ous biomedical applica ions (75–77).
Figu e. I.7. Schema ic diag am o a ypical e ical se up o elec ospinning
appa a us.
Elec ospinning echnique is go e ned by elec os a ic o ces o p oduce ibe s
wi h di e en diame e s om nanome e o mic ome e (76,77). To p oduce an
elec ospun ma e ial polyme solu ions a e pumped h ough a needle unde an
elec ic ield gene a ed by a ol age sou ce. When he elec ic ield applied o he
needle eaches a c i ical alue and he epulsi e elec ical o ces o e come he su ace
ension o ces, a Taylo cone is o med on he needle ip (Figu e I.7) (76,78–81).
Sol en e apo a ion o he polyme s solu ion occu s be ween he needle ip and he
collec o , lea ing a solid polyme in ibe o m on he collec o (76,77,82–86).
Se e al pa ame e s mus be aken in o accoun o p oduce elec ospun ma e ial,
such as solu ion p ope ies, p ocess pa ame e s, and en i onmen al condi ions

60
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
(76,86,87). All o hem a ec he p ocess in a di e en way, Table I.11 summa izes
hese e ec s.
Table I.11. E ec s and esul s o di e en pa ame e s on elec ospun mo phology
(76,83).
Pa ame e s
E ec on ibe mo phology
Resul s
Solu ion pa ame e s
Polyme
concen a ion
Di ec ly p opo ional.
Inc ease in polyme
concen a ion p oduce an
inc ease in ibe diame e .
Molecula weigh
(Mw) o polyme
Di ec ly p opo ional.
Numbe o beads and d ople s
a e educed wi h high Mw.
Viscosi y
Di ec ly p opo ional. Viscosi y,
polyme concen a ion and Mw
a e s ongly ela ed.
Op imal iscosi y ange is needed
o p oduce ibe s.
Su ace ension
I is no clea .
Ins abili y o je s is ob ained
when su ace ension is high.
Conduc i i y/su ace
cha ge densi y
In e sely p opo ional.
Dec ease in conduc i i y induces
an inc ease in ibe diame e .
P ocessing pa ame e s
Applied ol age
In e sely p opo ional.
Inc ease in ol age cause an
dec ease in ibe diame e .
Feed a e/Flow a e
Di ec ly p opo ional.
A dec ease in low a e gene a es
a dec ease in ibe diame e .
Pa icles can be ob ained i he
low dec eases conside ably.
Tip o collec o
dis ance
A minimum dis ance is
necessa y o acili a e he
sol en e apo a ion and
p oduce homogeneous ibe s.
Too la ge and oo small dis ance
can gene a e beads o ma ion.
Ambien pa ame e s
Humidi y
I mus con olled
A high humidi y can esul in
beads o ma ions and small
ci cula po es can appea on he
su ace o he ibe .
Tempe a u e
In e sely p opo ional.
Inc ease in empe a u e cause a
dec ease in ibe diame e .
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
61
Sca olds ob ained by elec ospinning o bone issue enginee ing ha e ecei ed
a special a en ion, due o i s high po osi y (> 90 % po osi y) and i s simila i ies wi h
he s uc u e and mo phology o he na i e ECM (85). A success ul deli e y o
di e en ia ion and p oli e a ion ac o s (g ow h ac o s, such as, BMP-2 and BMP7)
(88) wi h he elec ospun s uc u es composed by biodeg adable polyme illed o
imp egna ed wi h calcium phospha e could sol e he exigency o bone issue
enginee ing (89).
On he o he hand, elec ospun memb anes ha e shown a g ea capabili y o
wound d essing due o he ex emely high su ace a ea, allowing adjus men o he
wound mois u e. The elec ospun ma e ials p ese e he wound om bac e ial
in ec ions due o hei small po e size. The high po osi y allows a good exchange o
oxygen and wa e apo (75–77,82–88,90–94).In he elec ospinning p ocess, i is
also possible o load an ibio ics and an imic obial agen s o ob ain con olled deli e y
sys ems o one o mul iple bioac i e ac o s (20,76,88,94–99). Fu he mo e, hese
sys ems ha e he po en ial o be used as wound d essing ma e ials, bone issue
enginee ing sca olds, augmen a ion de ices, and an imic obial il e s
(29,30,34,37,75,100,101).
Mic o and/o nanopa icles can be ob ained by elec osp aying echnique, using
he same equipmen and go e ned by he same p inciple o elec ospinning (87,88).
Loaded and unloaded pa icles can be ob ained in a single s ep p ocess by
elec osp aying wi hou he use o any su ac an (87,93,102,103). Fo his pu pose,
he polyme solu ion should ul ill ce ain cha ac e is ics ela ed o low a e, polyme
concen a ion, iscosi y, and polyme molecula weigh Mw. On he o he hand, he
use o highly ola ile sol en s oge he wi h he app op ia e needle-collec o dis ance
could help he pa icles syn hesis(93,103–105). Pa icles ob ained by elec osp ay
ha e a highe encapsula e ion e iciency compa ed wi h o he pa icles p oduc ion
echnique (88,103).
Elec ospinning/Elec osp ayed echniques allow ob aining The polyme
solu ion can inco po a e mo e han jus one polyme and also o he ma e ials such as
62
CHAPTER I | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
d ugs, biomolecules, essen ial oils o ino ganic pa icles (dissol ed o dispe sed) wi h
he objec i e o ob ain an adequa e ma e ial o a speci ic applica ion (88). Wi h only
one needle i is possible o ob ain an app op ia e sca old o memb ane o bone
issue enginee ing o wound d essing. The use o coaxial o iaxial needles also he
simul aneous use o bo h echniques allows design e y speci ic ma e ials wi h
co e/shell s uc u es o biomedical applica ions (17,20,46,75–77,83–
85,87,88,90,93,94,96–98,105–129,129–136).
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER I
63
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133. Kim TG, Lee DS, Pa k TG. Con olled p o ein elease om elec ospun
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CHAPTER II
Composi e sca old ob ained by elec o-hyd odynamic echnique
o in ec ion p e en ion and ea men in bone epai
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107
machining is an e icien echnique o p epa e pa e ning s uc u es in elec ospun
polycap olac one (PCL) memb anes (10,11). I was ound ha elec ospun poly(l-
lac ide) sca olds wi h lase abla ed holes exhibi signi ican be e endo helial cell
ing ow h (5). McCullen e al. (12) used lase abla ion on elec ospun PCL sca olds
o a ou he adhesion and g ow h o human adipose-de i ed s em cells.
Elec ospun PCL/β-TCP ibe s we e also ea ed by lase i adia ion o ob ain
mic osized po es (13). Beside he meaning ul cellula ac i i y he ma e ial showed
enhanced comp essi e s eng h.
Na u ally occu ing polyme s a e well sui ed o a ious in i o applica ions,
p omo ing cell adhesion and g ow h. Howe e , sca olds ab ica ed om hese
polyme s could exhibi poo mechanical p ope ies o as deg ada ion a es.
Na i e p o eins, such as collagen and ib inogen, a e also mo e expensi e and mo e
di icul o sou ce han syn he ic polyme s (14). Among he syn he ic a ailable
polyme used o ab ica e h ee-dimensional (3D) sca olds alipha ic polyes e s
undoub edly ep esen so a he mos ex ensi e s udied class, since hey combine
good physico-chemical and mechanical p ope ies wi h assessed biocompa ibili y
(15). Pa icula ly PCL is a low cos , biocompa ible polyme ha has a slow
deg ada ion a e and dis inc heological and iscoelas ic p ope ies ha make i
sui able o speci ic long e m implan a ion (14). I s compa ibili y wi h a wide
ange o d ugs enables uni o m d ug dis ibu ion in he ma ix and i s long e m
deg ada ion acili a es d ug elease up o se e al mon hs (16). Poly inyl ace a e
(PVAc) polyme has also been applied in many medical ields because o i s s ong
biocompa ibili y. The hyd ogels con aining unc ional g oups such as COOH usually
show good biocompa ibili y wi h blood, body luids, and issues (17). This ine
polyme has he ad an age ha i does no induce a dele e ious eac ion in li ing
issue (18). Because o all hese cha ac e is ics PVAc has been used in many
medical ields, including d ug and cell ca ies and in issue enginee ing (19).
One impo an limi a ion in he use o syn he ic biodeg adable polyme s as
sca old ma e ials is he lack o bioac i i y, in pa icula o bone issue
applica ions (20). The main app oach o de elop bio eso bable and bioac i e
sca olds is he addi ion o bioac i e ma e ials o he polyme ma ix. Calcium

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CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
phospha e ce amics ha e been ex ensi ely in es iga ed o ab ica e highly po ous
sca olds o enginee bone due o hei nea simila composi ion o bone, including
excellen biocompa ibili y, os eoinduc i e and os eoconduc i e p ope ies (3). I
has been ound ha he addi ion o hyd oxyapa i e (HA) no only enhances he
ensile s eng h o PCL sca olds bu also ac s as a chela ing agen o accele a e he
mine aliza ion o human e al os eoblas cells o o m bone-like apa i e o bone
issue enginee ing (21).
Objec i e
The main objec i e o his wo k was o ob ain a ma e ial wi h app op ia e
ma ix a chi ec u e o a o cells adhesion and p oli e a ion composed o
biodeg adable polyme s, wi h mechanical esis ance, good biocompa ibili y and
high bioac i i y. PCL/PVAc and PCL/PCL memb anes wi h co e-shell s uc u e
loaded wi h syn he ic hyd oxyapa i e nanopa icles (HAn) o inc ease he
bioac i i y o he ma e ials we e ob ained by elec ospinning. In addi ion, he
memb anes ob ained we e ea ed by lase abla ion o c ea e desi ed mic oscale
opog aphical ea u es in o de o a o cell adhesion and g ow h.
III.2 Syn hesis o ino ganic nanopa icles and elec ospun sca olds
III.2.1 Syn hesis o hyd oxyapa i e nanopa icles
Syn hesis o HA (Ca10(PO4)6(OH)2) nanopa icles was conduc ed as
p e iously desc ibed (22). CaCO3 was used as calcium sou ces, main aining a Ca/P
a io o 1.67 du ing eac ion in solu ion wi h 0.3 M H3PO4.
III.2.2 P epa a ion o elec ospun sca olds
Elec ospun sca olds we e p epa ed using an Y low 2.2.D-500 elec ospinne
(Coaxial Elec ospinning Machines/R&D Mic oencapsula ion, Malaga, Spain). PCL
pelle s we e dissol ed in DCM/DMF (1:1) and PVAc was dissol ed in DMF, hese
solu ions we e s i ed o e nigh a oom empe a u e. To p epa e PCL-HAn
sca olds, HAn powde was dispe sed in DCM/DMF wi h he help o TWEEN® 80
by s i ing o e nigh a oom empe a u e, hen his solu ion was added o he PCL
solu ion and s i ed o e nigh a oom empe a u e. The polyme s solu ions we e
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER III
109
loaded in o 20 mL plas ic sy inges. The PCL-HAn suspension was ed h ough he
inne needle o he coaxial sys em and PCL o PVAc solu ions we e ed h ough he
ou e one. This las needle was connec ed o he posi i e ol age powe supply, a a
ol age anging om 7 o 13 kV. The shell and co e low a es and he spinning
dis ance we e ixed o 0.5 mL/h (in bo h needles) and 19 cm espec i ely. The
spun ibe s we e collec ed on a s a ic pla e (co e ed wi h aluminum oil)
connec ed o nega i e ol age powe supply, a a ol age anging om −2 o 4 kV
o 18 h. To c ea e mac opo es in he ib ous memb ane s uc u es, a pulse
Nd:YAG lase (TECHNOLOGY Q-Swi ch) was used. Di e en condi ions we e used
o each ma e ial: ou pu powe : 16 W, wa eleng h: 1064 nm o he PCL-HAn/PCL
ibe s and ou pu powe : 1 W, wa eleng h: 532 nm o PCL-HAn/PVAc ibe s.
The cha ac e iza ion echniques and di e en e alua ion me hods a e
desc ibed in Appendix 1.
III.3 Resul s and discussion
III.3.1 Hyd oxyapa i e cha ac e iza ion
The XRD pa e n o he syn hesized HA nanopa icles is shown in Figu e
III.1a, i could be seen ha all he peaks could be indexed o he hexagonal phase
hyd oxyapa i e (JCPDS N° 09-0432). The di ac og am do no show any o he
peaks co esponding o seconda y phases o in e media e compounds sugges ing
he o ma ion o pu e HAn phase. In ense di ac ion peaks wi h b oad wid h a e
indica i e o he c ys alline na u e o he p epa ed ma e ial and he small
c ys alli e size espec i ely (23). Inse o Figu e III.1a shows he peaks
co esponding o he (002) and (211) di ac ion planes o hyd oxyapa i e.
Acco ding o he JCPDS ca d 09-0432, he e e ence in ensi y a io o (211), (002)
is RI(211)/RI(002) = 2.61. The calcula ed deg ee o ex u e index (24) o he
syn hesized hyd oxyapa i e is 0.51 indica ing a p e e ed g ow h o ien a ion along
he c-axis (25). These esul s a e consis en wi h elec onic mic oscope images
(Figu e III.1b and III.1c), which show ib ous needle-like pa icles wi h mean
diame e s o 20 nm and leng h o app oxima ely 150 nm. The od-like shape o he
nanopa icles is mo e clea ly seen in TEM images (Figu e III.1c). The FTIR
spec um (Figu e III.1.d) o he ma e ial p esen s he cha ac e is ic bands o PO43-
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CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
appea ing a 472, 572 and 603 cm−1 ela ed o he asymme ic bending, and signals
assigned o symme ic and asymme ic s e ching posi ioned in he 900-1200 cm−1
ange (26). The b oad peak a ound 3455 cm−1 is due o he adso bed wa e on HAn
s uc u e and he abso p ion peak a 1635 cm−1 is a ibu ed o he bending mode
o OH− g oups (27). The abso p ion peak assigned o apa i e hyd oxyl bond is
obse ed a 3569 cm−1 (28). The shoulde a 878 cm−1 oge he wi h he double a
1415 and 1458 cm−1 indica e he exis ence o CO32− (29) p obably coming om he
a mosphe e ca bon dioxide du ing sample p epa a ion and would ha e been
inco po a ed in o he HAn c ys al s uc u e (30). All hese esul s imply ha he
syn hesis o od-like shaped hyd oxyapa i e nanopa icles was success ul.
Figu e III.1. Cha ac e iza ion o he syn hesized HAn: a) XRD, b) SEM images, c)
TEM mic og aphs, d) FTIR spec um.
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111
III.3.2 Sca olds cha ac e iza ion
III.3.2.1 As spun sca olds
The ma e ials p epa ed by elec ospinning a e lis ed in Table III.1 oge he
wi h posi i e and nega i e ol ages applied. Due o he p ope ies ( iscosi y,
su ace ension, conduc i i y) o he solu ions he ol ages equi ed o ob ain a
s able Taylo cone we e highe o p oduce ibe s con aining hyd oxyapa i e han
he ol ages used o he p is ine polyme ibe s (Table III.1).
Table III.1. P epa a ions condi ions and cha ac e iza ion esul s o he ob ained
elec ospun sca olds.
Sample (Co e/Shell)
PCL/PCL
PCL-HAn/PCL
PCL/PVA
c
PCL-HAn/PVAc
Applied ol age -/+ (k )
3.11/10.25
3.54/12.00
2.87/7.21
3.03/8.57
HAn loada (w %)
-
(14.2) (13.8)
-
(11.9) (12.7)
Fibe diame e b (nm)
206±62
210±64
264±56
225±47
Po osi y (%)
62.0
63.5
65.5
58.6
Po es diame e (nm)
232
210
209
188
a F om TGA esul s, heo e ical alue be ween b acke s.
b Ob ained by measu ing a leas 100 ibe s in di e en SEM images.
The HAn load in he ibe s was measu ed by he mog a ime ic analysis
(TGA, Figu e III.2) as he esidue a e comple e polyme s elimina ion a 600 °C
(Table III.1). The expe imen al alues o 13.8 and 12.7 w % o PCL-HAn/PCL and
PCL-HAn/PVAC espec i ely, we e close o he heo e ical ones (13.2 and
11.9 w %). The HAn load used was he maximum possible o ob ain a s able Taylo
cone; highe amoun o nanopa icles in he spinning solu ion inc ease excessi ely
he iscosi y and also hinde good nanopa icles dispe sion.
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CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Figu e III.2. TGA he mog am o PCL-HAn/PCL and PCL-HAn/PVAc ibe s.
Figu e III.3. XRD pa e ns o PCL/PCL, PCL/PVAc, PCL-HAn/PCL and PCL-
HAn/PVAc ibe s.

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113
XRD pa e ns o ibe s wi h HAn a e shown in Figu e III.3. The XRD
di ac g am o PCL-Han/PCL has peaks o bo h hyd oxyapa i e and PCL. Bo h he
polyme and ce amic e ain hei c ys alline beha io in he elec ospun ibe s
indica ing insigni ican change in hei c ys al s uc u e. This con i ms he ac ha
HAn is inely and uni o mly dispe sed in he ma ix (3). In PCL-HAn/PVAc pa e n
peaks a 25 and 35° es ablish he p esence o hyd oxyapa i e inco po a ed in he
sca old (inse o Figu e III.3).
Figu e III.4. SEM images o a) PCL/PCL ibe , b) PCL-HAn/PCL ibe s.
FE-SEM images o he elec ospun sca olds showed uni o m, beadless and
nano-scaled ib ous s uc u es andomly o ien ed o all he p epa ed ma e ials
unde he op imum spinning condi ions u ilized in each case. As an example Figu e
III.4a) shows SEM mic og aphs ob ained o ibe s wi h PCL in he co e and in he
shell. Hyd oxyapa i e nanopa icles a e mos ly encased inside he ibe s, bu some
agglome a es a e obse ed on he su ace (Figu e III.4b). The ibe s a e age
diame e was measu ed om SEM images (Table III.1). The p esence o
hyd oxyapa i e nanopa icles in he inne solu ion does no seem o ha e any
e ec on he PCL/PCL ibe diame e . I was p e iously obse ed o PCL ibe
wi hou de ec s, such as he ob ained in his wo k, ha o HA load highe han
5 w % he a e age diame e was simila o he p is ine ibe s (31). This esul o
b)
a)
50 µm
3 µm
500 nm
50 µm
3 µm
500 nm
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CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
high mass ac ion was a ibu ed o he inc eased iscosi y o he dispe sion
du ing elec ospinning. Fo he PCL/PVAc ma e ial he p esence o hyd oxyapa i e
leads o a sligh ly lowe ibe s mean diame e , p obably due o he highe ol age
needed o ob ain a s able cone in he case o he hyd oxyapa i e loaded ibe s (32).
The co e-shell s uc u e o he PCL/PVAc ibe s was in es iga ed by TEM
mic oscopy as shown in Figu e III.5a. The PVAc polyme ( he anspa en pa o
he ou side s uc u e o he ibe ) is he shell w apping uni o mly he PCL ibe
( he da ke pa o he inside s uc u e o he ibe ) as he co e. A TEM mic og aph
o a PCL/PCL ibe was added in he inse o compa ison pu pose, as expec ed
co e and shell a e indis inguishable in his case. The dis ibu ion o he HA
nanopa icles inside ibe s can be obse ed in Figu e III.5b, as men ioned be o e
mos o he pa icles a e encapsula ed in o he polyme s.
Figu e III.5. TEM images o a) PCL/PVAc ibe , inse PCL/PCL ibe , b) PCL-
HAn/PVAc ibe .
III.3.2.1 Lase ea ed sca olds
A e lase i adia ion he sca olds we e analyzed by SEM mic oscopy
(Figu e III.6). Fo bo h ma e ials he lase ene gy was no su icien o machine a
hole h ough he elec ospun ibe s. The images show ha he memb ane was no
signi ican ly a ec ed ou side he lase i adia ion a ea. Figu e III.6a shows SEM
200
nm
200
nm
200
nm
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER III
115
images o PCL-HAn/PCL abla ed sca olds, he mic opo es we e well ob ained wi h
diame e s in he ange 70–120 μm. I is impo an o obse e he mo phology o
he emaining ibe s since he aim is o imp o e he po osi y wi hou any
addi ional e ec (33). E en when some mel ing and coalescence o he ibe s can
be obse ed a ound he d illed holes, he su ace mo phology is only sligh ly
changed. Fo PCL-HAn/PVAC on he o he hand, he po es diame e a e in he 50–
90 μm ange and he change in he ibe s mo phology a ound he holes is mo e
impo an . Besides, in his ma e ial no all he holes o he ec angula pa e n
we e p oduced unde i adia ion. As men ioned in he expe imen al sec ion
di e en lase we e used in o de o ge he po es in each ma e ial.
Figu e III.6. SEM images and po e size dis ibu ion o lase ea ed a) and c) PCL-
HAn/PCL and b) and d) PCL-HAn/PVAc.
116
CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
III.3.3 In i o bioac i i y
Figu e III.7. SEM images o he p epa ed ma e ials a e imme sion in SBF
solu ion du ing di e en imes.
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123
Figu e III.13. Con ocal mic oscope images o sca olds su ace (le ) and iabili y
( igh ) o human os eoblas s seeded in PCL-HAn/PVAc sca olds no ea ed
(abo e) and ea ed wi h lase (below) a e 14 days o cell seeding. Scale ba s
100 μm.
P e ious s udies ha e epo ed he biocompa ibili y o PCL elec ospun 3D-
sca olds in a human os eoblas in i o model in which he high po osi y enabled
cells o pene a e in o he sca old (38). The g a ing o collagen and chond oi in
sulpha e on modi ied su ace PCL po ous sca olds syn hesized by pa icula e
leaching signi ican ly inc eased he in i o p oli e a ion o mu ine chond ocy es
ou weeks a e seeding hough po osi y was no al e ed (39) while chemically
c oss-linked PCL and HAn nanopa icles used o ab ica e nanocomposi e sca olds
loaded wi h he g ow h ac o BMP-2 also showed e y good cy ocompa ibili y in a
abbi bone ma ow s em cells in i o model (40), suppo ing PCL sui abili y as
biomedical ma e ial. In addi ion, he ab ica ion o PCL sca olds ea ed wi h a

124
CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
em osecond lase o c ea e po es, and he e o e o modi y he sca old su ace and
po osi y, has shown enhanced cellula ac i i ies compa ed o hose sca olds wi h
he same po e size and no ea ed wi h lase (41), indica ing ha lase ea men
may signi ican ly imp o e he po en ial o hese ypes o sca olds in bone
egene a ion as ou s udy shows. Though i is con o e sy ega ding he “ideal”
size po e o biomedical applica ions, in os eo egene a ion mos au ho s ha e
poin ed o 100–400 μm as ecommended o acili a e cell adhesion and g ow h
(42). Howe e , smalle po es a e able o inc ease sca olds su ace and lead o
highe cell a achmen while la ge po es acili a e cell mig a ion (41). Ou esul s
showed ha lase pulse on PCL-HAn/PVAc sca olds implied he o ma ion o
mic opo es o 50–90 μm while on PCL-HAn/PCL we e sligh ly la ge (70–120 μm).
These da a, oge he wi h he appa en ly highe cell densi y eco ded in PVAc
con aining sca olds, sugges ha smalle mic opo e sizes made easie cell
adhesion and p oli e a ion.
The inco po a ion o HAn in he syn hesis p ocess o poly(L/DL)-lac ide (43)
o polylac ic acid (PLA) (44) elec ospun sca olds o biomedical applica ions has
been epo ed in o de o imp o e cell a achmen and p oli e a ion. Since his
e ec is no clea in Figu es III.9-III.11, he cell iabili y 14 days a e seeding
NHOs on sca olds wi h and wi hou HAn was measu ed by image quan i ica ion
(Figu e III.12). Resul s show ha cell iabili y on samples wi h HAn a e in he
ange o he ones wi hou i . Besides, hey suppo ou obse a ions poin ing o a
highe iabili y o cells seeded on PVAc con aining sca olds, exe ing signi ican
di e ences among PCL-HAn/PVAc g oup ( ea ed and no ea ed wi h lase ) s
PCL/PCL and PCL-HAn/PCL g oups. Lase ea ed sca olds also displayed highe
iabili y pe cen ages han he no ea ed ones highligh ing he lase ea men as
an imp o emen o cell a achmen and iabili y.
To ou knowledge, his is he i s ime ha he inco po a ion o PVAc o PCL
elec ospun sca olds and ea ed wi h lase o enhance hei sui abili y in
biomedical applica ions has been shown hese p omising e ec s ega ding human
os eoblas s adhesion and p oli e a ion, poin ing o i s po en ial applica ion in bone
epai app oaches.
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125
III.4 Conclusions
Rod shaped hyd oxyapa i e nanopa icles we e success ully syn hesized and
inco po a ed in o co e-shell PCL/PCL and PCL/PVAc elec ospun nano ibe s. Only
HAn loaded ibe s p esen ed hyd oxyapa i e p ecipi a ion a e been soaked in SBF
o 14 and 30 days. The p esence HAn pa icles would be necessa y o he apa i e
o ma ion on he nano ibe su ace. Fibe s wi h PVAc shell su e some s uc u al
change bu hey s ill exhibi ed a well in e connec ed po e ne wo k s uc u e. E en
when human os eoblas s g ow h was obse ed on all seeded su ace, he lase
ea men o he su aces no ably inc eased he sca olds biocompa ibili y
obse ing highe iabili y and cell densi y. This e ec was mo e impo an on PCL-
HAn/PVAc sca olds wi h 50–90 μm mic opo es han on PCL-HAn/PCL (po es o
70–120 μm) sugges ing ha smalle mic opo e sizes a o cell adhesion and
p oli e a ion.
126
CHAPTER III | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
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CHAPTER IV
Polyme ic elec ospun sca olds o bone mo phogene ic p o ein
2 deli e y in bone issue enginee ing.
The con en s o his chap e ha e been adap ed om he ollowing published wo k:
Polyme ic elec ospun sca olds o bone mo phogene ic p o ein 2 deli e y in bone
issue enginee ing. Ja ie A agó n, Simóna Sale nó, Ló edana De Ba óló, Sil ia I us a and
G acia Mendóza. Jóu nal ó Cóllóid and In e ace Science, 531 (2018) 126–137.
DOI:10.1016/j.jcis.2018.07.029.
“The de elopmen o no el sca olds based on biocompa ible polyme s is o g ea
in e es in he ield o bone epai o ab ica ion o biodeg adable sca olds ha mimic he
ex acellula ma ix and ha e os eoconduc i e and os eoinduc i e p ope ies o enhanced
bone egene a ion.
Polycap olac one (PCL) and polycap olac one/poly inyl ace a e (PCL/PVAc) co e–
shell ibe s we e syn hesised and deco a ed wi h poly(lac ic-co-glycolic acid) [PLGA]
pa icles loaded wi h bone mo phogene ic p o ein 2 (BMP2) by simul aneous
elec ospinning and elec osp aying. Hyd oxyapa i e nano ods (HAn) we e loaded in o he
co e o ibe s. The ob ained sca olds we e cha ac e ized by scanning and ansmission
elec on mic oscopy, Fou ie - ans o m in a ed spec oscopy, and he mog a ime ic
analysis. The in i o po en ial o hese ma e ials o bone egene a ion was assessed in
biodeg ada ion assays, os eoblas iabili y assays, and analyses o exp ession o speci ic
bone ma ke s, such as alkaline phospha ase (ALP), os eocalcin (OCN), and os eopon in
(OPN).
PLGA pa icles we e homogeneously dis ibu ed in he en i e ibe ma . The g ow h
ac o load was 1.2–1.7 µg/g o he sca old whe eas he HAn load was in he 8.8–12.6 w %
ange. These sca olds we e able o suppo and enhance cell g ow h and p oli e a ion
acili a ing he exp ession o os eogenic and os eoconduc i e ma ke s (OCN and OPN).
These obse a ions unde line he g ea impo ance o he p esence o BMP2 in sca olds o
bone emodeling as well as he good po en ial o he newly de eloped sca olds o clinical
use in issue enginee ing.”
CHAPTER IV ................................................................................................................................................................... 130
Polyme ic elec ospun sca olds o bone mo phogene ic p o ein 2 deli e y in bone
issue enginee ing. ................................................................................................................................................... 130
IV.1 In oduc ion ...................................................................................................................................................... 133
Objec i e ...................................................................................................................................................................... 134
IV.2 Sca old ab ica ion ........................................................................................................................................ 135
IV.3 Resul s and discussion .................................................................................................................................. 137
IV.3.1 Sca old cha ac e iza ion .................................................................................................................... 137
IV.3.2 In i o p o ein elease ........................................................................................................................ 141
IV.3.3 In i o enzyma ic deg ada ion ........................................................................................................ 144
IV.3.4 Cell iabili y and mo phology .......................................................................................................... 148
IV.3.5 Os eogenic, os eoinduc i e, and os eoconduc i e ac i i ies o sca olds ....................... 150
IV.4 Conclusions ........................................................................................................................................................ 155
Re e ences ................................................................................................................................................................... 156
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139
was a sligh ly highe concen a ion in he shell, he luo escen compound was almos
uni o mly dis ibu ed inside he mic opa icles. No signi ican phase sepa a ion o he
polyme and/o d ug was obse ed.
HAn was also cha ac e ized by means o he SEM and TEM (Figu e IV.3). These
images con i med ou p e ious esul s (4), in which ib ous needle-like pa icles wi h a
od-like shape we e also seen (leng h and diame e o app oxima ely 150 and 20 nm,
espec i ely).
Figu e IV.3. SEM (a) and TEM (b) images o syn hesized HAn.
Once he pa icle syn hesis was op imized, he simul aneous elec osp ay-
elec ospinning p ocess was ca ied ou o gene a e he inal sca olds o HAn-loaded
PCL/PVAc ibe s deco a ed wi h PLGA pa icles (Figu e IV.4). The op imized
elec osp ay-elec ospinning p ocess p oduced ibe s wi h uni o m mo phology and a
andom dis ibu ion ha led o he c ea ion o a sca old wi h po osi y o ≈60–64%.
Because o he simul aneous syn hesis, PLGA pa icles wi h a mean diame e o 1.2 ± 0.5
µm we e homogeneously dis ibu ed in he en i e ibe ma . I is no ewo hy ha he
a achmen o pa icles on o ibe s did no in luence ibe mo phology (Figu e IV.4).
Howe e , some o he pa icles sligh ly de ia ed om he sphe ical shape (Figu es IV.2
and IV.4). The mean diame e o ibe s (380 ± 108 nm) o ming he sca olds was in he
ange o collagen ibe s ( om 50 o 500 nm), mimicking he nanos uc u es o he

140
CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
na u al ECM. SEM images clea ly e ealed he o ma ion o sca olds wi h an
in e connec ed ne wo k o la ge po es wi h size be ween 0.9 and 2.5 µm o PCL and
PVAc sca olds and smalle po es anging om 0.2 o 0.6 µm. The mean po e size was
1.14 µm o PVAc and 1.46 µm o PVAc:PLGA-BMP2 owing o he p esence o a
nano ib ous s uc u e ha con e s mic opo osi y on he sca old (Figu e IV.4). This
s uc u e p o ides a la ge su ace a ea- o- olume a io o cell a achmen as well as
su icien po osi y o nu ien di usion. Indeed, he measu ed hyd aulic pe meance
was 8.00 ± 0.30 and 6.60 ± 0.03 L/h m2 mba o PVAc and PCL sca olds, espec i ely.
An inc ease in hyd aulic pe meance by 16% was obse ed in he PVAc:PLGA-BMP2
sca olds (9.5 ± 0.4 L/h m2 mba ) wi h espec o he non-loaded sca old.
Figu e IV.4. SEM images o he syn hesised elec ospun ib es (le ) deco a ed wi h
BMP2-loaded PLGA elec osp ayed pa icles ( igh ).
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141
TGA analyses (Figu e IV.5) con i med HAn loads o 13.8 and 12.7 w % o PCL and
PVAc ibe s, espec i ely, in ag eemen wi h ou p e ious esul s (4). In PCL:PLGA-
BMP2 and PVAc:PLGA-BMP2 sca olds, owing o he p esence o he PLGA polyme , he
HAn load was educed o 12.6 and 8.8 w %, espec i ely. Howe e , BMP2 encapsula ion
e iciency was simila be ween he wo o mula ions, eaching pe cen ages o 39% ± 5%
o PCL:PLGA-BMP2 and 40% ± 11% o PVAc:PLGA-BMP2. A inal BMP2 concen a ion
o 39 and 40 µg/ml was achie ed in he sca olds, in line wi h he clinical equi ed dose
(21). These simila e iciency a es led o g ow h ac o loads o 1.2 and 1.7 µg/g in PCL
and PVAc composi e ibe s, espec i ely.
Figu e IV.5. TGA he mog am o he ou ypes o sca olds syn hesized: PCL,
PVAc,PCL:PLGA-BMP2 and PVAc:PLGA-BMP2.
IV.3.2 In i o p o ein elease
The kine ic p o ile o he BMP2 elease om bo h kinds o sca olds was
de e mined in he cou se o 28 days (Figu e IV.6a) and he mo phology o PVAc:PLGA-
BMP2 sca olds a e he elease o 28 days was also s udied unde he SEM (Figu e
IV.7).
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CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Figu e IV.6. BMP2 elease kine ic cu es ob ained o bo h ypes o sca olds (a) and
hei ma hema ical adjus men (b).
The pa e n o he g ow h ac o elease om bo h sca olds showed a s ong
ini ial bu s wi hin he i s 8 h, ollowed by a slow elease un il he end o he
expe imen . This beha io should syne gis ically enhance bone gene a ion because i
was sugges ed ha he ideal BMP2 elease s a egy includes bo h an ini ial bu s and a
subsequen sus ained elease. The eason is ha he o me helps o ec ui
os eop ogeni o cells o he deli e y sys em and he la e p omo es os eogenic
di e en ia ion (22). The bu s elease eached ≈ 68 % o he BMP2 load o PCL:PLGA-
BMP2 and only 40 % o PVAc:PLGA-BMP2. The di e ence could be due o he
abso p ion capaci y o PVAc (350 % wa e abso p ion in 24 h (23)) ha may e ain he
p o ein eleased om he PLGA pa icles du ing he swelling p ocess. I is impo an o
poin ou ha he hyd ophilici y o bo h memb anes is de e mined by he p esence o
HA, which u ns bo h sca olds in o highly hyd ophilic ma e ials. SEM images o sca olds
(Figu e IV.7) a e he elease (28 days) con i med his pa e n: he e we e no PLGA
pa icles on he su ace o he sca olds o only a ew damaged ones.
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143
Figu e IV.7. SEM images o PVAc:PLGA-BMP2 sca olds be o e and a e elease o 28
days.
The kine ic ma hema ical models we e i ed o he expe imen al esul s o he
elease (Table IV.2, Figu e IV.6b). P elimina y calcula ions (no shown) indica e ha he
Higuchi model was no sa is ac o y o i ing o he expe imen al elease da a. Gi en
ha some o hese models should be used only o he i s 60% o he elease, only he
da a ep esen ing he i s 24 h we e i ed because i was he elease om PCL:PLGA-
BMP2 achie ed a ha ime (24). The bes i solu ion was iden i ied by e alua ing
coe icien o co ela ion R2. The highes alues o he coe icien indica ed ha bo h
samples we e be e desc ibed by he Peppas–Sahlin elease kine ic model (Figu e
IV.6b). In his model, e m k1P n ep esen s he Fickian di usional con ibu ion o he
elease (F), whe eas e m k2 2n is he case-II elaxa ional con ibu ion (R). The nega i e
alues ob ained o k2 should be in e p e ed in e ms o a elaxa ion mechanism being
insigni ican compa ed o he di usion p ocess (25). In acco dance wi h he Peppas–
Sahlin equa ion, he alue o exponen n o he Fickian elease mechanism om a
polyme ic sys em wi h sphe e geome y should be ≈0.43 (26). The lowe alues ound
o PCL and PVAc samples may be ela ed o he wide pa icle size dis ibu ion
men ioned be o e (27).
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CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
Table IV.2. Release modeling pa ame e s.
Model
Pa ame e
pa ame e s
PCL:PLGA-
BMP2
PVAc:PLGA-
BMP2
Ko smeye -Peppas
k1K
0.42
0.21
n
0.12
0.18
R2
0.77
0.89
Peppas-Sahlin
k1P
0.50
0.23
k2
-0.11
-0.04
n
0.30
0.38
R2
0.91
0.98
IV.3.3 In i o enzyma ic deg ada ion
An ideal sca old o bone issue enginee ing should be biodeg adable and
bio eso bable while being able o suppo he g ow h o new bone. The deg ada ion
beha io o bioma e ials in physiological en i onmen s plays an impo an ole in he
enginee ing p ocess o a new issue. The g ea es ad an age o deg adable polyme s
such as PCL, PVAc, and PLGA is ha hey a e b oken down in o biologically accep able
molecules ha a e me abolized and emo ed om he body ia no mal me abolic
pa hways (28). To in es iga e he e ec o enzymes on he deg ada ion beha io o he
syn hesized sca olds, hey we e incuba ed in phospha e-bu e ed saline in he p esence
o lipase o lysozyme o di e en pe iods a he same concen a ions as hose ound in
human se um. The deg ada ion was ollowed by g a ime ic measu emen s (Figu e
IV.8). PVAc and PCL sca olds mani es ed simila beha io in he p esence o lysozyme,
hus eaching deg ada ion o 10 % ± 1 % and 13 % ± 1 %, espec i ely, a e 60 days.
This alue o PCL-based ibe s was highe han he one expec ed based on he esul s o
Bane jee el a . (29). These au ho s did no ind any signi ican PCL ilm deg ada ion in
he p esence o lysozyme. The g ea e deg ada ion obse ed in ou PCL sca olds could
be explained by he ib ous s uc u e and high po osi y ha inc ease he su ace a ea
exposed o he enzyme solu ion. In he p esence o lipase, he deg ada ion p o ile o all
sca olds was simila o ha obse ed in o he s udies, showing a signi ican weigh loss
wi h ime, which eached alues ≈ 90 % a e 60 days (29).

E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER IV
145
Figu e IV.8. Enzyma ic deg ada ion o memb anes by lipase (a) and lysozyme (b).
Table IV.3. A ea a io in lipase and lysozyme deg ada ion media.
SAMPLE
A ea a io
C=O/ PO4-3
C-O-C/PO4-3
PCL
2.9
0.7
PCL_LIPASE
0.5
0.2
PCL_LYSOZYME
2.2
0.6
PCL:PLGA-BMP2
2.1
0.6
PCL:PLGA-BMP2_LIPASE
0.2
0.1
PCL:PLGA-BMP2_LYSOZYME
1.9
0.6
PVAC
2.4
1.8
PVAC_LIPASE
1.7
1.7
PVAC_LYSOZYME
2.2
2.0
PVAC:PLGA-BMP2
2.1
1.4
PVAC:PLGA-BMP2_LIPASE
2.3
2.0
PVAC:PLGA-BMP2_LYSOZYME
2.2
1.6
The e ec s o lipase and lysozyme on he PCL we e co obo a ed by in a ed (IR)
spec oscopy (Figu e IV.9). Bands a 1725 and 1240 cm1, ela ed o C=O and C-O-C
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CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
bonds, espec i ely, sligh ly changed a e enzyma ic deg ada ion. Besides, he a ios
be ween signals assigned o PO43 (1040 cm1), owing o he p esence o HAn, and he
abo e-men ioned polyme bands indica e a mino change in sample polyme con en
a e he enzyma ic a ack (Table IV.3). E en when i is known ha PVAc can be
deg aded by mic oo ganisms, especially ilamen ous ungi (30,31), o ou knowledge,
he e is no published s udy abou PVAc deg ada ion by lysozyme. The mass dec ease
obse ed in he PVAc sca old may be ela ed only o he PCL deg ada ion.
Figu e IV.9. FTIR spec a o PCL (a, b) and PVAc (c, d) sca olds loaded (b, d) and no
loaded (a, c) wi h PLGA-BMP2 pa icles a e enzyma ic deg ada ion wi h lipase and
lysozyme.
In he p esence o lipase, he ibe s wi h PVAc in he shell showed lowe
deg ada ion han did he ibe s consis ing only o PCL (Figu e IV.8a). A e 40 days, PCL
los almos 75% o he mass, while PVAc sca olds los only 29% o hei mass, as
expec ed because PCL can be hyd oly ically and enzyma ically deg aded by lipase
h ough hyd olysis o he es e bond (32). On he o he hand, each lipase has dis inc
speci ici y owa ds he side chain hyd olysis o PVAc (33). The polyme backbone is no
hyd olyzed, and only he side chains con aining es e linkages unde go hyd olysis
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147
ca alyzed by lipase. The smalle mass loss o PVAc ibe s could be due no only o he
smalle amoun o PCL in he sample (48 and 86 w % o PVAc and PCL samples,
espec i ely) bu also o he epo ed educ ion in he deg ada ion o PCL wi h he
addi ion o PVAc in PCL–PVAc blends (34). PCL deg ada ion was also con i med by IR
spec oscopy (Figu e IV.5a), which e ealed ela i e enhancemen o a band a 1040
cm1 associa ed wi h HAn, he eby implying a polyme mass dec ease.
On he o he hand, he addi ion o PLGA pa icles o he ibe s sligh ly changed he
deg ada ion o PVAc sca olds in he p esence o lysozyme and did no a ec he
beha io wi h lipase. PLGA pa icles syn hesized om di e en polyme o mula ions
may possess di e en deg ada ion a es because he a e age molecula weigh and
lac ide:glycolide a io a ec he di usion a e and pe meabili y o he PLGA polyme ic
ma ix, which consequen ly a ec he deg ada ion a e (35). PLGA (75:25) equi es
mo e han 56 days o lose ≈ 8 % o i s mass in he p esence o lysozyme (36). The
polyme used in his wo k (50:50) is a mo e deg adable o mula ion because o he
p e e en ial deg ada ion o he glycolic acid domain owing o i s highe hyd ophilici y
(37). Howe e , i is equen ly used o encapsula e lysozyme, and he elease in PBS
akes place ia di usion h ough p e-exis ing po es and channels in he polyme ma ix
(38). This ac sugges s ha he deg ada ion o PLGA 50:50 should be e y slow, and
he e o e he elease o he emaining p o ein (32 % o PCL:PLGA-BMP2 and 60 % o
PVAc:PLGA-BMP2) may be sus ained long- e m while PLGA pa icle deg ada ion
p oceeds. This p ocess may enable a BMP2 elease in he nanog am ange as epo ed o
ake place du ing in i o no mal bone egene a ion (39) as well as du ing success ul in
i o bone epai in an expe imen al model (40). As a consequence, he p epa ed sca old
may mani es bo h equi ed elease ypes (an ini ial bu s ollowed by a sus ained
elease) o enhance bone egene a ion.
I has been ex ensi ely s udied and p o en ha biodeg ada ion o HAn is e y
limi ed. Howe e , due o he highly os eoconduc i e na u e o HAn, i s use in bone g a
subs i u es is c ucial o imp o ing he cellula esponse and he mine aliza ion p ocess
(41).
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CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
IV.3.4 Cell iabili y and mo phology
Human os eoblas s seeded on o he di e en ypes o he newly de eloped
sca olds adhe ed and sp ead, hus o ming (a e 2 weeks) a laye ha co e ed he
su ace as a esul o hei p oli e a ion (Figu es IV.10, IV.11 and IV.12). The nano ib ous
s uc u e o sca olds mimicking he a chi ec u e o he ECM a o ed adhesion and
p oli e a ion o os eoblas s as well as he main enance o hei pheno ype.
Figu e IV.10. Cell iabili y o os eoblas s seeded in a 2D sys em and on o PCL and PVAc
sca olds, deco a ed and no deco a ed wi h PLGA-BMP2 pa icles, a di e en ime
poin s. Mean ± SD (9 samples). Di e ences be ween g oups we e conside ed signi ican
when p < 0.05.
MTT assays (Figu e IV.10) showed cell iabili y and p oli e a ion du ing he
expe imen s o up o 4 weeks a e seeding. Os eoblas seeding in a 2D en i onmen
(di ec ly on o a cul u e pla e well) yielded sligh ly as e g ow h a e 2 and 3 weeks, as
compa ed o he abso bance eco ded in he i s week, and dec eased again a e 4
weeks. This dec ease is consis en wi h sa u a ion o he g ow h su ace, p e en ing cell
p oli e a ion and dec easing cell iabili y. Fu he mo e, he esul s ob ained clea ly
show he signi ican di e ences in cell iabili y and p oli e a ion be ween 2D and 3D
E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME) | CHAPTER IV
155
IV.4 Conclusions
I has been p e iously epo ed ha elec ospun PCL nano ibe s loaded wi h HA
p omo e apa i e o ma ion, whe eas he p esence o PVAc in he ibe shell inc eases
hyd ophilici y and a o s os eoblas adhesion and p oli e a ion (4). Gi en ha BMP2
could be an al e na i e way o inc ease os eogenic ac i i y (8), in his wo k, hose ibe s
we e decó a ed wi h PLGA‐BMP2 pa icles ób ained by elec ósp aying o
os eoinduc i e and os eoconduc i e pu poses in bone egene a ion. Fibe s and pa icles
kep hei size and s uc u e a e he pa icles we e elec osp ayed, hough HAn loading
was sligh ly lowe (≤ 3.9 %) when PLGA was p esen in he samples. BMP2 was
success ully loaded in o PLGA pa icles, and hei loading and ac i i y a e
elec osp aying we e con i med, yielding g ów h ac ó lóads up ó 1.7 μg/g in
PCL:PLGA-BMP2 and PVAc:PLGA-BMP2 sca olds wi h a inal BMP2 concen a ion o 39
and 40 µg/mL, espec i ely; a mid- ange concen a ion can use bone de ec s wi hou
ad e se e ec s (53). The BMP2 elease was 40–68 %, being lowe o PVAc-con aining
sca olds; his phenomenon may be a ibu ed o hei abso p ion capaci y. Enzyma ic
deg ada ion o he sca olds highligh ed hei good biodeg ada ion p o ile, suppo ing
hei good po en ial o bone egene a ion. In addi ion, os eoblas iabili y and
p oli e a ion inc eased when he FDA-app o ed g ow h ac o BMP2 was p esen in he
sca olds; he exp ession o bone o ma ion and ma u a ion ma ke s was also imp o ed
by BMP2. Taken oge he , hese esul s sugges ha BMP2 is a key ac o o he
po en ial imp o emen o PCL/PVAc sca olds. Besides, he combina ion o PCL, PVAc,
and PLGA-BMP2 is a no el and p omising he apeu ic app oach o bone epai .

156
CHAPTER IV | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
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CHAPTER V
Elec ospun asymme ic memb anes o wound d essing
applica ions
The con en s o his chap e ha e been adap ed om he ollowing submi ed wo k
(unde e iew):
Elec ospun asymme ic memb anes o wound d essing applica ions. Ja ie
A agó n, Cla inda Cós a, Isabel Cóelhósó, G acia Mendóza, Ana Aguia -Rica dó and Sil ia
I us a. Ma e ials Science and Enginee ing C (MSEC_2018_3013)
“To accomplish a apid wound healing i is necessa y o de elop an asymme ic
memb ane wi h in e connec ed po es consis ing o a op laye ha p e en s apid
dehyd a ion o he wound and bac e ia pene a ion and a sub-laye wi h high
abso p ion capaci y and bac e icidal p ope ies. Polycap olac one (PCL)/poly inyl
ace a e (PVAc) asymme ic memb anes loaded wi h he bac e icidal mono e pene
ca ac ol (CRV) we e syn hesized and cha ac e ized by scanning elec on
mic oscopy and Fou ie ans o m in a ed spec oscopy. Mechanical p ope ies in
d y and we condi ions and luid handling beha io we e also assessed. In addi ion,
biological s udies ega ding hei bac e icidal e ec s, cy ocompa ibili y and wound
closu e p ope ies we e also de eloped. Loading e iciencies o 40-50 % we e
achie ed in he p epa ed samples and 85-100 % o he loaded CRV was eleased in
simula ed wound pH e olu ion medium. The signi ican inhibi ion o G am nega i e
(Esche ichia coli S17) and G am posi i e (S aphylococcus au eus ATCC 25923)
bac e ia g ow h clea ly showed he sui abili y o he ab ica ed memb anes o
wound healing applica ions. Fu he mo e, cy ocompa ibili y o he loaded
memb anes was demons a ed bo h in 2D and 3D human de mal ib oblas cul u es,
as well as cell mig a ion was no impai ed by eleased ca ac ol om he
memb anes. These esul s highligh he po en ial o hese polyme ic elec ospun
memb anes o wound healing.”
CHAPTER V ......................................................................................................................................................... 161
Elec ospun asymme ic memb anes o wound d essing applica ions ............................. 161
V.1 In oduc ion ..................................................................................¡E o ! Ma cado no de inido.
Objec i e ................................................................................................¡E o ! Ma cado no de inido.
V.2 Memb ane p epa a ion ............................................................¡E o ! Ma cado no de inido.
V.3 Resul s and discussion .............................................................¡E o ! Ma cado no de inido.
V.3.1 Memb anes cha ac e iza ion by SEM and FTIR ....¡E o ! Ma cado no de inido.
V.3.2 Mechanical p ope ies .....................................................¡E o ! Ma cado no de inido.
V.3.3 Fluids handling p ope ies .............................................¡E o ! Ma cado no de inido.
V.3.4 Ca ac ol elease ................................................................¡E o ! Ma cado no de inido.
V.3.5 An imic obial p ope ies ................................................¡E o ! Ma cado no de inido.
V.3.6 Cy ocompa ibili y...............................................................¡E o ! Ma cado no de inido.
V.3.7 Cell sc a ch assay ...............................................................¡E o ! Ma cado no de inido.
V.4 Conclusions ...................................................................................¡E o ! Ma cado no de inido.
Re e ences .............................................................................................¡E o ! Ma cado no de inido.
190
GENERAL CONCLUSIONS | E asmus Mundus Doc o a e in Memb ane Enginee ing (EUDIME)
GENERAL CONCLUSIONS
The ma e ials p epa ed in his wo k ha e demons a ed he po en ial o
elec odynamic echnique o ob ain bioma e ials o wo di e en applica ions,
bone sca olds and wound d essing memb anes. The mos ele an conclusions o
each p epa ed ma e ial a e summa ized in his chap e .
Th ee polyme ic mul i unc ional sca olds we e de eloped using
elec ospinning and elec osp ay o bone epai and he p e en ion o ea men
o bone in ec ion.
 PCL and PCL/PVAc co e/shell ibe s ob ained by elec ospinning can
be deco a ed wi h RFP o BMP2 loaded PLGA mic opa icles using he
elec osp aying echnique.
 The inco po a ion o HAn pa icles in he polyme ibe s a o s he
apa i e o ma ion on nano ibe su ace.
 The combina ion o PCL, PVAc, and PLGA-BMP2 is a no el and
p omising he apeu ic app oach o bone epai . Gi en ha BMP2 could be
an al e na i e way o inc ease os eogenic ac i i y and he PVAc ibe shell
inc eases hyd ophilici y and a o s os eoblas adhesion and p oli e a ion.
 Enzyma ic deg ada ion o he sca olds highligh ed hei po en ial o
bone egene a ion.
 Lase ea men o he su aces no ably inc eases he sca olds
cy ocompa ibili y esul ing in highe iabili y and cell densi y.
 The syn hesized sca old shows a po ous ne wo k p o iding a la ge
su ace a ea- o- olume a io o cell a achmen as well as su icien po osi y
o nu ien di usion.
 The ob ained sca old p esen s a homogeneous dis ibu ion o RFP-
PLGA mic opa icles along he en i e sca olds he eby ensu ing a con inuous
elease o he RFP.