G een hemos a ic sponge-like sca old composed o soy p o ein and chi in
o he ea men o epis axis
Jon Jimenez-Ma in
a
,
1
, Ke in Las He as
a
,
c
,
1
, Alai z E xabide
d
, Jone U anga
d
,
Ko o de la Caba
d
,
e
, Ped o Gue e o
d
,
e
,
, Manoli Iga ua
a
,
b
,
c
, Edo a San os-Vizcaino
a
,
b
,
c
,
**
,
2
,
Rosa Ma ia He nandez
a
,
b
,
c
,
*
,
2
a
NanoBioCel Resea ch G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de La Uni e sidad 7, 01006
Vi o ia Gas eiz, Spain
b
Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Ins i u e o Heal h Ca los III, Mad id, Spain
c
Bioa aba, NanoBioCel Resea ch G oup, Vi o ia Gas eiz, Spain
d
BIOMAT Resea ch G oup, Uni e si y o he Basque Coun y (UPV/EHU), Escuela de Ingenie ía de Gipuzkoa, Plaza de Eu opa 1, 20018 Donos ia-San Sebas i
an, Spain
e
BCMa e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k, 48940, Leioa, Spain
P o einma Ma e ials SL, A enida de Tolosa 72, 20018 Donos ia-San Sebas ian, Spain
ARTICLE INFO
Keywo ds:
Epis axis
Nasal pack
Hemos asis
Sus ainabili y
ABSTRACT
Epis axis is one o he mos common o o hinola yngology eme gencies wo ldwide. Al hough he e a e cu en ly
se e al ea men s a ailable, hey p esen se e al disad an ages. This, in addi ion o he inc easing social need o
being en i onmen ally espec ul, led us o in es iga e whe he a sponge-like sca old (SP–CH) p oduced om
na u al by-p oduc s o he ood indus y —soy p o ein and β-chi in —can be employed as a nasal pack o he
ea men o epis axis. To e alua e he po en ial o ou ma e ial as a nasal pack, i was compa ed wi h wo o he
mos commonly used nasal packs in he clinic: a basic gauze and he gold s anda d Me ocel®. Ou SP-CH p e-
sen ed g ea physicochemical and mechanical p ope ies, los weigh in aqueous medium, and could e en
pa ially deg ade when incuba ed in blood. I was shown o be bo h biocompa ible and hemocompa ible in i o,
clea ing up any doub abou i s sa e y. I showed inc eased blood clo ing capaci y in i o, as well as inc eased
capaci y o bind bo h ed blood cells and pla ele s, compa ed o he s anda d gauze and Me ocel®. Finally, a a -
ail ampu a ion model e ealed ha ou SP-CH could e en educe bleeding ime in i o. This wo k, ca ied ou
om a ci cula economy app oach, demons a es ha a g een s a egy can be ollowed o manu ac u e nasal packs
using alo ized by-p oduc s o he ood indus y, wi h equal o e en be e hemos a ic p ope ies han he gold
s anda d in he clinic.
1. In oduc ion
Nasal hemo hage, also known as epis axis o nosebleed, is one o he
mos common o o hinola yngology eme gencies wo ldwide. I is es i-
ma ed ha 60% o he wo ld's popula ion will expe ience an epis axis
episode a leas once in hei li e ime, al hough only abou 6%–10% o
hem would seek medical a en ion [1]. Despi e being usually simple o
ea , se e e nosebleed migh cause se ious isks o he pa ien ,
especially o hose o e 70 yea s, o whom he incidence o hese epi-
sodes is highe [2]. In addi ion o his, nasal inju y is common a e nasal
su ge y, o which ea men mus be s a ed quickly. Se e al me hods
a e a ailable o ea epis axis, among which elec ocau e y, asocon-
s ic o s, su gical p ocedu es and nasal packing a ise as he mos popula
[3].
Nasal packing, which in ol es he inse ion o a kind o ampon in he
nasal ca i y, is o special in e es o he ea men o epis axis, o e ing
* Co esponding au ho . NanoBioCel Resea ch G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la
Uni e sidad 7, 01006 Vi o ia Gas eiz, Spain.
** Co esponding au ho . NanoBioCel Resea ch G oup, Labo a o y o Pha maceu ics, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la
Uni e sidad 7, 01006 Vi o ia Gas eiz, Spain.
E-mail add esses: edo a.san [email p o ec ed] (E. San os-Vizcaino), osa.he [email protected] (R.M. He nandez).
1
These au ho s con ibu ed equally o his wo k.
2
RM He nandez and E. San os-Vizcaino equally sha e c edi o senio au ho ship.
Con en s lis s a ailable a ScienceDi ec
Ma e ials Today Bio
jou nal homepage: www.jou nals.else ie .com/ma e ials- oday-bio
h ps://doi.o g/10.1016/j.m bio.2022.100273
Recei ed 1 Feb ua y 2022; Recei ed in e ised o m 25 Ap il 2022; Accep ed 26 Ap il 2022
A ailable online 30 Ap il 2022
2590-0064/©2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
Ma e ials Today Bio 15 (2022) 100273
ele a ed a es o success [4]. Ne e heless, he choice o he mos
adequa e nasal pack is i al o he ou come o he ea men . The ideal
nasal pack should p omo e hemos asis while being com o able o he
pa ien , as well as hinde ing mucosal damage [5]. The e o e, al e na i es
o cu en ly comme cialized nasal packs a e needed, since hey a e
commonly nonabso bable —causing pa ien 's discom o du ing emo al
—and ac jus by physical p essu e amponade, wi hou any in insic
hemos a ic e ec .
Addi ionally, sus ainabili y is a ac ing mo e and mo e a en ion in
all esea ch a eas —including heal h- ela ed a eas —wi h he aim o
educing he en i onmen al load associa ed o p ocesses and p oduc s. In
his sense, en i onmen al analysis is a use ul ool o measu e emissions
h oughou he p oduc li e. I s p incipal benefi is ha i iden ifies he
en i onmen al impac s wi hin he alue chain, which could be ede-
signed o educe he en i onmen al bu den. Mos en i onmen al as-
sessmen s a e s udies in which emissions and esou ce use a e classified
in o ca ego ies ha can po en ially ha m he en i onmen , such as global
wa ming po en ial [6]. Nume ous wo ks ha e been ca ied ou ela ed o
he p ocessing o pe ochemical-de i ed ma e ials, bu only a ew ela ed
o hose de i ed om biopolyme s [7–10].
In his con ex , na u al polyme s a e a ising as p omising compounds
o he de elopmen o hemos a ic ma e ials, being possible o manu-
ac u e hem as sca olds ha mimic he ex acellula ma ix (ECM),
which pe mi s hei eabso p ion by he body [11]. These biopolyme s
a e syn hesized by li ing o ganisms such as plan s, animals o mic oo -
ganisms, and hey gene ally o e good biocompa ibili y and biode-
g adabili y [12]. They migh also possess di e en mechanisms o
p omo e hemos asis.
Following his pa h, esidual o unused biomass is an excellen sou ce
o p o eins and polysaccha ides. In his ega d, soy p o ein (SP) is
ex ac ed om soybeans when soy oil is p oduced. In his p ocess, soy
flou is ob ained as by-p oduc , which is pu ified o ob ain soy p o ein
powde . Ou o e e y 3 kg o soybean, 1 kg o soy p o ein is ob ained
[13]. In his s udy, soy p o ein was ein o ced wi h chi in (CH), which
p o ides highe s abili y and be e mechanical p ope ies. Chi in can be
ob ained om ma ine was e, such as sh imp and c ab shells, ollowing
h ee p ocesses [14]: dep o eina ion in an alkaline medium o sepa a e
p o ein and polysaccha ide, demine aliza ion o elimina e ino ganic
ma e using an acidic medium, and decolo a ion o pigmen elimina-
ion. In e es ingly, using squid pens as a sou ce o chi in equi es nei he
demine aliza ion no decolo a ion s eps; hus, he ex ac ion ime, he
employmen o acid and basic sol en s, and he oluminous was ewa e
discha ge can be a oided, and he p oduc ion cos s, as well as he en i-
onmen al load, can be conside ably educed.
In his ega d, we ecen ly de eloped and cha ac e ized a b and new
sponge-like sca old (SP–CH) based on a g een s a egy [15], using
alo ized by-p oduc s o he ood indus y. SPI is an abundan
biopolyme ha shows biocompa ibili y and low immunogenici y, as
well as se e al pep ides wi h in e es ing biological unc ions, such as he
RGD-like sequence (A g-Gly-Asp) con aining pep ides, p o iding g ea
cell-adhesi e p ope ies [16]. Fo i s pa , CH —namely poly
(β-(1–4)-N-ace yl-D-glucosamine) —also shows low oxici y due o i s
na u al o igin [17], and is biodeg adable. These wo biopolyme s ha e
been selec ed due o hei b oad alo iza ion po en ial, since hey a e
non- oxic, biocompa ible, biodeg adable, enewable, abundan and
a ailable a ela i ely low-cos , cha ac e is ics ha p o ide hem wi h a
g ea alue o he de elopmen o no el biosys ems o heal h- ela ed
applica ions.
Al hough eeze-d ied sponge-like s uc u es wi h chi osan, c oss-
linked wi h glu a aldehyde, ha e shown blood-abso bing capaci y sui -
able o haemos asis [18], in his wo k, chi in was used as ein o cemen
in o de o p o ide soy p o ein wi h highe s abili y and be e me-
chanical p ope ies, a oiding he use o glu a aldehyde. Fu he mo e,
since a b and new sponge-like sca old (SP–CH) using alo ized
by-p oduc s o he ood indus y has been ecen ly de eloped and cha -
ac e ized in a p e ious wo k [15], he aim he e was o analyze i s
po en ial as a nasal pack o he ea men o epis axis. The ini ial cha -
ac e iza ion o ou SP-CH e ealed i s g ea physicochemical p ope ies,
wi h inc eased swelling capaci y and pa ial deg adabili y. Besides, he
mic os uc u e o he de eloped sca olds could p omo e app op ia e cell
adhesion and p oli e a ion, while emaining o ally biocompa ible. E en
i se e al biopolyme ic sponges has al eady been de eloped o he
ea men o hemo hagic wounds [19–21], ha dly any o hem seems o
be di ec ed owa ds he ea men o epis axis [22,23], and none o hem
employs alo ized p oduc s o i s manu ac u ing.
Conside ing he excellen biomedical p ope ies o he componen s o
ou SP-CH, we aimed o analyze i s po en ial as a nasal pack o he
ea men o epis axis, compa ing i wi h ma e ials widely used as nasal
packs du ing nosebleeds: a basic gauze and Me ocel®(MRC), he nasal
pack used wo ldwide as gold s anda d. We expec ou ma e ial o p esen
mo e adequa e mechanical p ope ies o be used as a nasal pack, in
addi ion o a g ea e hemos a ic capaci y, compa ed o he a o emen-
ioned widely used p oduc s. To his end, we fi s compa ed hei
po osi y, po e size, deg ada ion, swelling p ofile and mechanical p op-
e ies. Addi ionally, we pe o med in i o cy o oxici y s udies o confi m
he sa e y o ou SP-CH. Then, we in es iga ed hei hemos a ic po en ial,
including blood clo ing capaci y and he adhesion o e y h ocy es and
pla ele s o he ma e ials. Finally, we e alua ed he hemos a ic e ficacy o
each ma e ial in i o. On he o he hand, in addi ion o he physico-
chemical and biological aspec s, he en i onmen al assessmen was
conside ed in his wo k as a key ool o e alua e he impac s o he
sca olds and he p ocesses ollowed o hei manu ac u e. One o he
aims o using hese ma e ials was he alo iza ion o side s eams and bio-
based was e in o high alue-added p oduc s, p omo ing esou ce e fi-
ciency, in line wi h he ci cula economy s a egy, by c ea ing new alue
chains. A he same ime, he dependency on ossil-based polyme s and
GHG emissions a e educed in o de o mee sus ainable de elopmen
goals. The e o e, his wo k was ocused on esea ching unexploi ed op-
po uni ies o maximize he alo iza ion po en ial o biological esou ces.
2. Ma e ials and me hods
2.1. Ma e ials
Soy p o ein (SP), PROFAM 974, was supplied by ADM P o ein Spe-
cial ies Di ision (The Ne he lands). The amino acid analysis as well as
XPS and FTIR s udies we e pe o med in a p e ious wo k [15].
Chi in (CH) was ex ac ed om esh squid pens (Loligo sp.), which
we e ea ed wi h NaOH (1 M) a oom empe a u e unde con inuous
s i ing o 24 h; a e wa ds, he solid ac ion (CH) was washed wi h
dis illed wa e un il neu al pH. Finally, CH was eeze-d ied and milled
o ob ain he powde . Soy p o ein and chi in ba ches we e hose used and
cha ac e ized in a p e ious wo k [14].
Glyce ol, wi h a pu i y 99.5%, was supplied by Pan eac (Ba celona,
Spain). This polyol was employed as a plas icize in o de o dis up
in amolecula in e ac ions among p o ein chains and acili a e in e -
molecula in e ac ions be ween soy p o ein and chi in.
The p ope ies o ou SP-CH was compa ed wi h a s anda d gauze
(Medicomp®55 cm, Ha mann b , Nijmegen, The Ne he lands) and
wi h Me ocel®(Med onic Xomed, Jackson ille, Fla.). This comme cial
nasal pack, which is composed o hyd oxyla ed poly inyl ace a e, and
p esen ed as a comp essed and dehyd a ed sponge, is he p ima y
nonabso bable nasal pack in se e al eme gency depa men s a ound he
wo ld [24–27].
2.2. P epa a ion o he sponge-like sca old (SP–CH)
In o de o p epa e he SP-CH, 5 g o SPI we e ini ially mixed wi h 30
w % CH (based on he SPI d y basis), ollowed by he addi ion o 125 mL
o dis illed wa e o he mix u e. Nex , NaOH (1 M) was employed o
adjus he pH o 10, and he solu ion was hea ed a 80 C o 30 min
unde magne ic s i ing. Subsequen ly, 30 w % glyce ol (based on he
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
2
SPI d y basis) was added o he blend. So as o achie e a homogeneous
blend, i was main ained a 80 C o o he 30 min. The solu ion was hen
pou ed in o moulds and main ained o 48 h in a eeze a 22 C.
Finally, i was eeze-d ied o 72 h in o de o comple e he p epa a ion
o he SP-CH, which was e en ually cu in o discs using a hollow punch.
2.3. En i onmen al assessmen
En i onmen al assessmen was ca ied ou acco ding o ISO 14040
guidelines and ecommenda ions: goal defini ion, in en o y, impac
assessmen , and in e p e a ion. The main goal o he s udy was o assess
he en i onmen al impac o he ex ac ion o ma e ials, he
manu ac u ing o sca olds and biodeg ada ion as he end o li e. The
so wa e used o his analysis was SimaP o 9.2.0.1 (PR
e Consul an s,
The Ne he lands). The ma e ials used in he labo a o y, he ene gy
consump ion du ing he ex ac ion p ocess, and he anspo a ion o
ma e ials and esidues we e he esou ces and p ocesses conside ed o
de elop he in en o y phase. The en i onmen al bu den ela ed o he
p oduc ion o he sca olds was de e mined conside ing he ene gy
(elec ici y) and ma e ials (NaOH, glyce ol, wa e ) used in he ex ac ion
and manu ac u ing p ocesses. Also anspo a ion o ma e ials, dis illed
wa e p oduc ion and i s anspo a ion o he was e ea men plan a e
use we e conside ed. Da a we e ob ained om Ecoin en 3 da abase,
which p o ides da a ega ding ene gy p oduc ion, anspo and p o-
duc ion o chemicals. The unc ional uni selec ed was 1 g. Based on he
in en o y da a, en i onmen al impac s we e assessed acco ding o he
Hie a chis e sion o ReCiPe 2016, midpoin (H) 1.05. The impac
ca ego ies unde s udy we e global wa ming, wa e consump ion, land
use, human ca cinogenic oxici y, ozone o ma ion (human heal h),
e es ial eco oxici y, eshwa e eco oxici y, ma ine eco oxici y, human
non-ca cinogenic oxici y, fine pa icula e ma e o ma ion, e es ial
acidifica ion, eshwa e eu ophica ion, ma ine eu ophica ion, mine al
esou ce sca ci y, ossil esou ce sca ci y, ozone o ma ion ( e es ial
ecosys ems), s a osphe ic ozone deple ion, and ionizing adia ion.
2.4. Blood ob en ion
Blood was acqui ed om heal hy olun ee s acco ding o he p o-
ocols app o ed by he E hics Commi ee o Resea ching In ol ing
Biological Agen s &GMOs (P ocedu e numbe : M30/2021/257) and he
E hics Commi ee o Resea ch In ol ing Human Beings o he Uni e si y
o he Basque Coun y (P ocedu e numbe : M10/2021/256).
2.5. Po e size and po osi y
A Hi achi S-4800 field emission scanning elec on mic oscope (FE-
SEM) (Hi achi High-Technologies Co po a ion, Tokyo, Japan) was
employed o ob ain he scanning elec on mic oscopy (SEM) mic o-
g aphs, using a beam accele a ed ol age o 5 kV. All he es ed ma e ials
we e cu in o discs o 8 mm diame e , moun ed on a me al s ub wi h
adhesi e ape and coa ed wi h gold unde acuum (JFC-1100) unde an
a gon a mosphe e be o e es ing.
Image J so wa e was used o measu e po e diame e s om SEM
mic og aphs, in o de o de e mine po e sizes. Be ween 75 and 250
andomly selec ed po es we e measu ed o each ma e ial (n ¼3 samples
pe g oup), and he a e age po e size o each ma e ial was calcula ed.
To e alua e he po osi y o he samples (15–20 mg, n ¼3 samples pe
g oup), a liquid displacemen me hod was pe o med using 98% e hanol
as he liquid medium, since i can di use h ough he samples wi hou
causing swelling o sh inkage [28]. V1 was defined as he ini ial olume
o e hanol whe e he samples we e subme ged. Degassifica ion was
ca ied ou o 5 min using a acuum pump, so ha he samples could be
imp egna ed wi h he e hanol. V2 was defined as he o al olume —
e hanol plus soaked sample —a e degasifica ion. Las ,
e hanol-imp egna ed samples we e emo ed and V3 was defined as he
emaining olume o e hanol. The po osi y (
ε
) o each sample was
calcula ed using he equa ion below:
ε
ð%Þ¼V1V3
V2V3x100
2.6. Swelling capaci y and liquid e en ion a io unde p essu e (LRRP)
Swelling capaci y o he ma e ials (15–20 mg, n ¼4 samples pe
g oup) was calcula ed imme sing p eweighed samples (W
0
) o each
ma e ial in 5 mL o PBS. The samples we e weighed again (W
) a e 5 s,
15 s, 30 s, 1 min, 5 min and 15 min, o ob ain he swelling cu es o each
ma e ial. Swelling a each ime poin was calcula ed using he ollowing
equa ion:
Swellingð%Þ¼W W0
W0x100
Liquid e en ion a io unde p essu e (LRRP) was calcula ed in o de
o e alua e he abili y o each ma e ial o e ain he p eabso bed liquid
when a ce ain p essu e is applied. Samples o each ma e ial (20–25 mg,
n¼6 samples pe g oup) we e imme sed in PBS o abso b as much liquid
as possible, and hen hey we e weighed (W
0
). Nex , known weigh s —
10 g, 15 g, 30 g and 40 g —we e se ially placed upon he we ma e ial,
ca e ully collec ing he eleased liquid and weigh ing he sample again.
This p ocedu e was epea ed o each weigh (W
X
). Then, LRRP was
ob ained using he ollowing equa ion:
LRRPð%Þ¼Wx
W0x100
2.7. Weigh loss in aqueous medium
Weigh loss in aqueous medium was s udied by imme sing p e-
weighed (W0) samples (20–25 mg, n ¼4 samples pe g oup) o each
ma e ial in 2 mL PBS, and cul u ing hem a 37 C o di e en pe iods o
ime —2, 5, 9 and 14 days —. A e emo ing he samples om PBS, hey
we e fi s lyophilized and hen weighed (W ), in o de o calcula e he
emaining weigh (%). Weigh loss in aqueous medium was exp essed as
he emaining weigh o he incuba ed and lyophilized samples wi h
espec o hei ini ial weigh , acco ding o he ollowing equa ion:
Remaining weigh ð%Þ¼W
W0x100
2.8. Mechanical cha ac e iza ion
The mechanical p ope ies o he d y ma e ials we e es ed by
comp ession es and cyclic comp ession es (35–45 mg, n ¼5 samples
pe g oup). An Ins on 5969 mechanical es e —equipped wi h a 50 N
load cell —was used a a comp essi e a e o 0.5 mm s
1
up o 70%
s ain. As MRC is comme cialized in lyophilized o m, i was expanded
be o e he es , by we ing and comple ely d ying i o . Se e al pa am-
e e s we e calcula ed in o de o compa e he mechanical p ope ies o
he ma e ials:
The maximum s ess o each cycle ela i e o he maximum s ess o
he fi s cycle was calcula ed as ollows:
Rela i e s essð%Þ¼S essðcycle xÞ
S essðcycle 1Þx100
Young's modulus was calcula ed o all he s ains o he fi s cycle o
each bioma e ial, using he ollowing equa ion o each poin o he
s ess-s ain cu e:
Young0s modulusðkPaÞ¼ S essðkPaÞ
½S ainð%Þ=100
Damping coe ficien was calcula ed by means o he s ess-s ain
cu es o he ma e ials, bo h o cycle 1 and o cycle 10. The
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
3
ollowing equa ion was used:
Damping coe icien ¼D
U
whe e Dis he dissipa ed ene gy (a ea be ween he loading and
unloading cu es) and Uis he o al inpu ene gy (a ea unde he loading
cu e). All he a eas unde he cu e whe e calcula ed using he so wa e
G aphPad P ism 8.0.1.
Besides, an expansion o ce es was pe o med, hyd a ing samples o
d y ma e ials (35–45 mg, n ¼5 samples pe g oup) and eco ding he
upwa ds s ess displayed by he expansion o he ma e ial du ing wa e
abso p ion.
2.9. Cy o oxici y s udies
Cy o oxici y o he ma e ials was assessed acco ding o he ISO
10993-5:2009 guidelines o biological e alua ion o medical de ices.
Bo h di ec —di ec con ac wi h cells —and indi ec —cells exposed o
a condi ioned medium o each ma e ial —cy o oxici y assays we e
pe o med. In bo h cases iabili y o L-929 fib oblas s (ATCC®
30–2003™) was measu ed h ough CCK-8 eagen (Sigma-Ald ich,
Spain). A e incuba ing he cells wi h CCK-8 solu ion in medium (1:11)
o 4 h, he abso bance was ead wi h a pla e eade (Infini e®200 PRO
se ies, Tecan T ading AG, M€
annedo , Swi ze land) a 450 nm, using 650
nm as he e e ence wa eleng h. Cells ha we e no exposed o he ma-
e ials —100% iabili y —we e used as a con ol g oup.
In he di ec cy o oxici y assay, 35,000 cells we e seeded pe well in a
24-well pla e, using 500
μ
L/well o EMEM comple e medium. Nex , he
cells we e incuba ed o 24 h a 37 C. A e emo ing he medium, 1 mL
o esh medium was added and each ma e ial (10–15 mg, n ¼4 samples
pe g oup) was hyd a ed and placed in a answell o b ing i in o di ec
con ac wi h he bo om o he well. The ma e ials we e emo ed a e 48
h o incuba ion, eplacing he medium wi h 300
μ
L/well o CCK-8
eagen .
In he case o he indi ec cy o oxici y assay, L-929 fib oblas s we e
exposed o condi ioned medium o each ma e ial (n ¼5 samples pe
g oup), ob ained incuba ing 100 mg o each ma e ial wi h 500
μ
Lo
EMEM a 37 C o 24 h. Fi s ly, a 96-well pla e was used o seed 5000
cells/well in 100
μ
L/well o medium, and he pla es we e incuba ed o
24 h a 37 C. A e , he medium was eplaced wi h 100
μ
L/well o he
co esponding condi ioned medium, and he pla e was s aigh away
incuba ed a 37 C o 24 h. E en ually, he condi ioned medium was
emo ed, and 110
μ
L/well o CCK-8 eagen was added.
2.10. Blood swelling and deg ada ion in blood
Maximum blood swelling capaci y was es ed by imme sing p e-
weighed (W0) samples (15–20 mg, n ¼5 samples pe g oup) o he
ma e ials in 500
μ
L o blood. A e 2 min, o allow comple e blood ab-
so p ion, samples we e weighed again (W ). The maximum swelling ca-
paci y (%) was calcula ed using he ollowing equa ion:
Swellingð%Þ¼W
W0x100
To e alua e he deg ada ion o he ma e ials in blood, p eweighed
(W0) samples (15–25 mg, n ¼4 samples pe g oup) o each ma e ial we e
placed in 24-well pla es, using one pla e o each ime-poin es ed —2, 4
and 7 days —. Blood was added o each sample in an amoun o a 120% o
i s maximum blood swelling capaci y, and he pla es whe e incuba ed a
37 C. A each ime-poin , samples we e washed ou wi h PBS, lyophi-
lized and weighed (W ). Deg ada ion in blood was exp essed as he
emaining weigh o he incuba ed and lyophilized samples wi h espec
o hei ini ial weigh , acco ding o he ollowing equa ion:
Remaining weigh ð%Þ¼W
W0x100
2.11. Whole blood clo ing in i o
Hemoglobin con en o he samples was quan ified using a Hemo-
globin Assay Ki (Sigma-Ald ich, USA), by measu ing abso bance o he
samples (Is) wi h a pla e eade (Infini e®200 PRO se ies, Tecan T ading
AG, M€
annedo , Swi ze land) a 400 nm.
Fo he whole blood clo ing es , ci a ed whole blood —9:1 whole
blood o 3.8% sodium ci a e —was ob ained om a heal hy human
dono . Fi s , 0.2 mL o he ci a ed whole blood was added o p ehea ed
(30 min a 37 C) samples o he ma e ials disposed in a 24 well pla e
(25–35 mg, n ¼5 samples pe g oup). A e his, 20
μ
L o CaCl
2
(0.2 M)
we e added o s a coagula ion. The pla e wi h he ma e ials was incu-
ba ed unde 30 pm agi a ion o 10 min a 37 C. A e wa ds, 2 mL o
deionized wa e we e added in each well o hemolyze he ed blood cells
(RBCs) ha we e no wi hin he clo o med in he ma e ial. The deion-
ized wa e con aining he non-adhe ed and hemolyzed RBCs was
collec ed and i s hemoglobin con en was quan ified. 5
μ
L o CaCl
2
(0.2
M) and 50
μ
L o ci a ed whole blood we e added o 750
μ
L o deionized
wa e , and he abso bance o his solu ion was used as he e e ence alue
(I ). The abso bance o an emp y well was also measu ed (Io). Blood
Clo ing Index (BCI) o each sample was calcula ed using he ollowing
equa ion:
BCI ð%Þ¼ðIs IoÞ
ðI IoÞx100
wi h he pu pose o isually e alua ing he clo ing capaci y o each
ma e ial, 150 mg o each ma e ial we e disposed in Eppendo ubes,
subsequen ly adding 500
μ
L o ci a ed whole blood and 500
μ
L o CaCl
2
(10 mM). A e incuba ing he Eppendo ubes o 1 min a 37 C, hey
we e u ned upside down and gen ly shaken o obse e he o med clo s.
2.12. Hemolysis assay in i o
Fo he assessmen o hemocompa ibili y in i o, ci a ed whole blood
—9:1 whole blood o 3.8% sodium ci a e — om a heal hy dono was
ob ained and dilu ed 1:5 in no mal saline. Samples o each ma e ial
(10–15 mg, n ¼6 samples pe g oup) we e disposed in a 24-well pla e.
Dilu ed whole blood was added o each sample in an amoun o he 80%
o each ma e ial's swelling capaci y. The pla e was incuba ed o 1 h a
37 C. Samples we e mo ed o conical cen i uge ubes and cen i uged
o 10 min a 3000 pm. The supe na an o each sample was collec ed
and i s hemoglobin con en was quan ified as desc ibed in sec ion 2.8.
150
μ
L o whole blood was added o deionized wa e and o no mal sa-
line, in o de o use hese solu ions as posi i e (I ) and nega i e (Io)
con ols, espec i ely. Hemolysis a e was calcula ed using he ollowing
equa ion:
Hemolysisð%Þ¼ðIs IoÞ
ðI IoÞx100
2.13. Red blood cell adhesion in i o
To e alua e RBC adhesion o he ma e ial, ci a ed whole blood —9:1
whole blood o 3.8% sodium ci a e —was collec ed om a heal hy
dono . Samples o each ma e ial we e cu (15–20 mg, n ¼6 samples pe
g oup) and each sample was placed in a Pe i dish. Ci a ed whole blood
was added o each sample in an amoun o he 80% o each ma e ial's
swelling capaci y, and hey we e incuba ed o 5 min a 37 C. Nex ,
5–10 mL o deionized wa e we e gen ly added by he edge o he dish
un il he wa e ouched he sample and blood s a ed o flow. Mo e
deionized wa e was added un il a o al olume o 50 mL o each sample.
Each ma e ial was ca e ully mo ed o a clean Pe i dish, and he liquid in
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
4
each old Pe i dish, —con aining he RBCs ha could no adhe e o he
sample —, was collec ed. The hemoglobin con en o his solu ion was
quan ified as desc ibed in sec ion 2.8. This measu emen accoun s o
hemoglobin ou side he ma e ial, ha is, he RBCs ha could no adhe e o
he ma e ial.
A e wa ds, hemoglobin wi hin he ma e ials was measu ed, which
accoun s o he amoun o RBCs adhe ed o each sample. 10 mL o
deionized wa e we e added s aigh ly on he ma e ial —so ha he
p e iously adhe ed RBCs would be eleased —and he hemoglobin
con en in he esul an solu ion was quan ified as desc ibed in sec ion
2.8.
Fo bo h he hemoglobin ou side and wi hin he ma e ial, a posi i e
con ol was p epa ed by mixing 200
μ
L o blood and 50 mL o deionized
wa e . Hemoglobin concen a ion (mg dL
1
) o each sample was calcu-
la ed using he ollowing equa ion p o ided by he Hemoglobin Assay
Ki , and esul s o each sample we e no malized agains he blood olume
added o he con ol g oup:
½Hemoglobin¼ðIs IoÞ
ðI IoÞx100 mg
dL xd
whe e Is is he abso bance o he es ed solu ion; Io is he abso bance o
he blank (wa e ); I is he abso bance o he calib a o p o ided in he
Hemoglobin Assay Ki ; 100 mg/dL is he concen a ion o he dilu ed
calib a o ; d is he dilu ion ac o , which was calcula ed o each sample
depending on he olume o blood and deionized wa e added.
Ci a ed whole blood was also added o samples o each ma e ial (n ¼
3 samples pe g oup) and he adhesion o RBCs was isually e alua ed
using SEM mic og aphs, ob ained as desc ibed in sec ion 2.2.
2.14. Pla ele adhesion in i o
Adhesion o pla ele s o he ma e ials was s udied h ough a Lac a e
Dehyd ogenase (LDH) Assay Ki (Sigma-Ald ich, USA), which allows o
quan i y pla ele adhesion by measu ing he LDH eleased by pla ele s
when hey a e lysed, acco ding o a epo ed me hod [29]. B iefly,
ci a ed whole blood —9:1 whole blood o 3.8% sodium ci a e —was
ob ained om a heal hy dono . A e cen i uging i a 480 g o 10 min a
4C wi h 4 a/d, pla ele - ich plasma (PRP) was ob ained. The se um
abo e he bu y coa was collec ed om he cen i uged blood samples
and kep in ci a ed ubes. Samples o each ma e ial (15–20 mg, n ¼5
samples pe g oup) we e disposed in a 24-well pla e and PRP was added
o each sample in an amoun o he 80% o each ma e ial's swelling ca-
paci y, and hey we e incuba ed o 30 min a 37 C. No mal saline was
gen ly added o each well un il he samples we e imme sed, so ha he
non-adhe ed pla ele s could be emo ed om he ma e ial, and all
samples we e mo ed o a new 24-well pla e. 1 mL o T i on X-100
1%-PBS was added s aigh ly on he ma e ial, in o de o lyse he
pla ele s ha had adhe ed o he ma e ial. A e incuba ion o 1 h a 37
C, each solu ion o T i on and lysed pla ele s was collec ed and LDH
con en was measu ed acco ding o he p o ocol p o ided by he manu-
ac u e (Sigma-Ald ich, USA) o he LDH Ki . To p epa e he nega i e
(Io) and posi i e (I ) con ols, 200
μ
L o PRP we e added o 1 mL o PBS
and T i on X-100 1%, espec i ely. Abso bances o he lysed pla ele s o
each samples (Is) we e measu ed wi h a pla e eade (Infini e®200 PRO
se ies, Tecan T ading AG, M€
annedo , Swi ze land) a 400 nm, and
no malized agains he PRP olume added o he con ols. LDH elease
(%) ela i e o he posi i e con ol was calcula ed using he ollowing
equa ion:
LDH eleaseð%Þ¼ðIs IoÞ
ðI IoÞx100
PRP was also added o samples o each ma e ial (n ¼3 samples pe
g oup) and he pla ele adhesion was isually e alua ed using SEM mi-
c og aphs, ob ained as desc ibed in sec ion 2.4.
2.15. IN VIVO hemos a ic e ficacy
To e alua e he hemos a ic e ficacy in i o, a a - ail ampu a ion
model was used. This expe imen was conduc ed ollowing he p o ocols
app o ed by he Ins i u ional E hical Commi ee o Animal Expe imen-
a ion o he Uni e si y o he Basque Coun y (P ocedu e numbe : M20/
2021/362). Wis a a s —wi h weigh s be ween 250 and 300 g (Jan ie
Labs, Le Genes -Sain -Isle, F ance) —we e anes he ized wi h isoflu ane
(Isoflo®, Es e e, Spain), and 2.5 cm om he end o hei ail was cu
using a scalpel. The ail was immedia ely placed in ai o 10 s o gua -
an ee no mal blood loss, a e which he wound was b ough in o di ec
con ac wi h he co esponding p eweighed ma e ial (250–300 mg, n ¼6
a s pe g oup), holding i wi h a p eweighed gauze (900–950 mg). The
wound was unco e ed e e y minu e o check i bleeding pe sis ed. Fo
his eason, bleeding ime (min) was assessed by assigning a sco e o each
pe iod a which bleeding ceased, as ollows: 0–3 min: 1; 3–6 min: 2; 6–9
min: 3; >9 min: 4. The e o e, a lowe bleeding ime sco e ep esen s a
as e hemos a ic e ec . The blood-imp egna ed samples we e weighed
again o calcula e o al blood loss (g).
2.16. S a is ical analysis
Mean s anda d de ia ion was used o exp ess he esul s. In he case
o no mally dis ibu ed da a, esul s we e analyzed h ough a one-way
ANOVA es . Bon e oni o Tamhane pos -hoc analysis was applied
based on he Le ene es o he homogenei y o a iances. In e e se,
non-no mally dis ibu ed da a was analyzed by Mann–Whi ney's
nonpa ame ic analysis. Sample size o each expe imen is indica ed in
he co esponding ma e ials and me hods sec ion. p <0.05 was consid-
e ed s a is ically significan . Mo eo e , all he s a is ical compu a ions
we e pe o med using SPSS 25.0 (SPSS®, Inc., Chicago, IL, USA).
3. Resul s and discussion
3.1. En i onmen al assessmen
The inc easing en i onmen al conce n has led o assess he sui abili y
o biodeg adable polyme s ex ac ed om na u al and enewable e-
sou ces. The e o e, biopolyme s de i ed om esidual biomass ha e
become an a ac i e al e na i e due o hei abundance, cos and
biodeg adabili y. In his con ex , CH and soy p o ein we e used in his
wo k. On he one hand, CH can be ob ained om sh imp, c abs o squid
pens, among o he s. I should be no ed ha he CH con en a ies
significan ly depending on he sou ce employed o i s ex ac ion. When
CH is ob ained om c us acean shells, CH con en is a ound 20% a e a
demine aliza ion p ocess; howe e , he CH con en om squid pens can
each 50%, wi h a mine al con en lowe han 3% [30]. On he o he
hand, soy p o ein, a by-p oduc om soy oil p oduc ion, can be pu ified
o ob ain SPI. In his p ocess, soy flou is ob ained as by-p oduc , which is
pu ified o ob ain soy p o ein powde a e cen i uga ion, acidifica ion
and neu aliza ion p ocesses. Al hough he p ope ies o some p oduc s
based on na u al ma e ials di e be ween ba ches, in he case o he
ex ac ion o chi in om Loligo sp., he aceabili y o his p ocess has
been confi med. We ollow a s anda dized p ocess ha allows us o
ob ain sponges wi hou di e ences be ween ba ches, showing he same
deg ee o ace yla ion and c ys allini y [14]. To de e mine he en i on-
men al load associa ed wi h CH and soybean esidue alo iza ion, he
en i onmen al loads associa ed wi h hose p ocesses we e conside ed.
All he abo emen ioned p ocesses we e conside ed o ob ain he
global en i onmen al esul s o sca olds p oduc ion epo ed in Table 1.
The esul s showed ha e es ial eco oxici y, global wa ming and
ionizing adia ion caused low en i onmen al load in he p oduc ion o
sca olds, while he o he ca ego ies con ibu ed minimally o he o e all
en i onmen al bu den. We should no e ha he en i onmen al bu den o
e es ial eco oxici y is ela ed o soybean p oduc ion and he gene a ion
o ene gy needed o ob ain CH.
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
5
I is wo h no ing ha he use o squid pens as a sou ce o CH p o ided
en i onmen al benefi s. Due o he low amoun o ino ganic compounds
and he lack o pigmen s in squid pens, demine aliza ion and decolo -
iza ion p ocesses could be elimina ed om he CH ex ac ion p ocess
and, hus, he use o chemicals and ene gy consump ion associa ed wi h
hose p ocesses we e educed significan ly [14]. In addi ion, he elimi-
na ion o hose p ocesses en ailed economic benefi s, since he ex ac ion
p ocess implied he employmen o smalle amoun s o esou ces (ma-
e ials, ene gy, and ime) and, a he same ime, a highe yield o CH was
ob ained, compa ed o ha o CH ex ac ed om c us acean shells.
The ela i e con ibu ions in each impac ca ego y o he mos
ele an p ocesses in ol ed in he comple e li e cycle o he sca olds a e
shown in Fig. 1. The en i onmen al esul s ob ained a e shown in pe -
cen age p opo ions, whe e i can be seen ha he mos ele an
con ibu ing ac o was elec ici y consump ion in ex ac ion and
manu ac u ing p ocesses. We mus emembe ha he en i onmen al
impac calcula ed in his wo k was based on he esul s ob ained in he
labo a o y and ga e us an idea o which p ocesses should be op imized o
be able o educe he en i onmen al bu den, which could be mo e easily
educed a an indus ial scale.
In addi ion, Fig. 1 shows he en i onmen al esul s b oken down in o
pe cen ages o each impac ca ego y. In his way, he con ibu ion o
each s age on he final p oduc could be assessed. In his case, he con-
ibu ions o he di e en p ocesses and ac i i ies in ol ed h oughou
he li e cycle we e calcula ed as a unc ion o he o e all esul s. As shown
in Fig. 1, he s ages o ob aining CH equi ed ene gy and his ep esen ed
mo e han 80% o he o al impac , becoming he ac o wi h he g ea es
po en ial o imp o emen in decision making when scaling-up. In
pa icula , he ene gy consumed in he sca old p oduc ion, specifically
he consump ion o elec ici y, played a c i ical ole in he en i onmen al
impac , ega dless o he impac ca ego y conside ed. Since he sca olds
we e p oduced a labo a o y scale, scaling up he p ocesses would lead o
educing he a o emen ioned en i onmen al impac s.
3.2. Physicochemical cha ac e iza ion
3.2.1. Po e size and po osi y
Po e s uc u e o bioma e ials ha aim o be used as wound healing
agen s —ei he o ea hemo hages o o he ypes o wounds —is o
g ea impo ance. Di e ences in he s uc u e o he h ee ma e ials
employed du ing his s udy can be app ecia ed in he mac oscopic im-
ages (Fig. 2A). E en hough, di e ences in he mic oes uc u e a e o
g ea e impo ance. The excellen physicochemical p ope ies o ou
ma e ial we e al eady demons a ed in ou p e ious wo k [15], bu we
aimed o compa e some o hese p ope ies be ween he h ee ma e ials.
In his ein, he gauze was he ma e ial wi h he highes po e size —479
252
μ
m—, while MRC and SP-CH showed a e y simila po e size
dis ibu ion, 144 63 and 141 124
μ
m, espec i ely (Fig. 2B). MRC
and SP-CH showed no significan di e ence in mean po e size (p >0.05),
while bo h o hem showed significan di e ences wi h espec o gauze
(p <0.001). Po e size can be closely ela ed o he hemos a ic capaci y o
a ma e ial [31]. Ideally, a balance should be sough be ween la ge po e
sizes ha allow RBC and pla ele in e naliza ion and small po es ha
p o ide op imal su ace a ea, equi ed o app op ia e cell adhesion [32].
Po osi y is equi ed o be high o allow di usion o nu ien s and
oxygen [33]. Besides, high po osi y is ela ed o ele a ed su ace a ea,
which is o in e es o he abso p ion o wound fluids, ei he exuda es o
e en blood [34]. As a la ge su ace a ea o he ma e ial is also ela ed o
highe exposi ion o cells, i can influence cell adhesion and iabili y as
well [35]. In his con ex , MRC and SP-CH showed simila po osi y
(Figs. 2C), 92.97 4.39% and 86.95 1.58%, espec i ely. Meanwhile,
he gauze showed significan ly highe po osi y han SP-CH, 96.68
0.12%. The di ficul ies associa ed wi h a high po osi y migh be o e -
come by he weigh loss o pa ial deg ada ion o he nasal pack, which
would acili a e he painless emo al o he pack. In his sense, i is clea
ha he SP-CH o e ad an ages in he emo al o he pack compa ed o
MRC and he gauze, e en i he h ee o hem p esen high po osi ies. On
he o he hand, SEM mic og aphs (Fig. 2D) clea ly e ince ha in e nal
Table 1
Impac ca ego y alues measu ed in he p oduc ion o he sca olds.
Impac Ca ego y Uni To al
Global wa ming kg CO
2
eq 1.7517401
S a osphe ic ozone deple ion kg CFC11 eq 8.11E-07
Ionizing adia ion kBq Co-60 eq 0.99109783
Ozone o ma ion, human heal h kg NO
x
eq 0.00560783
Fine pa icula e ma e o ma ion kg PM
2.5
eq 0.00403105
Ozone o ma ion, e es ial ecosys ems kg NO
x
eq 0.005638093
Te es ial acidifica ion kg SO
2
eq 0.010339224
F eshwa e eu ophica ion kg P eq 0.00067464
Ma ine eu ophica ion kg N eq 6.30E-05
Te es ial eco oxici y kg 1,4-DCB 1.4278474
F eshwa e eco oxici y kg 1,4-DCB 0.023579723
Ma ine eco oxici y kg 1,4-DCB 0.033241366
Human ca cinogenic oxici y kg 1,4-DCB 0.061105474
Human non-ca cinogenic oxici y kg 1,4-DCB 1.3274998
Land use m
2
a c op eq 0.16276505
Mine al esou ce sca ci y kg Cu eq 0.001540514
Fossil esou ce sca ci y kg oil eq 0.48253294
Wa e consump ion m
3
0.012084432
Fig. 1. Rela i e con ibu ions in each impac ca ego y o he mos ele an p ocesses in ol ed in he en i e li e cycle o sca olds. Disagg ega ing en i onmen al
esul s a e displayed in pe cen age a ios o he mos ele an con ibu ing ac o s.
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
6
Fig. 2. Physicochemical cha ac e iza ion. (A) Mac oscopic images o he ma e ials: Gauze, MRC, and SP-CH. (B) Po e size dis ibu ion o he ma e ials. (C) Po osi y
(%) o each ma e ial. E o ba s, mean SD. *p <0.05; ***p <0.001 s gauze. N.S., nonsignifican (p >0.05). (D) SEM mic og aphs o d y ma e ials. (E) Swelling
p ofiles. E o ba s, mean SD. N.S., nonsignifican (p >0.05). (F) Liquid e en ion a io unde di e en p essu es. E o ba s, mean SD. &&&p<0.001 gauze s
MRC. #p <0.05; ##p <0.01; ###p <0.001 MRC s SP-CH. ***p <0.001 gauze s SP-CH. (G) Weigh loss in aqueous medium. E o ba s, mean SD. N.S.,
nonsignifican (p >0.05).
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
7
s uc u es o he h ee ma e ials di e . The gauze shows housands o
in e wined fibe s o ming a mesh, bu he space be ween he fibe s does
no p esen well defined po es, which a e equi ed o good liquid
e en ion. Fo hei pa , MRC and SP-CH show an in e nal mic os uc-
u e o well-defined in e connec ed po es, which in he case o MRC a e
mo e ci cula and in he case o SP-CH p esen a mo e amo phous
s uc u e. Toge he wi h a simila po e size and po osi y, his makes MRC
and SP-CH e y simila ega ding o hei in e nal mic oa chi ec u e,
which comple ely di e s om ha o he gauze.
3.2.2. Swelling capaci y and liquid e en ion a ion unde p essu e (LRRP)
The capaci y o bioma e ials o abso b fluids is impo an as i will
de e mine hei abili ies o abso b he blood p esen in hemo hages. We
es ed he wa e swelling p ofiles o he h ee ma e ials by measu ing
swelling a di e en ime poin s (Fig. 2E). The h ee ma e ials showed
simila wa e up ake capaci y, wi h he maximum swelling capaci y
anging be ween 1334 34% (MRC) and 1442 267% (SP–CH) o i s
ini ial weigh a e 15 min. Du ing ha pe iod o ime, he gauze
abso bed 1412 182% o i s ini ial weigh . E en i he maximum
abso p ion capaci y was simila o he h ee ma e ials, he swelling
p ofile u ned ou o be qui e di e en . While SP-CH and gauze achie ed
a 1383 168% and 1324 302% o hei ini ial weigh wi hin only 5 s,
espec i ely, MRC could only swell a 178 21% o i s ini ial weigh
du ing he same ime. This shows ha e en i all o hem displayed
simila maximum abso p ion capaci y, MRC showed delayed swelling,
only equaling SP-CH and gauze a e 60 s. The wa e abso p ion p ope y
appea s o be especially impo an in he ea men o epis axis as he
p incipal way in which nasal packs a es bleeding is by exe ing p essu e
on he damaged blood essels o he nasal mucosa o p omo e hemos asis.
A e being inse ed in he nos il, he nasal pack abso bs wound fluids
and expands, main aining p essu e on he wound si e. This p essu e
should be high enough o con ol he bleeding, bu no as high o damage
he nasal ca i y. Complica ions associa ed wi h excessi e p essu e
include mo emen o he pack om i s ini ial posi ion [36], obs uc ed
b ea hing and educed sense o smell [1], and e en nec osis o mucosa
and ca ilage [25].
Ne e heless, when he pack pu s p essu e on he nos il's walls, he
nos il's walls also pu p essu e on he pack, hus deso bing pa o he
Fig. 3. Mechanical cha ac e iza ion. (A) Scheme o he p ocedu e ca ied ou o he cyclic comp ession es . (B–D) Cyclic comp ession s ess-s ain cu es o gauze
(B), MRC (C) and SP-CH (D). (E) Rela i e s ess (%) o he ma e ials o 10 cyclic comp essions. E o ba s, mean SD. &&p<0.01 gauze s MRC. **p <0.01 by
gauze s SP-CH. (F) Young's modulus o he ma e ials a di e en s ains. (G) Damping coe ficien s o he ma e ial o cycles 1 and 10. E o ba s, mean SD. &&&p<
0.001 gauze s MRC. ***p <0.001 SP-CH s he o he g oups. $$$p <0.001 cycle 1 s cycle 10 o he same g oup. (H) Scheme o he p ocedu e ca ied ou o he
expansion o ce es . (I) Rela i e expansion s eng h cu es o he ma e ials. * indica es he momen a which wa e was added o each ma e ial. (J) Rela i e expansion
o ce o he ma e ials. E o ba s, mean SD. ***p <0.001 SP-CH s he o he g oups. N.S., nonsignifican (p >0.05).
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
8
p e iously abso bed fluids. Fo his eason, we e alua ed he capaci y o
he ma e ials o e ain he abso bed liquid unde p essu e. In his
expe imen (Fig. 2F) bo h MRC and SP-CH showed significan ly g ea e
(p <0.001) wa e e en ion capaci y compa ed o gauze, independen ly
o he p essu e applied. Besides, MRC displayed significan ly be e
e en ion a low p essu es compa ed o SP-CH, al hough bo h ma e ials
u ned ou o simila (p >0.05) e aining alues o p e-abso bed wa e
when he maximum weigh —40 g —was applied, wi h MRC main-
aining 49.58 4.37% and SP-CH 45.83 4.54% o hei maximum
swelling capaci y, while gauze main ained jus 34.44 2.35%. The e-
o e, e en i he h ee ma e ials showed simila wa e swelling capaci y,
gauze is no able o e ec i ely e ain he abso bed wa e when a p essu e
is applied, and hus loses i s abili y o exe p essu e o he bleeding si e,
likely due o i s in e nal mic os uc u e, as commen ed be o e. P obably
one o he majo d awbacks o he p essu e exe ed by he nasal packs is
he discom o caused o he pa ien , bo h wi h he pack in si u and du ing
i s emo al, wi h he la e e en causing ebleeding, mucosal ab asions,
demucosaliza ion and de achmen o sca s [37]. As p essu e is needed o
p omo e hemos asis, hese disad an ages always go oge he wi h
e ec i e nasal packs, as long as hey a e nonabso bable.
3.2.3. Weigh loss in aqueous medium
Weigh loss in aqueous medium is a p ope y o inc easing popula i y
among bioma e ials o nasal packings, as i educes bo h pa ien
discom o and ebleeding upon emo al, among o he ad an ages [38].
In his ein, bo h gauze and MRC showed no weigh loss du ing he 14
days o expe imen (Fig. 2G), while SP-CH los 25.96 3.22% o i s
ini ial weigh a e 48 h. A e ha measu emen , SP-CH showed no mo e
weigh loss on he ollowing days (p >0.05). These esul s clea ly show
ha bo h gauze and MRC a e o ally non-abso bable nasal packs, while
SP-CH can lose abou a 25% o i s ini ial weigh a e 48 h, due o he
glyce ol con en (30 w %) o he SP-CH dissol ing in wa e . This may
p obably be enough o elie e pa ien 's discom o and damage associ-
a ed wi h pack emo al, and o a oid ebleeding episodes, which would
suppose a g ea ad an age bo h o he pa ien and he clinician [39].
Besides, i is wo h men ioning ha in ou p e ious wo k abou he
SP-CH [15], we demons a ed ha i can be enzyma ically deg aded
wi hin 2 h, using collagenase P. Mo eo e , in his p e ious wo k we
pe o med cell-media ed deg ada ion s udies in an implan ed SP-CH,
obse ing comple e deg ada ion in i o. Howe e , since his enzyma ic
deg ada ion does no ep esen an epis axis scena io, we belie e ha in
his case weigh loss in aqueous medium and blood media ed deg ada-
ion a e mo e meaning ul.
3.3. Mechanical cha ac e iza ion
Fo app op ia e handling and clinical use nasal packs mus p esen
good mechanical p ope ies and esis ance o de o ma ion, hus alle i-
a ing pa ien 's conce ns abou discom o du ing he ea men and
p o iding mechanical s abili y [40].
We ca ied ou a cyclic comp ession s ess-s ain es wi h 10 cycles
(Fig. 3A) o cha ac e ize he mechanical p ope ies o he ma e ials
(Fig. 3B–D). These cu es show an ini ial pla eau egion, ollowed by a
hype elas ic egion a highe s ains, which is mo e no ewo hy o he
gauze and SP-CH han o MRC. Fo he h ee ma e ials he ela ionship
be ween s ess and s ain is nonlinea , exhibi ing nonlinea iscoelas-
ici y, simila ly o many body issues and ex acellula ma ix compo-
nen s [41]. This iscoelas ici y could be confi med by he loading and
unloading cu es o each cycle aking di e en pa hs, which occu due o
ene gy dissipa ion du ing de o ma ion o he ma e ial [42], p oducing an
hys e esis loop be ween loading and unloading cu es. The a ea unde
he loading cu e ep esen s he o al inpu ene gy o he cycle and he
a ea unde he unloading cu e ep esen s he elas ic s ain ene gy, while
he a ea be ween he wo cu es accoun s o he dissipa ed ene gy
du ing he comp ession cycle [43]. In addi ion o his, gauze showed he
highes s ess a a s ain o 70%, bo h o cycle 1 and o cycle 10, while
MRC showed he lowes , meaning ha gauze was he mos di ficul
ma e ial o de o m up o a s ain o 70%, and MRC he easies one, which
could be due o he in e nal mic os uc u e o each one. SP-CH equi ed
an in e media e s ess o be de o med up o a 70% o s ain.
Ne e heless, he cyclic comp ession es e ealed ha he s ess
needed o de o m he ma e ials dec eased subs an ially cycle a e cycle
(Fig. 3E), losing hei maximum abili y o esis de o ma ion. This phe-
nomenon, which is defined as cyclic so ening [44], is p obably a
consequence o i e e sible de o ma ions o he in e nal mic o-
a chi ec u e caused by he comp ession [45]. Resul s o he ela i e
maximum s ess (%) (Fig. 3E) show ha he gauze could only main ain an
80.2 2.0% o i s maximum s ess a e 10 cycles, compa ed o he
maximum s ess o cycle 1. This is significan ly lowe (p <0.01) han
MRC and SP-CH, ha displayed a ela i e s ess o 87.3 5.2% and 88.6
1.6% a e 10 cycles, espec i ely.
Young's modulus defines he abili y o a ma e ial o wi hs and
changes in leng h when subjec ed o comp essi e loads, and i is use ul o
measu e he s i ness o a ma e ial [46]: he highe he Young's modulus,
he highe he s i ness o he ma e ial. Since he h ee ma e ials p esen a
nonlinea beha io , Young modulus is no cons an bu changes
depending on he posi ion in he s ess-s ain cu e, and is defined by he
slope o he angen o he cu e [47]. Thus, we calcula ed he Young's
modulus o all he s ains o he fi s cycle o each bioma e ial (Fig. 3F).
Ini ially, he h ee samples p esen ed a high Young's modulus, which
apidly dec eased when s ain s a ed o inc ease. As s ained became
highe , he Young modulus o gauze and SP-CH s a ed o inc ease again
a s ains coinciding wi h he ones in which he hype elas ic egion o
each ma e ial began —a ound 25% o s ain o he gauze and 50% o
s ain o SP-CH —. This could indica e ha a 25% o s ain, all he
co on laye s o ming he gauze a e e y compac , and consequen ly he
s i ness o he sample s ongly inc eases. Meanwhile, he in e nal
mic os uc u e o he SP-CH would no ge comple ely compac un il a
s ain o 50%. MRC showed no meaning ul inc ease o i s Young's
modulus a highe s ains, indica ing ha i s s i ness emained low
h oughou all he comp ession p ocess. Summing up, while gauze be-
comes igid a low s ains, bo h MRC and SP-CH emain flexible un il a
s ain o 50% is achie ed.
As commen ed be o e, he cyclic loading and unloading p oduced
dissipa ion o ene gy wi hin he ma e ial, which is usually a esul o he
iscoelas ic beha io o he ma e ial o gene al damage o he s uc u e
[48]. Since iscoelas ic na u e o he h ee ma e ials could be p e iously
confi med by he p ofile o he s ess-s ain cu es and he p esence o
he hys e esis loop, ene gy dissipa ion can be a ibu ed o ha isco-
elas ic beha io . The a ea o his hys e esis loop — ha is, he a ea be-
ween he loading and unloading cu es —is di ec ly linked o he
damping capaci y o he ma e ial [49], which is defined by he damping
coe ficien —o mechanical loss coe ficien —, ha measu es o which
ex en a ma e ial can dissipa e ib a ional ene gy [50]. This p ope y
depends on ac o s such as he ype o ma e ial, in e nal o ces, sizes o
geome y and he su ace o he ma e ial [51], and seems o be o g ea e
impo ance in biological ma e ials o hei p o ec ion ia damping he
impac wa es [52]. Damping capaci y is commonly high in ma e ials
such as oams, elas ome s and polyme s [50], and he e o e hey a e
widely used as damping ma e ials [53]. Since a nasal pack is ine i ably
subjec ed o mechanical comp ession when inse ed in he nos il, we
e alua ed he damping coe ficien o he h ee ma e ials o cycle 1 and
10, calcula ing i as he a io o he ene gy dissipa ion —a ea o he
hys e esis loop —and he o al inpu ene gy —a ea unde de loading
cu e — o he gi en cycle [51]. Fo he fi s cycle, SP-CH p esen ed a
damping coe ficien o 0.72 0.02, which was significan ly highe (p <
0.001) han he ones ob ained o gauze and MRC, 0.59 0.05 and 0.29
0.01, espec i ely. Compa ing he fi s cycle's coe ficien o he cycle
10's, all he ma e ials significan ly (p <0.001) los damping capaci y,
p obably due o i e e sible al e a ions in hei in e nal mic os uc u e
caused by he cyclic comp ession. E en hough, SP-CH p esen ed a
damping coe ficien o 0.57 0.03 in cycle 10, which is s ill significan ly
J. Jimenez-Ma in e al. Ma e ials Today Bio 15 (2022) 100273
9
phospha idylse ine-exposing memb ane su ace, which is essen ial o
he o ma ion o h ombin [78]. In u n, h ombin ac i a es pla ele s —
h ough hyd olyza ion o G-p o ein- coupled p o ease-ac i a ed e-
cep o s (PAR) 1 and 4 [81]—and p omo es clo s abiliza ion by con-
e ing o fib inogen o fib in [82].
We hypo hesize ha his pla ele - h ombin in e ac ion, ha boos s
he o ma ion o he hemos a ic plug, can be influenced somehow by he
SP-CH. This is because SPI is a sou ce o phylloquinone — i amin K1 —
[83], which ce ainly is in ol ed in blood coagula ion. Vi amin K se es
as a co ac o o he endoplasmic enzyme g-glu ama e ca boxylase
(GGCX), which plays a i al ole in he ca boxyla ion o ce ain
p o ein-bound glu ama e esidues in o g-ca boxyglu ama e (Gla) [84].
Se en blood coagula ion ac o s —including p o h ombin, VII, IX, and
X [85]— equi e his K i amin-dependen glu ama e esidue ca box-
yla ion in o de o bind Ca
þ2
. The e o e, his ca boxyla ion is essen ial o
he o ma ion o ion b idges be ween he blood-clo ing enzymes and
phospholipids on pla ele s’memb anes [86], p omo ing he ac i a ion o
hese coagula ion ac o s.
In addi ion, i has also been men ioned ha he SPI p esen in he SP-
CH has RGD-mo i con aining pep ides [16], which could explain i s
abili y o bind pla ele s. This is because he RGD sequence is also p esen
in he
α
-chains o fib inogen [87], and i is one o he wo mo i s ha bind
o he
α
IIbβ3 in eg in ecep o o pla ele s o boos hei agg ega ion
[88]. The in e ac ion o hese RGD sequences wi h pla ele in eg in
α
IIbβ3 has been ecen ly desc ibed using op ical ap-based me hods, and
he impo ance o his mo i in he hemos a ic p ocess has been high-
ligh ed [89]. In ac , RGD sequences a e ecen ly being used o a ge
pla ele s wi h di e en objec i es [90]. Thus, we hypo hesize ha he
inc eased pla ele adhesion capaci y o ou SP-CH is due o he in e ac-
ion be ween he RGD-mo i s o he SPI and he
α
IIbβ3 in eg in ecep o
o he pla ele s.
3.8. IN VIVO hemos a ic e ficacy
A a - ail ampu a ion model (Fig. 8A) was used o e alua e he in i o
hemos a ic p ope ies o he h ee ma e ials. Pho og aphs o he
appea ance o he ma e ials (Fig. 8B) we e aken du ing he expe imen .
The gauze ob ained a mean bleeding ime sco e o 2.8 0.8 (Fig. 8C),
which was simila o MRC's, 2.5 1.0. Meanwhile, SP-CH ob ained a
significan ly lowe (p >0.05) bleeding ime sco e, 1.8 0.4, meaning
ha i could p omo e hemos asis in i o as e han bo h gauze and MRC.
Rega ding o blood loss (Fig. 8D), no significan di e ences we e ound
be ween g oups. The gauze p esen ed a mean blood loss o 1.1 1.0 g,
MRC 1.2 0.8 g and SP-CH 0.6 0.3 g. Howe e , i is no ewo hy ha
bo h gauze and MRC showed an inc eased de ia ion among eplica es,
while blood loss in he SP-CH g oup emained consis en in all he es ed
samples, gua an eeing a homogeneous hemos a ic e ficacy.
We conside ha a lack o a sui able epis axis animal model supposes
a limi a ion in ou s udy. Indeed, e en i in i o epis axis models exis [91,
92] hey ei he in ol e la ge size animals o ail o ai h ully ep oduce a
eal case o epis axis, since an ex e nal hemo hage is induced and he
ea men is no in oduced in he nasal ca i y. The e o e, o he ini ial
e alua ion o he gene al hemos a ic p ope ies o ou SP-CH, we aimed
o choose a model wi h an adequa e ela ionship be ween ele ance and
educed in asi eness. In his ein, he a - ail ampu a ion model has been
he animal model o choice o many impo an s udies in ol ing he
e alua ion o hemos a ic p ope ies o bioma e ials [93–96], and hence i
seems o be an app op ia e model o his objec i e. Undoub edly, a
bigge sample size would ha e allowed de ec ing bigge di e ences be-
ween he assayed g oups. We unde s and ha he expe imen migh be
unde powe ed, bu i is s ill o ally alid, and iable om an e hical poin
o iew, espec ing he p inciples o he 3R. In his con ex , he bleeding
ime esul s suppo hose ob ained in he in i o expe imen s, whe e
SP-CH showed supe io hemos a ic p ope ies compa ed o he gauze and
MRC.
4. Conclusions
In his s udy we demons a ed ha by-p oduc s o he ood indus y
a e a aluable and sus ainable sou ce o bioma e ials ha can be
employed o manu ac u e sa e and e ec i e nasal packs wi h g ea he-
mos a ic p ope ies. The de eloped SP-CH p esen s a g ea ly in e -
connec ed po ous mic os uc u e, g ea wa e and blood swelling
capaci y and app op ia e e en ion o he abso bed fluids. Besides, me-
chanical p ope ies o he bioma e ial demons a ed o be adequa e o i s
use as a nasal pack. The bioma e ial p esen ed excellen biocompa ibili y
and hemocompa ibili y in i o. In e es ingly, i was able o lose weigh in
aqueous medium and o deg ade pa ially wi hin a ew days when
incuba ed in blood, which is becoming mo e and mo e impo an cha -
ac e is ic o nasal packs. Mo eo e , he hemos a ic p ope ies o ou SP-
CH ou pe o med hose o he wo nasal packs used in he clinical ou ine
wo ldwide: a s anda d gauze and he comme cial syn he ic pack Me -
ocel®. Ou bioma e ial was ound o e ec i ely p omo e blood coagu-
la ion in i o, showing ou s anding RBC and pla ele binding p ope ies
compa ed o he gauze and MRC, likely due o he in insic hemos a ic
p ope ies o i s na u al componen s. Al hough u he esea ch o i s
hemos a ic e ec in i o is needed, SP-CH significan ly sho ened
bleeding ime in a a - ail ampu a ion model. Addi ionally, he s udy o
he en i onmen al loads associa ed wi h he ex ac ion o ma e ials and
he manu ac u ing o sca olds showed low en i onmen al impac s in all
he ca ego ies analyzed. All in all, ou SP-CH, p oduced om a enewable
and sus ainable sou ce o bioma e ials (by-p oduc s o he ood indus y),
showed supe io mechanical and hemos a ic p ope ies compa ed o
Me ocel®and a s anda d gauze. Thus, we ha e demons a ed ha a
g een and eco iendly s a egy can be ollowed o de elop a bioma e ial
ha ou pe o ms he gold s anda d in he ea men o epis axis. This
leads us o belie e ha ou SP-CH may be an app op ia e nasal pack
candida e o he ea men o epis axis.
CRediT au ho ship con ibu ion s a emen
Jon Jimenez-Ma in: Me hodology, In es iga ion, W i ing –o iginal
d a . Ke in Las He as: Me hodology, In es iga ion, W i ing –o iginal
d a . Alai z E xabide: In es iga ion. Jone U anga: In es iga ion. Ko o
de la Caba: Resou ces, Funding acquisi ion, W i ing – e iew &edi ing.
Ped o Gue e o: In es iga ion, W i ing – e iew &edi ing. Manoli
Iga ua: Resou ces, Funding acquisi ion, W i ing – e iew &edi ing.
Edo a San os-Vizcaino: Concep ualiza ion, Me hodology, Resou ces,
Supe ision, Funding acquisi ion, W i ing – e iew &edi ing. Rosa
Ma ia He nandez: Concep ualiza ion, Me hodology, Resou ces, Supe -
ision, Funding acquisi ion, W i ing – e iew &edi ing, P ojec
adminis a ion.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing financial
in e es s o pe sonal ela ionships ha could ha e appea ed o influence
he wo k epo ed in his pape .
Acknowledgmen s
Au ho s hank Eusko Jau la i za (G upos Consolidados, No e :
IT907-16) and MCI/AEI/FEDER, UE (RTI2018-097100-B-C22).
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