applied
sciences
A icle
In luence on Bone- o-Implan Con ac o
Non-The mal Low-P essu e A gon Plasma:
An Expe imen al S udy in Ra s
A u o Sanchez-Pe ez 1, Ana I. Nicolas-Sil en e 2,* , Ca men Sanchez-Ma as 3,
Fe nando Muñoz-Guzon 4, Ca los Na a o-Cuella 5and Geo gios E. Romanos 6
1Depa men o Pe iodon ology, Medicine and Den is y Facul y, Mu cia Uni e si y, 30008 Mu cia, Spain;
[email p o ec ed]
2
Depa men o Res o a i e Den is y, Medicine and Den is y Facul y, Mu cia Uni e si y, 30008 Mu cia, Spain
3Vi gen del Rocio Hospi al, 41013 Se illa, Spain; [email p o ec ed]
4Ve e ina y School, Uni e si y o San iago de Compos ela, 27002 Lugo, Spain; [email p o ec ed]
5Depa men o Su ge y, Complu ense Uni e si y, 28031 Mad id, Spain; cna a [email p o ec ed]
6Depa men o Pe iodon ology, School o Den al Medicine, S ony B ook Uni e si y, S ony B ook,
NY 11794-8712, USA; geo gios. omanos@s onyb ookmedicine.edu
*Co espondence: [email p o ec ed]; Tel.: +34-96824-7946
Recei ed: 15 Ma ch 2020; Accep ed: 25 Ap il 2020; Published: 28 Ap il 2020
Abs ac :
Roughness cha ac e is ics play an essen ial ole in osseoin eg a ion. Howe e , he e is
a conce n abou he suscep ibili y o hose su aces o bac e ial coloniza ion. New echniques o
cleaning and su ace ea men ha e appea ed ha could a o osseoin eg a ion wi hou he need o
c ea e su aces as ough. Such is he case o non- he mal low-p essu e a gon plasma (NTLP-A P).
One hund ed
and o y- ou implan s we e placed in he ibiae o 36 Sp ague Dawley a s, dis ibu ed
in ou expe imen al g oups: I: mechanized su ace; II: mechanized su ace ea ed wi h NTLP-A P, III:
eso bable blas media (RBM) su ace; and IV: RBM su ace ea ed wi h (NTLP-A P). Bone- o-implan
con ac (BIC) pe cen ages we e calcula ed by mic o omog aphic e alua ion and his ological analysis
a one, wo, and ou weeks a e implan placemen . ANOVA and Mann–Whi ney es s we e used
o s a is ical analysis, es ablishing p<0.05. No signi ican di e ences we e ound a one-week
compa isons. The g oups ea ed wi h NTLP-A P ob ained highe BIC% han hose no ea ed a
wo and ou weeks. Mechanized su aces ea ed wi h NTLP-A P ob ained BIC alues simila o
RBM su aces.
Keywo ds:
a gon plasma; bone- o-implan con ac ; implan den is y; mechanized su ace;
osseoin eg a ion; eso bable blas media su ace
1. In oduc ion
In ecen yea s, B anema k- ype implan s wi h a mechanized su ace ha e been conside ed he
gold s anda d in mode n implan ology, leading o a p edic able beha io [
1
]. Howe e , se e al s udies
demons a ed ha he biocompa ibili y and cell iabili y could be imp o ed [
2
] by modi ying he
oughness o he implan su ace using di e en echniques [
3
]. O he au ho s ha e inco po a ed
s em cells wi h he same objec i e [
4
]. Fu he mo e, bioma e ials and s em cells ha e been equen ly
combined, ep esen ing a common s a egy o bone issue enginee ing applica ions [5].
This oughness in he implan su ace seems o be one o he essen ial ac o s in achie ing
adequa e osseoin eg a ion [
6
,
7
]. The inc ease in oughness has been shown o lead o an inc ease in
he bone- o-implan con ac pe cen age (BIC) [
8
]. Wi h he c ea ion o hese new su aces, he ime
needed o achie e osseoin eg a ion was educed due o he imp o emen in os eoconduc ion and
Appl. Sci. 2020,10, 3069; doi:10.3390/app10093069 www.mdpi.com/jou nal/applsci
Appl. Sci. 2020,10, 3069 2 o 11
os eogenesis [
9
]. I was e en pos ula ed ha he inc ease in oughness could p e en bone loss [
10
].
Howe e , o he au ho s exp essed conce n abou he g ea e suscep ibili y o hese ough su aces o
bac e ial con amina ion [
11
]. Rega dless o he deg ee o oughness, ano he ac o o conside is ha
he implan su aces unde go changes om hei manu ac u e o hei clinical use.
One p ocedu e p oposed o imp o e he implan su ace is based on non- he mal low-p essu e
a gon plasma (NTLP-A P) [
12
]. Some au ho s ha e al eady shown ha NTLP-A P is capable o
enhancing he implan su ace cha ac e is ics, and he e o e, he ea lies biological esponse wi hou
al e ing he o iginal opog aphy [13].
One in e es ing s a egy o be e alua ed by he indus y would be he de elopmen o mechanized
su aces o which we can con e high osseoin eg a ion capaci y. The mechanized su ace would ecei e
a su ace ea men ha does no modi y i s oughness bu signi ican ly imp o es he osseoin eg a ion
capaci y. Wi h hese cha ac e is ics, we would ob ain be e beha io agains pe i-implan diseases by
no ha ing a ough su ace. The possibili ies o e ec i e ea men and simple main enance would
be inc eased.
Ou null hypo hesis was ha NTLP-A P does no ep esen an imp o emen in e ms o BIC
alues compa ed o su aces ea ed and no ea ed wi h eso bable blas media (RBM). The al e na i e
hypo hesis was he exis ence o di e ences be ween using o no using NTLP-A P on bo h su aces.
2. Ma e ial and Me hods
2.1. Sample Desc ip ion
Animal expe imen s we e conduc ed wi h 36 emale adul Sp ague Dawley a s wi h a mean
weigh o 619 g (478 g o 767 g) om he animal housing acili ies o he Uni e si y o San iago de
Compos ela (Spain). Each a was iden i ied wi h a numbe on an ea . This s udy was de eloped
in s ic acco dance wi h he guidelines o he use o animals o expe imen a ion pu poses [
14
].
The s udy
was app o ed by he e hics commi ee o he Uni e si y o San iago de Compos ela (Spain).
The p inciples o eplacemen euse and educ ion we e obse ed, and all pe sonnel in cha ge o he
expe imen al wo k we e e ed o hei quali ica ions.
2.2. Animals Keeping
Animals we e kep in indi idual cages in a oom wi h pho ope iod, ai enewal, and egula ed
empe a u e and humidi y. They we e ed wi h a speci ic g anula ed die o i s species and ee
wa e supply.
2.3. Implan Fea u es
144 Tica e sc ew- ype implan s wi h a diame e o 1 mm and leng h o 3 mm we e used (Tica e,
Mozo-G au, Valladolid, Spain). A o al o 72 implan s p esen ed a mechanized su ace and 72 implan s
a su ace ea ed by eso bable blas media (RBM) (Tica e, Mozo-G au, Valladolid, Spain). Hal o
hem (36 mechanized and 36 RBM) ecei ed a su ace ea men wi h a non- he mal low-p essu e
a gon plasma, and he o he hal we e placed as ecei ed by he manu ac u e ; hus, ou expe imen al
g oups we e es ablished:
- G oup I (M): (n =36) Mechanized implan s.
-
G oup II (M-P): (n =36) Mechanized implan s ea ed wi h non- he mal low-p essu e
a gon plasma.
- G oup III (RBM): (n =36) RBM implan s.
-
G oup IV (RBM-P): (n =36) RBM implan s ea ed wi h non- he mal low-p essu e a gon plasma.
Appl. Sci. 2020,10, 3069 3 o 11
2.4. Randomiza ion
Be o e su ge y, a andomiza ion sequence was pe o med using a compu e applica ion [
15
] and
he esul s we e concealed un il su ge y. A e anes he izing he animal, he implan s we e placed
ollowing he andom o de es ablished by he app, always p oducing he same epe i ions pe posi ion
and side.
2.5. Su ge y P o ocol
All su gical p ocedu es we e pe o med unde s e ile condi ions in an ope a ing oom and gene al
anes hesia using iso luo ane a a concen a ion o 1%–3% and oxygen wi h a mask. Be o e su ge y,
each a was p emedica ed wi h mede omidine in amuscula (i.m.) 50–100
µ
m/kg, ke amine i.m.
75–100 mg/kg, bup eno phine subcu aneous (s.c.) 0.01–0.05 mg/kg and induced by iso lu ane chambe .
Animals we e moni o ed du ing he su ge y in e en ion by an acc edi ed e e ina ian ained in
labo a o y animal science (ca ego y B).
A e seda ion and anes hesia, epila ion and su gical cleaning o he skin wi h chlo hexidine
5 mg/mL
in 70% e hanol we e pe o med. A medial app oach o he me aphysis was made o bo h ibias
h ough an an e omedial skin incision. Skin and pe ios eum we e e lec ed using pe ios eal ele a o s
and sepa a o s. In he p oximal a ea o each ibia, wo i anium implan s we e placed a e d illing
wi h an inc easing diame e d ills echnique. The incision was su u ed in laye s wi h a 5-0 eso bable
su u e. An ibio ic p ophylaxis was adminis e ed wi h en o loxacin, 100 mg/li e o d inking wa e .
Wa e was changed on a daily basis. Animals we e p o ec ed om ligh o h ee weeks.
Pos ope a i ely, he pain was con olled wi h bup eno phine o h ee days and meloxicam as an
analgesic o i e days, ollowing he animal s udy p o ocol USC-Lugo FMG a s Mozo-G au 2016.
2.6. Eu hanasia
Each expe imen al g oup was andomly subdi ided in o h ee g oups so ha he animals we e
eu hanized one week, wo weeks, and ou weeks a e su ge y, depending on he g oup assigned.
All animals
we e eu hanized by an o e dose o pen oba bi al (40–60 mg/Kg/I.V.), and ibia biopsies
we e ob ained o his ological p ocessing.
2.7. Sample P ocessing
2.7.1. Sample P epa a ion
Tibias we e dissec ed, and bone blocks con aining implan s we e ob ained. The bone blocks we e
ixed wi h a 10% bu e ed o maldehyde solu ion o se en days a 4
◦
C. Each sample was coded wi h
a numbe om 1 o 36, which indica ed he animal, and wo le e s, he i s le e e e ing o he
loca ion o he limb, being igh o le (R o L), and he second le e indica ing he posi ion o he
implan , being p oximal o dis al (P o D).
2.7.2. Mic o omog aphy
The samples we e scanned using high- esolu ion compu e ized mic o omog aphy equipmen
(Skyscan 1172, B uke mic oCT NV, Kon ig, Belgium). To cap u e he images, a 1 mm hick aluminum
il e was used, and he pa ame e s used we e 80 K and 124
µ
A wi h a pixel size o 6
µ
m. The samples
we e w apped in o malin soaked gauzed inside an Eppendo ube. The canning was ca ied ou wi h
a o a ion o 360
◦
, and he acquisi ion o he images was e e y 0.4
◦
. Image econs uc ion was ca ied
ou by Feld amp’s algo i hm using NRecon econs uc ion so wa e (B uke mic oCT NV, Kon ig,
Belgium). The same pa ame e s o all samples we e used, being: His og am 0-030; Smoo hing: 2;
Beam ha dening: 25% and Ring a i ac co ec ion: 5.
The images we e di ided in o wo sec ions: co ical and medulla . Due o he la ge medulla
ca i y, his a ea was no conside ed in his s udy. The analysis o he co ical po ion was pe o med
Appl. Sci. 2020,10, 3069 4 o 11
wi h he help o Da a Viewe So wa e (B uke mic oCT NV, Kon ig, Belgium). The co ical su ace was
posi ioned as pa allel as possible ega dless o he implan placemen , and he olume o in e es (VOI)
was selec ed. In his s udy, only VOIs we e conside ed whe e he comple e co ical appea ed: bo h he
p oximal and he dis al pa o he implan mus appea su ounded en i ely by co ical bone.
Once he
egions o in e es we e selec ed, da a we e analyzed using CTAn wi h a h eshold o 80–255 o he
implan and 30–255 o ha d issue (implan +bone). Bone- o-implan con ac (BIC) was e alua ed o
each sample (Figu e 1).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 o 11
was posi ioned as pa allel as possible ega dless o he implan placemen , and he olume o in e es
(VOI) was selec ed. In his s udy, only VOIs we e conside ed whe e he comple e co ical appea ed:
bo h he p oximal and he dis al pa o he implan mus appea su ounded en i ely by co ical
bone. Once he egions o in e es we e selec ed, da a we e analyzed using CTAn wi h a h eshold o
80–255 o he implan and 30–255 o ha d issue (implan + bone). Bone- o-implan con ac (BIC)
was e alua ed o each sample (Image 1).
Image 1. E alua ion on one sample by Da a Viewe So wa e o measu ing bone- o-implan con ac
(BIC) (co ical and medulla ).
2.7.3. His ological p epa a ion
- Block p epa a ion
A e cu ing he specimens in o smalle agmen s, he blocks we e dehyd a ed in inc easing
concen a ions o alcohol (80%, 96%, 100%, and 100%) e e y wo days. Inclusion was pe o med on
a ecip oca ing shake using a ligh -cu ing glycolme hac yla e based esin (Techno i 7200 VLC;
He aeus-Kulze GMBH, We heim, Ge many) a inc easing concen a ions in alcoholic solu ion e e y
wo days. The polyme iza ion ook place using high-in ensi y blue ligh wi h speci ic equipmen .
- Shee p epa a ion and e alua ion
The blocks ob ained we e sec ioned and oughened using Exak sys em machine y (Exak
Apa a ebau GMBH, No de s ed , Ge many). Once he hin shee s we e sliced, hey we e s ained by
Le ai Laczko s aining. S ained sec ions we e digi ally pho og aphed a 40× using a powe ed s age
ligh mic oscope wi h a digi al came a (BX51, DP71; Olympus Co po a ion, Tokyo, Japan). Images
we e colo ed using a digi al able and image ea men so wa e (Pho oshop, Adobe, San Jose, CA,
USA), di e en ia ing he implan , bone, and so issue.
The esul s we e quan i ied h ough an image analysis p og am (CellSens, Olympus
Co po a ion, Tokyo, Japan) by an obse e blind o he ype o implan . The su ace in con ac wi h
mine alized bone was called bone- o-implan con ac (BIC) and was calcula ed as a pe cen age.
2.8. S a is ical Analysis
The means, s anda d de ia ions, and medians o all he da a we e calcula ed using he s a is ical
analysis so wa e Sigma Plo 12.5 (So onic In e na ional, Ba celona, Spain). Di e ences be ween
expe imen al g oups we e analyzed using a one-way analysis o a iance (ANOVA). The no mali y
was e i ied h ough he Shapi o–Wilk es . The Mann–Whi ney es was used o disce n di e ences
o con inuous a iables. Signi ican di e ences we e conside ed wi h p < 0.05.
Figu e 1.
E alua ion on one sample by Da a Viewe So wa e o measu ing bone- o-implan con ac
(BIC) (co ical and medulla ).
2.7.3. His ological P epa a ion
- Block p epa a ion
A e cu ing he specimens in o smalle agmen s, he blocks we e dehyd a ed in inc easing
concen a ions o alcohol (80%, 96%, 100%, and 100%) e e y wo days. Inclusion was pe o med
on a ecip oca ing shake using a ligh -cu ing glycolme hac yla e based esin (Techno i 7200 VLC;
He aeus-Kulze GMBH, We heim, Ge many) a inc easing concen a ions in alcoholic solu ion e e y
wo days. The polyme iza ion ook place using high-in ensi y blue ligh wi h speci ic equipmen .
- Shee p epa a ion and e alua ion
The blocks ob ained we e sec ioned and oughened using Exak sys em machine y (Exak
Apa a ebau GMBH, No de s ed , Ge many). Once he hin shee s we e sliced, hey we e s ained by
Le ai Laczko s aining. S ained sec ions we e digi ally pho og aphed a 40
×
using a powe ed s age ligh
mic oscope wi h a digi al came a (BX51, DP71; Olympus Co po a ion, Tokyo, Japan). Images we e
colo ed using a digi al able and image ea men so wa e (Pho oshop, Adobe, San Jose, CA, USA),
di e en ia ing he implan , bone, and so issue.
The esul s we e quan i ied h ough an image analysis p og am (CellSens, Olympus Co po a ion,
Tokyo, Japan) by an obse e blind o he ype o implan . The su ace in con ac wi h mine alized
bone was called bone- o-implan con ac (BIC) and was calcula ed as a pe cen age.
2.8. S a is ical Analysis
The means, s anda d de ia ions, and medians o all he da a we e calcula ed using he s a is ical
analysis so wa e Sigma Plo 12.5 (So onic In e na ional, Ba celona, Spain). Di e ences be ween
Appl. Sci. 2020,10, 3069 5 o 11
expe imen al g oups we e analyzed using a one-way analysis o a iance (ANOVA). The no mali y
was e i ied h ough he Shapi o–Wilk es . The Mann–Whi ney es was used o disce n di e ences
o con inuous a iables. Signi ican di e ences we e conside ed wi h p<0.05.
3. Resul s
Du ing he expe imen al pe iod, he e we e no unexpec ed dea hs. Animals ha bi he su u e,
had a wound exposed, o had pa o he skin emo ed om he a ea we e selec ed o be pa o he
g oup ha was eu hanized one week a e implan placemen . No p oblems we e ound du ing he
inal sampling, and all he implan s could be eco e ed.
Rega ding he mic o omog aphy analysis, no signi ican di e ence was obse ed be ween he
expe imen al g oups.
In esul s a one week, when we used non- he mal low-p essu e a gon plasma (NTLP-A P) on a
mechanized su ace, he BIC% was imp o ed; on he o he hand, on RBM su aces we did no ob ain
an imp o emen . Howe e , hese di e ences had no s a is ically signi ican di e ence.
In he second week, ou esul s showed lowe BIC% alues when NTLP-A P was used on
bo h su aces.
The esul s in he ou h week showed somewha highe BIC alues when NTLP-A P was applied.
G oup IV (RBM-P) showed highe BIC alues han he es o he g oups. Howe e , no s a is ically
signi ican di e ence was obse ed be ween g oups o be ween ime in e als (p<0.05) (Table 1)
(Figu e 2).
Table 1.
BIC pe cen ages and s anda d de ia ion o each expe imen al g oup ob ained by
mic o omog aphy a 1, 2, and 4 weeks. No s a is ically signi ican di e ence was obse ed be ween
g oups o be ween ime in e als aking in o accoun he signi icance alue o p<0.05.
Expe imen al
G oup n1 Week 2 Weeks 4 Weeks
BIC (%) C.I. 95% BIC (%) C.I. 95% BIC (%) C.I. 95%
I (M) 12 70.67
61.09–80.25
76.97
73.42–80.52
76.16
71.05–81.27
II (M-P) 12 75.52
71.73–79.31
73.48
70.09–76.87
77.92
73.34–82.50
III (RBM) 12 77.85
74.38–81.38
75.60
72.11–79.09
75.81
71.48–80.14
IV (RBM-P) 12 73.78
68.13–79.43
74.83
69.61–80.05
78.80
72.95–84.65
Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 o 11
3. Resul s
Du ing he expe imen al pe iod, he e we e no unexpec ed dea hs. Animals ha bi he su u e,
had a wound exposed, o had pa o he skin emo ed om he a ea we e selec ed o be pa o he
g oup ha was eu hanized one week a e implan placemen . No p oblems we e ound du ing he
inal sampling, and all he implan s could be eco e ed.
Rega ding he mic o omog aphy analysis, no signi ican di e ence was obse ed be ween he
expe imen al g oups.
In esul s a one week, when we used non- he mal low-p essu e a gon plasma (NTLP-A P) on
a mechanized su ace, he BIC% was imp o ed; on he o he hand, on RBM su aces we did no ob ain
an imp o emen . Howe e , hese di e ences had no s a is ically signi ican di e ence.
In he second week, ou esul s showed lowe BIC% alues when NTLP-A P was used on bo h
su aces.
The esul s in he ou h week showed somewha highe BIC alues when NTLP-A P was
applied. G oup IV (RBM-P) showed highe BIC alues han he es o he g oups. Howe e , no
s a is ically signi ican di e ence was obse ed be ween g oups o be ween ime in e als (p < 0.05)
(Table 1) (G aphic 1).
Table 1. BIC pe cen ages and s anda d de ia ion o each expe imen al g oup ob ained by
mic o omog aphy a 1, 2, and 4 weeks. No s a is ically signi ican di e ence was obse ed be ween
g oups o be ween ime in e als aking in o accoun he signi icance alue o p < 0.05.
Expe imen al
G oup
n
1 Week
2 Weeks
4 Weeks
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
I (M)
12
70.67
61.09–80.25
76.97
73.42–80.52
76.16
71.05–81.27
II (M-P)
12
75.52
71.73–79.31
73.48
70.09–76.87
77.92
73.34–82.50
III (RBM)
12
77.85
74.38–81.38
75.60
72.11–79.09
75.81
71.48–80.14
IV (RBM-P)
12
73.78
68.13–79.43
74.83
69.61–80.05
78.80
72.95–84.65
G aphic 1. BIC pe cen ages wi h he C.I. (95%) o each expe imen al g oup ob ained by
mic o omog aphy a 1, 2 and 4 weeks.
The mos signi ican di e ences we e obse ed when ca ying ou he his ological s udy (Image
2). A one week, ou esul s did no show di e ences wi h a s a is ical signi icance (p < 0.05), al hough
he RBM and RBM-P g oups showed highe alues.
A wo weeks, bo h su aces, mechanized and RBM, showed be e BIC when hey we e ea ed
wi h NTLP-A P, showing s a is ical di e ences be ween g oups I and II (p = 0.018), be ween g oups
I and IV (p = 0.001). S a is ical di e ences also appea ed be ween g oups III and IV (p = 0.035).
A ou weeks, no di e ences we e ound on he mechanized su ace when ea ed o no ea ed
wi h NTLP-A P (g oups I and II). On RBM su aces, highe BIC was ound when ea ed wi h NTLP-
Figu e 2.
BIC pe cen ages wi h he C.I. (95%) o each expe imen al g oup ob ained by mic o omog aphy
a 1, 2 and 4 weeks.
The mos signi ican di e ences we e obse ed when ca ying ou he his ological s udy (Figu e 3).
A one week, ou esul s did no show di e ences wi h a s a is ical signi icance (p<0.05), al hough he
RBM and RBM-P g oups showed highe alues.
Appl. Sci. 2020,10, 3069 6 o 11
A wo weeks, bo h su aces, mechanized and RBM, showed be e BIC when hey we e ea ed
wi h NTLP-A P, showing s a is ical di e ences be ween g oups I and II (p=0.018), be ween g oups I
and IV (p=0.001). S a is ical di e ences also appea ed be ween g oups III and IV (p=0.035).
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 o 11
A P showing s a is ical di e ences when compa ing g oup IV wi h g oups I (p = 0.018) and wi h
g oup II (p = 0.024) (Table 2) (G aphic 2).
Table 2. BIC pe cen ages and s anda d de ia ions o each expe imen al g oup ob ained by
his ological e alua ion a one, wo, and ou weeks. No s a is ically signi ican di e ence was
obse ed be ween g oups in he i s week (p < 0.05). A wo weeks, he e we e signi ican di e ences
be ween (α): M and M-P (p = 0.018); (β): M and RBM-P (p = 0.001); and (Ω): RBM and RBM-P (p =
0.035). A ou weeks, signi ican di e ences appea ed be ween (Φ) M and RBM-P (p = 0.018) and (γ)
M-P and RBM-P (p = 0.024).
Expe imen al
G oup
n
1 Week
2 Weeks
4 Weeks
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
I (M)
12
66.20
51.89–80.51
69.06 α β
62.93–77.19
78.56 Φ
70.10–87.02
II (M-P)
12
69.27
62.20–76.34
79.66 α
74.75–84.57
79.92 γ
71.46–88.38
III (RBM)
12
73.90
65.27–82.53
73.97 Ω
68.34–79.60
83.91
78.20–89.62
IV (RBM-P)
12
71.92
62.55–81.29
83.35 β Ω
80.47–90.23
89.49 Φ γ
86.82–92.16
G aphic 2. BIC pe cen ages wi h he C.I. (95%) o each expe imen al g oup ob ained by his ological
e alua ion a one, wo, and ou weeks. p alues= (α) 0.018; (β) 0.001; (Ω) 0.035; (Φ) 0.018; (γ) 0.024.
Image 2. His ological p epa a ions a one, wo, and ou weeks a e implan placemen .
4. Discussion
The p esen s udy aimed o e alua e he in luence on he bone- o-implan con ac (BIC) o non-
he mal low-p essu e a gon plasma (NTLP-A P) o e wo di e en implan su aces: a mechanized
Figu e 3. His ological p epa a ions a one, wo, and ou weeks a e implan placemen .
A ou weeks, no di e ences we e ound on he mechanized su ace when ea ed o no ea ed
wi h NTLP-A P (g oups I and II). On RBM su aces, highe BIC was ound when ea ed wi h NTLP-A P
showing s a is ical di e ences when compa ing g oup IV wi h g oups I (p=0.018) and wi h g oup II
(p=0.024) (Table 2) (Figu e 4).
Table 2.
BIC pe cen ages and s anda d de ia ions o each expe imen al g oup ob ained by his ological
e alua ion a one, wo, and ou weeks. No s a is ically signi ican di e ence was obse ed be ween
g oups in he i s week (p<0.05). A wo weeks, he e we e signi ican di e ences be ween (
α
): M
and M-P (p=0.018); (
β
): M and RBM-P (p=0.001); and (
Ω
): RBM and RBM-P (p=0.035). A ou
weeks, signi ican di e ences appea ed be ween (
Φ
) M and RBM-P (p=0.018) and (
γ
) M-P and RBM-P
(p=0.024).
Expe imen al
G oup n1 Week 2 Weeks 4 Weeks
BIC (%) C.I. 95% BIC (%) C.I. 95% BIC (%) C.I. 95%
I (M) 12 66.20
51.89–80.51
69.06 α β
62.93–77.19
78.56 Φ
70.10–87.02
II (M-P) 12 69.27
62.20–76.34
79.66 α
74.75–84.57
79.92 γ
71.46–88.38
III (RBM) 12 73.90
65.27–82.53
73.97 Ω
68.34–79.60
83.91
78.20–89.62
IV (RBM-P) 12 71.92
62.55–81.29
83.35 βΩ
80.47–90.23
89.49 Φγ
86.82–92.16
Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 o 11
A P showing s a is ical di e ences when compa ing g oup IV wi h g oups I (p = 0.018) and wi h
g oup II (p = 0.024) (Table 2) (G aphic 2).
Table 2. BIC pe cen ages and s anda d de ia ions o each expe imen al g oup ob ained by
his ological e alua ion a one, wo, and ou weeks. No s a is ically signi ican di e ence was
obse ed be ween g oups in he i s week (p < 0.05). A wo weeks, he e we e signi ican di e ences
be ween (α): M and M-P (p = 0.018); (β): M and RBM-P (p = 0.001); and (Ω): RBM and RBM-P (p =
0.035). A ou weeks, signi ican di e ences appea ed be ween (Φ) M and RBM-P (p = 0.018) and (γ)
M-P and RBM-P (p = 0.024).
Expe imen al
G oup
n
1 Week
2 Weeks
4 Weeks
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
BIC (%)
C.I. 95%
I (M)
12
66.20
51.89–80.51
69.06 α β
62.93–77.19
78.56 Φ
70.10–87.02
II (M-P)
12
69.27
62.20–76.34
79.66 α
74.75–84.57
79.92 γ
71.46–88.38
III (RBM)
12
73.90
65.27–82.53
73.97 Ω
68.34–79.60
83.91
78.20–89.62
IV (RBM-P)
12
71.92
62.55–81.29
83.35 β Ω
80.47–90.23
89.49 Φ γ
86.82–92.16
G aphic 2. BIC pe cen ages wi h he C.I. (95%) o each expe imen al g oup ob ained by his ological
e alua ion a one, wo, and ou weeks. p alues= (α) 0.018; (β) 0.001; (Ω) 0.035; (Φ) 0.018; (γ) 0.024.
Image 2. His ological p epa a ions a one, wo, and ou weeks a e implan placemen .
4. Discussion
The p esen s udy aimed o e alua e he in luence on he bone- o-implan con ac (BIC) o non-
he mal low-p essu e a gon plasma (NTLP-A P) o e wo di e en implan su aces: a mechanized
Figu e 4.
BIC pe cen ages wi h he C.I. (95%) o each expe imen al g oup ob ained by his ological
e alua ion a one, wo, and ou weeks. p alues=(α) 0.018; (β) 0.001; (Ω) 0.035; (Φ) 0.018; (γ) 0.024.
Appl. Sci. 2020,10, 3069 7 o 11
4. Discussion
The p esen s udy aimed o e alua e he in luence on he bone- o-implan con ac (BIC) o
non- he mal low-p essu e a gon plasma (NTLP-A P) o e wo di e en implan su aces: a mechanized
implan su ace, wi h a smoo h su ace (Ra <0.5
µ
m) and a eso bable blas media (RBM) su ace ha
was mode a ely ough (Ra 1.0–2.0 µm) [16].
I is widely known ha he os eoblas ac i i y is inc eased wi h mic o- oughness anging be ween
1 and 100
µ
m when compa ed wi h un ea ed o smoo h su aces [
17
]. This is he eason why he
indus y de elops new su ace ea men echniques o achie e adequa e opog aphy, oughness,
o chemical composi ion cha ac e is ics o imp o e cell iabili y and biocompa ibili y [2].
Rega ding he chemical composi ion, Velasco-O ega e al. [
18
] showed ha he TiO
2
su ace
ea men and composi ion in luence he we abili y and su ace ee ene gy, a ec ing he p o ein
adso p ion. In he ci ed s udy, he machined (smoo h) su ace showed a minimal a ea occupied by cells
o connec i e issue g owing a ound. In addi ion, he cells cul u ed on oughe su aces p esen ed
a ibu es o mo e di e en ia ed os eoblas s [18].
Howe e , hese new su aces which allow quick osseoin eg a ion p esen some disad an ages,
like he p esence o ace elemen s, such as me als, ions, de e gen s, o lub ican s deposi ed on he
su ace [
19
] ha may a ec he body’s esponse du ing he osseoin eg a ion p ocess, leading o he
undesi able o ma ion o in lamma o y issue su ounding he implan [20,21].
Recen esea ch lines sugges he use o some echniques capable o enhancing he su ace
cha ac e is ics wi hou modi ying he opog aphy o oughness ea u es, as is he case wi h NTLP-A P.
Osseoin eg a ion ep esen s a dynamic p ocess bo h du ing i s es ablishmen and i s main enance.
Se e al ac o s ha e been ela ed o p ima y s abili y du ing he es ablishmen phase, including local
bone quali y and quan i y, and implan mac o-design [
22
–
24
]. All o hese come oge he in an adequa e
p ima y s abili y.
In he ea ly phase, he mac o-design o he implan plays an essen ial ole in p ima y s abili y.
This p ima y s abili y is due o he s ong con ac and ic ion be ween he h ead shape and he
bone [
25
]. The bone o ma ion begins wi h he angiogenesis p ocess, and is ini ia ed in he conca i ies
a he han con exi ies o he implan h eads due o he highe concen a ion o essels inside hese
conca i ies. Howe e , he p ecise mechanism is no ye comple ely elucida ed. I seems ha because
he en i onmen o med inside he ca i ies p omo es mechanical o ces, he concen a ion o g adien s
o chemo ac ic molecules and blood clo e en ion could d i e ascula and bone cell mig a ion [26].
The space c ea ed be ween he h eads (called he chambe ) is illed by he blood clo ha is
p og essi ely eplaced by a connec i e ma ix wi h mesenchymal cells and su ounded by blood
essels [27].
The h ead geome y no only a ec s he dis ibu ion o s ess o ces a ound he implan , bu i
also di ec ly in luences he size o he chambe and, indi ec ly, he bone healing. The e o e, he wid h,
he leng h, and he h ead shape all play an impo an ole [28,29].
In gene al, bone healing phases ha e been s udied in animal models [
25
,
30
–
34
]. In human s udies,
i was shown ha he pe cen age o newly o med bone a ached o he implan su ace inc eased wi h
ime [35,36].
One week a e inse ion, he os eoid deposi can be obse ed wi h a eas o newly o med bone.
This new bone g adually inc eases du ing he second week wi h he p esence o os eoblas s and blood
essels. Meanwhile, in some pi ch egions o he h ead (a eas ha we e esponsible o p ima y
mechanical s abili y), he bone issue exhibi ed signs o bone emodeling [25].
Some s udies ha e e alua ed how he mic o opog aphy may a ec he cellula esponse showing
ha local issue g ow h is p opo ional o he subs a e cu a u e, di ec ly a ec ing he os eoblas
beha io [
37
]. Magnaudeix e al. demons a ed in an
in i o
s udy ha he po e geome y in luences
coloniza ion by blood essels, due o he po e size and shape ha ing a c i ical ole in guiding cell
beha io [
38
]. This in luence was also demons a ed by Juigne e al. [
39
] in an
in i o
s udy g owing
mouse cal a ia cells o e hyd oxyapa i e ce amics wi h di e en mac o g oo e shapes, which ound
Appl. Sci. 2020,10, 3069 8 o 11
ha he o ganiza ion and g ow h a e p omo ed by a igh closu e angle o he subs a e geome y.
These au ho s desc ibed highe numbe s o cells a ached o g oo ed ce amics compa ed o la
con ols, sugges ing ha he mic oa chi ec u e p omo ed adhesion o he su ace. These esul s a e
in ag eemen wi h ou indings when compa ing he wo su aces (mechanized and RBM) wi hou
NTLP-A P ea men . The esul s also ag ee when compa ing bo h su aces a e NTLP-A P ea men ,
demons a ing ha he mic omo phology a ec s he BIC alues. None heless, he mic omo phology
no only in luences he issue esponse bu also he physical s imuli p oduced on he su ace by
NTLP-A P wi hou al e ing he su ace opog aphy.
The mos impo an ac o s a ec ing he complex p ocess o he in eg a ion o den al implan s o
he al eola bone a e he physical and chemical p ope ies o he su ace o he den al implan . Thus,
he su ace o a den al implan possesses impo an cha ac e is ics ha play a c ucial ole in bone
deposi ion and consequen healing and osseoin eg a ion.
The de elopmen o NTLP-A P allows o cleaning he implan su ace while modi ying he su ace
p ope ies wi hou al e ing hei mic o-design [
40
]. This imp o emen has mean ha mechanized
su aces can ha e a po en ial o osseoin eg a ion simila o ha o ough su aces, bu wi hou
hei d awbacks.
Ou s udy ocused on demons a ing whe he he e we e di e ences be ween mechanized implan
su aces and hose ea ed wi h RBM, bo h o hem ea ed o no wi h NTLP-A P, by compa ing he
BIC% a h ee di e en ime in e als: one week, wo weeks, and ou weeks a e implan placemen .
Ou esul s showed be e BIC% when he su aces we e ea ed wi h NTLP-A P in bo h mechanized
and RBM su aces. Su p isingly, he esul s o he mechanized su aces ea ed wi h NTLP-A P we e
simila o RBM su aces wi hou plasma in BIC alues.
The da a ob ained in his s udy a e in ag eemen wi h o he esul s published in p e ious animal
s udies, whe e NTLP-A P has been shown o imp o e he p ope ies o implan su aces [
41
], as well
as o he s udies ha con i m he esul s ob ained in cell cul u es [13,42].
The ac o highligh in ou esul s is ha he beha io o e e y expe imen al g oup was simila
du ing he i s week. This may be explained due o he nonspeci ic healing ha occu ed in he ini ial
s ages. These esul s imp o ed bo h in he second and he ou h week. The di e ences be ween he
expe imen al g oups again unde sco e he imp o emen wi h NTLP-A P applica ion in bo h su aces,
mechanized and eso bable blas media (RBM), a any poin in ime.
Finally, i should be men ioned ha he bene icial e ec s o NTLP-A P no only appea on i anium
su aces bu ha e also been desc ibed on di e en subs a es [31,35,36].
Sca ano e al. also e alua ed he e ec o he mal ea men o Ti6Al4V den al implan s on bone
healing in abbi s, showing esul s in ag eemen wi h ou s a emen s, and concluding ha he su aces
modi ied by he mal ea men imp o e he bioac i i y and BIC, a ac ha clinically means a sho ening
in he implan healing pe iod, especially in a eas wi h low-densi y bone [43].
Ou s udy could p esen some limi a ions, such as he apid healing in animals, which can make a
di e ence o humans. I is also limi ed by he ollow-up ime, which, in ou case, las ed up o ou
weeks. I would be in e es ing o e alua e he esul s a a longe ollow-up ime o check he s abili y
o he esul s. Ano he u u e esea ch issue would be he induc ion o pe i-implan i is, o assess he
esis ance o in ec ion and he e ec i eness o he ea men by he use o NTLP-A P.
5. Conclusions
Taking in o accoun he limi a ions o ou s udy, we con i m ou al e na i e hypo hesis wi h
he exis ence o di e ences be ween using o no using NTLP-A P on bo h su aces, and we can
conclude ha :
-
In he i s week o healing, he applica ion o NTLP-A P o e he implan su ace does no
ep esen any imp o emen among he expe imen al g oups.
- A wo weeks o healing, he g oups ea ed wi h NTLP-A P ob ained highe BIC alues.
Appl. Sci. 2020,10, 3069 9 o 11
-
A wo weeks o healing, he mechanized su ace ea ed wi h NTLP-A P ob ained simila BIC
alues as he RBM su ace.
-
A ou weeks o healing, he RBM-P g oup ob ained he highes BIC alues compa ed o he
o he expe imen al g oups
- These esul s should be implemen ed and e i ied wi h human clinical ials.
Au ho Con ibu ions:
All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Concep ualiza ion, A.S.-P.; me hodology, A.S.-P., F.M.-G. and C.N.-C.; alida ion, C.S.-M. and A.I.N.-S.; o mal
analysis, F.M.-G.; in es iga ion, A.S.-P. and A.I.N.-S.; esou ces A.S.-P.; da a cu a ion, F.M.-G. and C.N.-C.;
w i ing—o iginal d a p epa a ion, A.I.N.-S.; w i ing— e iew and edi ing, A.S.-P and G.E.R.; isualiza ion, G.E.R.
and A.S.-P.; supe ision, G.E.R. and A.S.-P.; p ojec adminis a ion, A.S.-P.; unding acquisi ion, A.S.-P.
Funding: This esea ch ecei ed no ex e nal unding.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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