pha maceu ics
Re iew
Ad ances in Use o Nanoma e ials o
Musculoskele al Regene a ion
Jose Jampilek 1,* and Daniela Placha 2,3,*


Ci a ion: Jampilek, J.; Placha, D.
Ad ances in Use o Nanoma e ials o
Musculoskele al Regene a ion.
Pha maceu ics 2021,13, 1994. h ps://
doi.o g/10.3390/pha maceu ics13121994
Academic Edi o s: Hansoo Pa k,
Won-Gun Koh and Kwang-sun Kim
Recei ed: 24 Oc obe 2021
Accep ed: 22 No embe 2021
Published: 24 No embe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Analy ical Chemis y, Facul y o Na u al Sciences, Comenius Uni e si y, Ilko ico a 6,
842 15 B a isla a, Slo akia
2Nano echnology Cen e, CEET, VSB-Technical Uni e si y o Os a a, 17. Lis opadu 2172/15,
708 33 Os a a-Po uba, Czech Republic
3Cen e ENET, CEET, VSB-Technical Uni e si y o Os a a, 17. Lis opadu 2172/15,
708 33 Os a a-Po uba, Czech Republic
*Co espondence: [email p o ec ed] (J.J.); [email p o ec ed] (D.P.)
Abs ac :
Since he wo ldwide incidence o bone diso de s and ca ilage damage has been inc easing
and adi ional he apy has eached i s limi s, nanoma e ials can p o ide a new s a egy in he
egene a ion o bones and ca ilage. The nanoscale modi ies he p ope ies o ma e ials, and many
o he ecen ly p epa ed nanocomposi es can be used in issue enginee ing as sca olds o he
de elopmen o biomime ic ma e ials in ol ed in he epai and healing o damaged issues and
o gans. In addi ion, some nanoma e ials ep esen a no ewo hy al e na i e o ea men and
alle ia ing in lamma ion o in ec ions caused by mic obial pa hogens. On he o he hand, some
nanoma e ials induce in lamma ion p ocesses, especially by he gene a ion o eac i e oxygen species.
The e o e, i is necessa y o know and unde s and hei e ec s in li ing sys ems and use su ace
modi ica ions o p e en hese nega i e e ec s. This con ibu ion is ocused on nanos uc u ed
sca olds, p o iding a close s uc u al suppo app oxima ion o na i e issue a chi ec u e o cells
and egula ing cell p oli e a ion, di e en ia ion, and mig a ion, which esul s in ca ilage and bone
healing and egene a ion.
Keywo ds:
nanoma e ials; nanocomposi es; ca ilage; bones; implan s; healing; musculoskele al diso de s
1. In oduc ion
Degene a i e diseases o he bones and join s a ec millions o people. F ac u es o
he hands, hips, and spine caused by os eopo osis a e associa ed wi h signi ican mo bidi y
and mo ali y. Des uc ion and de o mi y o he join s and o he complica ions caused
by a h i is no only make mo emen di icul , bu educe he abili y o pe o m ou ine
ac i i ies, which esul s in an o e all educed quali y o li e o pa ien s, among o he
hings. P olonged li e has a ec ed many aspec s o he e e yday li e o olde people, one
o which is he di icul y o mo emen common in olde people who su e om p oblems
such as ea o alling (81.7%), inabili y o s and wi hou a m suppo (81.1%), inabili y o
climb up he s ai s (81.3%), and slow walking speeds (71.7%), so hey p e e no o lea e
he la s (50%) [1–3].
Os eopo osis and a h i is a e among he mos common and se ious diseases o he
musculoskele al sys em [
4
–
6
]. Os eopo osis is a disease in which bone weakness inc eases
he isk o ac u es and is he mos common cause o ac u es ( e eb ae, o ea ms, and hip
bones) in he elde ly, especially women. Os eopo osis can also occu as a esul o a numbe
o diseases (alcoholism, ano exia, hype hy oidism, kidney disease, and su gical emo al
o he o a ies) o ea men (an ihype ensi es, chemo he apy, p o on pump inhibi o s,
selec i e se o onin eup ake inhibi o s, and glucoco icoids). I is cu en ly es ima ed ha
mo e han 200 million people su e om os eopo osis [
2
,
4
,
7
,
8
]. In u n, a h i is is an in-
lamma o y disease o he join s. I is mani es ed by swelling, edness, pain, and es ic ion
Pha maceu ics 2021,13, 1994. h ps://doi.o g/10.3390/pha maceu ics13121994 h ps://www.mdpi.com/jou nal/pha maceu ics
Pha maceu ics 2021,13, 1994 2 o 48
o mo emen . The e a e se e al ypes o a h i is, he mos common o which a e heuma-
oid a h i is (RA) and os eoa h i is (OA) [
5
,
6
,
9
]. OA is a degene a i e disease o he join s
in which he a icula ca ilage and he bones benea h i b eak down. I is he mos common
o m o a h i is, a ec ing abou 3.3% o he wo ld’s popula ion. The symp oms p og ess
slowly o e he yea s, bu only he join s a e a ec ed. The eal causes a e unknown [
9
–
13
].
A desc ip ion o he s uc u al al e a ion o ca ilage and chond ocy e-speci ic changes
in OA wi h he indica ed isk ac o s is shown in
Figu e 1[12].
The isk ac o s ha can
con ibu e o he de elopmen o OA a e named in Figu e 1; howe e , hey a e no he only
ac o s. In his igu e, a scheme o ca ilage s uc u al al e a ion and chond ocy e-speci ic
changes in OA is also depic ed including ca ilage b eakdown, subchond al bone hicken-
ing, o ma ion o os eophy es and co pus libe um, na owed join space, hickened and
ib o ic ligamen s, and join capsule hype ophy. A dec ease in chond ocy e numbe s in
ca ilage is seen by inc eased apop osis. Chond ocy es may be subjec o dedi e en ia ion
and o m he hype ophic and senescen pheno ypes. Senescence-associa ed sec e o y
pheno ype (SASP) is syn hesized and sec e ed by chond ocy es c ea ing a de imen al
en i onmen wi hin he join [
12
]. While he incidence o OA is much highe han ha o
RA (0.1–2.0% o he wo ld’s popula ion), he la e is a a mo e complex disease ha ing
o e 50 o ms wi h an o e iding and a iable in luence o in lamma ion and immune
media ed cellula e ec s in all o hese. RA is a sys emic in lamma o y au oimmune disease
ha leads o ch onic in lamma ion o he syno ial issue, causing i e e sible damage o
ca ilage and join bones. Howe e , in lamma ion can also a ec he eyes, hea , and lungs,
leading o ca dio ascula and espi a o y diseases. Thus, in addi ion o a adical educ ion
in quali y o li e, RA is associa ed wi h inc eased mo ali y. The causes o RA a e unknown.
Risk ac o s include gene ic p edisposi ion, excessi e smoking, alcoholism, obesi y, and
en i onmen al exposu e o asbes os [
5
,
14
–
16
]. An impo an dis inc ion be ween OA and
RA is ha OA is mechanically d i en wi h a mino in lamma o y componen compa ed o
RA; hence, OA p edominan ly a ec s he majo weigh bea ing join s such as he hip and
knee, while RA, which is p edominan ly d i en by in lamma ion and immune p ocesses,
also a ec s he smalle join s and is a symme ical disease (i.e., wi h he same in e pha-
langeal and me aca pal phalangeal join s o he inge s on bo h hands a ec ed). This is a
speci ic ea u e o RA no seen in OA. Fu he mo e, he ankle join s a e a ely a ec ed by
OA, while hey can be a ec ed by RA [5,6,16].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 2 o 47
hibi o s, selec i e se o onin eup ake inhibi o s, and glucoco icoids). I is cu en ly es i-
ma ed ha mo e han 200 million people su e om os eopo osis [2,4,7,8]. In u n, a h i-
is is an in lamma o y disease o he join s. I is mani es ed by swelling, edness, pain, and
es ic ion o mo emen . The e a e se e al ypes o a h i is, he mos common o which
a e heuma oid a h i is (RA) and os eoa h i is (OA) [5,6,9]. OA is a degene a i e disease
o he join s in which he a icula ca ilage and he bones benea h i b eak down. I is he
mos common o m o a h i is, a ec ing abou 3.3% o he wo ld’s popula ion. The symp-
oms p og ess slowly o e he yea s, bu only he join s a e a ec ed. The eal causes a e
unknown [9–13]. A desc ip ion o he s uc u al al e a ion o ca ilage and chond ocy e-
speci ic changes in OA wi h he indica ed isk ac o s is shown in Figu e 1 [12]. The isk
ac o s ha can con ibu e o he de elopmen o OA a e named in Figu e 1; howe e ,
hey a e no he only ac o s. In his igu e, a scheme o ca ilage s uc u al al e a ion and
chond ocy e-speci ic changes in OA is also depic ed including ca ilage b eakdown, sub-
chond al bone hickening, o ma ion o os eophy es and co pus libe um, na owed join
space, hickened and ib o ic ligamen s, and join capsule hype ophy. A dec ease in
chond ocy e numbe s in ca ilage is seen by inc eased apop osis. Chond ocy es may be
subjec o dedi e en ia ion and o m he hype ophic and senescen pheno ypes. Senes-
cence-associa ed sec e o y pheno ype (SASP) is syn hesized and sec e ed by chond ocy es
c ea ing a de imen al en i onmen wi hin he join [12]. While he incidence o OA is
much highe han ha o RA (0.1–2.0% o he wo ld’s popula ion), he la e is a a mo e
complex disease ha ing o e 50 o ms wi h an o e iding and a iable in luence o in-
lamma ion and immune media ed cellula e ec s in all o hese. RA is a sys emic in lam-
ma o y au oimmune disease ha leads o ch onic in lamma ion o he syno ial issue,
causing i e e sible damage o ca ilage and join bones. Howe e , in lamma ion can also
a ec he eyes, hea , and lungs, leading o ca dio ascula and espi a o y diseases. Thus,
in addi ion o a adical educ ion in quali y o li e, RA is associa ed wi h inc eased mo -
ali y. The causes o RA a e unknown. Risk ac o s include gene ic p edisposi ion, exces-
si e smoking, alcoholism, obesi y, and en i onmen al exposu e o asbes os [5,14–16]. An
impo an dis inc ion be ween OA and RA is ha OA is mechanically d i en wi h a mino
in lamma o y componen compa ed o RA; hence, OA p edominan ly a ec s he majo
weigh bea ing join s such as he hip and knee, while RA, which is p edominan ly d i en
by in lamma ion and immune p ocesses, also a ec s he smalle join s and is a symme -
ical disease (i.e., wi h he same in e phalangeal and me aca pal phalangeal join s o he
inge s on bo h hands a ec ed). This is a speci ic ea u e o RA no seen in OA. Fu he -
mo e, he ankle join s a e a ely a ec ed by OA, while hey can be a ec ed by RA [5,6,16].
Figu e 1. Risk ac o s (desc ibed in ed) con ibu ing o he de elopmen o os eoa h i is (OA). De-
sc ip ion o s uc u al al e a ion and chond ocy e-speci ic changes in os eoa h i is (desc ibed in
black). ECM: Ex acellula ma ix; SASP: Senescence-associa ed sec e o y pheno ype. Adap ed om
[12], MDPI, 2017.
Figu e 1.
Risk ac o s (desc ibed in ed) con ibu ing o he de elopmen o os eoa h i is (OA).
Desc ip ion o s uc u al al e a ion and chond ocy e-speci ic changes in os eoa h i is (desc ibed
in black). ECM: Ex acellula ma ix; SASP: Senescence-associa ed sec e o y pheno ype. Adap ed
om [12], MDPI, 2017.
Pha maceu ics 2021,13, 1994 3 o 48
Many di e en ea men app oaches a e being de eloped o he bu ning p oblem
o inc easingly common musculoskele al degene a i e diseases. T ea men op ions o he
musculoskele al sys em a e non-pha macological, pha macological, and su gical.
E o s o p e en ac u es in people wi h os eopo osis include die , exe cise, all
p e en ion, and li es yle changes ( educing smoking and alcohol). S anda d ea men is
ho mone eplacemen he apy (HRT: es ogen + p oges ogen, ibolone, aloxi ene, es os-
e one, calci onin), bisphosphona es ( ised ona e, e id ona e, alend ona e), e ipa a ide
( ecombinan pa a hy oid ho mone), s on ium anela e, denosumab, and i amin D sup-
plemen a ion [4,7,8,17–20].
A h i ides a e incu able diseases o his momen , so he goal o ea men is o
achie e emission o a low ac i i y o he disease. The la e he ea men is s a ed, he
wo se he esul s and he i e e sible damage o he join s a e. Non-pha macological
ea men is based on egula exe cise (weigh educ ion, physical ac i i y), ehabili a ion,
and manipula ion he apy o s eng hen muscles and main ain maximum mobili y and
join unc ionali y. In ad anced s ages o he disease, some damaged join s can be su gically
emo ed and eplaced wi h a i icial implan s (join endop os heses). Su gical ea men
o he pa ien also elie es pain in he a ec ed join . Pha macological ea men includes
wo basic g oups o d ugs, which a e usually combined: d ugs ha educe in lamma ion
and pain, and d ugs ha educe he p og ession o s uc u al damage (i.e., inhibi he
des uc ion o a icula ca ilage and induce he balance o i s me abolism). Non-s e oidal
an i-in lamma o y d ugs (nap oxen, ibup o en, and COX-2 selec i e inhibi o s), pa ace a-
mol, and weak opia es ( amadol) a e used o educe in lamma ion and pain. In he case
o acu e in lamma ion, glucoco icoids (hyd oco isone) can be gi en. Disease-modi ying
an i heuma ic d ugs educe he p og ession o s uc u al damage. They can be di ided
in o syn he ic and biological d ugs. Syn he ic d ugs a e u he di ided in o con en ional
syn he ics (me ho exa e, sul asalazine, le lunomide, hyd oxychlo oquine, chlo oquine,
and gold sal s) and a ge ed syn he ic JAK kinase inhibi o s (ba ici inib, o aci inib). Bio-
logicals a e an ibodies (e.g., adalimumab, e ane cep , in liximab, anakin a, ocilizumab,
aba acep , i uximab) agains p o-in lamma o y media o s/agen s o humo al and cell-
media ed immuni y [
5
,
16
,
21
–
26
]. In his con ex , i is necessa y o men ion ha he e a e
also many die a y supplemen s on he ma ke ha a e in ended o p e en o alle ia e
diseases o he musculoskele al sys em. Agen s ha inhibi he des uc ion o a icula ca i-
lage a e so-called chond op o ec i es; cu en ly ecommended a e glucosamine sul a e and
chond oi in sul a e (no o be chlo ide sal s), hyalu onic acid, a ocado-soybean unsaponi i-
ables, diace ein, Boswellia se a a ex ac , cu cumin, S-adenosyl me hionine, me hylsul onyl-
me hane, and ose hip. Al e na i ely, ish li e oil, omega-3 a y acids, i amins A, C, and
E in combina ion, i amin K, i amin D, ginge , Russian oli e
(Elaeagnus angus i olia),
ne le
(U ica dioica), and collagen/gela in a e lis ed as bene icial die a y supplemen s [
23
,
25
–
30
].
In addi ion o a ious ypes o d ugs including biological ea men s, mode n ma e-
ials made by nano echnology ha e begun o be used. These a e nanosys ems o d ug
deli e y wi h a ge ed dis ibu ion and modi ied d ug elease, nanodiagnos ics, and a i-
ous ma e ials wi h hei own an imic obial ac i i y [
31
–
38
]. In his con ex , i is necessa y
o men ion ha nanosupplemen s o os eopo osis managemen and o he supply o
i amins and many o he biologically ac i e agen s can be ound in he de elopmen and
on he ma ke [
30
,
39
–
41
]. I is mainly nanosized calcium in able s o nanopowde ed
eggshells, which is added o yogu o milk, esul ing in high-calcium yogu , calcium-
o i ied milk. Calcium ea ed in his way has conside ably inc eased bioa ailabili y and
e ec i ely se es as a suppo i e ea men o diseases ela ed o calcium de iciency in he
bones. Addi ionally, i amins and many o he biologically ac i e agen s a e e o mula ed
in o nanosys ems wi h inc eased bioa ailabili y and s abili y, especially wi h inc eased
dis ibu ion/ a ge ed deli e y o bone o ca ilage issue [31,33,34,39–42].
Ano he applica ion o nanoma e ials, nanocomposi es (NCPs), can be ound in issue
enginee ing, whe e hey began o be used as sca olds o he de elopmen o biomime ic
ma e ials in ol ed in he epai and healing o damaged issues and o gans [
43
–
47
]. The
Pha maceu ics 2021,13, 1994 4 o 48
e m “ issue enginee ing” was p oposed as ea ly as 1987 and is de ined by he Na ional
Science Founda ion as “
. . .
he applica ion o p inciples and me hods o enginee ing
and li e sciences owa d undamen al unde s anding he s uc u e– unc ion ela ionship
be ween no mal and mammalian issues and de elopmen o biological subs i u es o
es o e, main ain, and imp o e issue unc ions.” [
48
]. NCPs eplace damaged pieces
o issue and, in his case, a e designed o acili a e he g ow h o bone and ca ilage
cells. I is he e o e an inno a i e s a egy o u he help pa ien s a ec ed by he abo e-
men ioned diseases, which adically educe mobili y and quali y o li e. These NCPs
de eloped as eplacemen s o bones, join s, and ca ilage can be supplemen ed wi h
o he subs ances/d ugs ha ha e local an imic obial, an i-in lamma o y, o e en an i-
cance ac ion in o de o maximize he egene a i e e ec s o issues associa ed wi h he
musculoskele al sys em, which is pa hologically al e ed.
This con ibu ion is ocused on nanos uc u ed sca olds p o iding a s uc u al sup-
po app oxima ion o na i e issue a chi ec u e o cells and egula ing cell p oli e a ion,
di e en ia ion, and mig a ion, which esul s in ca ilage and bone healing and egene a ion.
2. Applied Nanoma e ials
Va ious ma e ials, cells, and bioac i e compounds a e s udied and assembled in
issue enginee ing o p o ide a h ee-dimensional (3D) sca old ha would be used o
induce and/o s imula e di e en ia ion signals and hus p omo e egene a ion o damaged
issue. The 3D s uc u e, ideally made o biomime ic ma e ials, is popula ed by cells
and mus he e o e p o ide a sui able en i onmen o cell g ow h, p oli e a ion, and
di e en ia ion. S em cells, undi e en ia ed cells wi h he abili y o di ide and gi e ise
o a ious o ms o specialized cells, a e ins alled in he 3D sca old. F equen ly, he
sca old con ains a a ie y o g ow h ac o s o di ec he beha io o he cells o he desi ed
p ocess, whe e he ul ima e goal is o c ea e a ully unc ional o gan/ issue. In o de o
a ma e ial o be used in issue enginee ing, i needs o mee basic equi emen s such as
(i) biocompa ibili y; (ii) bioin eg a ion; (iii) mechanical s abili y; (i ) easy p oduc ion and
handling; and ( ) low p oduc ion cos s [
49
–
51
]. A nice example is he use o ma e ials
based on chond oi in sul a e, hyalu onan, o collagen-binding pep ides o biosca olds.
All hese s uc u al mo i s con ibu e o he p oli e a ion and di e en ia ion o s em cells in
he sca old en i onmen and a e hus able o cause issue egene a ion and healing [
52
,
53
].
Nanoma e ials seem o be he ideal nanos uc u ed sca old in issue enginee ing
ha aims o eplace/ epai damaged issues in he human body. Nano echnologies a e
undoub edly one o he key echnologies o he new millennium. This ield is cons an ly
g owing and p o ides/specializes in he de elopmen o ma e ials wi h unique dimensions
and hus unique p ope ies ha ha e enabled signi ican b eak h oughs in many ields o
human ac i i y and ha e en e ed medicine and biological enginee ing. Nanoscale ma e ials
change hei physical and chemical p ope ies [
54
–
56
]; in his way, a numbe o indus-
ial, pha maceu ical, and medical p oduc s ha e been imp o ed and
inno a ed [57–67]
including a ious biocompa ible NCPs o he cons uc ion o medical implan s. The
success o all hese inno a i e biomedical applica ions is e lec ed in he size o he in e na-
ional nanomedicine ma ke , which is es ima ed a $293.1 billion in 2022 and g owing o
$350.8 billion
in 2025. On he o he hand, he e a e ba ie s o hei ull use, especially hei
oxicological p oblems [50,68,69].
A a ie y o biocompa ible ma e ials a e used o c ea e sca olds. Such a sca old is a
empla e o cell adhesion, di e en ia ion, p oli e a ion, and egene a ion/g ow h, which
means ha he sca old mus ha e a sui able mic oen i onmen o g owing cells. This can
be ensu ed by sui able su ace modi ica ion/ unc ionaliza ion o he chemical s uc u e o
p o ide minimal cy o oxici y, high biocompa ibili y, and adhesion o he cells o he whole
a i icial sca old. The e o e, i is no su p ising ha nanoma e ials ha e al eady been used
as su gical implan s o issue epai and egene a ion in den is y and o hopedics, he
p ope ies o NCPs o p omo e ca ilage and bone g ow h being used in combina ion wi h
hei abili y o ancho an i-in lamma o y, an i-in ec i e, o an i-cance d ugs [69–74].
Pha maceu ics 2021,13, 1994 5 o 48
NCPs used as implan s include o ganic–ino ganic, ino ganic–ino ganic, and bioino -
ganic nanoma e ials and a e, in gene al, based on he ollowing ma e ials: (i) polyme ic
(e.g., poly(L-lac ic acid) (PLLA), poly(D,L-lac ic-co-glycolic acid) (PLGA), polyme hyl
me hac yla e (PMMA), poly inyl alcohol (PVA), chi osan (CS), algina e (ALG), gela in
(GLT), collagen (CLG) glucan, hyalu onic acid); (ii) ca bon-based (g aphene-based ma-
e ials, ca bon nano ubes (CNTs), ca bon do s (CDs), g aphene oxide (GO), e c., (see
Figu e 2); (iii) ce amic (hyd oxyapa i e (HA), aluminosilica es); and (i ) me al (including
magne ic) [33,71,72,75–78].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 5 o 47
he whole a i icial sca old. The e o e, i is no su p ising ha nanoma e ials ha e al eady
been used as su gical implan s o issue epai and egene a ion in den is y and o ho-
pedics, he p ope ies o NCPs o p omo e ca ilage and bone g ow h being used in com-
bina ion wi h hei abili y o ancho an i-in lamma o y, an i-in ec i e, o an i-cance d ugs
[69–74].
NCPs used as implan s include o ganic–ino ganic, ino ganic–ino ganic, and bioino -
ganic nanoma e ials and a e, in gene al, based on he ollowing ma e ials: (i) polyme ic
(e.g., poly(
L
-lac ic acid) (PLLA), poly(
D
,
L
-lac ic-co-glycolic acid) (PLGA), polyme hyl
me hac yla e (PMMA), poly inyl alcohol (PVA), chi osan (CS), algina e (ALG), gela in
(GLT), collagen (CLG) glucan, hyalu onic acid); (ii) ca bon-based (g aphene-based ma e-
ials, ca bon nano ubes (CNTs), ca bon do s (CDs), g aphene oxide (GO), e c., (see Figu e
2); (iii) ce amic (hyd oxyapa i e (HA), aluminosilica es); and (i ) me al (including mag-
ne ic) [33,71,72,75–78].
Thei syn hesis/p oduc ion a ies depending on he s a ing ma e ial and applica-
ions and canno be b ie ly desc ibed. In he nex sec ions o his pape , whe e he indi-
idual ma e ials a e discussed, hei p epa a ion is desc ibed b ie ly.
Figu e 2. G aphene based ma e ials. G aphene is a basic uni om which o he o ms o a ious
shapes (nano ubes, onions, e c.) a e de i ed. G aphene oxide and educed g aphene oxide bea ox-
ygen-con aining g oups such as ca boxylic g oups (–COOH), hyd oxylic g oups (–OH), and e he
g oups (–O–). G aphi e consis s o indi idual g aphene shee s. Adap ed om [33], MDPI, 2019.
3. Nanoma e ials o Ca ilage Healing and Regene a ion
Ca ilage is an impo an issue p o iding he s uc u e and unc ion o suppo and
p o ec ion in he human body. I s degene a ion is induced i he ca abolic ac o s a e
highe han he anabolic ac o s. In he case o damage, he egene a ion abili y is poo
because o i s hypocellula and hypo ascula issue, and i is di icul o epai . T ea men
o i s inju y, degene a ion, and de ec s p esen s a meaning ul p oblem o clinical esea ch
because cu en ly a ailable ea men s do no p o ide a pe ec ly complian solu ion. Tis-
sue enginee ing wo king wi h so ma e ials is a e y p omising way o epai damaged
ca ilage and bone issue. I de elops sui able subs a es bea ing he equi ed physical,
chemical, and biological s imuli o cell p oli e a ion o di ec chond ogenesis [79–81].
Nano echnologies seem o be a s a egic ool in how o diagnose, p ognose, moni o ,
and/o clinically manage OA. Sma deli e y d ug sys ems, nano ubes, magne ic nano-
pa icles (NPs), NCPs, biological agen s, and biomime ic egene a i e pla o ms o sup-
po cell and gene he apies o s opping OA and p omo ing bone and ca ilage epai
Figu e 2.
G aphene based ma e ials. G aphene is a basic uni om which o he o ms o a ious
shapes (nano ubes, onions, e c.) a e de i ed. G aphene oxide and educed g aphene oxide bea
oxygen-con aining g oups such as ca boxylic g oups (–COOH), hyd oxylic g oups (–OH), and e he
g oups (–O–). G aphi e consis s o indi idual g aphene shee s. Adap ed om [33], MDPI, 2019.
Thei syn hesis/p oduc ion a ies depending on he s a ing ma e ial and applica ions
and canno be b ie ly desc ibed. In he nex sec ions o his pape , whe e he indi idual
ma e ials a e discussed, hei p epa a ion is desc ibed b ie ly.
3. Nanoma e ials o Ca ilage Healing and Regene a ion
Ca ilage is an impo an issue p o iding he s uc u e and unc ion o suppo and
p o ec ion in he human body. I s degene a ion is induced i he ca abolic ac o s a e
highe han he anabolic ac o s. In he case o damage, he egene a ion abili y is poo
because o i s hypocellula and hypo ascula issue, and i is di icul o epai . T ea men
o i s inju y, degene a ion, and de ec s p esen s a meaning ul p oblem o clinical esea ch
because cu en ly a ailable ea men s do no p o ide a pe ec ly complian solu ion.
Tissue enginee ing wo king wi h so ma e ials is a e y p omising way o epai damaged
ca ilage and bone issue. I de elops sui able subs a es bea ing he equi ed physical,
chemical, and biological s imuli o cell p oli e a ion o di ec chond ogenesis [
79
–
81
].
Nano echnologies seem o be a s a egic ool in how o diagnose, p ognose, moni o , and/o
clinically manage OA. Sma deli e y d ug sys ems, nano ubes, magne ic nanopa icles
(NPs), NCPs, biological agen s, and biomime ic egene a i e pla o ms o suppo cell
and gene he apies o s opping OA and p omo ing bone and ca ilage epai ha e been
desc ibed in many esea ch s udies. Nanoma e ials and NCPs can be combined wi h
a ious cell, gene, and biological he apies and o m a new gene a ion o u u e OA
he apeu ics. The physical and mechanical p ope ies o he sca olds can be enhanced using
a ious me hods such as inco po a ion o nano pa icles, c oss-linking, and o he s [37].

Pha maceu ics 2021,13, 1994 6 o 48
Cu en ly, he e is no de ini i e ea men o a icula ca ilage de ec s de eloped.
T ea men p ocedu es o en end wi h an a i icial knee join eplacemen [
82
]. Thus, a icu-
la ca ilage egene a ion is a challenge o esea ch in o hopedics and issue enginee ing.
The ollowing o e iew shows many ways in helping o epai and egene a e i .
A biological issue can be accu a ely imi a ed using 3D p in ing. I s cons uc ion
includes seed cell laye s, biological ac i i y ac o s, and biologically compa ible sca olds.
GO in he amoun o 10 w % was success ully es ed o he p epa a ion o a 3D-p in ed
sca old wi h chond ocy e-p oli e a ion po en ial (see Figu e 3). The newly o med ca ilage
ma ix ex ended along he sca old and he bo de o he ca ilage and ma u ed as con i med
by scanning elec on mic oscopy, immuno luo escence, and
in i o
esea ch. I was isible
ha he sca old was en wined in a ne . The mic o-GO lakes wi h he size o less han
100
µ
m we e localized inside he c oss-linked sca old s uc u e. The ca ilage g ow h on
he 3D-p in ed GO sca old was hicke han ha on he 3D-p in ed sca old wi hou GO,
which con i med GO po en ial o a ca ilage ma ix [83].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 6 o 47
ha e been desc ibed in many esea ch s udies. Nanoma e ials and NCPs can be combined
wi h a ious cell, gene, and biological he apies and o m a new gene a ion o u u e OA
he apeu ics. The physical and mechanical p ope ies o he sca olds can be enhanced
using a ious me hods such as inco po a ion o nano pa icles, c oss-linking, and o he s
[37].
Cu en ly, he e is no de ini i e ea men o a icula ca ilage de ec s de eloped.
T ea men p ocedu es o en end wi h an a i icial knee join eplacemen [82]. Thus, a ic-
ula ca ilage egene a ion is a challenge o esea ch in o hopedics and issue enginee -
ing. The ollowing o e iew shows many ways in helping o epai and egene a e i .
A biological issue can be accu a ely imi a ed using 3D p in ing. I s cons uc ion in-
cludes seed cell laye s, biological ac i i y ac o s, and biologically compa ible sca olds.
GO in he amoun o 10 w % was success ully es ed o he p epa a ion o a 3D-p in ed
sca old wi h chond ocy e-p oli e a ion po en ial (see Figu e 3). The newly o med ca i-
lage ma ix ex ended along he sca old and he bo de o he ca ilage and ma u ed as
con i med by scanning elec on mic oscopy, immuno luo escence, and in i o esea ch.
I was isible ha he sca old was en wined in a ne . The mic o-GO lakes wi h he size
o less han 100 µm we e localized inside he c oss-linked sca old s uc u e. The ca ilage
g ow h on he 3D-p in ed GO sca old was hicke han ha on he 3D-p in ed sca old
wi hou GO, which con i med GO po en ial o a ca ilage ma ix [83].
Figu e 3. 3D-p in ed sca old con aining g aphene oxide (GO) o he ca ilage laye cons uc ion.
(a) Scheme o 3D-p in ed sca old con aining GO and chond ocy es (cells), (b) SEM and immuno-
luo escence in i o e alua ion o mic o-GO p esence in sca old. The sca old (in blue colo ) o ms
ne s, in which mic o-GO (in ed colo ) and chond ocy es a e localized. Mic o-GO a e lakes wi h
leng h less han 100 µm. Toluidine blue and 1,1-dioc adecyl-3,3,3,3- e am-e hylindoca bocyanine
pe chlo a e (DiI; Sigma, S . Louis, MO, USA) we e used o label mic o-GO lakes wi hin he sca old.
Adap ed om [83], BMC, 2020.
Rajze e al. [84] u ilized a 3D p in ing p ocess o p oduce a polycap olac one
(PCL)/g aphene (GR) sca olds wi h an imic obial p ope ies using sho ilamen s icks.
New ilamen ma e ials wi h GR nanopla ele s in concen a ion o 0.5, 5, and 10 w % we e
p epa ed using injec ion molding. The p esence o GR enhanced he mechanical p ope -
ies o ilamen s. The ilamen s we e used in a comme cial 3D p in e o p in sca olds o
nasal ca ilage eplacemen , and he p oli e a ion o chond ocy es was p o en [84]. A GO-
modi ied 3D acellula ca ilage ex acellula ma ix (ACM) sca old o ca ilage epai
Figu e 3.
3D-p in ed sca old con aining g aphene oxide (GO) o he ca ilage laye cons uc ion.
(
a
) Scheme o 3D-p in ed sca old con aining GO and chond ocy es (cells), (
b
) SEM and immuno lu-
o escence
in i o
e alua ion o mic o-GO p esence in sca old. The sca old (in blue colo ) o ms
ne s, in which mic o-GO (in ed colo ) and chond ocy es a e localized. Mic o-GO a e lakes wi h
leng h less han 100
µ
m. Toluidine blue and 1,1-dioc adecyl-3,3,3,3- e am-e hylindoca bocyanine
pe chlo a e (DiI; Sigma, S . Louis, MO, USA) we e used o label mic o-GO lakes wi hin he sca old.
Adap ed om [83], BMC, 2020.
Rajze e al.[
84
]u ilizeda3Dp in ingp ocess op oduceapolycap olac one(PCL)/g aphene
(GR) sca olds wi h an imic obial p ope ies using sho ilamen s icks. New ilamen
ma e ials wi h GR nanopla ele s in concen a ion o 0.5, 5, and 10 w % we e p epa ed using
injec ion molding. The p esence o GR enhanced he mechanical p ope ies o ilamen s.
The ilamen s we e used in a comme cial 3D p in e o p in sca olds o nasal ca ilage
eplacemen , and he p oli e a ion o chond ocy es was p o en [
84
]. A GO-modi ied 3D
acellula ca ilage ex acellula ma ix (ACM) sca old o ca ilage epai was p epa ed
by GO and ACM c osslinking using 1-e hyl-3-(3-dime hylaminop opyl)ca bodiimide hy-
d ochlo ide and N-hyd oxy succinimide. GO addi ion enhanced he in e nal s uc u e and
mechanical p ope ies o he sca old. Cell adhesion, cell p oli e a ion, and chond ogenic
di e en ia ion
in i o
we e p omo ed, and good biocompa ibili y and mild in lamma o y
Pha maceu ics 2021,13, 1994 7 o 48
esponse we e p o en wi h subcu aneous implan a ion in a s. A e 12 weeks o implan-
a ion, he composi e sca old loaded wi h bone ma ow mesenchymal s em cells (MSCs)
comple ely b idged he ca ilage de ec s in he abbi knee wi h hyaline ca ilage [
85
].
Se icin/ educed g aphene oxide ( GO) NCPs wi h se icin/ GO a ios 10:1, 50:1, and 100:1
we e s udied in [
86
]. The NCPs p omo ing glycosaminoglycan and CLG le els can be
p omising in epai ing a icula ca ilage in knee join s in nu sing ca e. 3D biop in ed
GO-doped GLT based sca olds o p omo ing chond ogenic di e en ia ion o human bone
ma ow MSCs we e p epa ed using GO–GLT me hac yla e (GLT-IMA)-poly(e hylene gly-
col)diac yla e (PEGDA) as a biocompa ible pho opolyme izable bioink. The s uc u e o 3D
p in ed sca old was GLT–IMA–PEGDA–GO, and he sca old inc eased he glycosamino-
glycan and CLG le els a e chond ogenic di e en ia ion o hMSCs [
87
]. Bio-inks o 3D
bio-p in ing o os eochond al sca olds wi h di e en a ios o CS, GLT, and hyalu onic
acid we e p epa ed also con aining GR wi h he a io o 0.024, 0.06, and 0.1 w % o imp o e
he mechanical p ope ies o he biosca old. To s udy he biocompa ibili y o he sca olds,
bone MSCs we e inocula ed on o he biosca olds. CS/GLT/hyalu onic acid sca olds
con aining GR showed a good 3D po ous s uc u e; po osi y was mo e han 80%; he
mechanical s eng h was imp o ed; po e walls we e smoo he and hicke ; and bone MSCs
success ully g ew on he sca olds [80].
PCL sca olds wi h g id-like s uc u e and pe iodic la ice con aining GR nanopla ele s
we e p epa ed o ca ilage issue applica ions. The po ous sca old cons uc ion was
conduc ed using a laye -by-laye assembly. The GR/PCL composi e sca olds showed
good cy ocompa ibili y and non- oxici y using mouse bone ma ow MSCs ha p oli e a ed
well on he sca olds and con i med a chond ogenic di e en ia ion [
88
]. Using he plasma
a c discha ge me hod, Holmes e al. [
79
] p epa ed a ca bon nanoma e ial mix u e con ain-
ing GR nanopla ele s and single-walled ca bon nano ubes. The mix u e was added in o
elec ospun PCL mic o ib ous sca olds wi h o wi hou poly-L-lysine su ace coa ing. The
sca olds con aining ca bon nanoma e ial showed highly enhanced mechanical p ope ies
and imp o ed s em cell adhesion, p oli e a ion, and chond ogenic di e en ia ion, hus he
ma e ial is p omising o ca ilage o ma ion in clinical applica ions [
79
]. A mac opo ous
polyme ic sca old o chi in and PCL was p epa ed by he lyophiliza ion echnique. T ans-
o ming g ow h ac o -
β
(TGF-
β
) was encapsula ed in chond oi in sul a e (ChS) NPs and
inco po a ed in he chi in-PCL sca old o s udy a p olonged TGF-
β
elease. TGF-
β
-ChS
NPs we e cha ac e ized using a dynamic ligh sca e ing pa icle size and SEM, and i was
p o en ha hey we e sphe ical pa icles o a 230
±
20 nm. The composi e sca old was
s able in swelling and deg ada ion s udies. The p esence o TGF-
β
posi i ely in luenced
he a achmen and p oli e a ion o abbi adipose de i ed MSCs. In addi ion, an inc eased
p o eoglycan deposi ion was con i med in he p esence o TGF-β[81].
Umbilical co d MSCs loaded wi h GO g anula lub ican we e used o ea a knee
OA animal model. Me hods o ea men o 24 New Zealand abbi models o knee
OA we e es ablished. The models we e di ided in o he blank g oup, he GO g oup,
he umbilical co d MSCs g oup, and he GO + umbilical co d MSC g oup, each g oup
including six animal models. The bes esul s o NO, IL-6, TNF-
α
, GAG, and COL-II
we e ob ained in he case o he GO + umbilical co d MSC g oup. Ca ilage epai was
con i med in his g oup [
89
]. Shamekhi e al. [
90
] p epa ed sca olds based on CS con aining
di e en amoun s o ex olia ed GO NPs ( om 0 o 0.3 w %). The physical and mechanical
p ope ies o he p epa ed samples we e enhanced wi h he inc easing GO con en . The
human a icula chond ocy es we e seeded on he sca olds, and a highe p oli e a ion
was obse ed in samples wi h highe GO pe cen age [
90
]. A GO-doped elec ospun PLGA
nano ib ous memb ane was p epa ed using he elec ospinning echnique, and
in i o
cell
assays we e used o i s e alua ion using abbi models. The e was no change in he 3D
mic os uc u e o ilamen a e GO mixing wi h PLGA. By means o an
in i o
e alua ion,
i was p o en ha he GO-PLGA memb ane suppo ed he p oli e a ion o bone MSCs and
hei os eogenic di e en ia ion. The local applica ion o he GO-PLGA memb ane o he
space be ween he bone and he endon in a abbi model imp o ed he healing en hesis,
Pha maceu ics 2021,13, 1994 8 o 48
inc eased new ca ilage and bone gene a ion, and imp o ed he CLG a angemen and
biomechanical p ope ies compa ed o he use o a PLGA memb ane [91].
CLG is commonly used o ca ilage epai , bu chond ogenesis is dis a o ed by i s
low s i ness and apid deg ada ion. An injec able hyd ogel was p epa ed using bio-
compa ible CDs and CLG, which we e c osslinked by genipin (CLG-genipin-CDs, CGC)
wi h highe s i ness. Using pho odynamic he apy (PDT), a mode a e amoun o eac-
i e oxygen species (ROS) was gene a ed, which suppo ed chond ogenic di e en ia ion
o bone ma ow-de i ed MSCs and subsequen ly imp o ed ca ilage egene a ion. The
deg ada ion a e o CGC was 39% lowe and he comp ession modulus was 21- old highe
compa ed o he pu e CLG hyd ogel. The CGC hyd ogel in combina ion wi h PDT en-
hanced he bone MSCs p oli e a ion by 50.3%, and he ca ilage egene a ion was less
han eigh weeks [
92
]. A ype II CLG–CS/PLGA sca old was used o he cul i a ion
o abbi chond ocy es labelled by magne ic NPs o p epa e cul u es wi h isible cells o
s udy hei g ow h, di e en ia ion, and egene a ion. The SEM image showed no cell
a achmen on he sca old a e one day; he cells we e only collec ed on he sca old
su ace. A e se en days, he cells began o adhe e and p oli e a e deep in o he su ace
o he sca old. A e 14 days, a sec e ion o ex acellula ma ix was isible oge he wi h
he accumula ion on he sca old su ace (see Figu e 4). Magne ic NPs did no a ec he
chond ocy e pheno ype o p o ein and gene exp ession. Inc easing gene exp ession o
agg ecan and ype II CLG indica ing chond ogenesis was obse ed. The di e en ia ion
was associa ed wi h os eogenesis [93].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 8 o 47
assays we e used o i s e alua ion using abbi models. The e was no change in he 3D
mic os uc u e o ilamen a e GO mixing wi h PLGA. By means o an in i o e alua-
ion, i was p o en ha he GO-PLGA memb ane suppo ed he p oli e a ion o bone MSCs
and hei os eogenic di e en ia ion. The local applica ion o he GO-PLGA memb ane o he
space be ween he bone and he endon in a abbi model imp o ed he healing en hesis,
inc eased new ca ilage and bone gene a ion, and imp o ed he CLG a angemen and bio-
mechanical p ope ies compa ed o he use o a PLGA memb ane [91].
CLG is commonly used o ca ilage epai , bu chond ogenesis is dis a o ed by i s
low s i ness and apid deg ada ion. An injec able hyd ogel was p epa ed using biocom-
pa ible CDs and CLG, which we e c osslinked by genipin (CLG-genipin-CDs, CGC) wi h
highe s i ness. Using pho odynamic he apy (PDT), a mode a e amoun o eac i e oxy-
gen species (ROS) was gene a ed, which suppo ed chond ogenic di e en ia ion o bone
ma ow-de i ed MSCs and subsequen ly imp o ed ca ilage egene a ion. The deg ada-
ion a e o CGC was 39% lowe and he comp ession modulus was 21- old highe com-
pa ed o he pu e CLG hyd ogel. The CGC hyd ogel in combina ion wi h PDT enhanced
he bone MSCs p oli e a ion by 50.3%, and he ca ilage egene a ion was less han eigh
weeks [92]. A ype II CLG–CS/PLGA sca old was used o he cul i a ion o abbi chon-
d ocy es labelled by magne ic NPs o p epa e cul u es wi h isible cells o s udy hei
g ow h, di e en ia ion, and egene a ion. The SEM image showed no cell a achmen on
he sca old a e one day; he cells we e only collec ed on he sca old su ace. A e se en
days, he cells began o adhe e and p oli e a e deep in o he su ace o he sca old. A e
14 days, a sec e ion o ex acellula ma ix was isible oge he wi h he accumula ion on
he sca old su ace (see Figu e 4). Magne ic NPs did no a ec he chond ocy e pheno ype
o p o ein and gene exp ession. Inc easing gene exp ession o agg ecan and ype II CLG
indica ing chond ogenesis was obse ed. The di e en ia ion was associa ed wi h os eo-
genesis [93].
Figu e 4. SEM images p esen ing chond ocy es (bea ing magne ic nanopa icles) g own on he bi-
phasic ype II collagen–chi osan/polylac ic-co-glycolic acid (CLG–CS/PLGA) sca old a 1, 7, and 14
days a e seeding. (A) No a achmen o he cells on he sca old a e one day was obse ed, only
hei collec ion on he sca old su ace was indica ed, (B) a e se en days, cells s a ed o adhe e
and p oli e a e deep in o he sca old, (C) a e 14 days, he ex acellula ma ix was sec e ed wi h a
highe accumula ion on he sca old su ace. Cell adhesion and sp eading on he po es o he sca -
old is clea ly isible. Magni ica ion 500×. Adap ed om [93], MDPI, 2017.
The mosensi i e CS-based composi es chemically modi ied wi h CLG and con ain-
ing bioac i e glass NPs we e used o he p epa a ion o injec able nanohyb ids o egen-
e a i e medicine. The he mosensi i e esponse o he hyd ogel was app oxima ely 37 °C,
which co esponds o he human body empe a u e. The CS hyd ogels we e cha ac e ized
by 3D-po ous s uc u es; he p esence o CLG inc eased he a e age po e size and o-
ge he wi h he p esence o bioac i e glass imp o ed he mechanical p ope ies. The ad-
di ion o 2 w % o bioac i e glass NPs led o an app oxima ely 39% inc ease in s i ness
compa ed o pu e CS, while he addi ion o 30 w % o CLG inc eased he s i ness by 95%.
No oxic e ec o he composi es on he human os eosa coma cell cul u e and kidney cells
Figu e 4.
SEM images p esen ing chond ocy es (bea ing magne ic nanopa icles) g own on he
biphasic ype II collagen–chi osan/polylac ic-co-glycolic acid (CLG–CS/PLGA) sca old a 1, 7, and
14 days a e seeding. (
A
) No a achmen o he cells on he sca old a e one day was obse ed, only
hei collec ion on he sca old su ace was indica ed, (
B
) a e se en days, cells s a ed o adhe e
and p oli e a e deep in o he sca old, (
C
) a e 14 days, he ex acellula ma ix was sec e ed wi h a
highe accumula ion on he sca old su ace. Cell adhesion and sp eading on he po es o he sca old
is clea ly isible. Magni ica ion 500×. Adap ed om [93], MDPI, 2017.
The mosensi i e CS-based composi es chemically modi ied wi h CLG and con aining
bioac i e glass NPs we e used o he p epa a ion o injec able nanohyb ids o egene a i e
medicine. The he mosensi i e esponse o he hyd ogel was app oxima ely 37
◦
C, which
co esponds o he human body empe a u e. The CS hyd ogels we e cha ac e ized by
3D-po ous s uc u es; he p esence o CLG inc eased he a e age po e size and oge he
wi h he p esence o bioac i e glass imp o ed he mechanical p ope ies. The addi ion o
2 w %
o bioac i e glass NPs led o an app oxima ely 39% inc ease in s i ness compa ed o
pu e CS, while he addi ion o 30 w % o CLG inc eased he s i ness by 95%. No oxic e ec
o he composi es on he human os eosa coma cell cul u e and kidney cells line o human
emb yo (HEM293T) was ound using MIT (170
µ
L, 5 mg/mL; Sigma-Ald ich,
S . Louis,
MO, USA) and Li e/Dead
®
assays (Li e Technologies o B azil L da, São Paulo, B azil) [
94
].
Fo ca ilage epai , hyd ogels ha ing enhanced biocompa ible, bio ibological, and
biomechanical p ope ies also seem o be pe spec i e ma e ials. Physically c oss-linked
PVA-nHA/(2-hyd oxyp opyl ime hyl ammonium chlo ide CS) hyd ogels wi h a double
ne wo k we e de eloped ia a eezing/ hawing echnique and an imme sing p ocess. The
esul ing hyd ogel wi h an op imized HA con en exhibi ed ou s anding mechanical p op-
Pha maceu ics 2021,13, 1994 9 o 48
e ies such as ac u e ensile s ess (2.70
±
0.24 MPa), oughness
(14.09 ±2.06 MJ/m3),
and comp essi e modulus (0.88
±
0.09 MPa) accompanied wi h no able an i- a igue p op-
e y, excep ional sel - eco e y, and he abili y o ene gy dissipa ion, which was caused
by hese c oss-linked s uc u es. The con en o nHA posi i ely in luenced he low
alue o he ic ion coe icien and he excellen cy ocompa ibili y [
95
]. A bi-polyme ic
PVA/poly inylpy olidone hyd ogel composi e wi h inco po a ed s ick-like TiO
2
nanos-
uc u es was designed. The esul ing hyd ogel composi es had an imp o ed su ace
opog aphy, and mo e la ed cell mo phologies and enhanced os eoblas a achmen
we e obse ed. The s ick-like TiO
2
NCPs and c ys alline bone p omo ed he bioac i -
i y ia lamellipodia and ilopodia ex ension o os eoblas cells because o hei excellen
in e cellula connec ion and egula ed cell esponses. An an ibac e ial ac i i y agains
S aphylococcus au eus and Esche ichia coli bac e ial s ains was also ound [96].
Biocomposi es o glycol (GLY)–CS ma ices con aining nHA wi h he a e age size
o 74
±
15 nm we e ab ica ed by an eco- iendly chemical colloidal p ocess in aqueous
media, sol en cas ing, and e apo a ion a oom empe a u e. I was ound ha he GLY–
CS ligand had a majo ole in he nuclea ion, g ow h, and colloidal s abiliza ion o nHA.
nHA pa icles we e homogenously dispe sed in he GLY–CS ma ix. An adequa e cell
iabili y esponse and non-cy o oxic beha io owa d os eoblas ic-like and emb yonic cell
lines (HEK293T) we e p o en. Based on he os eogenic di e en ia ion es s, i is ob ious
ha he nHA/GLY–CS composi es a e os eoinduc i e o human bone MSCs and can
be es ed o bone, ca ilage, and pe iodon al egene a ion [
97
]. No el in si u o ming
composi e hyd ogels based on CS and GLT biopolyme s associa ed wi h bioac i e glass
NPs we e syn hesized and cha ac e ized by he ze a po en ials a 37
◦
C anging om
+3.1 ±1.4 mV
o +6.9
±
3.2 mV. The ca ionic na u e o hese biocomposi es was con i med
wi h he abili y o in e ac ion wi h anionic compounds con ained in he na i e ex acellula
ma ix. FTIR spec a showed ha he hyd ogels o m a ne wo k mainly h ough molecula
in e ac ions. The elas ic modulus (G) inc eased om 5.4 Pa o pu e CS hyd ogels o
12.4 Pa o he composi es wi h highe GLT and bioac i e glass con en s. All o mula ions
we e injec able and cy ocompa ible, as con i med by he li e cell iabili y esponses o he
human os eosa coma cell line [
98
]. ChS loaded zein NPs (~150 nm) we e inco po a ed
in a hyd ogel based on a biphasic semi-in e pene a ing polyme ne wo ks o med by
blending ALG wi h PVA and calcium c osslinking. The inal hyd ogel sys em was used
o unc ional a icula hyaline ca ilage es o a ion. The hyd ogel was cha ac e ized by
a po ous mic os uc u e wi h a 39.9
±
5.8
µ
m po e diame e and 57.7
±
5.9% po osi y,
swellabili y o 92%, and an elas ic modulus highe han > 350 Pa. Compa ibili y wi h
p ima y chond ocy es, in e ac ion o chond ocy es wi h he ma ix, and cell–cell clus e ing
we e s udied; p oli e a ion was de e mined; and posi i e in luence o ChS on chond ocy es
was p o en [
99
]. Yang e al. [
100
] used he nonp o ein compound ka ogenin (KGN), which
is able o p omo e he di e en ia ion o bone ma ow-de i ed MSCs in o chond ocy es.
KGN was ancho ed on o he su ace o modi ied supe pa amagne ic i on-oxide (SPIO)
and inco po a ed wi h cellulose nanoc ys al/dex an hyd ogels, which se ed as a ca ie
o SPIO–KGN as well as a ma ix o he epai o ca ilage. I was ound ha KGN is
eleased s able in he long un, in akes endogenous hos cells, and p omo es bone MSCs
o di e en ia e in o chond ocy es. Thus, i is sui able o a ca ilage egene a ion. The
egene a ed ca ilage issue was simila o a na u al hyaline ca ilage [100].
All o he abo e-men ioned NCPs and hei p ope ies a e summa ized in Table 1.
Pha maceu ics 2021,13, 1994 16 o 48
sca olds ho ough elec ospinning. A educ ion in c ys allini y was obse ed wi h he
inc easing nHA concen a ion. The e was no cy o oxici y ound in he es s o all sca olds,
and all he p epa ed PBAT/nHA sca olds suppo ed bone epai [117].
Me al Doped Hyd oxyapa i e
AuNP-loaded HA NCPs we e ab ica ed o con ol he os eogenic di e en ia ion o
human MSCs ia he syne gis ic e ec s o bo h AuNPs and HA. The HA–AuNPs exhib-
i ed a good cy ocompa ibili y and we e in e nalized in o human MSCs. The inc ease in
human MSC os eogenic di e en ia ion was con i med by he inc eased ALP p oduc ion
le el, calcium mine aliza ion deposi ion, and he ypical os eogenic gene exp ession. The
Au inco po a ion ac i a ed he Wn /
β
-ca enin signaling pa hway. A syne gis ic e ec
on human MSC os eogenic di e en ia ion was exe ed using he HA–Au NPs [
118
]. Mi-
c osphe es (COS–Ag–ALG-HA) wi h size anging om 1.5
±
0.5 o 4.0
±
0.5 mm and
in ol ing chi ooligosaccha ide (COS) coa ed AgNPs wi h ALG and HA we e designed
and p epa ed. The p epa ed mic osphe es we e igid wi h mu ual chemical in e ac ions
be ween indi idual pa s. High an imic obial ac i i y was obse ed agains S. au eus
oge he wi h he biocompa ibili y wi h os eoblas -like cells [
119
]. Zinc-doped HA could
be used as a g a bioma e ial o bone egene a ion, bu he Zn e ec on os eoconduc i i y
has s ill been unknown due o he Ca, P, and Zn elease and eso p ion in g a -implan ed
de ec s. Mic osphe es consis ing o ALG and non-doped ca bona ed HA o ALG and
nanoc ys alline 3.2 w % zinc-doped HA (Zn–HA) we e inse ed in c i ical-sized cal a ial
de ec s in Wis a a s o one, h ee, and six mon hs. Any signi ican di e ence in he
new bone quan i y was no de e mined be ween hese wo ma e ials, and hey bo h e-
leased high Ca, P, and Zn quan i ies, which we e dis ibu ed in he de ec i e a ea. Zn was
s ongly adso bed by he HA su ace. Phospho us was eso bed as e han Ca. Zn and
Ca showed equi alen elease p o iles, which con i ms hei s oichiome ic dissolu ion
and non-p e e en ial Zn eso p ion. The high nanome ic Ca and Zn accumula ion in he
de ec in luenced os eoconduc ion, inhibi ing and impai ing bone epai [
120
]. The po ous
sca olds o he Z O
2
/HA composi e we e o med by he digi al ligh p ocessing (DLP)
echnology wi h a posi i e e ec on cell p oli e a ion and di e en ia ion. The sca old
con aining 10 w % HA had he bes comp essi e capaci y. A e he sca old was imme sed
in he simula ed body luid, i s comp essi e s eng h dec eased wi hin he i s 14 days and
hen inc eased p obably due o he deg ada ion o calcium phospha e componen s and he
deposi ion o apa i e. On day 28, he comp essi e s eng h eached app ox. 20 MPa and
was close o ha o he sca olds made o Z O2(25 MPa) [121].
A p o ein co ona o ma ion can help o unde s and he mechanisms o immune-
modula ed bone wound healing. An
in i o
dynamic model o he p o ein co ona o
magne ic HA sca olds was designed o s udy he co ela ion be ween he in lamma o y
eac ion and bone wound healing oge he wi h he unde lying mechanism con olling his
p ocess. The le els o some p o eins ela ed o he immune esponse and in lamma ion,
bone and wound healing, ex acellula ma ix, cell beha io , and signaling we e inc eased
in he p o ein co ona o he MNP-in il a ed sca olds in a ime-dependen manne . The
immune esponse and in lamma ion p o eins adso bed on he magne ic HA sca olds co -
ela ed well wi h he bone wound healing p o eins. The p esence o MNPs supp essed he
ch onic in lamma o y esponses, bu highly p omo ed he acu e in lamma o y esponses.
The ac i a ion o acu e in lamma o y eac ions induces he ec ui men o immune cells
and emodeling o he ex acellula ma ix, which leads o accele a ed bone healing [
122
].
A NCP sca old was syn hesized using bac e ial cellulose (BC) wi h magne i e (Fe
3
O
4
)
and HA NPs using ul asound. The esul ing sca old (BC–Fe
3
O
4
–HA) had homogenous
dispe sion o he NPs in he BC ma ix wi h a Ca/P a io o 1.63 and 1.56 o he su ace and
c oss sec ion, espec i ely. The BC c ys allini y index was lowe ed in he composi e ( om
82.5% o 62%). A dec ease in sa u a ion magne iza ion om 15.84 o 3.94
µ
/g a
±10 kOe
was ound a e he deposi ion o HA wi h supe pa amagne ic cha ac e is ics oge he
wi h signi ican lowe ing in swelling abili y a e he inco po a ion o he NPs and high

Pha maceu ics 2021,13, 1994 17 o 48
po osi y deg ee (a ound 80%). The sca old was non- oxic o mouse ib oblas L929 cells
and biocompa ible o os eoblas (MC3T3-E1 cell line) a achmen and p oli e a ion [123].
A comp ehensi e e iew o he cu en issues o p epa a ion and p ope ies o mag-
ne ic HA and he applica ion o hese NCP ma e ials in biomedicine as implan s o bone
egene a ion wi h an imic obial ac i i y, con olled d ug/gene deli e y, and magne ic
hype he mia ea men was ecen ly published by Mush aq e al. [
124
]. Addi ionally,
Scialla e al. [
125
] ob ained posi i e esul s o he use o a NCP composed o magne ic i on
oxide g a ed wi h dex an in combina ion wi h nHA in bone issue enginee ing [125].
All o he abo e-men ioned NCPs and hei p ope ies a e summa ized in Table 2.
Table 2. Summa y o composi e ma e ials con aining nano-hyd oxyapa i e as basic ille (only any o he ille s a e lis ed).
Ma ix Fille Tes ed Cells In Vi o In Vi o Re .
Ca boxyme hyl chi osan Sodium algina e - MTT assay, li e/dead
assays - [108]
Poly(D-lac ic acid) Bone mesenchymal
s em cells
P oli e a ion assay,
li e/dead assays,
os eogenic
di e en ia ion
- [109]
Poly(L-lac ic acid)
Mesopo ous silica
San a Ba ba a
Amo phous-15
MG63 os eoblas cells
MTT assay, cell
p oli e a ion,
os eogenic
di e en ia ion,
- [110]
Poly (lac ide-co-p opylene
glycol-co-lac ide)
dime hac yla e
Hyd oxye hyl
me hac yla e
Long- e m elease
BMP-2
Biocompa ibili y in
a mesenchymal
s em cells, li e/dead
assays, p oli e a ion
cell, os eogenesis,
gene exp essions o
os eogenesis- ela ed
ma ke s
Rabbi emo al
condyle de ec
animal model,
mic o-CT,
his ological
obse a ions
[111]
Poly(lac ic-co-glycolic acid) Poly(γ-benzyl-L-
glu ama e)
Mouse p eos eoblas
cells MC3T3-E1
Cell cul u e, iabili y
and mo phology
assay using mouse
p eos eoblas cells,
MTT assay, ALP
assay
Repai o abbi
adius de ec , X- ay,
mic o-CT es s
[112]
Ac yla ed epoxidized soybean
oil, polye hylene glycol
diac yla e, phenylbis(2,4,6-
ime hylbenzoyl)phosphine
oxide
- - - [113]
Chi osan, poly(me hyl inyl
e he -al -maleic anhyd ide
SD a bone ma ow
mesenchymal s em
cells
Biocompa ibili y,
iabili y o cells - [114]
hyd oxyl-capped poly(lac ide),
ca boxyl-capped aniline
pen ame ,
poly(lac ide-co-glycolide)
L-Lac ic acid
oligome
Mouse p eos eoblas
cells MC3T3-E1
MC3T3-E1 cell
p oli e a ion ac i i y
wi h and wi hou
elec ical s imula ion,
MTT assays
In amuscula
implan a ion in o
abbi s do sal
muscles,
implan a ion o
epai o adius
de ec s in abbi s and
o ibia de ec s in
sheep
[115]
Poly( hioke al u e hane) - -
Femo al de ec s in
New Zealand Whi e
abbi s
[116]
Poly(bu ylene-adipa e-co-
e eph hala e)
G aphene
nano ibbons - -
Implan a ion in o
c i ical ibia de ec s
in a s, adiog aphy
analysis, omog aphy,
bone emodeling,
biomechanical
p ope ies
[117]
Pha maceu ics 2021,13, 1994 18 o 48
Table 2. Con .
Ma ix Fille Tes ed Cells In Vi o In Vi o Re .
- Gold nanopa icles
Human bone
ma ow-de i ed
mesenchymal s em
cells
Cell iabili y,
p oli e a ion by
CCK-8 assay,
Aliza in ed S
s aining, RT-PCR,
wes e n blo ing
- [118]
Algina e
Chi ooligosaccha ide
coa ed sil e
nanopa icles
MG-63 cells
An imic obial es ing,
MTT assay, cell
iabili y and
p oli e a ion,
Hoechs 33342
s aining assay
- [119]
Algina e Zinc
Mu ine os eoblas ic
mycoplasm- ee cell
line
Cy ocompa ibili y
assay, cell iabili y,
cy o oxici y assays
Wis a a s:
implan a ion in o
c i ical-sized cal a ial
de ec s, his ological
p epa a ion,
his omo phome ic
e alua ion,
deg ada ion,
bioa ailabili y
[120]
- Zi conia Mouse os eoblas
p ecu so cell line
Cell
cy ocompa ibili y,
adhesion,
p oli e a ion and
di e en ia ion
- [121]
- Magne i e Mouse p eos eoblas
cells MC3T3-E1
Cell p oli e a ion and
mo phology
Female SD a s:
p o ein co ona
o ma ion and
de e mina ion
[122]
Bac e ial cellulose Magne i e Mouse ib oblas
L929 cells
Cell
cy ocompa ibili y on
MC3T3-E1,
p oli e a ion
- [123]
Dex an-g a ed i on oxide Human-de i ed
os eoblas -like cells
Cell
cy ocompa ibili y,
gene exp ession,
RNA isola ion and
e e se ansc ip ion,
RT-PCR
- [125]
4.2. Ca bon-Based Nanoma e ials
A sca old based on ca bon nanoma e ials such as GO, CNTs, CDs, and hei de i a-
i es, has become one o he key ma e ials, which, depending on hei unc ionaliza ion,
has ema kable abili ies o in luence bone egene a ion, e ec i e cell p oli e a ion, and
os eogenic di e en ia ion. Thus, CNTs and GR-based nanoma e ials ha e o en been es ed
as nano ein o cemen s in bone issue enginee ing due o hei unique mechanical, elec i-
cal, and biological p ope ies, allowing hem o be seconda y-phase ein o cemen s. In
addi ion, NCPs con aining CNTs and GR demons a ed be e os eoblas cell adhesion,
leading o he p omo ion o bone issue o ma ion
in i o
; hus, hey a e expec ed o
b ing g oundb eaking echnologies o egene a i e medicine and bone issue enginee -
ing [
33
,
126
]. Fu he mo e, hey p o ide an imic obial p ope ies and can ein o ce he
mechanical p ope ies [
104
]. On he o he hand, he p oblem is ha he cy ocompa ibili y
o CNTs and GR is s ill a con o e sial opic [33,126].
4.2.1. Ca bon Nano ubes
A PLLA/CNT sca old is a p omising candida e as a bone implan . PLLA is a p omis-
ing implan ma e ial due o i s biocompa ibili y and deg adabili y; howe e , he insu icien
Pha maceu ics 2021,13, 1994 19 o 48
mechanical s eng h is no sui able o bone epai applica ion. The c ys allini y o PLLA
sca olds con aining CNTs inc eased signi ican ly because CNTs p omo e o de ly s acking
o PLLA chains. Mo eo e , CNTs ac ed as a b idge ac oss he c acks. The comp essi e
s eng h, Vicke s ha dness, and ensile s eng h o he sca old we e enhanced by 22.7%,
58.8%, and 17.6%, espec i ely [
127
]. Sca olds based on elec ospun PLLA ma ix co e ed
wi h hyb id composi es o CNT/g aphene nano ibbons (GNRs) and nHA we e p epa ed
and s udied using a ious me hods (SEM, EDS, and AFM). The GNRs showed a oxici y
and cy o oxici y a he concen a ions o 60 and 120
µ
g/mL, and nei he oxici y no cy-
o oxici y was de e mined a he concen a ion o 30
µ
g/mL using he Allium cepa assay.
The hemolysis es de e mined ha he sca olds wi h he concen a ion o 0.3 mg/cm
2
we e no oxic, and co obo a ing da a om he biochemical ma ke s glu amic py u ic
ansaminase, glu amic oxaloace ic ansaminase, and u ea showed no cy o oxici y, geno-
oxici y, o mu agenici y [
128
]. O de ed CNT–HA sca olds wi h imp o ed mechanical
p ope ies and accele a ed cell g ow h
in i o
o
in i o
we e p epa ed using aga ose
gel elec opho esis o imi a e a pa e n o CLG and HA hyd ogel sca olds (AG-CLG-o-
CNT). The enhanced p oli e a ion and di e en ia ion o bone MSC lines was p o en, and
he bone de ec s we e epai ed a e 28 and 56 days
in i o
[
129
]. Os eogenic pep ides
ha e he os eogenic abili y o a i icial bone ma e ials. CNTs wi h ca boxyl and amino
g oups we e used as a nano ein o cemen o syn he ic sca old ma e ials, in which hey
we e co alen ly a ached o he RGD/BMP-2 os eogenic pep ide. MC3T3-E1 cells we e
subsequen ly cul u ed on hese sca olds. The pep ide bound ia amino g oups could
p omo e cell unc ions mo e e icien ly han ha bound h ough ca boxyl g oups, p obably
due o he posi i e cha ges o he amino g oups on he CNT su aces, leading o changes
in he pep ide con o ma ion, p o ein adso p ion, and a ge ing os eogenic e ec s [
130
].
An injec able CNT and wo-dimensional (2D) black phospho us (BP) gel wi h enhanced
mechanical s eng h, elec ical conduc i i y, and con inuous phospha e ion elease was
p epa ed. Biodeg adable oligo(poly(e hylene glycol) uma a e) polyme was used as a
hyd ogel c oss-linking ma ix oge he wi h he c oss-linkable CNT–poly(e hylene gly-
col)ac yla e (CNT–PEGA) o imp o e he mechanical p ope ies and elec ic conduc i i y.
The BP–CNTPEGA gel enhanced he adhesion, p oli e a ion, and os eogenic di e en ia ion
o MC3T3 p eos eoblas cells. The os eogenesis o p eos eoblas cells was imp o ed wi h
elec ic s imula ion [131].
Du e al. [
132
] ound ha mul i-walled ca bon nano ubes (MWCNTs) could be mo e
e ec i e o enhanced bone o ma ion han nHA. They s udied he os eogenic abili y o
MWCNTs and nHA o he
in i o
cul u e o human adipose-de i ed MSCs. No signi ican
di e ence be ween he MWCNTs and he nHA was ound in he cell adhesion amoun ;
howe e , he cell a achmen s eng h and p oli e a ion o he MWCNTs we e be e . The
MWCNTs also showed be e induc ion o he HASC os eogenic di e en ia ion han he
nHA, and unlike he nHA, hey could induce ec opic bone o ma ion
in i o
. I is assumed
ha MWCNTs concen a e mo e p o eins such as speci ic bone-inducing p o eins, which
a e sec e ed om M2 mac ophages, and he e o e s imula e inducible cells in issues o
o m induc i e bone be e han nHA [
132
]. 3D conduc i e sca olds made om PCL and
MWCNTs we e p oduced using ex usion-based addi i e manu ac u ing o ea la ge
cal a y bone de ec s in a s. Based on his ology esul s, i was ound ha a combina ion
o PCL/MWCNTs sca olds and exogenous elec ical s imula ion induced hicke and
inc eased bone issue o ma ion wi hin he bone de ec suppo ed by p omo ed angio-
genesis and mine aliza ion wi h he concen a ion o MWCNTs o 3 w % and elec ical
s imula ion. The a a e- esis an acid phospha ase posi i e cell o ma ion was p omo ed.
While he os eoclas ogenesis was inhibi ed using MWCNTs, he use o ES p omo ed i [
133
].
Simila ly, a 3D p in ed po ous sca old wi h aligned MWCNTs and nHA was p epa ed by
Huang e al. [134].
MWCNTs wi h simila dimensions as CLG ibe s coupled wi h nHA
we e mixed wi h a PCL ma ix. I was con i med ha MWCNTS we e aligned in he PCL
ma ix, and he sca old was simila o he na i e bone nanos uc u e [
134
]. PCL sca olds
wi h double ille s, MWCNTs and eggshell, wi h imp o ed mechanical and os eogenic
Pha maceu ics 2021,13, 1994 20 o 48
p ope ies we e p epa ed. I was ound ha eggshell imp o ed he PCL/eggshell/MWCNT
sca old hyd ophilici y and biocompa ibili y, whe eas MWCNTs enhanced hei comp es-
sion and ensile s eng h [135].
A ough polyion complex (PIC) hyd ogel con aining MWCNTs was syn hesized o
o m a PIC/MWCNT biohyb id hyd ogel, which was used o he ab ica ion o 3D sca -
olds by ex usion-based 3D p in ing. The esul ing sca olds had a good biocompa ibili y
wi h a bone ma ow-de i ed MSCs and enhanced hei os eogenic di e en ia ion. A
highe deg ee o os eogenic di e en ia ion was ob ained by using PIC/MWCNT sca olds
han PIC sca olds. In addi ion, he PIC/MWCNT sca olds signi ican ly p omo ed he
egene a ion o cal a ial de ec healing [136].
Nanoc ys alline cellulose is a widely a ailable na u al ma e ial on Ea h. I is isola ed
om lignocellulosic plan s o om ag icul u al was e using he acid hyd olysis me hod. I
can be cha ac e ized by ou s anding physicochemical p ope ies, low oxici y, and eco oxi-
cological isks owa d li ing cells. Due o hese ac s, i has o en been used in designing
ma e ials o bone sca olds [
137
]. A no el NCP sca old based on ni ogen-doped MWCNTs,
cellulose, and nHA was designed by Xing e al. [
138
]. The mechanical p ope ies o he
hyb id sca old con aining 1 w % N-MWCNTs we e signi ican ly imp o ed, and i s su ace
mo phology was ough and po ous.
In i o
cellula a achmen , p oli e a ion, iabili y,
and mine aliza ion o bone MSCs was also con i med. The p esence o N-MWCNTs in
he sca old induced he p e e en ial di e en ia ion o bone MSCs o os eogenic lineage,
which was accompanied by inc eased ALP ac i i y and he exp ession o key os eogenic
genes. No only was he in e ace bonding wi h he bone issue accele a ed, bu new bone
o ma ion and egene a ion we e also con i med [138].
4.2.2. G aphene-Based Ma e ials
GR and GO a e able o suppo cell g ow h and p oli e a ion, cell a achmen , and
cy oskele on de elopmen and o ac i a e os eogenesis and bone de elopmen . They also
ha e posi i e e ec s on a polyme ma ix causing mo e o de ed mo phologies, g ea e
su ace a ea, and highe o al po osi y, which a e a o able sca old p ope ies acili a ing
cell a achmen and mig a ion [139].
HA/hyd ophilic GR (hGR) composi es wi h a highe s abili y we e p epa ed wi hou
ex a ion in oduc ion using he sel -assembling me hod. The c osslinked s uc u e was
o med due o he in e nal in e ac ion be ween HA and hGR, and he composi e oughness
and hyd ophilic abili y could be ailo ed using an inc eased hGR con en . The composi e
HA/5%hGR demons a ed a highe cell p oli e a ion a e (264.81%) and suppo ed he
sp eading and g ow h o MC3T3-E1 cells compa ed o he pu e HA [
140
]. Polyme ic hyb id
NCPs con aining ca ageenan/ac ylic-acid/GR/HA and mimicking he s uc u al and
chemical composi ion o na u al bone we e syn hesized using ee- adical polyme iza ion
and in ended o ac u ed bone egene a ion. S uc u al p ope ies, su ace mo phology,
hyd ophilici y, biodeg adabili y, and swelling o he NCPs oge he wi h he cell iabili y,
cell cul u e, and p oli e a ion agains mouse p eos eoblas (MC3T3-E1) cell lines we e es ed.
Op imum po osi y o 49.75% and po e size o 0.41
×
10
3µ
m
2
, mechanical p ope ies such
as comp ession s eng h o 8.87 MPa and elas ic modulus o 442.63 MPa, swelling o 70.20%
a 27
◦
C, 77.21% a 37
◦
C, and biodeg ada ion o 23.8% we e con i med [
103
]. GR pla ele s
as ille s, NaCl as a po ogenic ma e ial and PCL as a ma ix we e used o p oduce po ous
sca olds using he sol en -cas ing/pa icula e-leaching me hod. The p epa a ion p ocess
and p oduc s a e shown in Figu e 6[
141
]. The comp essi e s eng h, po osi y, con ac angle,
weigh loss, and a ia ions in pH alues in deg ada ion es s as well as he biocompa ibili y
by seeding os eoblas -like (MG-63) cells
in i o
we e s udied, and i was con i med ha
he mechanical p ope ies, cell a achmen , and p oli e a ion we e imp o ed wi h a highe
a io o GR [141].
Pha maceu ics 2021,13, 1994 21 o 48
Pha maceu ics 2021, 13, x FOR PEER REVIEW 21 o 47
and i was con i med ha he mechanical p ope ies, cell a achmen , and p oli e a ion
we e imp o ed wi h a highe a io o GR [141].
Figu e 6. Illus a ions o polycap olac one/g aphene pla ele sca olds p epa a ion. G aphene (GR) pla ele s we e p epa ed
using g aphi e ia in e cala ion a 700 °C o 60 s. The o med GR pla ele s we e dispe sed in ichlo ome hane and mixed
oge he wi h NaCl (p ogen) wi h a polycap olac one solu ion (PCL), s i ed o 2 h, and pou ed in o a o m. A e d ying,
he sys em was imme sed in deionized wa e (D.I.) o emo e NaCl and consequen ly d ied o o m a po ous sca old.
Va ious GR amoun s we e used o he sca old p epa a ion. (a) Scheme o PCL/GR sca old p epa a ion and (b) sca old
samples wi h a ious con en s o GR pla ele s I. 0.05 w %, II. 0.1 w %, III. 0.5 w %, IV. 1.0 w %, and V. 2.0 w %. Adap ed
om [141], Else ie , 2020.
GR nanoshee s and polye he e he ke one (PEEK) we e used o he p epa a ion o
mul i unc ional NCPs wi h 12 o de s o magni ude inc ease in elec ical conduc i i y due
o he o ma ion o an elec ical pe cola ion ne wo k and π–π* bonds be ween GR and
PEEK. This suppo ed elec opho e ic deposi ion o a bioac i e/an ibac e ial coa ing con-
sis ing o s ea yl ime hylammonium chlo ide-modi ied HA. The esul ing coa ed im-
plan demons a ed signi ican boos ing o bone MSC p oli e a ion in i o wi h he pho-
o he mal con e sion e ec o he GR nano ille s. I is usable o pho o he mal applica-
ions such as inc easing bac e ial e adica ion, umo cell inhibi ion, o bone issue egen-
e a ion in i o [142].
PEEK NCPs wi h a ious GO loading we e p epa ed by injec ion molding. The GO
loading in luenced selec ed mechanical p ope ies, and he g ea es elonga ion a b eak
(86.32% highe han ha o pu e PEEK) was in he case o 0.5% GO p obably due o he
well dispe sed GO o ming π–π* bonds wi h PEEK. The inc easing GO con en (>0.5%)
induced GO agglome a ion and, consequen ly, he de e io a ion o some mechanical
p ope ies. The addi ion o GO in o PEEK suppo ed he adhesion and sp eading o bone
MSCs [143]. Huang e al. [144] desc ibed a NCP composed o PEEK, in which GO and HA
we e inco po a ed. A e lase ea men , he composi e had su ace mac opo es wi h di-
ame e s om 200 µm o 600 µm, which imp o ed cell adhesion and p oli e a ion o con-
s an cells and hus o e all biocompa ibili y and u iliza ion [144]. Lopes e al. [145] p e-
pa ed HA–GO NCPs wi h he addi ion o 0.5 w %, 1.0 w %, and 1.5 w % o GO. HA NPs
we e adhe ed o he su ace o he GO shee s, and he a ini y be ween HA and GO in-
c eased om 0.5 w % o 1.5 w % GO in he HA–GO NCPs. The bioac i i y p ope ies o
HA–GO NCP and indi ec cy o oxici y connec ed wi h a dec ease in he human den al
pulp s em cells iabili y and p oli e a ion occu ed when GO concen a ion inc eased o
1.5 w %. Thus, he 0.5 w % HA–GO NCP was a p omising bioma e ial o bone issue
egene a ion compa ed o he pu e HA [145]. The GO/CS/nHA sca old was p epa ed ia
he e ec i e egula ion o CS unc ionalized wi h a GO ne wo k ma ix and demons a ed
enhanced p ope ies such as 3D po ous bone-like hie a chical s uc u e, p ope mechani-
cal p ope y, and biodeg ada ion as well as sui able wa e up ake and e en ion a io. The
biomime ic mine aliza ion and cell cul u e expe imen s demons a ed ha he hyb id
Figu e 6.
Illus a ions o polycap olac one/g aphene pla ele sca olds p epa a ion. G aphene (GR) pla ele s we e p epa ed
using g aphi e ia in e cala ion a 700
◦
C o 60 s. The o med GR pla ele s we e dispe sed in ichlo ome hane and mixed
oge he wi h NaCl (p ogen) wi h a polycap olac one solu ion (PCL), s i ed o 2 h, and pou ed in o a o m. A e d ying,
he sys em was imme sed in deionized wa e (D.I.) o emo e NaCl and consequen ly d ied o o m a po ous sca old.
Va ious GR amoun s we e used o he sca old p epa a ion. (
a
) Scheme o PCL/GR sca old p epa a ion and (
b
) sca old
samples wi h a ious con en s o GR pla ele s I. 0.05 w %, II. 0.1 w %, III. 0.5 w %, IV. 1.0 w %, and V. 2.0 w %. Adap ed
om [141], Else ie , 2020.
GR nanoshee s and polye he e he ke one (PEEK) we e used o he p epa a ion o
mul i unc ional NCPs wi h 12 o de s o magni ude inc ease in elec ical conduc i i y due
o he o ma ion o an elec ical pe cola ion ne wo k and
π
–
π
* bonds be ween GR and
PEEK. This suppo ed elec opho e ic deposi ion o a bioac i e/an ibac e ial coa ing con-
sis ing o s ea yl ime hylammonium chlo ide-modi ied HA. The esul ing coa ed implan
demons a ed signi ican boos ing o bone MSC p oli e a ion
in i o
wi h he pho o he -
mal con e sion e ec o he GR nano ille s. I is usable o pho o he mal applica ions
such as inc easing bac e ial e adica ion, umo cell inhibi ion, o bone issue egene a ion
in i o [142].
PEEK NCPs wi h a ious GO loading we e p epa ed by injec ion molding. The GO
loading in luenced selec ed mechanical p ope ies, and he g ea es elonga ion a b eak
(86.32% highe han ha o pu e PEEK) was in he case o 0.5% GO p obably due o he well
dispe sed GO o ming
π
–
π
* bonds wi h PEEK. The inc easing GO con en (>0.5%) induced
GO agglome a ion and, consequen ly, he de e io a ion o some mechanical p ope ies.
The addi ion o GO in o PEEK suppo ed he adhesion and sp eading o bone MSCs [
143
].
Huang e al. [
144
] desc ibed a NCP composed o PEEK, in which GO and HA we e
inco po a ed. A e lase ea men , he composi e had su ace mac opo es wi h diame e s
om 200
µ
m o 600
µ
m, which imp o ed cell adhesion and p oli e a ion o cons an cells
and hus o e all biocompa ibili y and u iliza ion [
144
]. Lopes e al. [
145
] p epa ed HA–GO
NCPs wi h he addi ion o 0.5 w %, 1.0 w %, and 1.5 w % o GO. HA NPs we e adhe ed o
he su ace o he GO shee s, and he a ini y be ween HA and GO inc eased om
0.5 w %
o
1.5 w % GO in he HA–GO NCPs. The bioac i i y p ope ies o HA–GO NCP and indi ec
cy o oxici y connec ed wi h a dec ease in he human den al pulp s em cells iabili y and
p oli e a ion occu ed when GO concen a ion inc eased o 1.5 w %. Thus, he 0.5 w %
HA–GO NCP was a p omising bioma e ial o bone issue egene a ion compa ed o he
pu e HA [
145
]. The GO/CS/nHA sca old was p epa ed ia he e ec i e egula ion o CS
unc ionalized wi h a GO ne wo k ma ix and demons a ed enhanced p ope ies such as
3D po ous bone-like hie a chical s uc u e, p ope mechanical p ope y, and biodeg ada ion
as well as sui able wa e up ake and e en ion a io. The biomime ic mine aliza ion
and cell cul u e expe imen s demons a ed ha he hyb id sca old possessed supe io
bioac i i y and cell p oli e a ion abili y
in i o
. In addi ion, he a cal a ial de ec epai
models and issue pa hological cha ac e iza ion u he p o ed ha he hyb id sca old

Pha maceu ics 2021,13, 1994 22 o 48
had excellen biocompa ibili y and he capabili y o induce bone egene a ion in si u. The
p epa ed sca old migh be an excellen candida e o endogenous bone epai [
146
]. The
nanohyb ids o 2D od-like nHA loaded on a low-concen a ion GO shee (GO–nHA)
we e inse ed in o spe mine-based high-s eng h he moplas ic polyu e hane-u ea (PUU)
ma ices using an in si u echnique o ab ica e po ous sca olds. The sca olds wi h he
con en o 1 w % GO–nHA showed imp o ed physico-mechanical p ope ies. Cy o oxici y
es s using os eoblas cells such as he MG-63 cell line con i med cell iabili y abo e 95%
and imp o ed p oli e a ion o e a pe iod o wo weeks o cul u e. Type I CLG exp ession
was posi i e, and pe ec ma u a ion and biomine aliza ion o os eoblas s was indica ed
by os eocalcin (OCN) p esence [
147
]. GO and isocyana e we e used o p epa e a GO
shape-memo y polyu e hane composi e wi h imp o ed mechanical and shape-memo y
e ec s. The modulus o app ox. 339 MPa and he shape eco e y a io o 98% we e
ob ained. A e being implan ed in a de ec i e bone ia a minimally in asi e ea men , he
composi e ensu ed a gene a ed o ce du ing he eco e y p ocess and seemed o p o ide a
new possibili y o a p ac ical applica ion o shape-memo y polyme s and composi es in
he ield o bone epai [
148
]. nHAp/CNTs wi h GO and e med GNRs composi es had
good bioac i i y and osseoin eg a ion p ope ies o bone egene a ion. Th ee di e en
con en s o GNR (1, 2, and 3 w %) in nHAp/GNRs we e used. The assessmen was made
in i o
using 36 Wis a a s wi h os eopo osis induced by oopho ec omy in emale a s
p io o implan a ion. The e alua ion was made a e days 21 and 45, when his ological,
biochemical, and adiog aphic analyses (DIGORA me hod) we e done and e alua ed
h ough ANOVA, he Tukey’s es , and he Kolmogo o –Smi no es wi h s a is ical
signi icance a p< 0.05. The os eoconduc i e ac i i y o nHAp and GNRs was obse ed in
dependence on GNR concen a ion in he ollowing o de : 3 > 2 > 1 w % [149].
I is known ha he s on ium (S )-subs i u ed HA sca old canno p ope ly i he
equi ed mechanical p ope ies. GO- ein o ced S HA NPs we e p epa ed using a hy-
d o he mal me hod. GO easily sel -assembles in o a laye ed s uc u e in he dispe sion,
which helps o egula e he S HA deposi ion on he GO su ace. The S HA/GO NPs
we e hen used o inco po a ion in o CS and qua e nized chi osan (qCS) mixed solu-
ions o p epa e he sca old by a eeze-d ying me hod. The comp essi e modulus o
he CS/qCS/S HA/GO sca old achie ed 438.5 kPa, being 4- old highe han ha o he
CS/qCS sca old. In addi ion,
in i o
mine aliza ion le els and ALP ac i i y we e in-
c eased [
150
]. S –GO NCPs allowing o he long- e m elease o S ions we e ab ica ed
and subsequen ly used o ein o ce CLG sca olds. The esul ing S –GO–CLG sca olds
showed high wa e e en ion a es and excellen mechanical p ope ies. They displayed
a s ong e ec on adipose-de i ed s em cells, which was ob ious om cell adhesion and
os eogenic di e en ia ion, and p omo ed he sec e ion o angiogenic ac o s o s imula e
he
in i o
ube o ma ion o endo helial cells. The angiogenic ascula endo helial g ow h
ac o (VEGF) and os eogenic BMP-2 p o ein sec e ion we e inc eased due o syne gis ic
e ec s o GO and S . I ansplan ed in o a c i ical-size cal a ial bone de ec s, he bes bone
egene a ion and angiogenesis we e obse ed a 12 weeks. In addi ion, esul s showed ha
he S –GO–CLG g oup achie ed comple e de ec b idging wi h he newly o med bone
issue, and he esidual S –GO NPs we e phagocy osed and deg aded by mul inuclea ed
gian cells [151].
Ae ogels based on na u al polyme s ha e high po osi y and g ea biocompa ibili y;
howe e , hei mechanical p ope ies a e ex emely poo when using hem as an a i i-
cial g a o bone epai . A highly po ous and hyd ophilic ae ogel was o med by GO
and ype I CLG using he sol-gel p ocess (GO con en : 0, 0.05, 0.1, and 0.2% w/ ). The
comp essi e modulus inc eased wi h he highe GO con en . The 0.1% GO–CLG showed
be e biomine aliza ion a e and cell compa ibili y
in i o
, while a be e bone epai
e ec compa ed o ha o CLG ae ogel was obse ed in a c anial de ec models in he
in i o
s udy [
152
]. A bioac i e PLGA-
α
- icalcium phospha e (
α
-TCP) composi e sca old
con aining GOs and BMP-2 pep ide (PTG/P) was p oduced by a c yogenic 3D p in ing
me hod o epai a c i ical-sized bone de ec . The sca olds we e compa able o human
Pha maceu ics 2021,13, 1994 23 o 48
cancellous bone wi h i s mechanical p ope ies and hie a chical po osi y. GO enhanced he
sca old we abili y and mechanical s eng h; he pep ides ensu ed biological ac i i y. The
a bone ma ow-de i ed MSC ing ow h in o he PTG/P sca old and enhanced os eogenic
di e en ia ion we e p omo ed [
153
]. Silk ib oin (SF) is a na u al p o ein wi hou any phys-
iological ac i i y, which has good biocompa ibili y, is easily p ocessed, and causes minimal
in lamma o y eac ions in he body. SF elec ospun sca olds con aining GO unc ional-
ized wi h BMP-2 polypep ide we e p epa ed ia elec os a ic in e ac ions. The esul ing
sca old showed a be e biocompa ibili y, p omo ing cell adhesion and p oli e a ion and
enhancing
in i o
he os eogenic di e en ia ion o bone ma ow s omal cells using ei he
an os eogenic o non-os eogenic medium.
In i o
bone o ma ion in c i ical-sized cal a ial
bone de ec s was also p o en [
154
]. Mic oRNAs (miRNAs) a e impo an o egula ing
os eogenic di e en ia ion and bone o ma ion. A polye hylenimine (PEI)- unc ionalized
GO complex was loaded wi h he miR-214 inhibi o in o SF/HA sca olds. SF/HA/GO
sca olds showed high mechanical s eng h, and cell adhesion and g ow h we e p omo ed.
The SF/HA/GO–PEI sca olds loaded wi h miR-214 inhibi o (SF/HA/GPM) enhanced
os eogenic di e en ia ion by inhibi ing he exp ession o miR-214 and a same ime by
inc easing he exp ession o ac i a ing ansc ip ion ac o 4 (ATF4) and ac i a ing he Ak
and ERK1/2 signaling pa hways in mouse os eoblas ic cells (MC3T3-E1) in i o [155].
Polyme based ink ma e ials a e cha ac e ized by insu icien mechanical s eng h, low
sca old ideli y, and loss o os eogenesis induc ion. A human MSC-laden GO/ALG/GLT
composi e bioink was p epa ed o o m 3D bone-mimicking sca olds using a 3D biop in -
ing echnique. The GO composi e bioinks con aining highe GO concen a ions (0.5, 1,
and 2 mg/mL) imp o ed he biop in abili y, sca old ideli y, comp essi e modulus, cell
p oli e a ion, os eogenic di e en ia ion, and ex acellula ma ix mine aliza ion compa ed
o he pu e ALG/GLT sys em, while he bioink wi h GO concen a ion 1 mg/mL was
he op imal ille [
156
]. A sca old composed o mesopo ous bioac i e glasses and GO
was in es iga ed o local angiogenesis and bone healing. I had be e cy ocompa ibili y
and highe os eogenesis di e en ia ion abili y wi h a bone BMSCs compa ed o he pu e
mesopo ous bioac i e glass sca old. I also suppo ed ascula ing ow h and enhanced
bone epai a he de ec si e in a a c anial de ec model. The newly o med bone was
in eg a ed no only on he pe iphe y, bu also in he sca old cen e [157].
Fe
3
O
4
/GO NCPs we e added in o
α
-TCP/calcium sul a e (CaS) biphasic bone cemen
o p epa e injec able magne ic bone cemen (
α
-TCP/CaS/Fe
3
O
4
/GO,
α
-CFG) o he appli-
ca ion in bone umo minimally in asi e su ge y. The magne o he mal p ope ies o he
α
-CFG bone cemen could be well adjus ed by changing he Fe
3
O
4
/GO NCP con en and
he magne ic ield pa ame e s. The mos s able bone cemen wi h excellen magne o he mal
pe o mance was in he case o 10 w % con en o Fe
3
O
4
/GO. The
α
-CFG bone cemen
enabled bone egene a ion and demons a ed umo ea men e ec s [158].
The hie a chical po ous HA/ GO composi e sca olds we e p epa ed using a so
empla e me hod wi h nanosu ace mo phology, sui able po osi y and po e size, and good
biomechanical s eng h. The loaded GO p omo ed he adhesion, p oli e a ion, and spon a-
neous os eogenic di e en ia ion o bone MSCs. The sca old is g adually deg aded and
newly o med bone eplaces i [
47
]. A zinc-doped HA NCP deco a ed on GO, named as
G
3
H
ap
Z, showed i s os eoconduc i e po en ial in biomine aliza ion s udies. The os eoin-
duc i e abili y was es ed on MSC di e en ia ion o os eogenic lineage and exp ession
o os eogenic ma ke s including un - ela ed ansc ip ion ac o 2 (RUNX-2), ALP, ype
I CLG, BMP-2, OCN, and os eopon in (OPN). Thus, he G
3
H
ap
Z NCPs we e es ed as
o hopedic bone g a s o accele a e bone egene a ion [
159
]. Shape memo y polyme s
(SMPs) ha e a g ea po en ial o applica ions in he a ea o minimally in asi e su ge y.
HA/ GO nano ille s we e inse ed in o shape memo y polyu e hane (SMPU) o imp o e
i s mechanical p ope ies. This NCP was u he modi ied using a ginyl-glycyl-aspa ic
acid (AGA) o imp o e cellula adhesion. I was obse ed ha he mechanical p ope ies o
SMPU/HA/ GO/AGA NCP we e signi ican ly imp o ed (e.g., a 200% inc ease in Young’s
modulus and >300% enhancemen in ensile s eng h compa ed o he unmodi ied SMPU).
Pha maceu ics 2021,13, 1994 24 o 48
Rabbi bone MSCs we e adhe ed on he NCP su ace. The excellen shape memo y be-
ha io (e.g., shape ixi y a io 97.3% and shape eco e y a io 98.2%) was con i med [
160
].
Me hyl anilla e (MV) is known o p omo e he Wn /
β
-ca enin signaling pa hway and
induce os eoblas di e en ia ion. GLT- GO o MV deli e y was p epa ed o ealize he
e ec i e os eogenesis o bone epai . The biocompa ibili y o GLT- GO was p o en by
easy cell abso p ion and dis ibu ion in he nucleus and cy oplasm. The MV has a pos-
i i e in luence on he BMSC os eogenesis in a concen a ion-dependen manne wi h a
signi ican imp o emen a he concen a ion le el o 1
µ
g/mL. I was con i med by he
ALP assay, Aliza in ed S s aining, immuno luo escence s aining, and gene exp ession
o ela ed os eogenic ma ke s ha he MV/GLT- GO weigh a io o 1:1000 ob iously
inc eased BMSC os eoinduc ion [161].
4.2.3. O he Ca bon-Based Nanoma e ials
An elec ospun nano-bio memb ane om PVA, nano-demine alized bone ma ix,
and ca bon NPs was p epa ed using an elec ospinning machine. Tes s wi h he MG63
os eoblas cell line showed 100% biocompa ibili y, and mo e iable cells p esen in he
nano-biomemb ane we e obse ed as well as mo e apa i e o ma ion was con i med
using SEM. The con en o ca bon NPs (0.6 w %) in luenced mechanical p ope ies, which
we e imp o ed, achie ing a ensile s eng h o 14.58
±
0.13 MPa, elonga ion a b eak
13.87 ±0.05%, and wa e abso p ion 36.84 ±0.11% [162].
CDs a e ano he ca bon ma e ial ca ying a ious eac i e g oups on he su ace.
They p o ide a unique su ace o anspo he apeu ic genes and p omo e os eogenic
di e en ia ion. MiR-2861 has e ealed os eogenic di e en ia ion e ec s. Bu e al. [
163
]
c ea ed asco bic acid-PEI CDs loaded wi h miR-2861 by he mic owa e-assis ed py olysis
me hod. The esul ing CDs had excellen luo escence s abili y u ilizable o luo escence
imaging
in i o
and
in i o
. The CDs we e inco po a ed in o bone ma ow s omal
cells (BMSCs) and dis ibu ed in he mi ochond ia, endoplasmic e iculum, lysosome,
and nucleus. I was p o en ha he CDs e icien ly ans e ed miR-2861 in o bone MSCs
in i o
, and he CDs wi h miR-2861 (CD@miR) had he s onges os eogenic e ec s because
hey ac ed syne gis ically. No cy o oxici y was ound [
163
]. Zn
2+
-passi a ed CDs showed
good os eogenic ac i i y
in i o
and
in i o
. A 5 mm diame e cal a ial bone de ec model
was c ea ed in a s and he Zn-CDs we e used o he ea men o he c i ical bone de ec .
I was ound ha zinc glucona e (Zn-G) and he Zn-CDs p omo ed he su i al o BMSCs
when he Zn
2+
ion concen a ion was 10
−4
mol/L (Zn-G: 45.6
µ
g/mL) and 10
−5
mol/L
(Zn-CDs: 300
µ
g/mL) o below, espec i ely. Compa ed o he os eogenic capabili y, he
ALP ac i i y induced by he Zn-CDs was be e han ha by Zn-G. The a ea o calci ied
nodules was inc eased in he Zn-CD g oup. Thus, Zn-CDs eached he highes os eogenic
e ec a he concen a ion o 10
−5
mol/L wi hou a ec ing cell p oli e a ion in long- e m
s imula ion [164].
Lai e al. [
165
] ound ha ca bon nanocages (CNCs) could imp o e he os eogenesis
o BMP-2 (0.8
µ
g/mL) a he concen a ion o 20–80
µ
g/mL in a dose-dependen manne .
A composi e po ous SF/CNC sca old was in es iga ed o he con olled deli e y o
BMP-2.
An ini ial bu s elease o 23.9% and a elease o e he subsequen six days o abou
47.7% we e de e mined. The p omo ion o he os eogenic di e en ia ion o bone MSCs
was e alua ed as dose-dependen , and i was ound ha he BMP-2/SF/CNC sca old
signi ican ly imp o ed he os eogenic di e en ia ion o bone MSCs
in i o
and p omo ed
new bone o ma ion in i o [165].
Ca bon nano ibe (CNF)/AuNPs conduc i e sca olds we e p epa ed using blending
elec ospinning and elec ospinning/elec osp aying. The elec ospun and elec osp ayed
nano ibe s had dec eased diame e s: om 178.66
±
38.40 nm o 157.94
±
24.14 nm and
120.81
±
13.77 nm, espec i ely. Elec ical conduc i i y was inc eased by up o 29.2% and
81% by elec osp aying and blending elec ospinning, espec i ely. Nei he signi ican
oxici y no in luence on cell p oli e a ion was obse ed. Cell a achmen and sp eading on
Pha maceu ics 2021,13, 1994 25 o 48
p epa ed sca olds a e shown in Figu e 7; hei ypical mo phology and a achmen ha e
been ound o be e y p omising o u u e s udies [166].
Pha maceu ics 2021, 13, x FOR PEER REVIEW 25 o 47
nano ibe s had dec eased diame e s: om 178.66 ± 38.40 nm o 157.94 ± 24.14 nm and
120.81 ± 13.77 nm, espec i ely. Elec ical conduc i i y was inc eased by up o 29.2% and
81% by elec osp aying and blending elec ospinning, espec i ely. Nei he signi ican
oxici y no in luence on cell p oli e a ion was obse ed. Cell a achmen and sp eading
on p epa ed sca olds a e shown in Figu e 7; hei ypical mo phology and a achmen
ha e been ound o be e y p omising o u u e s udies [166].
Figu e 7. SEM images o MG-63 cell a achmen and sp eading on: (a) ca bon nano ib ous (CNF)
sca olds wi hou gold nanopa icles (CNF sca old), (b) CNF sca olds con aining 2.5 w % o gold
nanopa icles (CNF/2.5% AuNPs), and (c) CNF sca old wi h 5 w % o sp ayed AuNPs. Scale ba =
10 µm. Adap ed om [166], Else ie , 2020.
Highly po ous PEEK/HA bioNCP sca olds ein o ced wi h CNF and CNTs we e
p epa ed using sal po ogen (size 200–500 µm) leaching me hods. The NCP showed con-
olled po e size and dis ibu ion, enabling be e cellula in il a ion and he bioin eg a-
ion o NCPs wi hin human issue. I was p o en ha he NCPs we e non- oxic wi h e y
good cell iabili y, and bone ma ow cell g ow h was con i med, while he p esence o
CNTs (0.5 and 1.0 w %) and CF (0.5 w %) inc eased cell a achmen compa ed o he nea
PEEK/HA composi es, and he mechanical and biological p ope ies we e imp o ed [167].
All o he abo e-men ioned NCPs and hei p ope ies a e summa ized in Table 3.
Table 3. Summa y o composi e ma e ials con aining nanoca bons de eloped o bone healing and egene a ion.
Ma ix Fille Tes ed Cells In Vi o In Vi o Re .
Poly-(L-lac ic acid) Ca bon nano ubes
Human os eosa -
coma MG63 os e-
oblas cells
Cell mo phology, ia-
bili y, p oli e a ion - [127]
Poly(L-lac ic acid)
G aphene nano ib-
bons, nano-hyd ox-
yapa i e
- Allium cepa es , hemol-
ysis
Female Wis a Ra s: su -
gical de ec s in ibias,
come assay, bone egen-
e a ion
[128]
Collagen Ca bon nano ubes,
hyd oxyapa i e
Ra bone mesen-
chymal s em
cells
Cell mo phology, ia-
bili y, p oli e a ion,
BMP-2 le el, insulin-
like g ow h ac o 1 e-
cep o
Female SD a s: X- ay,
mason s aining and oxi-
cology expe imen s
[129]
A ginine-glycine-aspa -
ic acid/BMP-2 pep ides,
poly-(L-lac ic acid)
Ca bon nano ubes
wi h ca boxyl and
amino g oups
Mouse p eos eo-
blas cells
MC3T3-E1
Cell adhesion, p oli e -
a ion, di e en ia ion,
mine aliza ion
- [130]
Oligo(poly(e hylene gly-
col) uma a e), poly(e h-
ylene glycol)ac yla e
Ca bon nano ubes,
black phospho us
MC3T3 p eos eo-
blas cells
Cell adhesion, p oli e -
a ion, os eogenic di e -
en ia ion, elec ic s im-
ula ion enhanced os eo-
genesis
- [131]
Figu e 7.
SEM images o MG-63 cell a achmen and sp eading on: (
a
) ca bon nano ib ous (CNF)
sca olds wi hou gold nanopa icles (CNF sca old), (
b
) CNF sca olds con aining 2.5 w % o
gold nanopa icles (CNF/2.5% AuNPs), and (
c
) CNF sca old wi h 5 w % o sp ayed AuNPs.
Scale ba = 10 µm. Adap ed om [166], Else ie , 2020.
Highly po ous PEEK/HA bioNCP sca olds ein o ced wi h CNF and CNTs we e p e-
pa ed using sal po ogen (size 200–500
µ
m) leaching me hods. The NCP showed con olled
po e size and dis ibu ion, enabling be e cellula in il a ion and he bioin eg a ion o
NCPs wi hin human issue. I was p o en ha he NCPs we e non- oxic wi h e y good
cell iabili y, and bone ma ow cell g ow h was con i med, while he p esence o CNTs (0.5
and 1.0 w %) and CF (0.5 w %) inc eased cell a achmen compa ed o he nea PEEK/HA
composi es, and he mechanical and biological p ope ies we e imp o ed [167].
All o he abo e-men ioned NCPs and hei p ope ies a e summa ized in Table 3.
Table 3. Summa y o composi e ma e ials con aining nanoca bons de eloped o bone healing and egene a ion.
Ma ix Fille Tes ed Cells In Vi o In Vi o Re .
Poly-(L-lac ic acid) Ca bon nano ubes
Human os eosa coma
MG63 os eoblas cells
Cell mo phology,
iabili y, p oli e a ion
- [127]
Poly(L-lac ic acid)
G aphene
nano ibbons,
nano-hyd oxyapa i e
-Allium cepa es ,
hemolysis
Female Wis a Ra s:
su gical de ec s in
ibias, come assay,
bone egene a ion
[128]
Collagen Ca bon nano ubes,
hyd oxyapa i e
Ra bone
mesenchymal s em
cells
Cell mo phology,
iabili y,
p oli e a ion, BMP-2
le el, insulin-like
g ow h ac o 1
ecep o
Female SD a s:
X- ay, mason s aining
and oxicology
expe imen s
[129]
A ginine-glycine-aspa ic
acid/BMP-2 pep ides,
poly-(L-lac ic acid)
Ca bon nano ubes
wi h ca boxyl and
amino g oups
Mouse p eos eoblas
cells MC3T3-E1
Cell adhesion,
p oli e a ion,
di e en ia ion,
mine aliza ion
- [130]
Oligo(poly(e hylene
glycol) uma a e), poly(e hylene
glycol)ac yla e
Ca bon nano ubes,
black phospho us
MC3T3 p eos eoblas
cells
Cell adhesion,
p oli e a ion,
os eogenic
di e en ia ion,
elec ic s imula ion
enhanced
os eogenesis
- [131]
Pha maceu ics 2021,13, 1994 32 o 48
halloysi e nano ubes (gHNTs) was p epa ed o combine he p in abili y o PLLA, he
pe ec os eogenic ac i i y o gMgOs, and he excellen ein o cemen and oughening o
gHNTs. The composi e sca olds wi h la ge and small po es and honeycomb s uc u e
showed inc eased hyd ophilici y, ensile, and comp essi e p ope ies, oge he wi h a ini y
and os eogenic ac i i y, imp o ed mechanical p ope ies, and p omising cell adhesion,
p oli e a ion, and mig a ion [183].
4.4. Me al-Based Nanoma e ials
4.4.1. Magnesium-Based Nanoma e ials
Magnesium (Mg) and i s alloys ha e demons a ed sui able biocompa ibili y and
mechanical s eng h o medical applica ions. The p oblem is low Mg co osion esis ance
in a physiological en i onmen . nHA coa ings can dec ease deg ada ion a es and imp o e
he mechanical s eng h o Mg based implan s, and bone healing is enhanced due o hei
os eoinduc i i y and os eoconduc i i y. Con o mal nano- o-submic on HA coa ings de-
posi ed on Mg pla es and ods ia he ansonic pa icle accele a ion p ocess we e s udied
o desc ibe hei e ec s on Mg deg ada ion
in i o
. The co osion esis ance o Mg was
imp o ed, and he coa ings e ained 86–90% o he inal comp essi e s eng h a e
in i o
imme sion in simula ed body luids o six weeks, while uncoa ed Mg e ained only 66%
o s eng h. The deg ada ion o he ods was slowe han ha o he pla es. Be e cell
adhesion densi ies we e ound unde indi ec con ac condi ions han unde di ec con ac
condi ions o he HA coa ed Mg, which educed he adhesion densi ies o bone ma ow-
de i ed MSCs on he su ace, bu inc eased hem unde indi ec con ac . Howe e , i was
also ound ha Mg-based pla e and sc ew de ices could be di e en ly deg aded, e en i
hey we e ea ed wi h he same coa ings and implan ed a he same o simila ana omical
pa s [
184
]. Mg-based alloys a e known o hei apid deg ada ion and high co osion
a e; o his eason, hey a e associa ed wi h
in i o
in ec ions and implan ailu e. The
s abili y and an i-in lamma o y p ope ies o Mg alloys can be imp o ed by hei modi i-
ca ion wi h GR NPs. Low quan i ies o GR (0.18 and 0.50 w %) we e success ully added
by spa k plasma sin e ing (SPS) in o a Mg alloy wi h Al (1 w %) and Cu (0.25 w %). The
deg ada ion a e o Mg-based alloys dec eased app ox. 4- old, and he bac e icidal ac i i y
inc eased up o 5- old when 0.18 w % GR was used. This NCP showed he comp essi e
p ope ies co esponding o hose o na i e cancellous bone (modulus app ox. 6 GPa). A
high cy ocompa ibili y oge he wi h excellen os eogenic p ope ies was p o en in
in i o
s udies wi h human MSCs [
185
]. Mg-based NCPs we e p oduced using HA bioce amic
nano ein o cemen . The addi ion o HA inc eased he yield s eng h o he alloy ma ix
and exhibi ed supe io s eng h and duc ili y e en ion pos co osion o 21 days. The
p esence o HA also imp o ed he hyd ophilici y and biocompa ibili y o he alloy ma-
ix wi h enhanced co osion esis ance, non-cy o oxici y, and high cell a achmen [
186
].
Khalili e al. [187]
s udied he e ec s o ho isos a ic p essing and su ace anodizing on he
p ope ies o an in si u su ace modi ied magnesium ma ix NCP wi h di e en pe cen age
by weigh o HA by s i -cen i ugal cas ing. They wan ed o educe he de ec s and o
eplace he Mg/HA su ace wi h a ce amic ma ix NCP laye o MgO/HA, which was
con i med by ene gy dispe si e spec oscopy and X- ay di ac ion. The 1.8 w % nHA
was homogeneously dis ibu ed in he MgO ma ix wi h a well-a anged nanos uc u e
on he su ace, which educed he H2 elease and co osion a e. The lowes he mody-
namic endency o co osion (
−
1.345 V) and he co osion a e o
3.8388 mm/yea
wi h
he highes p o ec ion e iciency o 42.26% in compa ison wi h he as-cas pu e magne-
sium we e obse ed. Thus, i is a p omising ma e ial o bone implan s [
187
]. HA–MgO
NCPs we e ab ica ed wi h di e en bioac i e composi ions. The e we e changes in he
composi ion o NCPs sin e ed a 1200
◦
C because nHA was pa ially decomposed in o
β
- icalcium phospha e (
β
-TCP). The NCP densi y was in he ange om 2.72
±
0.066 o
3.03 ±0.093 g/mL,
depending on he MgO con en
(0.0–2.0 w %).
An ob ious inc ease in
he mechanical p ope ies o he composi e was achie ed wi h inc easing MgO amoun s.
The bes mechanical p ope ies we e ound o he NCP HA–1.0 MgO (e.g., comp essi e

Pha maceu ics 2021,13, 1994 33 o 48
s eng h 111.20
±
5 MPa, ac u e oughness 136.98
±
5 MJ/m
3
) compa ed o he pu e
n-HA. The NCP su ace had a hyd ophilic na u e, and biocompa ibili y in e ms o cell
iabili y was also epo ed [188].
MgO NPs we e modi ied wi h poly(L-lac ic acid-co-malic acid) (PLMA) o suppo
he in e acial compa ibili y in he PLLA sca old. PLMA has a hyd ophilic end (comes
om he ca boxylic g oups o malic acid) and an L-lac ic acid chain. Hyd ogen bonds we e
o med be ween he ca boxylic g oups and MgO-NPs, and he L-lac ic acid chain hyd oxyl
g oups eac ed wi h he PLLA ca boxyl g oups. Comp essi e s eng h and modulus o he
ab ica ed sca old we e signi ican ly enhanced by 47.1% and 237.7%, espec i ely [
189
].
NCP ma e ials o PLA/s ea ic acid-modi ied MgO (1 w %) we e p epa ed using blend-
ing ex usion. I was ound ha he long- e m deg ada ion o NCPs depended on he
ille shape and was accele a ed by an inc ease in he wa e up ake a e o he PLA ma-
ix. The MgO NCP was a ec ed signi ican ly by he inc eased hyd ophilici y. Thus, he
PLA/MgO ma e ials can e icien ly egula e he PLA ma ix deg ada ion and enhance i s
bioac i i y [190].
A NCP consis ing o PLGA,
β
- icalcium phospha e (
β
-TCP), and Mg(OH)
2
was
designed o p omo e bone epai h ough os eoinduc i e, os eoconduc i e, and an i-
in lamma o y abili ies. The PLGA/
β
-TCP/Mg(OH)
2
NCP inc eased he bone egene a ion
a e o ully epai bone de ec healing wi h supp essed in lamma o y esponses [
191
]. A
polybu ylene succina e (PS: 50 w %), magnesium phospha e (MP: 40 w %), and whea
p o ein (WP: 10 w %) composi e (PMWC) sca old was p epa ed wi h in e connec ed
mac opo es (400
µ
m o 600
µ
m) and mic opo es (10
µ
m o 20
µ
m) on he mac opo e walls.
The p esence o MP imp o ed he apa i e mine aliza ion o he PMWC sca old in simula ed
body luid, and he addi ion o WP imp o ed he PMWC deg adabili y in PBS compa ed
o he sca old o he PS/P composi e and PS alone. The PMWC sca old suppo ed he p o-
li e a ion and di e en ia ion o mouse p eos eoblas ic cell line (MC3T3-E1) cells, inc eased
new bone o ma ion and ing ow h, and p omo ed os eogenesis and ascula iza ion, which
was con i med
in i o
[
192
]. The majo ino ganic componen o eggshells is CaCO
3
. Thus,
MgO NPs-coa ed eggshell pa icles (deno ed CaCO
3
/MgO NCPs) we e p epa ed and used
o subsequen ab ica ion o a biomime ic ac i e sca old based on he chemical c osslink-
ing o he CaCO
3
/MgO NCP, CM–CS, and BMP-2 (see Figu e 8). The esul ing composi es
CaCO
3
/MgO/CM–CS/BMP-2 demons a ed a highe modulus and comp essi e s eng h
han he CM–CS sca old. The CaCO
3
/MgO/CM-CS/BMP-2 sca old also showed mine -
aliza ion abili y, os eogenic di e en ia ion po en ial, and he abili y o elease Mg
2+
ions
and BMP-2, which could ac i a e he phospho yla ion o he ERK1/2 and Ak pa hways
and p omo e os eogenesis ia he c oss alk o mul iple pa hways. Ou s anding esul s in
bone epai we e p o en using an in si u a cal a ial de ec epai expe imen [193].
3D mesopo ous biocomposi es we e designed o den al o bone implan applica-
ions based on Z O
2
–MgO–hBN con aining highly po ous nano ubes o hexagonal bo on
ni ide (hBN). These we e cha ac e ized by low densi y, high s eng h, and mesopo ous
in e connec ed a chi ec u e. The ma e ials exhibi ed in e es ing p ope ies due o hei
s abili y in wa e , minimum essen ial medium eagle-
α
modi ica ion (
α
-MEM), acids, and
oils (e.g., sui able p oli e a ion o os eoblas such as MG63 cells o il a ion o E. coli om
wa e [
194
]). Magnesium-en iched GO nanosc olls (MgNPs@GNSs) we e designed o
he combina ional modula ion o he in lamma o y esponse. I was p o en ha GO ac-
i a es in lamma o y M1 mac ophages and ha Mg
2+
acili a es he epola iza ion o M1
mac ophages o he p o-healing M2 pheno ype. Thus, wi h he sus ained elease o Mg2+,
he MgNPs@GNS nanopla o m can induce syne gic ype 1 and ype 2 in lamma o y e-
sponses. Mg
2+
ions dec eased he GO in e naliza ion and down egula ed he nuclea ac o
κ
B pa hway in ol ed in he in lamma o y p ocess. The o de ed in lamma o y esponses
s imula ed
in i o
angiogenesis and os eogenesis h ough chemo ac ic, mi ogenic, and
mo phogenic ac ions, and ascula ized bone egene a ion was achie ed in a a c anial
bone de ec model [195].
Pha maceu ics 2021,13, 1994 34 o 48
Pha maceu ics 2021, 13, x FOR PEER REVIEW 34 o 47
Figu e 8. Scheme o he p epa a ion o calcium ca bona e/magnesium oxide/ca boxyme hyl chi-
osan/bone mo phogene ic p o ein 2 (CaCO3/MgO/CMC/BMP2) sca olds and hei applica ions in
i o. Eggshell mic opowde nanopa icles modi ied using MgO (CaCO3/MgO) we e p epa ed in
si u and hen mixed wi h bone mo phogene ic p o ein 2 (BMP2) and ca boxyme hyl chi osan
(CMC). A po ous sca old was p epa ed ia chemical c osslinking. The os eoinduc i i y o his sca -
old was cha ac e ized a he molecula , cellula , and animal le els. The os eogenic mechanism o
he p epa ed sca old was ela ed o he elease o Mg2+ and BMP2, and i could signi ican ly p o-
mo e seed cell os eogenic di e en ia ion h ough he ERK1/2 and Ak pa hways. Adap ed om
[193], Else ie , 2020.
4.4.2. Ti anium-Based Nanoma e ials
Ti anium (Ti) is a widely used su gical ma e ial ha is also used o implan s. Un o -
una ely, i s bioine ness may be impai ed due o poo an ibac e ial p ope ies. These can
be imp o ed, o example, by su ace co e age o i anium implan s, o example, by a
combina ion o HA and an ibac e ially ac i e CS, as desc ibed by Li e al. [196]. Such a
HA–CS composi e coa ing imp o ed cell adhesion and inhibi ed bac e ial g ow h, and he
au ho s desc ibed i s applica ions in o hopedics and den is y [196].
On he i anium implan Ti-6Al-4V, a hie a chical mic o/nano-s uc u ed su ace
suppo ing he p oli e a ion and os eogenic di e en ia ion o p eos eoblas cells (MC3T3-
E1) was c ea ed by ul asonic acid e ching and anodic oxida ion by imp o ing su ace
hyd ophilici y and bioac i i y compa ed o implan s wi h a polished Ti su ace, so ha
he bioac i i y and os eogenic p ope ies we e imp o ed [197]. In addi ion, g aphdiyne-
modi ied i anium implan s ha e good biocompa ibili y and os eoinduc i e capabili ies
o cell adhesion and di e en ia ion as well as signi ican an ibac e ial p ope ies [198].
The Ti6Al4V ma e ial coa ed wi h a composi e composed o nHA and GR showed high
co osion esis ance and good biocompa ibili y [199]. Ano he way o imp o e he p op-
e ies o Ti (Ti–6Al–4V) implan s is o co e hei su ace wi h GR o CNTs [200]. A
HA/TiO2/CNT NCP p epa ed by he hyd o he mal me hod showed good mechanical and
physicochemical p ope ies and p o ed o be sui able o he g ow h o he human cell
line MDA-MB-231 [201].
The AgNP/GO su ace- ea ed i anium Ti–6Al–7Nb implan had signi ican an ibac-
e ial ac i i y agains E. coli and S. au eus and high os eoblas cy ocompa ibili y [202]. In
addi ion, a 0.1 mm hick diamond ca bon-coa ed Ti–6Al–7Nb honeycomb s uc u e ex-
hibi ing excellen in i o p ope ies and high bone g ow h suppo has been desc ibed by
Kawaguchi e al. [203]. The i anium implan Ti16Nb coa ed wi h GO, HA, and CLG had
inc eased we abili y, which was e lec ed in inc eased cell adhesion and ib oblas iabil-
i y [204].
Ti anium coa ed wi h a laye o CaO NPs was highly ac i e in i o agains me hicil-
lin- esis an S. au eus (MRSA) and a he same ime, signi ican ly p omo ed os eogenic
Figu e 8.
Scheme o he p epa a ion o calcium ca bona e/magnesium oxide/ca boxyme hyl chi-
osan/bone mo phogene ic p o ein 2 (CaCO
3
/MgO/CMC/BMP2) sca olds and hei applica ions
in i o
. Eggshell mic opowde nanopa icles modi ied using MgO (CaCO
3
/MgO) we e p epa ed in
si u and hen mixed wi h bone mo phogene ic p o ein 2 (BMP2) and ca boxyme hyl chi osan (CMC).
A po ous sca old was p epa ed ia chemical c osslinking. The os eoinduc i i y o his sca old
was cha ac e ized a he molecula , cellula , and animal le els. The os eogenic mechanism o he
p epa ed sca old was ela ed o he elease o Mg
2+
and BMP2, and i could signi ican ly p omo e
seed cell os eogenic di e en ia ion h ough he ERK1/2 and Ak pa hways. Adap ed om [
193
],
Else ie , 2020.
4.4.2. Ti anium-Based Nanoma e ials
Ti anium (Ti) is a widely used su gical ma e ial ha is also used o implan s. Un-
o una ely, i s bioine ness may be impai ed due o poo an ibac e ial p ope ies. These
can be imp o ed, o example, by su ace co e age o i anium implan s, o example, by
a combina ion o HA and an ibac e ially ac i e CS, as desc ibed by Li e al. [
196
]. Such a
HA–CS composi e coa ing imp o ed cell adhesion and inhibi ed bac e ial g ow h, and he
au ho s desc ibed i s applica ions in o hopedics and den is y [196].
On he i anium implan Ti-6Al-4V, a hie a chical mic o/nano-s uc u ed su ace
suppo ing he p oli e a ion and os eogenic di e en ia ion o p eos eoblas cells (MC3T3-
E1) was c ea ed by ul asonic acid e ching and anodic oxida ion by imp o ing su ace
hyd ophilici y and bioac i i y compa ed o implan s wi h a polished Ti su ace, so ha
he bioac i i y and os eogenic p ope ies we e imp o ed [
197
]. In addi ion, g aphdiyne-
modi ied i anium implan s ha e good biocompa ibili y and os eoinduc i e capabili ies o
cell adhesion and di e en ia ion as well as signi ican an ibac e ial p ope ies [
198
]. The
Ti6Al4V ma e ial coa ed wi h a composi e composed o nHA and GR showed high co osion
esis ance and good biocompa ibili y [
199
]. Ano he way o imp o e he p ope ies o Ti (Ti–
6Al–4V) implan s is o co e hei su ace wi h GR o CNTs [
200
]. A HA/TiO
2
/CNT NCP
p epa ed by he hyd o he mal me hod showed good mechanical and physicochemical
p ope ies and p o ed o be sui able o he g ow h o he human cell line MDA-MB-
231 [201].
The AgNP/GO su ace- ea ed i anium Ti–6Al–7Nb implan had signi ican an ibac-
e ial ac i i y agains E. coli and S. au eus and high os eoblas cy ocompa ibili y [
202
]. In
addi ion, a 0.1 mm hick diamond ca bon-coa ed Ti–6Al–7Nb honeycomb s uc u e ex-
hibi ing excellen
in i o
p ope ies and high bone g ow h suppo has been desc ibed
by Kawaguchi e al. [
203
]. The i anium implan Ti16Nb coa ed wi h GO, HA, and CLG
had inc eased we abili y, which was e lec ed in inc eased cell adhesion and ib oblas
iabili y [204].
Pha maceu ics 2021,13, 1994 35 o 48
Ti anium coa ed wi h a laye o CaO NPs was highly ac i e
in i o
agains me hicillin-
esis an S. au eus (MRSA) and a he same ime, signi ican ly p omo ed os eogenic di -
e en ia ion o bone ma ow MSCs [
205
]. nHA coa ed wi h polyamide 66 wi h AgNPs
and TiO
2
NPs showed high an ibac e ial ac i i y agains E. coli and S. au eus and good
biocompa ibili y. The NCP p omo ed cell adhesion and cell p oli e a ion o p eos eoblas s.
Thus, he whole sys em seems o be sui able o he ea men o os eomyeli is [206].
An elec oac i e biocomposi e o PLGA mixed wi h gadolinium-doped ba ium i ana e
NPs (Gd-BTO NPs) was s udied o es ablish he ela ionship be ween su ace po en ial and
os eogenic ac i i y. The in oduc ion o he Gd-BTO NPs imp o ed he elas ic modulus
and was sui able o MRI and X- ay dual imaging. The elec ical p ope ies o hese NPs
(e.g., dielec ici y, piezoelec ici y, and su ace po en ial elec ical cha ac e is ics) we e
e ec i ely imp o ed. The nega i e su ace po en ial o poled Gd-BTO/PLGA signi ican ly
inc eased cell a achmen and os eogenic di e en ia ion, induced in acellula Ca
2+
ion con-
cen a ion oscilla ion, and imp o ed os eogenic di e en ia ion ia he calcineu in/NFAT
signal pa hway [207].
4.4.3. O he Me al-Based Nanoma e ials
Highly-c ys alline, ound-shaped ZnO nanoc ys als (ZnO NCs) o 20 nm in diame e
we e es ed in bone implan applica ions
in i o
in a o m o s able colloidal solu ion in
e hanol. The NCs we e also pa ially unc ionalized by ancho ing amino-p opyl g oups o
he ZnO su ace (ZnO-NH
2
NCs). The es s o biocompa ibili y owa d p eos eoblas cells,
p omo ion o cell p oli e a ion and di e en ia ion, and an imic obial ac i i y agains G am-
posi i e and -nega i e bac e ia showed ha ZnO-NH
2
NCs a e applicable o he ea men
o implan - ela ed in ec ious diseases and could be used as a highly biocompa ible and
os eoinduc i e nanoan ibio ic agen o bone issue enginee ing [
208
]. The sca olds wi h
high po osi y (app ox. 93%) and po e size anging om 100 o 400
µ
m based on a poly(D,L-
lac ide acid) (PDLLA) ma ix con aining undoped and Cu-, Zn-, and CuZn-doped bioac i e
glass pa icles we e p oduced by eeze-d ying and sal -leaching me hods. Imp o emen s
in he elas ic moduli we e as high as 130%, and he apa i e o ma ion on he sca old
su ace was induced. The sca old deg ada ion showed he highes a e in he case o he
PDLLA/undoped glass sca old. Thus, he inco po a ion o undoped and me al-doped
bioac i e glasses inc eased he mechanical s eng h, p omo ed he bioac i i y, and modi ied
he deg ada ion p o ile o he sca olds [209].
A 3D po ous i on (Fe) sca old wi h skele on diame e 143
µ
m, in e connec ed po es,
a e age po e size 345
µ
m, po osi y > 90%, and yield s eng h 3.5 MPa was p epa ed ia
a empla e-assis ed elec odeposi ion me hod. S on ium inco po a ed oc acalcium phos-
pha e (S -OCP) was used as a coa ing o he Fe sca old skele on o ensu e he biocompa i-
bili y. The coa ing was in he o m o nanowhiske s wi h he mean diame e o 300 nm and
he leng h o 30
µ
m and dec eased he elease a e o Fe ions o a le el sa e o he human
body. The cell adhesion and biocompa ibili y we e enhanced [
210
]. Polyme /phospha e
glass/Fe
3
O
4
MNP (CG/PG/MNP) composi e sca olds we e de eloped using a eeze
d ying echnique. The sca olds we e highly po ous con aining in e connec ed po es o he
size anging om 20 o 150
µ
m. Thei swelling and deg ada ion beha io we e in luenced
by he in eg a ion o Fe
3
O
4
MNPs, and hey demons a ed sligh e omagne ic p ope ies.
The comp essi e modulus inc eased wi h inc easing MNP con en . Good bioac i i y and
cy ocompa ibili y we e con i med [211].
Ce ium oxide (CeO
2
) NPs ha e ee adical sca enging capabili ies. CeO
2
NPs we e
inco po a ed in o GLT–ALG sca olds o ob ain NCP sca olds (GAC) by eeze d ying.
Va ious CeO
2
NP concen a ions we e used, and hei in luence on he physicochemical and
biological p ope ies o he NCP sca olds was e alua ed. The mechanical p ope ies and
bio-mine aliza ion we e imp o ed; he swelling and
in i o
weigh loss o he sca olds
dec eased. The ALP ac i i y, RUNX-2, and OCN exp ession s udy indica ed ha he GAC
sca olds suppo MSC di e en ia ion in o os eoblas s. In addi ion, he GAC a e capable o
educing ee adicals [212].
Pha maceu ics 2021,13, 1994 36 o 48
4.5. Polyme s and O he O ganic Ma e ials
Due o hei unique 3D ne wo k s uc u e, high con en o wa e , and unc ional
p ope ies, hyd ogels a e he nex p omising candida es o bone issue enginee ing. Many
s udies in oduce syn hesis me hods (e.g., 3D-p in ing echnology) o p epa e implan ed
hyd ogel sca olds wi h op imal p ope ies. CLG, hyalu onic acid, CS, polye hylene glycol
(PEG), and o he biocompa ible ma e ials a e used as injec able hyd ogels in minimally
in asi e su ge y. They ha e adjus able physicochemical p ope ies and can ill i egula
shapes o de ec si es and elease d ugs o g ow h ac o s ia di e en s imuli (pH, em-
pe a u e, edox, enzyme, ligh , magne ic, e c.) [
213
]. P ima y MSCs along wi h BMP-2
we e inco po a ed in o he ALG skele on. By eleasing BMP-2 in o he de ec i e bone
mic oen i onmen , i caused os eogenic di e en ia ion, leading o he apid o ma ion o
ma u e bone [
214
]. Simila ly, Jin e al. [
215
] p epa ed a NCP composed o PEI-ALG, in
which BMP-2 was inco po a ed. Tes s showed ha he composi e eleased BMP-2 p o ein
o a leas 14 days and ha os eogenesis was p omo ed. A he same ime, ALP ac i i y
and calcium s o age we e inc eased [
215
]. An enginee ed implan able sca old sus ainably
eleasing alend ona e (Aln) o os eopo o ic bone de ec s was in es iga ed. Aln was added
in o 2% CLG solu ion, and hen he mix u e was used o ob ain CLG–Aln sca olds. I was
ound ha Aln was eleased o one mon h wi h he a e age a e o 2.99
µ
g/d wi hin he
i s eigh days. The CLG and CLG–Aln sca olds we e implan ed in o 5 mm c anial de ec s
in o a iec omized a s. Be e bone egene a ion in de ec a ea (11.74
±
3.82%) a e h ee
mon hs was ound in he de ec implan ed wi h he CLG–Aln sca olds compa ed o he
CLG sca old (5.12
±
1.15%) (p< 0.05) [
216
]. CS/SF/glyce ophospha e (GP) composi es,
o which coppe -con aining bioac i e glass NPs (Cu–BG NPs) we e inco po a ed, we e
p epa ed o p oduce injec able hyd ogels o cell- ee bone epai . The highly po ous
Cu–BG/CS/SF/GP gels we e well injec ed, and gela ion a physiological empe a u e
and pH was apid. They could adminis e Si, Ca, and Cu ions a sa e doses. The g ow h
o seeded MC3T3-E1 and HUVECs was suppo ed, and we e sui able o os eogenesis
and angiogenesis. The Cu–BG/CH/SF/GP gel could ully epai he bone de ec , which
was ob ious hanks o he o ma ion o ascula ized bone issue and mine alized CLG
deposi ion du ing eigh weeks wi h no cells and/o g ow h ac o s being used [217].
An injec able luminescen hyd ogel composi e composed o PLGA–PEG–PLGA i-
block copolyme and NaYF
4
: Yb
3+
, E
3+
hollow mic o ubes was ab ica ed o nonin asi e
bone egene a ion moni o ing. The o med hyd ogel had much oughe su ace, enhanced
mechanical p ope ies and b igh luminescence, sui able d ug elease p ope y o p o ein
d ugs, good cellula compa ibili y, and MSC adhesion. The composi e hyd ogel wi h
loaded ecombinan human bone mo phogene ic p o ein 2 (os eogenic induc ion ac o )
was implan ed in o he ibial de ec o a s o e alua e he bone epai . The hyd ogel
sca old deg ada ion was comple ed a e ou weeks; he epai o he ibial de ec was
inished a e six weeks; and he biological sa e y was epo ed [
218
]. NCPs based on
polyu e hane (PU), ghee, and p opolis we e de eloped using he elec ospinning ech-
nique. The PU/ghee (817
±
138.39 nm) and PU/ghee/p opolis (576
±
144.96 nm) NCPs
had smalle ibe diame e han he pu e PU memb ane (890
±
116.911 nm). The con ac
angle aised in PU/ghee (122
±
1
◦
) showed hyd ophobic p ope ies, while i was lowe
in PU/ghee/p opolis (55
±
1
◦
) wi h hyd ophilic beha io . The su ace oughness o he
NCPs dec eased, and hei he mal s abili y was imp o ed. The NCPs we e cha ac e ized
by be e blood compa ibili y wi h non-hemoly ic and non- oxic p ope ies and imp o ed
sa e y o RBCs [219].
O he a ac i e ma e ials o he ab ica ion o unc ional sca olds a e poly( inylidene
luo ide) (PVDF) and poly( inylidene luo ide- i luo oe hylene) (P(VDF-T FE) wi h ex-
cellen piezoelec ici y and good biocompa ibili y. Elec ospun PVDF and P(VDF-T FE)
sca olds p oduced elec ical cha ges du ing mechanical de o ma ion; hus, hey s imula ed
bone de ec s and damaged ne e cells epai ing. Bone and neu al cells we e p omo ed
o adhe e, p oli e a e, and di e en ia e on hei su aces. Addi ionally, neu i e g ow h
along he di ec ion o ibe o ien a ion was enhanced by he aligned PVDF and P(VDF-
Pha maceu ics 2021,13, 1994 37 o 48
T FE) ib ous s uc u e. Small po e sizes p e en ed he in il a ion o bone and neu onal
cells in o he sca olds, which led o he o ma ion o a single cell laye on he sca old
su aces [
220
]. Magne oac i e 3D po ous sca olds based on PVDF and magne os ic i e
pa icles o CoFe
2
O
4
we e ab ica ed using a sol en cas ing me hod wi h nylon empla e
s uc u es and h ee di e en ibe diame e s (60, 80, and 120
µ
m). The magne oac i e
composi es had a s uc u e e y simila o ha o he abecula bone wi h po e sizes om
5 o 20
µ
m due o he c ys alliza ion o PVDF in he p esence o he NPs, in e connec ed
wi h bigge po es ha we e o med a e he emo al o he nylon empla es. The ma e ials
c ys allized in he PVDF elec oac i e
β
-phase, and he p oli e a ion o p eos eoblas s was
p omo ed ia he applica ion o magne ic s imuli [221].
A new class o ci a e-based ma e ials wi h glyce ophospha e sal s,
β
-glyce ophospha e
disodium (
β
-GP-Na) and glyce ophospha e calcium (GP-Ca) was used o he p epa a ion
o he esul an poly(oc ame hylene ci a e glyce ophospha e). The ensile s eng h o POC–
GP–Na and POC–GP–Ca was app ox. 28.2
±
2.44 MPa and 22.76
±
1.06 MPa, espec i ely.
The ini ial modulus anged om 5.28
±
0.56 MPa o 256.44
±
22.88 MPa. POC–GP–Ca
exhibi ed highe cy ocompa ibili y and he co esponding composi e POC–GP–Ca/HA
showed enhanced os eogenic di e en ia ion o human MSCs
in i o
compa ed o POC–GP–
Na/HA and POC/HA. The be e
in i o
pe o mance o he POC–GP-Na/HA sca olds
was con i med in a abbi emo al condyle de ec model [222].
5. C i ical View and Pe spec i es
I is impo an o no e ha he diseases discussed abo e a e caused by sys emic
imbalances (in he case o os eopo osis) and whole-join degene a i e disease (in he
case o os eoa h i is) in ol ing all join issues including a icula ca ilage, subchond al
bone, in apa ella a pad, syno ium, ligamen s/ endons, and menisci. The e o e, join
eplacemen o imp o ed/ acili a ed healing (ca ilage/bone issue egene a ion) wi h
nanocomposi es is no eally a cu e, bu only an a emp o co ec he consequences o he
disease, which does no add ess he unde lying cause o he disease. The apeu ic ea men
wi h “classical” d ugs ocuses p ima ily on ca ilage damage as he p ima y lesion wi hou
aking in o accoun o he join issues and hei impac on he pa hology o join diseases.
Un o una ely, as his o y and he p esen show, hese app oaches a e no en i ely success ul,
and managing a h i is is s ill a majo challenge.
Cu en ly, he e a e no app o ed d ugs ha could igo ously al e he cou se o degen-
e a i e join diseases and cause long- e m, clinically signi ican bene i s. Fo a he apeu ic
in e en ion o be e ec i e, disease-modi ying d ugs able o modula e many di e en cell
ypes p esen in he join s a e needed o be de eloped, so ha many pa hophysiological p o-
cesses can be co ec ed and a “global” he apy canac ually be
o med [6,10–13,16,21–24,27,223].
As ou lined, indi idual and combina o ial app oaches o he ea men exis o a e being
de eloped, and many ma e ials a e being p epa ed o eplace degene a e issues using 3D
p in ing and biosca olds. Simila ly, anspo sys ems ha e been de eloped o a numbe
o chemical and biological he apeu ics.
Since 2010, when he p i a e sec o became in e es ed in issue enginee ing again
a e he c isis, he i s comme cial p oduc s ocused on so issue eplacemen s such
as a i icial skin applicable as a coa ing o bu ns appea ed on he ma ke . O he p od-
uc s can be ound in he cosme ics indus y. I is also posi i e ha he cu en egula o y
amewo k, especially in he U.S., acili a es he as e comme cializa ion o issue engi-
nee ing p oduc s [
224
,
225
]. In he Eu opean Union, hei in oduc ion in o he ma ke
is mo e complica ed; howe e , he issue enginee ing ma ke in he EU is es ima ed o
g ow, especially o companies ope a ing in he 3D biop in ing ma ke , hanks o g owing
go e nmen suppo and g owing demand o cosme ic su ge y. Implan s sui able o
ee h and jawbones and os eo ansplan s o bone su ge y applica ions can be ound on he
ma ke . Os eoinduc i e p oduc s con aining collagen, g ow h ac o s, and human cells ha
induce bone g ow h and ha e he po en ial o s eng hen damaged o weakened bones o
c ea e new bones ha e been ma ke ed [
226
,
227
]. Un o una ely, despi e all he expec a ions,

Pha maceu ics 2021,13, 1994 38 o 48
p omising ad ances, and an inc edible inc ease in p o essional s udies, issue enginee ing
based on nanocomposi e sca olds aces nume ous challenges (i.e., ansla ing all expec-
a ions and plans in o eali y is a complica ed ask). Al hough nano echnology, h ough
nanopa icle enginee ing, is expec ed o ha e he huge po en ial o sol e all p oblems, he e
a e s ill obs acles o he clinical and widesp ead use o nanocomposi es due o he inabili y
o a i icial solid ma e ials o mimic he na u al p ope ies o issues.
6. Conclusions
Biomedical applica ions o nanoma e ials ha e a signi ican ly inc easing end. In ad-
di ion o hei use as d ug ca ie s and diagnos ics, in ecen yea s, a ious nanocomposi es
ha e been used as sca olds in issue enginee ing o help o p epa e biomime ic eplace-
men s o epai damaged issues and o gans. As he numbe o degene a i e diseases o he
musculoskele al sys em inc eases and he adi ional he apy has eached i s limi s, he use
o hese implan s in o hopedics and heuma ology, whe e a ious nanocomposi es o he
healing o ca ilages in join s and damaged bones a e used o in es iga ed, is no su p ising.
Cu en ly, he mos p e e ed a e polyme ic ma e ials, hyd oxyapa i e, and ca bon- o
silica-based ma e ials. These a e a iously unc ionalized o acili a e he p oli e a ion o
heal hy cells in he a ec ed issues, inc ease biocompa ibili y, and educe oxici y. Func ion-
aliza ion can also se e o encapsula e d ugs (i.e., he implan can also ha e an ibac e ial,
an i-in lamma o y, o an i-cance p ope ies). On he o he hand, many nanoma e ials a e
known o cause in lamma o y p ocesses and issue damage. These nega i e phenomena
can be elimina ed p ecisely by unc ionaliza ion, ca e ul es ing, and uncomp omising
selec ion o he mos sui able, s able, and biocompa ible nanoma e ials. Thus, he ad anced
design o nanos uc u ed sca olds has he ambi ion o p o ide syn he ic nanocomposi es
wi h mo e ad an ageous p ope ies o na i e issue, which in u n will lead o inc eased
and accele a ed healing and he o ma ion o unc ional egene a ed issues.
Au ho Con ibu ions:
J.J. and D.P. composi ion and w i ing. All au ho s ha e ead and ag eed o
he published e sion o he manusc ip .
Funding:
This s udy was suppo ed by he Slo ak Resea ch and De elopmen Agency (APVV-
17-0373) and p ojec no. CZ.02.1.01/0.0/0.0/17_049/0008441 “Inno a i e The apeu ic Me hods o
Musculoskele al Sys em in Acciden Su ge y” wi hin he Ope a ional P og am Resea ch, De elopmen .
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : Da a is con ained wi hin he a icle.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Abb e ia ions
2D ( wo-dimensional); 3D ( h ee-dimensional);
α
-MEM (minimum essen ial medium eagle-
α
);
α
-TCP (
α
- icalcium phospha e);
β
-GP-Na (
β
-glyce ophospha e disodium);
β
-TCP (
β
- icalcium
phospha e); ACM (acellula ca ilage ex acellula ma ix); AFM (a omic o ce mic oscopy); AGA
(a ginyl-glycyl-aspa ic acid); ALG (algina e); Aln (alend ona e); ALP (alkaline phospha ase); AMP
(an imic obial pep ide); ATF4 (ac i a ing ansc ip ion ac o 4); AuNPs (gold nanopa icles); BC (bac-
e ial cellulose); BCN (bo oca boni ides); BMP-2 (bone mo phogene ic p o ein-2); BMSCs (bone ma -
ow s omal cells); BP (black phospho us); BSiC (bo osilica e gel); CA (ca echol); CCK-8 (Cell Coun -
ing Ki -8); CDs (ca bon do s); ChS (chond oi in sul a e); CLG (collagen); CM (ca boxyme hyl); CNCs
(ca bon nanocages); CNF (ca bon nano ibe ); CNTs (ca bon nano ubes); CNT-PEGA (poly(e hylene
glycol)ac yla e); –COOH (ca boxylic g oups); COS (chi ooligosaccha ide); CS (chi osan); DLP (dig-
i al ligh p ocessing); EDS (ene gy-dispe si e X- ay spec oscopy); Gd-BTO (gadolinium-doped
ba ium i ana e); GLT (gela in); GLY (glycol); GNRs (g aphene nano ibbons); GO (g aphene ox-
ide); GP-Ca (glyce ophospha e calcium); GR (g aphene); HA (hyd oxyapa i e); hBN (hexagonal
bo on ni ide); HEMA (hyd oxye hyl me hac yla e); hGR (hyd ophilic GR); HRT (ho mone e-
Pha maceu ics 2021,13, 1994 39 o 48
placemen he apy); HUVECs (human umbilical ein endo helial cells); KGN (ka ogenin); LAP
(laponi e); miRNA (mic oRNA); MMT (mon mo illoni e); MRSA (me hicillin- esis an S. au eus);
MSCs (mesenchymal s em cells); MSN (mesopo ous silica e nanopa icle); MV (me hyl anilla e);
MWCNTs (mul i-walled ca bon nano ubes); NCPs (nanocomposi es); NCs (nanoc ys als); nHA
(nano-hyd oxyapa i e); NPs (nanopa icles); –O– (e he g oups); OA (os eoa h i is); OCN (os eocal-
cin); –OH (hyd oxylic g oups); OPN (os eopon in); PBAT (poly(bu ylene adipa e-co- e eph hala e));
PBLG (poly(
γ
-benzyl-L-glu ama e)); PBT (poly(bu ylene e eph hala e)); PCL (polycap olac one);
PDLLA (poly(D,L-lac ide acid)); PDT (pho odynamic he apy); PEEK (polye he e he ke one); PEG
(polye hylene glycol); PEGDA (poly(e hylene glycol)diac yla e); PEI (polye hylenimine); PEOT
(poly(e hylene oxide e eph hala e)); PIC (polyion complex); PLGA (poly(D,L-lac ic-co-glycolic acid));
PLLA (poly(L-lac ic acid)); PLMA (poly(L-lac ic acid-co-malic acid)); PMMA (polyme hyl me hac y-
la e); P(MVE-al -MA (poly(me hyl inyl e he -al -maleic anhyd ide)); POC-GP (poly(oc ame hylene
ci a e glyce ophospha e)); PS (polybu ylene succina e); PTKUR (poly( hioke al u e hane)); PU
(polyu e hane); PUU (polyu e hane-u ea); PVA (poly inyl alcohol); PVDF (poly( inylidene luo-
ide)); P(VDF-T FE (poly( inylidene luo ide- i luo oe hylene)); qCS (qua e nized chi osan); RA
( heuma oid a h i is); GO (g aphene oxide); ROS ( eac i e oxygen species); RUNX-2 ( un - ela ed
ansc ip ion ac o 2); RT-PCR ( eal- ime polyme ase chain eac ion); SASP (senescence-associa ed
sec e o y pheno ype); SBA15 (San a Ba ba a Amo phous-15); SD a s (Sp ague–Dawley a s); SEM
(scanning elec on mic oscope); SF (silk ib oin); SiCs (nanosilica es); SMPs (shape memo y polyme s);
SMPU (shape memo y polyu e hane); SPIO (supe pa amagne ic i on-oxide); SPS (spa k plasma
sin e ing); S -OCP (s on ium inco po a ed oc acalcium phospha e); TGF-
β
( ans o ming g ow h
ac o -
β
); VEGF ( ascula endo helial g ow h ac o ); WP (whea p o ein); ZIF-8 (zeoli ic imidazola e
amewo k-8 NPS).
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