nanomaterials

Review
Novel Inorganic Nanomaterial-Based Therapy for Bone
Tissue Regeneration
Yu Fu 1 , Shengjie Cui 2 , Dan Luo 3, * and Yan Liu 2, *

1 Fourth Clinical Division, Peking University School and Hospital of Stomatology; National Engineering
Laboratory for Digital and Material Technology of Stomatology, Beijing Key Laboratory of Digital
Stomatology, Beijing 100081, China; [email protected]
2 Laboratory of Biomimetic Nanomaterials, Department of Orthodontics, Peking University School and
Hospital of Stomatology, National Engineering Laboratory for Digital and Material Technology of
Stomatology; Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China; [email protected]
3 CAS Center for Excellence in Nanoscience, Beijing Key Laboratory of Micro-nano Energy and Sensor, Beijing
Institute of Nanoenergy and Nanosystems, Chinese Academy of Sciences, Beijing 100083, China
* Correspondence: [email protected] (D.L.); [email protected] (Y.L.)

Abstract: Extensive bone defect repair remains a clinical challenge, since ideal implantable scaf-
folds require the integration of excellent biocompatibility, sufficient mechanical strength and high
biological activity to support bone regeneration. The inorganic nanomaterial-based therapy is of
great significance due to their excellent mechanical properties, adjustable biological interface and
diversified functions. Calcium–phosphorus compounds, silica and metal-based materials are the
most common categories of inorganic nanomaterials for bone defect repairing. Nano hydroxyapatites,
similar to natural bone apatite minerals in terms of physiochemical and biological activities, are



the most widely studied in the field of biomineralization. Nano silica could realize the bone-like


hierarchical structure through biosilica mineralization process, and biomimetic silicifications could
Citation: Fu, Y.; Cui, S.; Luo, D.; Liu,
stimulate osteoblast activity for bone formation and also inhibit osteoclast differentiation. Novel
Y. Novel Inorganic
metallic nanomaterials, including Ti, Mg, Zn and alloys, possess remarkable strength and stress
Nanomaterial-Based Therapy for
absorption capacity, which could overcome the drawbacks of low mechanical properties of polymer-
Bone Tissue Regeneration.
based materials and the brittleness of bioceramics. Moreover, the biodegradability, antibacterial
Nanomaterials 2021, 11, 789. https://
doi.org/10.3390/nano11030789
activity and stem cell inducibility of metal nanomaterials can promote bone regeneration. In this
review, the advantages of the novel inorganic nanomaterial-based therapy are summarized, laying
Academic Editor: Maria J. the foundation for the development of novel bone regeneration strategies in future.
Blanco Prieto
Keywords: inorganic nanomaterials; bone regeneration; nano hydroxyapatites; nano silica; metal-
Received: 15 December 2020 lic nanomaterials
Accepted: 16 March 2021
Published: 19 March 2021

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in Bone consists of inorganic minerals and organic matrix, and its highly hierarchical
published maps and institutional affil- structure ensures excellent mechanical properties to withstand stress [1]. Although bone
iations.
can heal itself through dynamic remodeling when suffering minor damage, bone lesions of
critical size that cannot be cured spontaneously are quite common in daily life [2]. Until
now, autologous transplantation is still the gold standard for the treatment of large bone
defects [3]. However, the high incidence of donor sites mobility and the limited volume of
Copyright: © 2021 by the authors. autologous bone grafts limit the large-scale promotion of bone replacement surgery [4]. The
Licensee MDPI, Basel, Switzerland. ideal bone graft substitutes should not only imitate the extracellular matrix (ECM) of natural
This article is an open access article bone to achieve excellent biocompatibility, more importantly, they must provide strong
distributed under the terms and mechanical support for the defect tissue. Inorganic nanomaterials possess better mechanical
conditions of the Creative Commons
strength than natural and synthetic polymer scaffolds and can maintain stability for several
Attribution (CC BY) license (https://
weeks in vivo to support the bone healing process in the early stage of regeneration, making
creativecommons.org/licenses/by/
them the most promising candidate for bone graft substitutes. Calcium–phosphorus
4.0/).

Nanomaterials 2021, 11, 789. https://doi.org/10.3390/nano11030789 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2021, 11, x FOR PEER REVIEW 2 of 18

Nanomaterials 2021, 11, 789 2 of 18

process in the early stage of regeneration, making them the most promising candidate for
bone graft substitutes. Calcium–phosphorus compounds, silica and metal-based materi-
als are the most
compounds, common
silica categoriesmaterials
and metal-based of inorganic nanomaterials
are the most common for categories
bone defect ofrepairing.
inorganic
Among them, nano hydroxyapatites (nHAs) are widely studied due
nanomaterials for bone defect repairing. Among them, nano hydroxyapatites (nHAs) to their high simi-
are
larity with natural bone apatite [5]. Nano silica has been proven to establish
widely studied due to their high similarity with natural bone apatite [5]. Nano silica has hierarchical
structure
been provenandtopromote bone
establish regeneration
hierarchical through
structure anda promote
biosilicification process [6]. through
bone regeneration For metala
materials, traditional bulk metal scaffolds have been used for permanent and temporary
biosilicification process [6]. For metal materials, traditional bulk metal scaffolds have been
orthopedic applications
used for permanent and[7]. Recently,orthopedic
temporary biodegradable metallic [7].
applications nanomaterials are able to be
Recently, biodegradable
manufactured through aare
metallic nanomaterials 3Dable
printing
to betechnology,
manufactured namely additive
through a 3Dmanufacturing (AM),
printing technology,
to produce
namely personalized
additive orthopedic
manufacturing (AM), implants [8]. According
to produce personalized to orthopedic
computer-aided design
implants [8].
data, the mechanical properties, pore size, porosity and surface characteristics
According to computer-aided design data, the mechanical properties, pore size, porosity of the im-
plant can becharacteristics
and surface perfectly controlled [9]. In this
of the implant can review, the controlled
be perfectly advantages[9]. and
In applications
this review, theof
the above three kinds of inorganic nanomaterials in bone tissue engineering
advantages and applications of the above three kinds of inorganic nanomaterials in bone will be re-
viewed (Figure 1). will be reviewed (Figure 1).
tissue engineering

Figure 1. The main inorganic nanomaterials used for bone tissue regeneration [10–14].
Figure 1. The main inorganic nanomaterials used for bone tissue regeneration [10–14].
2. Nano Hydroxyapatites
2. Nano Hydroxyapatites
Natural mature bone is composed of minerals, type I collagen, water, small amounts
Natural
of other mature
collagen bone
types, is composed ofproteins
noncollagenous minerals,
andtype I collagen, water,
proteoglycans. small amounts
The minerals mainly
of other collagen types, noncollagenous proteins and proteoglycans.
refer to thin plate-shaped carbonated calcium-deficient hydroxyapatites (50 nm The× minerals
25 nm in
mainly refer
size, 1.5–4 nmtothick),
thin plate-shaped carbonated
distributed along calcium-deficient
the collagen hydroxyapatites
fibrils [1]. Hydroxyapatite (50 nm
(HA) has ×a
25 nm ratio
Ca/P in size, 1.5–4higher
of 1.67, nm thick), distributed
than that along the collagen
of calcium-deficient fibrils [1]. Hydroxyapatite
hydroxyapatites, and is insoluble
(HA) hasHA
in vivo. a Ca/P ratiotoofdegrade
is hard 1.67, higher
and than that of calcium-deficient
has favorable hydroxyapatites,
mechanical properties and
and chemical
is insoluble in vivo. HA is hard to degrade and has favorable mechanical properties
binding ability, thus it is mostly used as a nanofiller in polymers to improve the mechanical and
chemical binding ability, thus it is mostly used as a nanofiller
strength or coating onto metallic implants to impart bioactivity [15]. in polymers to improve the
mechanical strength or coating onto metallic implants to impart bioactivity [15].
2.1. nHA/Polymer Composites
Inspired by the composition and structure of bone, researchers try to mineralize or-
ganic matrix with calcium phosphates, especially nHAs, which are the most stable and
Nanomaterials 2021, 11, 789 3 of 18

similar member with natural mineral phase in vivo. It has been shown that mineralization
with nHAs enhanced Young’s modulus of type I collagen from 0.2–7.8 GPa to 11.1–16.3 GPa
(Figure 2a) [16]. In addition to the improvement of mechanical properties, the mineralized
fibrils showed rough surface topography and could promote the osteogenesis of mesenchy-
mal stem cells (MSCs) in vitro and bone defect regeneration in vivo [17,18]. Even though
chitosan had much higher compressive modulus (around 27 kPa) and compressive strength
(around 36 kPa) compared to those of collagen (around 2 kPa and 3 kPa, respectively), nHAs
were still able to significantly increase the mechanical properties of chitosan with compres-
sive modulus of around 43 kPa and compressive strength of 40 kPa, respectively [19]. In
another study, the Young’s modulus of pure chitosan was reported to be approximately
3.0 GPa, while that of nHA/chitosan composite increased to over 3.5 GPa [20]. Similarly,
the addition of nHAs could elevate the ultimate compressive strength of chitosan both in
wet and dry conditions and the fabrication methods of nHA/chitosan composites further
affected the mechanical strength [21]. Silk fibroin is also a natural polymer with compres-
sive modulus of 0.43 MPa, and after being assembled with nHAs, its compressive modulus
became 4 times higher (Figure 2b) [22]. Aside from the abovementioned natural polymers,
the synthetic polymers also need nHAs in the fabrication process. It has been shown that
HA nanorods can increase the elastic modulus and strength of polycaprolactone by approx-
imately 50% and 26%, respectively [23]. However, there was an exception when nHAs were
used to fabricate nHA/poly (ester urea) composite scaffold through 3D printing technology.
The addition of nHAs had no significant influence on compressive modulus, which may re-
sult from the balance between the reinforcement effect and nonoptimized nHA/poly(ester
urea) interaction [24]. Unlike the controversial effect on polymers’ mechanical performance,
nHAs can delay the degradation of poly(D, L-lactic acid)/poly(D, L-lactic- co-glycolic acid)
blends [25]. Impressively, the effect of nHAs on the degradation rate of chitosan was even
higher than that of nanobioglass, which may be attributed to the fine dispersion of nHAs
in composites [26]. The addition of nHAs not only affects the mechanical performance
and degradation rate of organic polymers, but also releases Ca2+ and PO4 3 − ions and
regulates osteogenesis and bone regeneration [27]. For instance, the appearance of nHAs
in poly(ester urea) scaffold significantly increased the alkaline phosphatase (ALP) activ-
ity, bone sialoprotein (BSP) and osteocalcin (OCN) expression and calcium deposition of
MC3T3-E1 preosteoblast cells (Figure 2d) [24]. Gonzalez Ocampo et al. also proved nHAs
promoted the cell spreading, ALP activity and matrix mineralization of human osteoblasts
seeded on kappa-carrageenan (Figure 2c) [28]. Furthermore, nHA/polymer composites can
promote bone regeneration in both calvarial defect (Figure 2e) [22] and long bone defect
models [29]. Based on the inspiration from cortical bone and nacre, Feng et al. fabricated
a “brick and mortar” multilayer nHA-based scaffold, which induced not only osteogen-
esis, but excellent angiogenesis as well (Figure 2f,g) [29]. The effect of nHAs coating on
metal-based nanomaterials will be briefly discussed in Section 4.

2.2. 3D Printed nHA-Based Inorganic Nanomaterials

The latest emerging 3D printing technology makes nHAs and other inorganic nano-
materials easy to shape and may help to solve the poor biodegradability and excessive
mechanical properties of biomaterial scaffolds consisting only or mainly of nHAs. Com-
pared with the commercially available particle-type bone substitutes OSTEON 3 (Genoss® ),
the 3D printed customizable HA/tricalcium phosphate scaffolds promoted more new
bone formation (Figure 3a) [30]. Even using the same raw materials calcium phosphate
cement and polyvinyl butyral, different solvent of polyvinyl butyral, such as ethanol and
tetrahydrofuran could lead to quite different geometry, microstructure, mechanical proper-
ties and osteoconductivity [31]. nHA/polyvinyl butyral composite scaffold fabricated in
ethanol had 2-fold higher ultimate tensile strength and 3.4-fold higher ultimate compres-
sive strength and promoted the osteogenesis of human primary osteoblasts, compared with
that made in tetrahydrofuran. In addition to internal structure, surface topography and
chemical characteristics also contribute to the bone reparation effect. Wei et al. reported
Nanomaterials 2021, 11, 789 4 of 18

that the hexagon-like column array topography of 3D printed HA scaffold promoted the
osteogenic differentiation of human adipose-derived stem cells [32]. Furthermore, this
research group found that the surface modification with strontium ion substitution can
enhance the effect of HA porous scaffold on osteogenesis [33]. Similarly, the ECM derived
from bone marrow MSCs (BMSCs) modified the surface chemistry of 3D HA scaffold
and then improved the ALP activity, osteogenesis-related mRNA expression and calcium
deposition of BMSCs, which finally promoted the bone reparation in rat skull defects
(Figure 3b) [34]. As for the pore size, the HA-based 3D printed scaffolds with 1.4 mm and
1.2 mm pore sizes can promote bone regeneration at 4 weeks in rabbit calvarial defects
while decreasing the mechanical strength, compared with those scaffolds with 0.8 mm and
1.0 mm pore sizes. However, the effect of pore size on bone regeneration was diminished
as time went on to 8 weeks (Figure 3c) [35]. Future research is required to explore better
methods to accurately determine the topography of nHA-based 3D printing scaffolds.

a) COL IMC b) c) control control

Calcium Deposition (mg/dL)

nHAs/0.5% κ-CG nHAs/0.5% κ-CG

ALP activity (μmol/(L*min))

nHAs/1% κ-CG nHAs/1% κ-CG
nHAs/1.5% κ-CG nHAs/1.5% κ-CG

EMC

7 14 21 7 14 21
COL EMC IMC Time (Days) Time (Days)
d) 2w 4w e)

blank
f) CTS g)
CIS

blank
CTS
CIS
Positive pixels (%)
H&E Vascularity/

Figure 2. The mechanical and biological properties of nHA/polymer composites. (a) The highly ordered deposited nano
hydroxyapatites (nHAs) provided intrafibrillarly mineralized collagen (IMC) with much higher Young’s modulus than
that of pure collagen (COL) and extrafibrillarly mineralized collagen (EMC) [16]. (b) The addition of nHAs increased
the compressive modulus of silk fibroin significantly [22]. (c) nHA/kappa-carrageenan (κ-CG) enhanced the alkaline
phosphatase (ALP) activity and calcium deposition of human osteoblasts, regardless of the concentration of κ-CG [28].
(d) With the increase of nHAs content, the MC3T3-E1 preosteoblast cells expressed more osteocalcin (OCN) and bone
sialoprotein (BSP) [24]. (e) The nHA/silk fibroin composites promoted the bone regeneration in rat calvarial defects [22]. The
co-inspired scaffold (CIS) which consisted of nHAs and chitosan induced both new bone formation (f) and vascularization
(g) compared with blank and pure chitosan [29]. *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Nanomaterials 2021, 11, 789 5 of 18

a) b)
4 weeks

Bone graft
New bone
8 weeks

+ Total
Bone graft
New bone

c)
4 weeks
8 weeks

Figure 3. The properties affecting bone regeneration of nHA-based 3D printing scaffolds. (a) Histomorphometric examina-
tion demonstrated that the 3D printed HA/ tricalcium phosphate scaffold (3DS) had better bone reparation effect than both
negative control (NS) and positive control (particle-type bone substitutes, PS) in beagle dog mandibular bone defects [30].
(b) Radiographic analysis showed that 3D hydroxyapatite/extracellular matrix (HA/ECM) composites promoted bone
regeneration in rat calvarial defects after 12 weeks [34]. (c) Both radiographic and histological evaluations exhibited more
new bone formation for 3D printed nHA-based scaffolds with higher pore size only at 4 weeks [35]. *: p < 0.05, **: p < 0.01,
***: p < 0.001.

3. Nano Silica
Nano silica is an important component of bioceramics, and it is widely applied in
bone tissue engineering. According to the definition of the International Federation of
Applied Chemistry, mesoporous materials have a pore size between 2 nm and 50 nm.
Sol-gel method was initially used to synthesize regular pore structure in mesoporous silica
nanoparticles (MSNs) with adjustable pore size [36]. The mesoporous structure offers
good biocompatibility and biodegradability among inorganic nanomaterials [37]. Based
on the exciting characteristics, MSNs are drawing more and more interest in the basic
research of bone tissue engineering. MSNs serve as a kind of drug carrier with excellent
release efficiency, and they also have the biological activity of promoting bone formation.
Therefore, the researchers mainly focus on two aspects of modification: one is to modify the
pore size adapted to functional factors that the mesoporous silicon material can load; the
second is to explore different composite scaffold materials to improve the physiochemical
property and bioactivity of MSNs.
The mechanisms of MSNs that promote bone defect reparation are as follows: (1) the
silicon ions released by hydrolysis can promote the expression of osteogenic-related genes
in osteoblasts (Figure 4a) [13]; (2) the mesoporous structure helps the deposition of HA,
which further promotes mineralization [38]; (3) MSNs induce efficient macrophage uptake
and promote immunomodulatory effects, which are conducive to osteogenic differenti-
ation [39]. Moreover, the efficient uptake is good for drug delivery in bone defect area
in vivo. The efficiency of drug delivery relies on the pore size and superficial chemical
modification. As the controllability of MSN fabrication based on various synthesis methods
has already been testified to load different types of drugs, the characteristics of modified
MSN-drug releasing system are used more and more often in basic research with or without
combined scaffolds.
Nanomaterials 2021, 11, 789 6 of 18

MSN loaded-drugs including traditional chemicals, protein and peptides and nu-
cleotides exert positive effects on bone formation. Combining dexamethasone-loaded
MSNs with mineralized porous biocomposite scaffolds induced bone regeneration by
enhancing the osteogenic activity of host BMSCs [40]. Alvarez et al. demonstrated that
ibandronate sodium-loaded MSNs incorporated into the collagen gel had continuous
drug release around 10 days, which effectively inhibited the function of osteoclasts and
promoted the osteogenic differentiation of MSCs [41]. 17-beta estradiol-loaded MSNs
with EDTA modified surface significantly enhanced the efficiency of hormone therapy
for osteoporosis (Figure 4b) [42]. Furthermore, injectable miR222/MSN/aspirin hydrogel
promoted mandibular bone regeneration and induced neurogenesis in the defect area [43].
Therefore, the effectiveness of drug delivery of MSNs is demonstrated by numerous studies
and is a promising strategy for drug-MSN-scaffold-induced bone regeneration. To acquire
maximum loading capacity of drugs, researchers modified the structure of MSNs with
rough or hydrophilic surface, which enhanced the adhesion of the target drug [44]. Based
on previous studies, hollow structure has enough space for drug loading and storage, thus
hollow mesoporous silica materials provide new insight for mesoporous silica-based drug
delivery [45]. However, the simple modification of surface or pore size of MSNs is not
sufficient to realize precise releasing system. Therefore, the incorporation of biomacro-
molecules (organic polymers, macrocyclic molecules, etc.) and MSNs enhances the efficacy
of MSN-drug releasing system.

3.1. Modification of MSN-Based Scaffolds

In order to acquire enough strength, proper morphology and enhanced bioactivity of
MSNs for bone formation, the composites of MSNs and organic/inorganic scaffolds have
been fabricated. Considering the importance of nHAs during biomineralization, numerous
studies have adopted the combination of MSNs with nHAs and testified to the synergistic
effects on bone formation. He et al. showed that MSN/nHA composites enhanced the
adhesion and proliferation rate and osteogenesis-related gene expression of rabbit BMSCs
and new bone formation in rabbit femur defects, compared to MSNs or nHAs alone [46].
Similar co-enhancement effect on osteogenic differentiation of MG-63 cells was found by
Shuai et al. [47]. Furthermore, the existence of nHAs endowed discontinuous pore surface
of MSNs, which induced rapid drug releasing of ciprofloxacin from 32% to 93% in 24 h [48].
Along with nHA-silica nanomaterials, the natural components of extracellular matrix
can induce an increase of cell–scaffold interaction and result in better biocompatibility
and bioactivity of nano silica. In turn, the application of silica reinforced the mechanical
strength, enhanced the water uptake capacity, and fastened degradation rate of matrix
scaffolds [49], which benefited repair of large-scale bone defects. Thus, the application
of matrix components as silica-based scaffolds is attracting the interest of researchers.
Gaihre et al. reported that injectable nano silica–chitosan microparticles performed sig-
nificant enhancement of ALP activity during osteogenic induction of osteoblasts [50]. In
addition, collagen-alginate-nano-silica microspheres improved the osteogenic potential of
human osteoblast-like MG-63 cells by promoting the expression of OCN and BMP-2 [51].
Intrafibrillar silicified collagen scaffold promoted in situ bone regeneration via p38 sig-
naling pathway in monocytes and recruited host MSCs (Figure 4c) [52]. Therefore, it
is a promising method to fabricate nano silica with natural polymer scaffolds for bone
regeneration applications.
Nanomaterials 2021, 11, 789 7 of 18

a) b)

c) i d) i

ii

Bone mineral density (mg/cc)

Bone volume (mm3)
ii
CMMS 4w CMMS 12w

CMMS/rhBMP-2 4w CMMS/rhBMP-2 12w

iii

Figure 4. The application of nano silica in bone tissue engineering. (a) The effect of Si ions on osteogenesis-related gene
expression of human bone marrow mesenchymal stem cells (BMSCs). A: blank control (DMEM); B: 10 µg/mL Si ions
in DMEM; C: 50 µg/mL Si ions in DMEM; D–F: A–C solution plus 35 µg/mL dimethyloxaloylglycine, respectively.
*: Comparison between blank and other groups; #: comparison between group B and E, C and F, respectively;
p < 0.05 [13]. (b) Mesoporous silica nanoparticles (MSNs) assisted E2 sustained release, which prevented osteoporosis in vivo.
OVX = ovariectomy; UCHRT = NaLuF4 :Yb,Tm@NaLuF4 @mSiO2 -EDTA-E2 nanocomposites. *: p < 0.05, **: p < 0.01 [42].
(c) Nano silica incorporated collagen fibrils promoted bone regeneration in rat femoral defects. SCS = silicified collagen
scaffold; NC: negative control; SCS + PD: silicified collagen scaffold plus PD098059; SCS + SB: silicified collagen scaffold
plus SB203580; PD and SB are MAPK inhibitors. (i) Micro-CT scans and quantitative results. (ii) van Geison staining.
Bar = 1 mm. (iii) Ponceau trichrome staining. Bar = 200 µm [52]. (d) The osteoconductivity of calcium/magnesium-doped
rhBMP-2-incorporated MSN scaffold in vitro and in vivo. CMMS= calcium/magnesium-doped silica-based scaffolds.
(i) rhBMP-2 increased the osteogenesis-related gene expression of rat BMSCs. (ii) CMMS/rhBMP-2 scaffold promoted bone
regeneration in rabbit femoral defects as demonstrated by micro-CT analysis and histological evaluation. M: materials, B:
bone, F: fibrous tissue. *: p < 0.05; #: p < 0.05 [53].

Furthermore, some studies focus on doped metal ions in nano-silica-based materials.

Dai et al. synthesized magnesium-doped mesoporous silica materials containing recombi-
nant human bone morphogenetic protein-2 (rhBMP-2) with macroporous and mesoporous
structure using polyurethane foam as a template (Figure 4d) [53]. This magnesium-doped
silica material induced osteogenic differentiation of rat BMSCs in vitro and ectopic osteo-
genesis in vivo and also showed a good repair effect in a rabbit femoral defect model with
diameter of 5 mm and depth of 5 mm. Shi et al. demonstrated that copper-doped MSNs
induced robust immunomodulatory effects of murine-derived macrophage cell line RAW
264.7 and promoted osteogenesis of human BMSCs [39]. Recently, rare earth elements, such
as lanthanum- [54], europium- [55] and gadolinium-doped [56] MSNs showed the exciting
ability in osteogenesis of BMSCs and bone regeneration in rat skull bone defects. Despite
the positive results in osteogenesis in vitro, the application of metal ions in nano silica
materials still need more in vivo evidence to demonstrate the effectiveness and biosafety
of such modification of nano silica.
Nanomaterials 2021, 11, 789 8 of 18

4. Metallic Nanomaterials
Most traditional bulk metals exhibit much higher mechanical properties and worse
bioactivity than natural bone, which may lead to bone resorption and poor osteointegration
and osteogenesis [9]. Surface modification at nanoscale would help to improve the surface
topography and chemistry of metal implants. In order to balance the gap in mechanical
properties, the researchers tried to build a micro-nano structure to increase the porosity of
metal materials to more than 50% [57]. In the meanwhile, porous materials can promote
the penetration of cells and nutrients and the regeneration of bone and blood vessel when
the pore size exceeds 300 µm [58]. In addition, the metallic nanomaterials are preferably
biodegradable, which means they are expected to gradually corrode in vivo, and the
corrosion products are metabolized or absorbed by cells and/or tissues. After the defects
are repaired, the implants are completely dissolved without residue. In the following
sections, four widely studied metal nanomaterials are introduced.

4.1. Ti-Based Nanomaterials

4.1.1. Nanoscale Surface Modification of Ti-Based Biomaterials
Titanium (Ti)-based biomaterials are widely used as permanent implants in orthopedic
surgery and dental implantation because of their high load-bearing properties, nondegrad-
ability and good biocompatibility [59]. Due to the superior corrosion resistance of bulk
Ti and its alloy, they often exhibit slow biological response, low osseointegration rate
and lack of antibacterial properties. Nanoscale surface modification strategies, such as
coating and doping, are effective means to solve the above problems and endow the im-
plants with functionalization. In order to enhance the osteoconductivity, Ding et al. mixed
strontium-incorporated lysozyme solution with Ti substrates and spontaneously formed a
2D nanofilm which could promote the ALP activity and osteogenesis-related genes expres-
sion of BMSCs [60]. The sustained release of Sr2+ from lysozyme nanofilm further facilitated
osteointegration of Ti implants in rat femur bone defect model. Similarly, nano-graphene
oxide (GO) was deposited on Ti surface through ultrasonic atomization spraying technique.
This nano-GO coating induced the osteogenic differentiation of rat BMSCs via FAK/P38
signaling pathways and further accelerated bone regeneration in vivo [61]. Furthermore,
lanthanide mineral-substituted hydroxyapatite nanorods were coated on Ti substrates by
electrophoretic deposition method to mimic the topography and composition of natural
bone [62]. The lanthanide mineral-substituted hydroxyapatite nanorods modified Ti im-
plants had better osteogenic effect in vitro and bone regeneration effect in rat tibia defects.
Compared to the above-mentioned inorganic coatings, human MSC-derived extracellular
vesicles (MSC-EVs) were difficult to be anchored onto pristine Ti. Chen et al. successfully
self-assembled human MSC-EVs onto biotin-doped polypyrrole titanium, which exhibited
osteoinductivity in nude mice ectopic bone formation model with the help of osteoinduc-
tive miRNAs tested in MSC-EVs [63]. As for the antimicrobial activity, Zhang et al., inspired
by the adhesion mechanism of mussel, developed a novel silicon-doped calcium phos-
phate composite coating (Van-pBNPs/pep@pSiCaP) loaded with vancomycin on porous Ti
scaffold via modified surface mineralization process [64]. The functionalized biomimetic
Ti scaffold can prevent the adhesion and proliferation of staphylococcus epidermidis. In
addition to antibiotics, metal ions also have bactericidal activity. Huang et al. fabricated
Cu-containing micro/nanotopographical bioceramic surface through micro-arc oxidation.
The subsequent hydrothermal and final heat treatment can assist Ti implants to induce
proinflammatory M1 macrophage polarization via Cu-transport signaling pathway and
enhance bacterial phagocytosis. [65]. In addition to chemical antibacterial, the researchers
prepared zinc oxide@collagen type I coating to achieve a broad-spectrum antibacterial
effect through the photothermal effect of zinc oxide [66]. Surface modification materials can
also cooperate with metal matrix to promote bone tissue repair. Fu et al. proved that the
silicon-doped hydroxyapatite coating could demonstrate not only enhanced osteogenesis,
but also promote angiogenesis [67].
Nanomaterials 2021, 11, 789 9 of 18

4.1.2. Additive Manufacturing of Ti-Based Biomaterials

Traditional bulk Ti implants have the advantages of easy commercial availability and
wide clinical acceptance, but their much higher stiffness than natural bone may cause stress
shielding, leading to bone resorption and implant failure [68]. Although investigators are
committed to improving the surface topography and chemical structure of bulk Ti implants,
the porous structure, essential to the mechanical properties of natural bone, could not be
simulated through traditional manufacture methods. The emergence of AM technologies
is expected to solve this problem fundamentally. The computer-controlled and bottom-up
fabrication process of AM technologies can more easily realize the control of chemical
composition and micro-nano structure, so as to obtain better biomechanical properties to
promote bone regeneration [69]. The elastic modulus of Ti–6Al–4V–10Mo [70], Ti–35Nb [71]
and Ti–50Ta [72] (in wt%) nanomaterials prepared by 3D printing was significantly reduced
to 73–84.7 GPa. The 3D printed, customized Ti implants have been used in craniofacial and
orthopedic applications [73]. A clinical study involving 21 patients demonstrated good
fixation between bone and custom-made 3D-printed Ti implants (with surface area ranging
from 12,146 to 24,980 mm2 ) and satisfactory skull-shape symmetry without any compli-
cations during the 6 to 24 months follow-up period (Figure 5a) [74]. For tongue cancer
excision with a tumor recurrence, Lee et al. utilized Ti-6AL-4V-ELI medical grade powder
to fabricate a Ti implant with four dental implants based on computer-aided design data
and restored the facial symmetry and occlusion at 5 months after surgery [75]. Impressively,
Ti-6AL-4V-based AM implant with a total volume of 30.7 cm3 was able to successfully re-
place the complicated bone defects of distal tibia and foot caused by motor vehicle collision,
and the patients could basically return to normal life and walk without ambulatory aids
after 6 months [76]. Although there are still early and late complications, cranioplasty based
on AM Ti implants has a higher success rate compared with other techniques [77]. In order
to modify porous Ti nanomaterials based on AM, geometrical cues and surface structure de-
sign were investigated. As the porosity of AM Ti implants increased from 55.51% to 76.14%,
the yield compressive strength decreased from 186.05 ± 1.85 MPa to 51.95 ± 0.62 MPa, and
the elastic moduli decreased from 6.74 ± 0.47 GPa to 0.98 ± 0.03 GPa [78]. Nonetheless,
the sharp decline of mechanical strength did not affect the bone regeneration, which may be
attributed to the optimum porosity. Interestingly, the regeneration results of implanted non-
load-bearing bones (such as the skull) were much worse than those of load-bearing bones
such as the femur, which indicated the importance of stress stimulation [78]. In contrast,
Maietta et al. demonstrated that with the help of finite element analysis, the Ti-6AL-4V
architectural characteristics, such as pore size and shape, could be changed without signifi-
cant alteration in mechanical properties [79]. For biological applications, Zhu et al. proved
that shape- and size-controlled microgroove-patterned Ti surface structure manufactured
by a combination of photolithography and inductively coupled plasma-based dry etching
was beneficial to osteogenesis and bone regeneration [80]. Specifically, R3G7 (ridge width
of 3 µm, groove width of 7 µm and depth of 2 µm) was the most effective micropattern
to promote the osteogenic differentiation of MC3T3-E1 cells and bone regeneration in rat
calvarial defects (Figure 5d). In addition to porosity and micropattern, roughness also
plays an important role. Saruta et al. found that submicro-rough surface (with roughness of
24 ± 1.2 nm) promoted the attachment, proliferation and calcium deposition of osteoblasts,
while micro-rough surface (with roughness of 123 ± 6.15 nm) had the strongest bone–
implant integration in rat femurs [81]. Surface biofunctionalization is also well investigated
to modify AM porous Ti-based nanomaterials. Several kinds of coating including silk
fibroin, calcium phosphate and tricalcium phosphate can promote bone integration and
regeneration both in normal [64,82] and osteoporotic bone defect models [83] (Figure 5b,c).
The vancomycin coating helps to prevent the bacteria colonization caused by the high
surface area to volume ratio of porous Ti implants [82]. On the other hand, a high sur-
face area/volume ratio is beneficial for drug sustained release, such as BMP-2 and silver
nanoparticles [84].
Nanomaterials 2021, 11, 789 10 of 18

AM Ti + SF +
AM Ti AM Ti + SF AM Ti + SF + Vancomycin AM Ti AM Ti + SF
a) b) c) Vancomycin

d) i iii iv
v

v
ii v

v v

Figure 5. The biological properties of AM Ti-based implants. (a) Custom-made Ti-6Al-4V-ELI AM implants helped to
reconstruct the skull defect of 21 patients [74]. (b) Silk fibroin and vancomycin coating promoted the survival of MC3T3-E1
cell line seeded on 3D printed Ti implants [82].(c) Silk fibroin and vancomycin coating promoted the calcium deposition
of MC3T3-E1 cell line seeded on 3D printed Ti implants [82]. (d) The micropattern of Ti substrates induced osteogenesis.
(i) SEM images and 3D surface profile of different micropatterns. (ii) MC3T3-E1 preosteoblasts aligned along the ridges in
R3G7 micropattern. (iii) The R3G7 microgroove pattern promoted osteogenesis of MC3T3-E1 cells. (iv) and (v) The R3G7
microgroove pattern induced bone regeneration in rat skull defects [80]. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

As mentioned above, AM Ti-based nanomaterials would have broad application

aspects in bone tissue regeneration. The excellent chemical stability of titanium makes it
difficult to degrade in vivo. Although titanium-based biomaterials are still the mainstream
of current clinical applications, the developments of biodegradable metallic nanomaterials
are promising and necessary.

4.2. Mg-Based Biomaterials

4.2.1. Nanoscale Surface Modification of Mg-Based Biomaterials
Magnesium is the most studied biodegradable metal ion. It has been confirmed that
magnesium ions can induce bone marrow MSCs to differentiate into osteoblast lineage
through canonical Wnt signaling pathway [85]. In distraction osteogenesis model, high-
purity magnesium pins promoted angiogenesis and bone consolidation [86]. Moreover, the
elastic modulus of magnesium is much lower than that of Ti, preventing bone resorption
and implant failures induced by stress shielding. Additionally, the excellent degradability
of Mg makes it an excellent temporary bone fixation device and possible low load-bearing
bone substitute [87]. Degradability is a double-edged sword. In most cases, pure Mg
implants degrade too fast to fully support new bone formation and the rapid release of
hydrogen may interfere with the local microenvironment and hinder bone regeneration.
Therefore, alloys of magnesium with calcium [7], zinc [7], strontium [88] and rare earth (RE)
Nanomaterials 2021, 11, 789 11 of 18

elements [89] have been developed to solve the above-mentioned problems. A long-term
clinical study has confirmed the early bone healing and the complete replacement of Mg-
5wt%Ca-1wt%Zn alloy by new bone in the late stage [7]. Fifty-three patients with hand and
wrist fractures were involved in this study. All the patients returned to normal life with
no sign of pain and the Mg-5wt%Ca-1wt%Zn implants were degraded, as confirmed by
radiographic examination within 1 year, demonstrating the controlled degradation of this
kind of Mg-based alloy. Furthermore, in order to reduce the corrosion rate of Mg, various
nanoscale surface coating strategies have been applied to provide Mg-based alloys with
a protective layer [90]. Zhang et al. developed calcium-phosphate-coated Mg−Zn−Gd
scaffolds via chemical deposition method, which could not only repair rat cranial defects
of critical size, but also promote osteogenesis, angiogenesis and the production of neu-
ropeptide calcitonin gene-related peptide from trigeminal neurons in the orbital bone
defect model of beagle dog [91]. Similarly, polycaprolactone and nHA nanocomposites
dual coating via dip-coating and electrospinning could induce bone regeneration in rabbit
femoral defects [92]. Kang et al. fabricated poly(ether imide)–SiO2 /nHA-coated porous Mg
scaffold through dip-coating technique, and repaired the bone defect in the femoropatellar
groove model of rabbits due to the proper corrosion rate and enhanced mechanical strength
of hybrid scaffold [12]. In addition, the surface modification by inorganic nanomaterials
can also bring new functions to the implanted scaffold. For example, a functionalized 70-
nm-thick TiO2 /Mg2 TiO4 nanolayer was fabricated by plasma ion immersion implantation
technique on WE43 Mg-based alloy. The TiO2 /Mg2 TiO4 coating can promote osteogenesis
of MC3T3-E1 cells and induce twice and six times higher levels of new bone volume (175%)
than those of pristine WE43 alloy (88%) and blank control (28%) in rat femoral defect model.
In the meanwhile, TiO2 /Mg2 TiO4 nanolayer suppressed bacterial infection and controlled
the degradation behavior [93]. As for the immumodulatory effect, the combination of
fibrinogen and magnesium can lead macrophages to M2 polarization and further promote
osteogenic differentiation of MSCs [94]. In some special bone defect models (such as
osteoporotic fracture), biodegradable Mg-based implants may serve as a sustained drug
delivery system. The calcium phosphate nanocoating ensured the suitable degradation rate
and structural integrity of Mg-based alloy, and the co-delivered magnesium degradation
products and zoledronic acid modulated bone formation and resorption [95].

4.2.2. Additive Manufacturing of Mg-Based Biomaterials

Compared to bulk Mg-based biomaterials, the AM of biodegradable Mg implants is
still quite difficult due to the intrinsic properties of magnesium. Salehi et al. developed a
novel 3D printing technique followed by sintering process to fabricate the Mg-5.9Zn-0.13Zr
alloy with high precision at nano- and micron-scale. This AM Mg-based alloy possessed an
average pore diameter of 15 µm, compressive strength of 174 MPa and elastic modulus of
18 GPa, which were quite similar to those of human cortical bone [96]. Until now, only a
few research groups fabricated Mg alloys through selective laser melting technique [97]
and investigated their mechanical and degradation properties. The biological performances
of AM Mg-based alloys, for instance biocompatibility and regenerative effect in vivo, need
to be further investigated.

4.3. Zn-Based Biomaterials

Zinc is an essential element for humans and plays an important role in many physio-
logical activities. Zinc ions can enter human MSCs with the help of TRPM7 and GPR39, and
then activate cAMP-PKA pathway, thereby ultimately enhancing cell survival/proliferation,
differentiation, ECM mineralization and osteogenesis [98]. The degradation rate of Zn-
based biomaterials is slower than that of Mg, and matches the tissue healing speed. The
mechanical properties of Zn materials are between Ti and Mg, similar to natural bone [99].
Biodegradable Zn alloys, such as Zn-0.8%Li- (Mg, Ag), Zn–2Ag–1.8Au–0.2V and Zn-1Ag
have better mechanical properties, corrosion rates and antibacterial activities than pure
Zn implants, while only certain alloys have modest biocompatibility [100]. After the first
Nanomaterials 2021, 11, 789 12 of 18

published work in 2017 [101], a growing number of AM Zn-based nanomaterials appeared.

The mechanical properties of AM porous Zn are similar to those of cancellous bone, and
the degradation rates allow satisfactory bone substitution [102]. Although Zn exhibits
mild cytotoxicity, it is still acceptable [103]. Like Mg, the biological evaluation of Zn-based
biomaterials, especially AM porous Zn and alloys, remains blank.

4.4. Au-Based Biomaterials

Gold nanoparticles (AuNPs) have been widely investigated in biomedical applications,
especially in antitumor therapy and bone regeneration therapy, due to their good biocom-
patibility, photothermal stability and near-infrared absorbance. The size and morphology
of gold nanomaterials are important factors affecting their biological functions. Celentano
et al. have successfully synthesized stable ultra-small gold nanoparticles, anisotropic
gold nanoflowers and twisted gold nanorods by a simple green method and confirmed
the biocompatibility of these AuNPs [104–106]. The addition of AuNPs into poly(methyl
methacrylate)-based bone cement can significantly improve the punching performances
while maintaining stable compressive properties [107]. In addition to better mechanical
properties, as an efficient photothermal agent, AuNPs can eradicate residual tumor cells
after the solid tumor is removed through photothermal therapy [108]. The theory of
photothermal therapy can also be used to treat osteomyelitis. Gold-nanocage-containing
aspirin can convert laser light into heat and realize controlled release of aspirin, which
can perform anti-inflammatory effects on monocytes and promote bone regeneration after
the monocytes eliminate infection [109]. Similarly, Sanchez-Casanova et al. entrapped
heat-activated transgenic cell constructs in near-infrared-responsive hydrogels contain-
ing AuNPs, which can conditionally produce BMP-2 and promote bone regeneration
in vivo [110]. Despite the above progress, it is still necessary to clarify how to co-assemble
AuNPs and biomolecules and form biomimetic hierarchical scaffolds to achieve better
outcomes in the field of bone regeneration therapy.

5. Concluding Remarks and Future Perspectives

Trauma, infection, tumor, degenerative and congenital diseases are the major causes of
excessive bone defects. The current bone tissue engineering is trying to develop new mate-
rials to replace autologous bone grafts to achieve bone regeneration. Novel therapies based
on inorganic nanomaterials, providing excellent mechanical properties and abundant phys-
ical/chemical/biological functions, are complementary to natural biomacromolecules and
polymeric-based materials (Table 1). The development of inorganic nanoparticle/polymer
composites can effectively integrate the respective advantages of inorganic and organic
phases, bringing unlimited possibilities for the development of novel bone substitute mate-
rials. Biomimetic mineralization utilizing nano hydroxyapatites and nano silica is currently
one of the most successful organic material modification processes. Furthermore, with the
progress of technology, precisely designed porous inorganic nanomaterials appear to have
lower stiffness and elastic modulus than traditional bulk materials, making them much
closer to natural bone. Additive manufacturing is the most promising technology to real-
ize computer-aided design and personalized customization. The 3D printed nHA-based
nanomaterials and Ti-, Mg-, Zn-based metals would be able to act as temporary fixations
or permanent implants according to their own characteristics, and eventually be replaced
by regenerated bone tissue. Finally, inorganic nanoparticles are excellent carriers of drugs,
growth factors and genes. nHAs, MSNs and AuNPs are commonly used to carry drugs
into scaffolds and realize sustained release with the controllable degradation of scaffolds.
Nanomaterials 2021, 11, 789 13 of 18

Table 1. The advantages and drawbacks of novel inorganic nanomaterial.

Type Advantages Drawbacks

(1) Enhanced mechanical performance of polymers [16,19–23]

nHA/polymer composites (2) Delayed degradation rate of polymers [25,26] Not custom-made
(3) Enhanced osteogenesis [24,27–29]

3D printed nHA-based (1) Customized [30] Immature design and manufacturing

inorganic nanomaterials (2) Flexible mechanical and biological properties [31,32] methods [35]

(1) Good biocompatibility [37]

(2) Good biodegradability [37]
MSNs (3) Sustained release of silicon ions [13], drugs [40–42], Seldom used alone in bone regeneration
cytokines and miRNAs [43]
(4) Immunomodulatory effects [39]

(1) High load-bearing properties [59]

(2) Good biocompatibility [59] (1) Nondegradability [59]
Ti-based nanomaterials (3) AM Ti nanomaterials could change the exorbitant elastic (2) Poor biological response and
modulus of traditional Ti materials [69–72] and replace anti-bacterial properties [59]
complicated bone defects [74–76].

(1) Fast degradation and hydrogen

release rate
(1) Biodegradability [86] (2) Low elastic modulus for
Mg-based biomaterials (2) Osteoinductivity [85,86] load-bearing bone defects [87]
(3) Immature AM technology of
Mg-based biomaterials

(1) Biodegradability and suitable degradation rate (1) Mild cytotoxicity [103]
Zn-based biomaterials (2) Suitable mechanical properties similar to natural bone [99] (2) Immature AM technology of
Zn-based biomaterials

(1) Seldom used alone in

(1) Good biocompatibility [108] bone regeneration
AuNPs (2) Photothermal stability [108] (2) The co-assembly of AuNPs and
(3) Near-infrared absorbance [108] biomolecules lacks
hierarchical structure

Author Contributions: D.L. and Y.L. designed, prepared and revised the manuscript; Y.F. and S.C.
wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: The authors are grateful for financial support from the Projects of Ten-Thousand Talents Program
No. QNBJ2019-2 (Y.L.), Key R&D plan of Ningxia Hui Autonomous Region No. 2020BCG01001 (Y.L.),
National Natural Science Foundations of China No. 81871492 (Y.L.), No. 51902344 (D.L.) and
No. 81901053 (Y.F.), the National Key R&D project from Minister of Science and Technology, China
No 2016YFA0202703 (D.L.).
Conflicts of Interest: The authors declare no conflict of interest.

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