Pharmaceutical Nano Technology

Review
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Nanomaterials for bone tissue regeneration:

updates and future perspectives
Michael J Hill1 , Baowen Qi2 , Rasoul Bayaniahangar1 , Vida Araban3 , Zahra Bakhtiary4 ,
Michael R Doschak5 , Brian C Goh6 , Mohammadreza Shokouhimehr7 , Hojatollah Vali8 , John
F Presley8 , Amir A Zadpoor9 , Mitchel B Harris*,10 , Parisa PSS Abadi**,1 & Morteza
Mahmoudi***,11
1
Department of Mechanical Engineering – Engineering Mechanics, Michigan Technological University, Houghton, MI 49931, USA
2
Center for Nanomedicine & Department of Anesthesiology, Brigham & Women’s Hospital Harvard Medical School, Boston, MA
02115, USA
3
School of Engineering, University of British Columbia, Kelowna, BC V1V 1V7, Canada
4
Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
5
Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2R3, Canada
6
Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA
7
Department of Materials Science & Engineering, Seoul National University, Seoul 08826, Republic of Korea
8
Department of Anatomy & Cell Biology & Facility for Electron Microscopy Research, McGill University, Montreal, QC H3A 0G4,
Canada
9
Department of Biomechanical Engineering, Delft University of Technology (TU Delft), Delft, The Netherlands
10
Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA
11
Precision Health Program & Department of Radiology, Michigan State University, East Lansing, MI 48824, USA
*Author for correspondence: mbharris@mgh.harvard.edu
**Author for correspondence: pabadi@mtu.edu
***Author for correspondence: mahmou22@msu.edu
Joint replacement and bone reconstructive surgeries are on the rise globally. Current strategies for im-
plants and bone regeneration are associated with poor integration and healing resulting in repeated
surgeries. A multidisciplinary approach involving basic biological sciences, tissue engineering, regenera-
tive medicine and clinical research is required to overcome this problem. Considering the nanostructured
nature of bone, expertise and resources available through recent advancements in nanobiotechnology
enable researchers to design and fabricate devices and drug delivery systems at the nanoscale to be more
compatible with the bone tissue environment. The focus of this review is to present the recent progress
made in the rationale and design of nanomaterials for tissue engineering and drug delivery relevant to
bone regeneration.
First draft submitted: 17 November 2018; Accepted for publication: 18 September 2019; Published
online: 29 November 2019
Keywords: bone • drug delivery • nanomaterials • nanotechnology • tissue engineering
In the USA, an aging population has led to an increasing need for joint replacement and (post-traumatic) re-
constructive surgeries [1]. The most complex lesions may require both hardware implants as well as substituting
the bone with either autologous or allogenic grafts. While small bone defects may heal without any interventions
beyond fixation, critical-sized bone defects that are too large to regenerate naturally are becoming increasingly more
common [2]. This is partially due to the more frequent use of advanced surgical techniques such as limb salvage
following bone tumor resection, motor vehicle accidents with bone loss, bone excision after chronic infection or
gunshot wounds to the extremities [2]. Furthermore, comorbidities such as osteoporosis, osteoarthritis or trauma
impair the bone-healing potential, necessitating bone augmentation [3,4].
Mechanically stiff inorganic materials such as commercially pure titanium (Ti), Ti alloys, cobalt alloys, ceramics
or organic materials, such as ultra-high molecular weight polyethylene, are typically used alone or in combination
with each other to structurally augment or replace bone and joints [5–7]. However, osteolysis, mechanical wear and
the creation of inflammatory debris (e.g., foreign particle disease), as well as redox corrosion products are a major
problem for tissue healing in addition to the primary issue of poor biomaterial osseointegration [6,8–11]. For instance,
10.2217/nnm-2018-0445
C 2019 Future Medicine Ltd Nanomedicine (Lond.) (Epub ahead of print) ISSN 1743-5889
Review Hill, Qi, Ahangar et al.
there is a growing awareness of the accumulation of particles in extraneous tissues of the body due to the mechanical
wearing of artificial joint articulations under heavy loads or even from nonload bearing surfaces of the implant [12].
The eventual biological effects of such particle accumulation is not completely elucidated but particle-induced
osteolysis with subsequent aseptic implant loosening is a well-known complication and a particularly difficult
scenario to resolve [13–15]. In addition, bone serves as a reservoir for bone marrow and production of blood cells
making the replacement of marrow space with nondegradable synthetic materials less than ideal [16].
Therefore, enhancing the native regenerative capacity of bone tissue or replacing diseased bone with a living graft
is highly desirable. The current state of the art in replacing bone defects with living tissue is an autologous bone
graft (or autograft), where bone is taken from another part of the patient’s body, or an allograft (i.e., devitalized
donor bone from another individual), that can be used to fill the void [17]. Although the poor integration of
bone grafts does not lead to infection, bone grafts will be resorbed in the presence of an infection which causes
immune reactions, or death of the graft and donor site morbidity [1,18]. Other disadvantages include the pain
associated with the implant and the massive shortage of donors [19,20]. Bone tissue engineering (TE) and bone
regenerative medicine are strategies that have emerged to replace diseased bone using patient-specific cells grown on
mechanically supportive scaffolds in vitro or to enhance the native regenerative capacity of bone using engineering
and pharmacological interventions [21,22].
The paradigm of TE has been to use engineering strategies to control the scaffold microenvironment to
recapitulate natural developmental processes that result in mature and functional tissues [23]. Different cell types
such as progenitor or stem cells populate the supportive scaffolds, and multidimensional cues guide the cells toward
a differentiated and mature phenotype [24]. Multipotent mesenchymal stem cells (MSCs) and induced pluripotent
stem cells (iPSCs) are two promising patient-specific cell sources for bone TE. MSCs can be isolated from the
patient’s bone marrow due to the ability of those cells to adhere to tissue culture plastic relative to other bone
marrow cells, and iPSCs can be reprogrammed from a variety of patient cell sources, including skin, blood – among
others [24,25]. Both stem cell types have the capacity to differentiate toward the osteogenic lineage in vitro. MSCs
have been most frequently used in bone TE, but the more recent iPSCs have the advantage of unlimited self-renewal
and one study showed increased osteogenic differentiation relative to adipose derived MSCs (AD-MSCs) [26]. While
the differentiation of stem cells toward the osteogenic lineage can be achieved using cytokine and growth factor
cocktails under traditional culturing conditions, those in vitro conditions often fail to recapitulate the complex in
vivo milieu and result in inferior regenerated tissue properties or reduced regenerative potential [1,27,28].
The multiscale structure of bone

The complex native structure of bone consists of two main architectural tissue types, namely spongy inner cancellous
bone and dense outer cortical bone. Cancellous bone, otherwise known as trabecular bone, is porous or spongy,
and is located within the internal volume of bones. It is particularly important in mineral homeostasis due to the
collectively expansive surface area. On the other hand, cortical bone is dense, compact and is the primary structural
component that makes up the outer layer of bones (Figure 1A–D). Compact bone consists of lamellar structures
of collagen called osteons (∼200 μm diameter and 2 cm in length) that surround a central cavity (Haversian
canal), which is a conduit for blood vessels, lymph vessels and nerves [29,30]. In terms of proportion, cortical bone
is known to comprise approximately 80% of all bone volume in the human skeleton. However, in terms of bone
metabolism (i.e., resorptive removal and formative replenishment of new bone by bone-resident cells, a process
known as remodeling), it accounts for only 20% of total bone turnover [31].
The three principal types of differentiated bone cells involved in bone remodeling are the bone resorbing
osteoclasts, the bone-forming osteoblasts and the mechanosensing osteocytes [34]. Following an initiating stimulatory
signal issued by the osteocytes, the multinucleated giant osteoclast cells are formed through the fusion of precursor
macrophage lineage cells after which they in effect ‘burrow a cylindrical hole’ in the mineralized bone matrix, using
a combination of acid hydrolases and proteolytic enzymes, at the same time liberating stores of calcium, phosphate,
magnesium and other organic growth factors and molecules into the microenvironment [31,35]. Those biochemical
cues differentiate and recruit the mesenchymal derived osteoblast cells to the site of osteoclast resorptive activity, and
in a process known as reversal, the osteoblasts synthesize a layer of new bone to ‘fill in the hole’, initially in the form
of a loose layer of type I collagen known as osteoid [36]. Due to subsequent events, including the secretion of mineral-
binding glycoproteins and pH-modifying enzymes by the osteoblast creating an alkaline microenvironment, the
osteoid layer mineralizes by the binding of calcium and phosphate in a six to four ratio. The mineralization process
forms the rigid hydroxyapatite (HA) lattice that surrounds the organic collagen ground substance, resulting in a
10.2217/nnm-2018-0445 Nanomedicine (Lond.) (Epub ahead of print) future science group

Nanomaterials for bone tissue regeneration: updates & future perspectives Review
10-1 10-3 10-6 10-9
(m)
Lacunar-canicular
space
Acc.V Spot Magn Det WD 10 µm

10.0 kv 3.0 2000x SE 12.3
Nanoparticles
Figure 1. The multiscale architecture of a typical long bone. (A) Wolff’s law that bone fibers remodel along the lines
of applied stress is visualized in the proximal femur, where arcade-like trabecular bone (shown as red lines) support
the principal load axis of bodyweight at the hip. (B) Bone consists of two major structural types, outer cortical
(compact) bone and spongy inner cancellous (trabecular) bone. (C) The cylindrical structure of osteons in the outer
compact bone. Surrounding the central red-colored Haversian canal (filled with blood vessels, nerves, bone-resorbing
osteoclast and bone-forming osteoblast cells) are the concentric layers of lamellar bone, populated by
mechano-sensing osteocyte cells (thin red cells). Despite being embedded in microscopic bone spaces (known as
lacunae), osteocytes are capable of communicating with each other by connecting cellular processes through
nanoscopic fluid filled channels known as canaliculi. (D) The structure of triple helical collagen fibers in parallel
networks (black circle represents crosslink) and mineralized hydroxyapatite platelets (grey) with high aspect ratio are
the basic ‘building blocks’ of the bone. The dimensions of the mineral phase are of the utmost importance for bone’s
unique mechanical properties according to the Jaeger–Fratzl equation (Eq. 1) [32]. (E) Nanoparticles can diffuse within
the nanoscale extracellular matrix of bone cells such as lacunar-canaliculi space. (F) Nanomaterials and nanomaterial
composites have similar dimensions to the components of native bone and can be used as biomimetic scaffolds for
bone regeneration. (G) Scanning electron microscopy image of the nanoscale fibers of bone which show alternating
alignment directions in consecutive layers, as shown by yellow arrows.
Panel (G) is reprinted with permission from [33] C Springer Nature (2015).
replenished pocket of bone tissue – a process critical for bone micro-crack repair and lifelong structural skeletal
health. As a consequence of osteoid mineralization, osteoblasts become embedded between successive layers of
newly mineralized bone layers (lamellae), where they remain trapped as bone-resident osteocytes [36]. However,
within the basic cylindrical structural unit of bone tissue (namely, the osteon), the osteocytes embedded between
concentric circular layers of lamellar bone tissue are situated in spaces known as lacunae, with their cellular processes
interconnected to each other by narrow nanoscopic fluid filled channels known as canaliculi (Figure 1E). In terms of
bone tissue microstructure, the mineralized collagen lamellae themselves are 3–5 μm thick and have a plywood-like
organization [29]. Numerous strategies on the macro-, micro- and mesoscale have been developed for fabricating
scaffolds to mimic these complex structures or to deliver agents to bone or stem cells to enhance regeneration or
medical imaging capabilities [37–40]. On the macroscale, it is now well accepted that the average Young’s modulus
of a TE scaffold will influence the differentiation of stem cells, with a greater modulus promoting osteogenic
differentiation. In addition, microparticles have been used as agents to deliver growth factors, cytokines or small
molecules and to give stem cells osteogenic cues [41]. However, more recently, a trend to engineer biological systems
down to the smallest length scales has been embraced in the form of bionanotechnology.
future science group 10.2217/nnm-2018-0445

Over the past 50 years, as the field of nanotechnology has advanced, structural analogs to the biological
components of bone tissue have become apparent (Figure 1F). An important example is the ability to create
synthetic 1D nanofibers or nanotubes that mimic the dimensional nature of nanofibrous collagen I matrices,
comprising approximately 90% of the organic phase of bone tissue. Collagen is a polypeptide rich in glycine,
proline and hydroxyproline that assumes a triple-helical conformation and undergoes both enzymatic crosslinks
that stabilize the networks of fibrils and nonenzymatic crosslinking processes (e.g., advanced glycation end products)
in vivo [42]. The dimensions of a single collagen molecule (tropocollagen) are comparable with that of a nanotube
at approximately 1.5 nm diameter and 300 nm in length. These molecules tend to assemble into 100 nm diameter
bundles or fibrils (Figure 1G) [43]. An additional advantage of engineered nanoparticles (NPs) is that they can
further encapsulate drugs or biological molecules intended to accumulate in specific tissues to direct drug release
kinetics, or be geometrically imprinted with biomolecules as templates allowing for biorecognition as ‘artificial or
plastic antibodies’ [44,45]. The overall ethos of nanotechnology of designing materials by manipulating their smallest
constituents to control the structure, and resultant properties readily finds application in bone regeneration due
to the nanoscale structural features of natural bone tissue that are essential to its unique properties. For instance,
the body naturally plays the role of nanotechnologist by controlling the placement and morphology of nanoscopic
collagen fibers in bone to reinforce them against stress gradients in accordance with Wolff ’s law as shown in
Figure 1A. This process is performed by the perpetual interplay of osteoclasts and osteoblasts that results in the
continuous process of bone remodeling in healthy adults.
In addition to collagen networks, the mineral fraction of bone is large at approximately 65% of the total dry
weight [46]. The combination of rigid mineral and soft matrix gives bone its paradoxical high compressive strength
and toughness, a combination difficult to achieve with synthetic materials [47]. The mineral fraction of tissues such
as bone, tooth enamel and other hard biomaterials, such as nacre (hexagonal platelets of aragonite found on the
inner surface of some mollusc shells), avian egg shells – among others, consists of particles with high aspect ratios
that vary between a few nanometers and hundreds of nanometers in diameter in some cases displaying helical
organization with chiral characteristics, the handedness of which can vary based on the geographical location of
the organism (Figure 1D) [32,48,49]. The nanostructure–property relationships of natural materials such as bone
can guide the design of synthetic composites. As an example, the mineral crystals in bone are platelet-shaped HA
particles and reside in the soft, hydrated collagenous aligned fiber matrix as clustered helical arrays and are 20–30 nm
wide, a few nanometers in thickness, with an aspect ratio of approximately 30 (100 nm in length) [32,50]. The
self-affine helical structures in bone creates a fractal-like geometry with self-similarity extending over approximately
ten orders of magnitude, from the bottom up, beginning at the collagen triple helix, extending to the organization
of HA particles and finally to the macroscopic helical curving shape of many whole bones [50]. This length scale
and aspect ratio are important according to the Jaeger–Fratzl model (Eq. 1) where the Young’s modulus of the bone
is proportional to the square of the aspect ratio of the mineral platelets (Figure 1D). In Eq. 1, Gcoll is the Shear
modulus of the collagen network, Emineral is the Young’s modulus of the mineral platelets, ρ is the aspect ratio of
platelets, π is the volume fraction of mineral, and E is the Young’s modulus of the bone.
1 4(1 − ) 1
= + (Eq. 1)
E G coll ρ
2 2 E min eral
This unique nanostructure allows the load to be carried by the high compressive strength mineral phase and
transferred between mineral domains via shear by the tough collagen network [32]. Therefore, the Young’s modulus
of the mineral domain and the shear modulus of the collagen network are both important parameters in determining
the fracture properties of the bone [47]. Such findings indicate that mimicking bone mechanical properties will
require optimizing the correct parameter for both hydrogel and nanomaterial phases. In addition, the mineral
domains to exceed an estimated critical thickness of approximately 30 nm (i.e., larger than the nanoscale), the
respective fracture strength would be more sensitive to any defects in the structure, which would cause stress
concentrations, invariably increasing fracture risk. Therefore, both mineral platelet nanoscale thickness and aspect
ratios are extremely important and biologically controlled. For example, higher aspect ratio of mineral HA platelets
is associated with a compensatory mechanism for the loss of bone density in osteoporosis, making the size and
shape of the nanoscale building blocks extremely important to the emergent mechanical properties [32,51,52].
Not only can an engineered tissue’s mechanical properties be intimately dependent on the scaffold nanostructure,
but cells also interact with nanoscale cues to guide signaling and growth processes. Receptor–ligand interactions

Table 1. Bone substitute materials in clinical use.

Bone-substitute materials Source Clinical application Pros Cons Ref.
Demineralized bone Procurement of bone Already being used by Osteoinductive and No structural support [61]
powder from donors veterinarians to avoid rapidly induces new bone
harvesting autograft formation
Hydroxiapetite Teeth and bone; synthetic Cranioplasty, open-wedge Biocompatible and Low ductility and fracture [62,63]
tibial osteotomy and porous toughness
hand surgery
Coral Species other than human Contained bone defects Osteoconductive, low rate Low resorption in [64]
of diseases and infections coralline hydroxyapatite
state
Calcium sulfate Synthetic Spine fusion, fracture Rapid graft resorption Inflammatory reaction [63,64]
nonunion, contained
bone defects
Nonresorbable calcium Bone mineral and tooth Injectable calcium A fibrous reaction to the No cartilage or bone [65]
phosphate enamel phosphate was used to implantation of developed
repair human periodontal nonresorbable calcium
intrabony defects phosphate occurs and
does not evoke a foreign
body reaction
Resorbable calcium Bone mineral and tooth Easy adaptation and A bone substitute should Little bone formation, [66]
phosphates enamel fixation, reduced surgical be replaced by mature inferior mechanical
time, favorable esthetic bone without transient properties
results and minimal waste loss of mechanical
products support
Polymethylmethacrylate A synthetic material Limited in clinical use due Separated from the Can lead to recurrent [67]
generated from the to poor osseointegration, reactive bone with a infections and secondary
polymerization of methyl monomer toxicity, and fibrous layer contamination; generally
methacrylate tissue necrosis is utilized as a temporary
spacer
␤-tri-calcium phosphates Synthetic Open-wedge tibial Osteoconductive and Strength not as high as [63]
and hydroxiapetite osteotomy, contained improved bone formation cortical bone’s strength
bone defects, and spine
fusion
Poly-ethylene (Medpo) A synthetic resin The porous polyethylene The interstitial space Bone filling and bridging [68]
is safe and effective with consisted of connective was significantly reduced
low morbidity in clinic use tissue with small blood
vessels and cells
are the most paradigmatic example due to the scale of protein molecular recognition sites being approximately
50 nm2 . Another example is the ionic fluid flow through nanoscopic pores in bone (∼16 nm in HA phase and
∼100 nm in lacunar–canicular space) in response to loads that is responsible for the strain generated potential
in bone, which may play a role as a cue for bone remodeling in addition to mechanical fluid shear forces [53,54].
In addition, as colloidal materials approach dimensions of approximately 100 nm or less, their interactions with
proteins and cells exhibit unforeseen properties. To demonstrate this concept, it is well known that several naturally
occurring surfaces have bactericidal properties when bacterial cells come into physical contact with them. Such
surfaces include dragonfly wings, cicada wings and gecko skin. These surfaces have in common nano-needle like
structured surfaces, which can penetrate the bacterial cell wall, causing cell death [55]. On smoother surfaces of
similar chemistry or when the dimensions of nanopatterns deviate from a specific range, the bacteria would not be
damaged and could form biofilms [56]. For bone tissue, a similar concept will apply to the interactions of osteoblasts
with nanostructured versus smooth surfaces of similar chemistry [57]. For instance, Tsimbouri, et al. developed
substrates of Ti with TiO2 surface nanowires, which could provide cues for bone marrow mesenchymal stromal
cells (BM-MSCs) and bone marrow hematopoietic stem cells differentiation (BM-HSCs) toward the osteoblast
lineage while simultaneously reducing the viability of bacteria [58]. Such a nanoengineering strategy could lead to
marked improvements in bony ingrowth and, thus, implant fixation, thereby reducing the likelihood of both septic
and aseptic implant loosening.
The challenges of mimicking the structural properties of bone on the nanoscale and providing stem cells with
osteogenic cues are not the only remaining issues facing effective bone biomaterials. There are some traditional
materials currently in use that are associated with various pros and cons as explained in Table 1. However, thus
far, no materials in clinical use can mimic the multidimensional, multiscale natural microenvironment of bone

tissue. For example, bone tissue must also have its own vasculature system consisting of endothelial and other cell
types, which also interact with the uniquely nanostructured bone extracellular matrix (ECM) [59]. Therefore, the
native arrangement of numerous cell types must be considered. The mechanosensing bone cells (i.e., osteocytes)
are embedded in the matrix and control the remodeling process by signaling osteoblast and osteoclasts, which will
depend on their spatial arrangement and the geometry of the ECM. Osteocytes have a pericelluar region <100 nm
in thickness through which fluid flow and shear stress in response to loading is sensed possibly by glycocalyx
deformation as well as by other parts of the cell, and integrin-based signaling is likely to be involved [47,54,60]. This
fluid flow response to loading is considered more important than mechanical stretching on osteocyte signaling,
making the arrangement of cells and continuity of pores and canaliculi for fluid flow of the utmost importance for
normal tissue functioning [47]. For instance, in osteoporosis, the changing pore structure of bone tissue can result
in alteration of the interstitial fluid flow, in turn reducing bone apposition.
Numerous nanomaterials and nanofabrication techniques have been discovered over the past half century and are
now being used to meet these challenges and enhance bone regeneration by recapitulating the native bone milieu
with biomimetic engineering solutions. Materials such as mesoporous silica NPs, nano HA, carbon nanotubes
(CNTs) and graphene have all recently received attention as novel nanoscale materials for reinforcing scaffolds or
as carriers for drug delivery [69–74]. This review will focus on current solutions to overcoming the discussed barriers
to bone regeneration using nanotechnology.
Advances in nanomaterials for bone regeneration

The scaffold is an essential part in TE approaches where its key function is to provide a temporary mechanical
support in the defect site and a suitable environment for the cells to grow [75–78]. This specific role of scaffolds is of
paramount importance in bone TE, as bone is among the hardest load-bearing tissues in the human body with a
significant Young’s modulus (E = 17.5–18.9 GPa) [79,80]. The design and selection of materials used for successful
tissue growth are therefore a significant challenge. Ideally, a scaffold should possess the major characteristics
of being 3D, porous, bioconductive, biocompatible and bioresorbable, with mechanical properties comparable
with the native bone tissue and having suitable surface properties for facilitating cell attachment, proliferation
and differentiation [81]. These material characteristics such as three dimensionality and porosity are important to
mimic the native cell arrangements (and thus cell–cell communication) and diffusion characteristics to prevent
tissue necrosis, while bioresorbable scaffolds which mimic native tissue mechanical properties can substitute and
eventually be replaced by native ECM molecules [76]. The need for bioresorbable and biocompatible scaffolds
restrains the range of the material that can be used.
Despite their utility, polymers usually lack the required mechanical properties that are ideal for bone tissue
scaffolds. Discoveries of nanomaterials have paved the way for achieving that goal. Adding nanomaterials as a filler to
these matrix polymers can augment the mechanical properties of the scaffolds [82]. As a result, different nanomaterials
are being studied as scaffold fillers to investigate their effect on cells. To that end, ceramic nanomaterials such as
HA and bioactive glass, carbon-based materials like CNTs and graphene, and metallic based NPs such as gold and
titanium oxide (TiO2 ) have been widely investigated as interesting potential candidates for scaffold filler materials
(Figure 2A).
In addition to their role in enhancing the mechanical properties of scaffolds, it has also been suggested that
nanoscale materials can have specific influences on cell signaling pathways as shown schematically in Figure 2B &
C. For instance, it was suggested that nanomaterial based scaffolds can modify the adhesion of proteins to the
scaffolds [83–86]. Zhu et al. showed magnetic NPs increase the amount and variation of cytokines in the protein
corona compared with a scaffold without NPs [85]. It was shown that these proteins enhanced the MAPK/ERK
signaling pathway resulting in enhanced cell proliferation. In addition, Liu et al. studied the effects of hydroxyapatite
NPs (HANPs) in electrospun chitosan (CHI) matrix scaffolds, where they found that the NPs enhanced BM-MSCs
proliferation through activation of the (BMP/Smad) signaling pathway [86].
HANPs are the major inorganic part of bone tissue [87]. They are therefore a very attractive biomaterial to be
employed in scaffold design due to the ability to mimic the natural inorganic phase of bone relative to organic
polymeric NPs. The addition of HANP fillers to matrix materials for the fabrication of composite scaffolds has
been studied intensively as an approach for osteogenesis enhancement. Polymers such as CHI [88], gelatin [89],
polycaprolactone (PCL) [90], poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) [91] are among the
matrix materials used in combination with HANP. Maji et al. utilized gelatin-carboxymethyl CHI incorporated
HANP scaffolds and cultured mesenchymal stem cells (MSC) spheroids on it [92]. High speed stirring was used

i ii
0-D Increased roughness BMP/Smad signaling Improved cell proliferation
pathway proteins
1-D
Enhanced porosity
2-D
Figure 2. Nanoparticles in bone tissue engineering scaffolds. (A) Various nanomaterial structures are used as the
filler material in scaffolds. 0D materials such as AuNP, magnetic nanoparticles (NPs), etc.; 1D NPs such as carbon
nanotubes, gold nanorod; 2D structures such as graphene. (B) Manipulating scaffold design with incorporation of
nanomaterials to introduce surface roughness, nanoporosity and higher stiffness for mimicking native bone
tissue, and NPs modify the protein corona formed around the scaffold, where these proteins activate the BMP
signaling pathway for stem cells. (C) Enhanced cell proliferation achieved in a scaffold incorporated with NPs as a
result of mechanical and biological mechanisms induced by them.
to generate foaming and the foams were freeze dried to fabricate scaffolds with macroporous sizes. Mechanical
properties were significantly improved compared with conventional freeze drying. Improvement of viability, greater
penetration depth of tissue into the scaffolds, and increased activities of alkaline phosphatase (ALP) and osteocalcin
(OCN) (i.e., early and late biomarkers for osteogenic activities, respectively) were observed relative to control
culture conditions.
In an attempt to mimic the inorganic part of bone, scaffolds consisting of HANP and nano whitlockite particles
(WHNP) have been developed [93,94]. Cheng et al. used a solution of ultrasonically dispersed HANP and WHNP
in gelatin methacryloyl (GelMA). Human MSCs were cultured on the HANP-WHNP-GelMA scaffolds and 30 s
of UV light exposure crosslinked the solution with an intensity that did not damage cells. Addition of WHNP to
HANP decreased the Young’s modulus of the crafted scaffolds substantially (depending on particle concentration
by ∼20%), demonstrating that the HANP are more effective in providing mechanical support to the scaffold.
While the result showed that the GelMA-100% WHNP scaffolds significantly increased osteogenic activity in
comparison with GelMA-100% HANP scaffolds, the optimum results were achieved with the HANP to WHNP
ratio of 3:1, a similar ratio as in native bone, which suggested the synergistic effects of both HANP and WHNP.
ALP and OCN activities were higher in GelMA-75% HANP-25% WHNP as compared with both GelMA-100%
HANP and Gelma-100% WHNP in both DMEM and osteoinductive media.
Bioactive glass ceramic nanoparticles (nBGC) incorporated with natural biopolymers are another group of
nanomaterials that has been studied extensively due to the ability to better mimic the natural mineral phase of
bone, relative to metallic NPs. For example, silk fibroin and bioactive glass/polyvinyl alcohol (PVA) have been
electrospun to form a bilayer scaffold, which has been shown to result in improved proliferation and differentiation
of MSCs compared with scaffolds that lacked bioactive glass [95]. In another study, scaffolds made of 10% gelatin
and 30% nBGC powder were fabricated by freeze drying. Different kinds of MSCs, human bone marrow (BM-
MSCs), umbilical cord Wharton’s jelly (UC-MSCs) and adipose derived (AD-MSCs) were cultured on scaffolds
and their potential for healing was investigated in critically sized rat bone defects in vivo. The results showed that
all the scaffolds had high cell viability, where the BM-MSCs had the greatest potential for formation of de novo
bone and UC-MSCs showed the highest potential for neovascularization [96]. The use of gelatin [97,98], CHI [99,100],
alginate [101] and CHI-alginate containing nBGC scaffolds have also been reported. Nikpour et al. devised a scaffold
made of crosslinked dextran with nBGC [102]. Sonically dispersed nBGC particles in an aqueous alkaline solution of
dextran were crosslinked and freeze dried to fabricate the scaffolds. Addition of up to 2% nBGC initially increased
the compressive modulus; however, increasing the amount to 16% led to a significant decrease in the compressive
modulus that was attributed to the agglomeration of the nBGC particles. Human osteoblasts were cultured on
the fabricated scaffolds where significantly improved cell viability and ALP activity were observed in scaffolds
containing 8 and 16% of nBGCs. PCL [90,103,104], PCL-CHI [105], PLLA [106,107] and PLGA [108,109] were also

investigated as the matrix material in combination with nBGC for fabrication of scaffolds in bone TE applications,
where enhancement of either some or all of the crucial criteria, such as mechanical properties, hydrophilicity, water
absorption, cell attachment, viability, differentiation – among others, were observed.
CNTs possess exceptional mechanical properties, high aspect ratios and thermal/electrical conductivity. As a
result, they have been an attractive component for enhancing composite scaffolds properties. Jing et al. incorporated
multiwall carbon nanotubes (MWCNT) and HANP in a collagen matrix [110]. The freeze dried scaffolds showed
improved mechanical properties (i.e., Young’s modulus), cell spreading and proliferation response of rat BM-MSCs
for scaffolds containing 0.5% MWCNT. Reverse-transcription PCR (RT-PCR) results for expression of bone
sialoprotein and OCN indicated significantly higher amounts in all the scaffolds containing MWCNT (from 0.25
to 2%), with the optimum result at 0.5%. Furthermore, scaffolds containing 0.5% MWCNT were examined in
vivo in a rat calvarial bone defect model for healing 8 mm diameter defects. In this model, inflammation was scarcely
observed. After 12 weeks of healing, newly formed bone and connective tissue was observed, which were more
abundant when compared with collagen-HA and control groups. Other molecules such as CHI have been used
with MWCNTs for fabricating scaffolds for bone TE [111,112]. In addition, some studies investigated functionalized
or pristine MWCNT-CHI along with HA in their scaffold composites [113–118].
The use of graphene (another high aspect ratio nanomaterial) in the fabrication of scaffolds for bone TE has been
extensively reviewed in other works [119,120]. Raucci, et al. devised scaffolds of graphene oxide (GO) and HANP
via a sol-gel method and scaffolds of GO were coated with amorphous calcium phosphate by immersing them in
supersaturated simulated body fluid, which was called the biomimetic approach [121]. Composite scaffolds showed
a higher amount of cell viability and osteogenic differentiation than controls for human MSC (hMSC). Another
study, evaluated the scaffolds of HANP and GO nanoribbons (GNR) in vitro and in vivo, which proved to have
promising results in healing the defects [122]. GO was also incorporated with nBGC for fabricating scaffolds [123]
and very recently, HA nanowhiskers were grown on the surface of GO mixed in a poly lactic acid (PLA) matrix [124].
Reduced GO (RGO) was also used in combination with other materials like HANP [125], and zinc silicate–calcium
silicate for developing scaffolds [126]. HA has favorable interactions and nucleation ability with GO which it renders
more biocompatible, while zinc and silicon are important trace elements in bone and combined with GO rendered
the scaffold electrically conductive for promoting osteogenesis. In each case the GO or RGO enhanced scaffolds
resulted in enhanced cell viability or differentiation into the osteogenic lineages as judged by expression of key
markers.
Metallic NPs incorporated in scaffolds are also being developed for bone TE applications. Due to their excellent
mechanical properties, they can be an exceptional candidate as a filler in the composites; however, the challenge is
to find biocompatible particles. Gold (Au), silver (Ag) and TiO2 have long been known as interesting candidates
in the field of TE. Silver nanoparticles (AgNP) have been incorporated in scaffolds for enhancing osteogenic
performance [127–129]. Gold NPs (AuNPs) were incorporated in scaffolds, which showed exceptional potential
in enhancing cell differentiation [130–132]. Studies were done on the effects of gold particle size in regulating cell
activities [133,134]. For primary osteoblasts, it has been shown that a diameter of 20 nm exhibits the highest osteogenic
effect, while for the human adipose-derived stem cells, 30–50 nm particles performed the best. In another study,
the effects of AuNP with three different functional groups, amine (AuNP-NH2), carboxyl (AuNP-COOH) and
hydroxyl (AuNP-OH) were investigated on human BM-MSCs [135]. There was no negative effect on cell viability
after 21 days of culture. While the hydroxyl functional group showed the highest ALP activity, the other two groups
revealed a lower amount of ALP compared with the control group. AuNPs were utilized in US FDA approved
polymers such as PLGA and PCL [132,136] or hydrogels such as GelMA [131], as well. TiO2 NPs were utilized
in combination with a variety of polymers for the fabrication of scaffolds. For instance, natural polymers such
as CHI [137], silk fibroin [138,139] and biodegradable synthetic polymers, such as PCL [140] and PLGA [141], were
investigated for enhancing the osteogenic performance of scaffolds.
As mentioned, though MSCs have been most widely used, iPSCs have also been used recently for bone TE
using nanostructured scaffolds. However, for applications such as cellular therapy or bone TE, IPSCs must first be
differentiated into mature cells to avoid the risk of teratoma formation [142]. Wang et al. used a scaffold composed of
the M12 phage, a virus that is harmless to humans and is approximately 880 nm long and 6.6 nm in diameter [143].
The phage can be produced by infecting bacteria and then be self-assembled into different patterned scaffolds. Since
the peptide sequence of the protein coating had little effect on self-assembly, the influence of peptide recognition
sequences (by fusing them to the coat protein by genetic insertion) versus nanotopography could be investigated.
Four peptides including two adhesive domains from fibronectin as well as a domain from osteogenic growth peptide

and bone BMP-2 were displayed on the side-wall of the phage. The adhesive characteristics and morphology of
iPSCs were independent of the recognition sequences and were mostly a function of the alignment of the fibers.
However, after differentiating iPSCs into mesenchymal progenitor cells, which subsequently underwent osteoblastic
differentiation, the recognition sequences resulted in varying levels of ALP activity, OCN and OPN expression
(growth factor sequences > fibronectin). All such effects were greater on oriented fibers as compared with random
fiber controls demonstrating the interplay and synergism between nanotopography and molecular recognition in
the differentiation of iPSC toward the osteogenic lineage.
IPSCs have also been utilized in two studies with nanofibrous polyether sulfone (PES)-based electrospun
scaffolds, in one case with gas plasma surface modification [144,145]. PES is a biocompatible polymer with many
biomedical applications such as dialysis or blood purification. It was shown that additional gas plasma treatment
and modification with collagen could enhance the osteogenic differentiation relative to pristine PES, as measured
by increased mineralization, upregulation of osteogenic markers, and improved bone regeneration in a rat calvarial
bone defect model. Electrospinning has also been used to create composite nanofibrous scaffolds of calcium deficient
HANPs and PLLA [146]. Murine embryonic stem cells and murine iPSCs have been tested against human BM-
MSCs for osteogenic differentiation, while the HANP-PLLA composites have been tested against PLLA controls.
For each stem cell type, osteogenic differentiation was promoted on HANP-PLLA nanofiber scaffolds relative to
PLLA, with concentrations as low as 1% weight particles showing the enhanced effect.
NPs present great possibilities for appropriate and local delivery of growth factors in the bone healing process.
Relative to many small molecules, NPs can easily bypass the barrier of the cell membrane via specific, receptor
mediated processes or nonspecific processes, depending on NP surface chemistry, shape, and size [147]. Mechanisms
for particle internalization (endocytosis) include phagocytosis (cell eating), and pinocytosis (cell drinking). Phago-
cytosis typically occurs with microparticles, while pinocytosis occurs for NPs. Thereafter intracellular trafficking
takes place typically to endosomes and then to lysosomes (for receptor-mediated endocytosis), where the particle
protein corona will be degraded after which the cytoplasm will be exposed to the NPs [148,149]. NPs can also localize
to other cellular organelles, depending on their surface properties and shape [147].
BMP, TGF-β, bFGF, VEGF, PDGF, IGF and TNF are all growth factors that NPs have introduced new ways
for delivering [150]. The basic concept for drug delivery in bone tissue is shown in Figure 3A–E. BMPs are growth
factors belonging to the superfamily of TGF-β [151]. BMPs exhibit significant osteogenic activities and promote
MSC differentiation into osteoblasts for bone formation. For example, it has been shown that BMP-2 is a potent
osteogenic factor, which is necessary for the induction of bone formation [152]. The FDA has approved use of
BMP-2 and BMP-7 for spinal fusion procedures and long-bone fractures [153,154]. BMPs are pleiotropic proteins
that can have adverse effects when administered systematically. These effects include life-threatening events, implant
displacement or infection [155]. Also, their rapid clearance makes conducting further studies essential for applying
them for the aim of bone fracture healing.
Given that BMP-2 is an expensive and potentially toxic protein, introducing novel factors for endogenous
stimulation of BMP is of great importance [155]. Several studies have shown the stimulating effects of statins on
BMP-2 production from bone cells and their potential for use in fracture healing [162–164]. Due to their first pass
metabolism, oral administration of statins is not suitable for the aim of increasing fracture healing as very large doses
are required. In another study done by Budge et al., they prepared simvastatin-loaded solid lipid NPs with a binary
mixture of glyceryl monooleate and emulsifying wax [165]. An increased level of ALP showed the utility of these NPs
for osteoblast differentiation. Treatment of preosteoclasts with RANKL differentiates them to osteoclasts, which
can be seen by their increased level of tartrate-resistant acid phosphatase (TRAP) production [165]. Tartrate-resistant
acid phosphatase was meaningfully lower in cells that were treated with both RANKL and simvastatin-loaded NPs
in comparison with the cells that were treated with just RANKL, which shows the ability of these simvastatin-loaded
NPs for inhibition of osteoclast differentiation.
As all the expectations from a scaffold cannot be typically met by using a single polymer, several studies are
focusing on optimizing their scaffold by using combinations of polymers and nanomaterials [166,167]. Rezk et al.
prepared a three-layered nanofiber scaffold using electrospinning for controlled release of simvastatin with diffusion
processes. The first layer that serves as a mechanical stabilizer for the scaffold is made of PCL nanofibers. The
middle layer is made of a PCL-cellulose acetate-β-tri calcium phosphate (PCL-CA-β-tcp) layer, which supports
osteogenic differentiation and mineralization in the later stages of the healing process. The final layer is composed
of poly(vinyl acetate) (PVAc) crosslinked to a PVA nanofiber layer, which is loaded with simvastatin. The sustained
release of simvastatin up to 7 days was observed from this three-layered scaffold. In vitro studies were performed

Mi-RNA Dex
BMP-2
TGF-β
NPs Cells
Phospholipid cell membrane
Figure 3. Drug delivery for bone regeneration. (A) Various NPs have been used for encapsulating drugs for directed
delivery to bone. Important factors in osteogenesis such as BMP-2 and TGF-β as well as miRNAs and small molecules
like dexamethasone have been successfully encapsulated with controlled release kinetics (molecular images of
3BMP [156], 5VQF [157] and 4GXY [158] from the protein databank [159] visualized using visual molecular
dynamics [160]. (B) NPs diffuse in the extracellular space and can more easily enter the cell than small molecules by
interacting with the cell membrane via receptor mediated or nonspecific processes. (C) In the process of endocytosis
inward budding of the cell membrane encapsulates NPs inside vesicles (lipid structure from [161] and visualized with
visual molecular dynamics [160]). (D) Sliced image of NPs encapsulated inside a spherical lipid vesicle which are
subsequently internalized by the budding off of the vesicle from the cell membrane. (E) NPs are then trafficked in the
cytoplasm to lysosomes (yellow) which begin the process of NP degradation and also release of NPs into the
cytoplasm and other organelles, occasionally the nucleus (blue), depending on the specific NP properties.
NP: Nanoparticle.
on preosteoblast cells, and the examination of cellular morphology showed significant cell attachment to different
layers and the increased ALP activity confirmed that this scaffold is able to induce osteogenic differentiation. In
addition, simvastatin or lovastatin delivery by PLGA based NPs has recently shown increased fracture healing or
osteoblast differentiation including increased expression of BMP-2 [168–171].
As described in previous sections, HA is the main component of bone and several studies have used scaffolds
based on it. However, its low mechanical stability and osteo-conductivity have necessitated further modifications
for its use in 3D scaffolds. In a study done by Kim et al., for achieving HA scaffolds, calcium sulfate hemihydrate
powder was used, which transformed to HA using hydrothermal treatment. To improve the osteo-conductivity and
mechanical strength of the scaffold, BMP-2 PLGA NPs were incorporated in the scaffold using PCL. Continuous
30-day release of BMP-2 was achieved, which increased the cell proliferation and ALP activity of hMSC [172].
In another study done by Gaihre et al., BMP-2 was encapsulated in CHI/ HANPs and released in a 3-week
time period mostly by diffusion in vitro [173]. In addition, thermosensitive NPs of poly(phosphazene) and BSA
NPs stabilized with polyethyleneimine-poly(ethylene glycol) coatings have been loaded with BMP-2 as injectable
formulations for increasing bone regeneration. In addition to BMP-2 itself, several scaffolds and NP systems have
been used for loading and delivery of BMP [174–183]. In a study done by Raftery et al., a gene-activated scaffold
was designed by incorporating BMP-2 modified plasmid loaded CHI NPs in a collagen HA scaffold. This scaffold
showed high transfection of MSCs. Micro-CT analysis of bone defects in a rat model confirmed its potential for
further investigation in bone healing [178].

VEGF is one of the most important growth factors for bone healing and promotion of regeneration processes.
Also, due to the important role of VEGF in osteoclastogenesis, it affects the bone remodeling process in the final
stage of bone healing. Regarding the short half-life of VEGF, successful delivery of VEGF with optimal doses
can improve the impaired vascularization and regeneration process in fractured bones which is the aim of several
researches [184–187]. In a study done by Lu et al., VEGF was immobilized to PLGA nanofibers by biomimetic
conjugation with heparin, which shows the sustained release of VEGF for up to 31 days [184].
In a different study, the controlled release profile of VEGF was obtained by designing a 3D scaffold in which
the release profile of VEGF NPs is based on the cell-contact to the implant. In this study, the VEGF molecule
was encapsulated in CHI NPs by using an inotropic gelling method. The nanoreservoir of VEGF NPs was created
by using CHI and these nanoreservoirs were incorporated in a 3D scaffold made of PCL through layer-by-layer
electrospinning. The obtained scaffold was then preseeded with osteoblasts followed by the seeding of endothelial
cells. Using osteoblastic cells helps anchoring of the endothelial cells to the scaffold and degrading the nanoreservoirs
of VEGF, resulting in its release and a subsequent increase in vascularization by endothelial cells.
MiRNAs are a family of short noncoding RNAs (20–24 nucleotides), which are important players in post-
transcriptional regulation of gene expression. MiRNAs usually repress their specific target genes by inhibiting
translation of mRNA or promoting mRNA degradation by binding to their 3 -UTR. It has been estimated that
around 3% of all human genes encode for miRNAs, and the expression of approximately 40–90% of proteins
are regulated by miRNAs [188]. Therefore, miRNAs are involved in multiple biological processes, such as cell
differentiation, proliferation and apoptosis, just to name a few and not surprisingly, there is a large body of evidence
supporting the role of miRNAs in the regulation of osteogenesis and osteoclastogenesis [189]. A recent paper has
reviewed the multitude of miRNAs involved in bone remodeling [189,190].
As a result, several studies have focused on the delivery of miRNAs for modulation of bone repair [191,192].
For instance, miRNAs have been delivered in a gene therapy format. Liao et al. engineered hASCs with miRNA-
expressing baculovirus vectors, for the ultimate goal of repairing large calvarial bony defects, which is a challenging
task for orthopedic surgeons. Four different baculoviruses expressing individual human miRNAs (miR-26a, miR-
29b, miR-148b and miR-196a) all enhanced the osteogenesis of hASCs, but miR-148b and miR-196a had a higher
potency than that of the others. Moreover, the authors discovered that combined miR-148b and BMP-2 expression
vectors were synergistic in induction of osteogenic differentiation in vitro. Finally, the hASCs coexpressing BMP-
2/miR-148b were implanted into calvarial bone defects in nude mice and were shown to accelerate and potentiate
bone healing. Using micro-CT, histology and immunohistochemical staining, they showed that around 94% of
the defect area and 89% of the defect volume could be filled within 12 weeks, demonstrating that miRNAs and
growth factor delivery can synergistically stimulate osteogenesis and promote bone healing.
Another drug frequently used in bone TE, is dexamethasone (DEX). DEX is a type of corticosteroid that is
routinely used for the treatment of disorders such as rheumatoid arthritis, skin diseases such as psoriasis, severe
allergies, asthma and chronic obstructive lung disease. Research has demonstrated that delivering DEX to osteoblasts
can enhance osteoblast differentiation and accelerate bone regeneration [193]. In this line, Qi Gan et al. have shown
that a suitable dosage of DEX can enhance BMP-2-induced osteoblast differentiation. To test the osteoinductive
effect of DEX, pH-responsive CHI-functionalized mesoporous silica NPs (CHI-MSNs) were designed for dual-
delivery of BMP-2 and DEX. The MSNs were prepared by a CTAB-templated sol-gel method and further
functionalized by CHI by glycidoxypropyltrimethoxysilane crosslinking. Since DEX is a small molecule, it was
encapsulated in the mesopores, leaving BMP-2 to be in the CHI coating. The CHI-MSNs were shown to release
bioactive BMP-2. The released DEX was efficiently endocytosed. Furthermore, the release of DEX was controlled
with intracellular pH. This dual delivery system employed the synergism of BMP-2 and DEX outside and inside
the cell, and was shown to significantly stimulate osteoblast differentiation and bone regeneration in vitro and in
vivo [194].
Future perspective
An increasing awareness of how nanoscale features of bone tissues are essential to their unique properties has led to
vast efforts to use cutting-edge nanotechnology for replacing, imaging or regenerating bone. These efforts have led
to new breakthroughs in understanding and engineering bone tissue. However, numerous challenges remain.
Generation of vascularized and innervated bone tissue is considered one of the greatest hurdles, which current
nanofabrication techniques still fail to address. Mimicking the multiscale ‘organized chaos’ of bone tissues including
cells and the hierarchical ECM requires the use of nanomaterials and nanotechnologies to be combined with

advanced fabrication techniques such as multimaterial bioprinting. The challenges in the way of such combinations
are the different mechanical characteristics of relatively hard materials, which are appropriate for bone ECM
modeling and hydrogels commonly used in bioprinting. The advancement of high-resolution printing techniques
and development of novel bioinks are expected to cause major advancement in the field of bioprinting of hierarchical
bone tissue in the future.
It is now well accepted that in the future, medicine will be more patient-specific and personalized. Currently,
internet companies harvest massive quantities of information on consumer populations with powerful algorithms
for influencing advertising and subsequent buying decisions; however, medical treatment decisions are made based
on comparatively miniscule information. Combining big-data science, genomics, proteomics – among others, with
advances in nanotechnology could change the field of TE in unforeseen ways [195]. For instance, in immunology, the
unique biology of a patient is inescapable, but small molecule drugs are developed for diseases rather than patients.
The use of patient specific cells such as MSCs and the currently understudied iPSCs for bone TE is a first step
in the direction of personalized medicine and as more patient data can be collected and processed at lower costs,
bone TE approaches will become more tailored to the individual. In addition, engineering at scales lower than
10-9 m by influencing the electronic environment of atoms and molecules, otherwise known as picotechnology,
may be important for achieving the proper organization of collagen and mineral phases to mimic the natural
complex structure of bone [196]. Recently, it was discovered that addition of chiral amino acids to calcium carbonate
could influence crystal morphology and handedness, similar to what is seen in the natural helical structures of
bone [48]. Addition of small molecules with different electron distributions could be considered a primitive example
of engineering nanostructures, from ‘below’.
The personalization of medicine and picotechnology will likely go hand in hand for enhancing the success of
nanomedicine and reducing the potential toxicity of nanomaterials. For instance, the spontaneously formed protein
corona around NPs will drastically influence their biological identity and fate as vehicles for drug delivery. NPs
which favor adsorption of complement proteins will be eliminated prior to reaching targeted tissues relative to NPs
with some measure of stealth ability due to their surface properties, such as electron distribution (picotechnology).
The recent finding that NP corona composition varies by patient and disease type highlights the complexity that
nanomedicine researchers increasingly must embrace [197]. More patient-specific information as well as knowledge
of NP properties down to the smallest scales will change nanomedicine approaches in the future.
At last, since bone TE scaffolds have yet to mimic the unique mechanical properties of bone that result from
its multiscale structure the appeal of decellularized bone matrices in contrast to bottom-up scaffold design has
emerged [198]. An increased understanding of the dispersal of nanomaterials in a solid phase and the interfacial
relationships between both phases that result in force transmission will be required to create tissues that can
mimic the natural strength and toughness of bone, from the bottom (nanoscale) up, possibly with the aids of
picotechnology, advanced computer simulations and data science.
To validate the compatibility and efficacy of the nanoengineered systems for bone engineering, enhanced
evaluation techniques are required. To mimic the in vivo conditions, testing under more realistic conditions
including interstitial flow, multiple cell types with 3D organization, and amalgams of body liquids such as growth
factors and serum proteins as opposed to relying solely on defined medium, will surely increase the likelihood of
success of therapeutics in vivo.
Financial & competing interests disclosure

The authors gratefully acknowledge funding from American Heart Association (grant number 17SDG33660925) (PPSS Abadi).
MJ Hill was supported by American Heart Association (grant number 19POST34450219). M Mahmoudi thanks The Gillian Reny
Stepping Strong Center for Trauma Innovation and Precision Health Program at Michigan State University. The authors have no
other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.

Executive summary
The multiscale structure of bone
• Bone has a complex nanostructured architecture consisting of a collagen and hydroxyapatite composite, which
serves as extracellular matrix for bone and bone forming cells.
• This natural matrix can serve as inspiration for bone tissue engineering and nanomaterials can be used to
enhance local imaging or drug delivery in regenerating bone.
Advances in nanomaterials for bone regeneration
• 1D, 2D and 3D nanoparticles have been used as filler materials to enhance the mechanical properties of polymers
and to mimic those of the natural bone.
• Incorporating nanoscale architecture is important to mimic the natural combined stiffness and toughness of bone.
• Numerous nanoparticulate strategies have been developed for delivering bone regenerative molecules such as
BMP2, VEGF, dexamethasone, statins or resveratrol.
• Controlled release of these nanoencapsulated agents alone or in combination therapies has resulted in increased
osteogenic differentiation or bone defect healing.
Future perspective
• Complex fabrication techniques such as bioprinting require the discovery of new methodologies for printing
mixtures of disparate materials that can mimic the native structures of bone but will play an increasing role in
bone regeneration in the near future.
• More attention must be paid to the nanoparticle protein corona and nanoparticle biological identity in realistic
clinical situations.
• More realistic in vitro culturing conditions will become more widespread with the increasing awareness that flat
smooth culturing surfaces, quiescent flow conditions and defined mediums cannot recapitulate the in vivo milieu.
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Nanomaterials for bone tissue regeneration:

Keywords: bone • drug delivery • nanomaterials • nanotechnology • tissue engineering

The multiscale structure of bone

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10-1 10-3 10-6 10-9

Acc.V Spot Magn Det WD 10 µm

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Table 1. Bone substitute materials in clinical use.

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Advances in nanomaterials for bone regeneration

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Phospholipid cell membrane

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Financial & competing interests disclosure

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