Materials Today Bio 22 (2023) 100741

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Materials Today Bio

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Microenvironment-targeted strategy steers advanced bone regeneration

Shuyue Hao a, Mingkai Wang a, Zhifeng Yin c, Yingying Jing a, ***, Long Bai a, **, Jiacan Su a, b, *
a
Institute of Translational Medicine, Shanghai University, Shanghai, 200444, China
b
Department of Orthopedic Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200444, China
c
Department of Orthopedics, Shanghai Zhongye Hospital, Shanghai, 201941, China

A R T I C L E I N F O A B S T R A C T

Keywords: Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery.
Bone regeneration Traditional strategies in bone tissue engineering have focused primarily on mimicking the extracellular matrix
Physiological microenvironment (ECM) of bone in terms of structure and composition. However, the synergistic effects of other cues from the
Chemical microenvironment
microenvironment during bone regeneration are often neglected. The bone microenvironment is a sophisticated
Physical microenvironment
Biomaterials
system that includes physiological (e.g., neighboring cells such as macrophages), chemical (e.g., oxygen, pH),
and physical factors (e.g., mechanics, acoustics) that dynamically interact with each other. Microenvironment-
targeted strategies are increasingly recognized as crucial for successful bone regeneration and offer promising
solutions for advancing bone tissue engineering. This review provides a comprehensive overview of current
microenvironment-targeted strategies and challenges for bone regeneration and further outlines prospective
directions of the approaches in construction of bone organoids.

1. Introduction is a complex structural and biological system that can be distinguished

into three parts according to its function and components it contains:
With the development of an aging society, the incidence of bone- physiological microenvironment, chemical microenvironment, and
related diseases has dramatically increased worldwide [1]. Bone has physical microenvironment (Fig. 1). Physiologically, it contains immune
the potential to heal and regenerate, but the inability to complete bone cells and endothelial cells, and the physiological microenvironment
healing on its own in large segmental bone defects (>2 cm critical size or supports the effective functioning of bone tissue through the integration
>50% loss of bone girth) due to trauma, infection, tumor resection or of cellular energy metabolism exhibiting a tendency to immunosup­
developmental malformations is a serious problem in orthopedic treat­ pression and protection of hematopoietic stem cell components. Recent
ment [2]. Although autologous bone grafting is the “gold standard” for studies have shown that M2-type macrophages can improve the physi­
clinical bone repair, it still suffers from secondary surgical injuries, se­ ological microenvironment at the site of injury and promote osseointe­
vere donor area injuries and complications [3]. Bone tissue engineering gration by converting the inflammatory microenvironment into an
to design material scaffolds, cells and signaling molecules to induce new anti-inflammatory microenvironment [6]. Chemically, nutrients such
bone tissue formation by eliminating risks associated with autografting as oxygen and pH as well as various signaling molecules released by
has been a key approach in current bone repair treatments [4]. A variety different cell types are essential for cell survival and function. Li et al.
of different cells in bone (including bone-associated cells, immune cells, attempted to enhance the oxygen supply to bone defect areas, and the
endothelial cells, etc.) share the same microenvironment and play an hypoxic environment was improved, showing a significant increase in
important role in osteogenesis. However, traditional bone tissue engi­ osteogenic capacity [7]. Physically, appropriate external stimuli can also
neering studies have focused only on the effects of extracellular matrix influence cell fate, providing new ideas for bone tissue engineering to
(with collagen and hydroxyapatite as the main components [5]) on treat bone defects. By comparing osteoblasts in static culture to those
osteogenesis, ignoring the effects of other cellular and non-cellular cues subjected to mechanical stimulation, Brady found that paracrine factors
in the local microenvironment on osteogenesis. Bone microenvironment secreted after mechanical stimulation significantly enhanced migration,

* Corresponding author. Institute of Translational Medicine, Shanghai University, Shanghai, 200444, China.
** Corresponding author.
*** Corresponding author.
E-mail addresses: jingy4172@shu.edu.cn (Y. Jing), bailong@shu.edu.cn (L. Bai), drsujiacan@163.com (J. Su).

https://doi.org/10.1016/j.mtbio.2023.100741
Received 11 May 2023; Received in revised form 26 June 2023; Accepted 19 July 2023
Available online 21 July 2023
2590-0064/© 2023 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Hao et al. Materials Today Bio 22 (2023) 100741

Fig. 1. The diagram of bone tissue cell microenvironment. Bone microenvironment can be divided into three parts: physiological microenvironment, chemical
microenvironment and physical microenvironment.

proliferation, and osteogenesis [8]. In conclusion, engineering of bone variety of tissue cells such as adipose, bone, cartilage, muscle, nerve,
microenvironment has emerged as a promising research direction for the cardiac muscle, endothelium, etc. [14]. It is also the most widely studied
design of bone biomaterials. In this review, we elucidate the dynamic seed cell in bone tissue engineering. Research has supported that bio­
regulatory role of cellular and peripheral cues in the bone microenvi­ materials can achieve stem cell osteogenic differentiation by modulating
ronment, systematically classify the various cues of the bone microen­ nanoscale surface morphology in the absence of additional osteogenic
vironment, and summarize the regulatory role of each class of cues on supplements [15]. For example, Zhang et al. designed a nanocomposite
bone repair. Finally, we discuss the challenges faced in constructing membrane that mimics endogenous potentials, and a proper electrical
bone microenvironments and future directions for incorporating bone microenvironment greatly promotes bone regeneration [16]. In this
organoids. This work provides solid theoretical support for the con­ review, we focus on the field of biomaterials to modulate heterogeneous
struction of advanced bone microenvironments and offers new avenues osteogenic differentiation. The composition of bone tissue ECM can be
for the development of bone tissue engineering. categorized into organic and inorganic components. The organic
component primarily consists of collagen, while the inorganic part is
2. Traditional strategy mimicking ECM mainly composed of hydroxyapatite (HA). Together, these components
constitute the ECM and possess specific biological functions [17].
ECM is regarded as a novel regenerative material that not only Mimicking the ECM of bone tissue to promote cellular function and bone
provides a physical scaffold for cells in tissues but also regulates regeneration is a reasonable and feasible approach [18]. In this section,
numerous cellular processes, including growth, migration, differentia­ the traditional ECM-based repair strategy will be discussed in detail.
tion, and morphogenesis [9–12]. The ECM is adynamic structure that
undergoes controlled remodeling continually. Maintaining its integrity
and homeostasis is essential for tissue growth and organ physiology, 2.1. Mimicking component of ECM
while the loss of ECM components or structural changes may result in
disease development. A key distinction between bone and other tissues 2.1.1. Collagen
lies in the high percentage of matrix components and the low percentage The ECM of bone tissue is abundant in collagen, which can be cross-
of cellular components in bone. Heterogeneity is one of the fundamental linked via functional groups to generate materials with tailored me­
features of ECM, and numerous studies have shown that different chanical or biological properties, making it highly applicable in tissue
physical properties of biomaterials can influence the differentiation fate engineering [19,20]. Collagen scaffolds exhibit favorable biological
and cellular behavior of stem cells [13]. Mesenchymal stem cells have properties, such as hydrophilicity, low antigenicity, and biodegrad­
multidirectional differentiation potential and can differentiate into a ability [21,22]. However, singular collagen scaffolds lack sufficient
mechanical strength, leading to the incorporation of other biomaterials

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Fig. 2. Biomaterials mimic the component and structure of ECM for bone repair. A) Collagen-HA co-assembled scaffolds loaded with immune ions are pro­
grammed to promote osteogenic gene expression in BMSCs. Reproduced and adapted with permission [25]. Copyright 2022, Elsevier. B) Bionic HA scaffold regulates
the cellular activity of MSCs and enhances the ossification process to promote bone repair. Reproduced and adapted with permission [37]. Copyright 2021, Elsevier.
C) Age-specific dECM provides different local environments for BMSCs and promotes microfracture repair in vivo. Reproduced and adapted with permission [40].
Copyright 2021, American Chemical Society. D) HA scaffold with natural bone-mimicking nanopores to improve bone regeneration efficiency. Reproduced and
adapted with permission [41]. Copyright 2022, Springer Nature.

like calcium phosphate, bioceramics, and polymers during scaffold unique bilayer structure that effectively inhibits epithelial cell infiltra­
design [23,24]. Zhong et al. developed a collagen-HA co-assembled tion into the defect site and fosters osseointegration [30,31]. To enhance
scaffold, which exhibited strong bioactivity and increased the osteo­ the resistance to degradation and to expand the efficacy of absorbable
genic differentiation potential of BMSCs with a similar composition to collagen membranes, many chemical and physical cross-linking
ECM (Fig. 2A) [25]. Xia’s group further combined collagen with calcium methods have been applied to manufacture cross-linked collagen fi­
phosphate to create a biomimetic composite coating that enhanced bers, including UV, glutaraldehyde, hexamethylene diisocyanate,
cellular affinity and in vivo absorption, supporting osteoblast activity diphenylphosphoryl azide, and enzymatic cross-linking [32]. The prin­
and new bone formation [26]. ciple is to extract collagen into single fibers, which are then recon­
Numerous membrane types have been utilized in guided bone stituted and cross-linked [33].
regeneration techniques. Collagen membranes, the most prevalent, offer
resorbability and in vivo degradation, minimizing inflammatory re­ 2.1.2. Hydroxyapatite
sponses associated with foreign body scaffolds [27,28]. Presently, HA possesses exceptional biocompatibility and mechanical proper­
collagen membranes are frequently employed in the restoration of ties and demonstrates stable interfacial binding within bone tissue [34,
periodontal defects, obviating the need for secondary surgery [29]. 35]. HA surfaces can directly bind to new bone, support osteoblast
Bio-Gide®, a prominent commercial collagen membrane, exhibits a adhesion, growth, and differentiation, and facilitate new bone

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deposition by means other than adjacent living bone [36]. Conse­ superior physicochemical properties in terms of bionanotechnology, and
quently, HA is extensively employed as a bone substitute, metal implant nanoscale ECM scaffolds are emerging as a developmental direction to
coating, tissue engineering scaffold, and drug delivery carrier, earning promote bone repair potential [48]. Li et al. introduced HA nano­
the designation of a “bioactive material.” Recent studies have revealed particles into bone graft materials by varying the sintering temperature
that HA scaffolds can function as osteogenic mediators during osteo­ to form nanoscale pore scaffolds similar to natural bone [41]. The results
genesis via the ZBTB16 and WNT signaling pathways [37]. This phe­ demonstrated that the mechanical strength, cell proliferation, and dif­
nomenon may be attributed to the integrated microenvironment of ferentiation rates of the scaffolds with nanopores were significantly
specific bioactive materials providing an osteogenic advantage for os­ increased (Fig. 2D).
teoblasts (Fig. 2B). Ohgushi et al. further corroborated the interaction Electrospinning, a versatile technique facilitating the fabrication of
between HA and bone tissue, demonstrating that the HA surface can nanofibers with controlled diameters and distinct structures, is
support cell differentiation and facilitate cell and bone formation [38]. employed to construct polymeric nanofiber scaffolds for bone tissue
HA coating combined with metal implants is a widely employed bone engineering due to its structural resemblance to tissue ECM, straight­
repair strategy. Yamada et al. successfully generated forward setup, and cost-effective operation [49]. Fibers generated
nano-polycrystalline HA on the surface of micro-coarsened titanium (Ti) through this method exhibit high homogeneity and mechanical strength,
[39]. During the healing phase, the HA-coated Ti significantly enhanced forming porous scaffolds with an elevated surface-to-volume ratio. A
the strength of bone-implant integration compared to uncoated diverse array of synthetic biodegradable polymers and natural macro­
micro-roughing Ti. Thus, the osteogenic scaffold can be optimized by HA molecules have been utilized to create fibrous scaffolds. While electro­
coating to improve bone-implant integration and pinpoint specific bone spun natural polymers demonstrate enhanced hydrophilicity, synthetic
morphogenesis parameters. polymers possess greater robustness and superior mechanical properties.
Synthetic electrospun fiber scaffolds can be further functionalized to
augment cellular activities by incorporating compounds or morphogens
2.2. Mimicking the structure of ECM
such as HA, glycosaminoglycan, and recombinant human BMP-2.
Controlled release of these compounds from the scaffolds can be ach­
2.2.1. Decellularized ECM scaffold
ieved through the meticulous blending of various synthetic biodegrad­
The decellularized extracellular matrix (dECM) is the extracellular
able polymers. Providing both topographical and biochemical signals,
part of the tissue that is highly bioactive, low immunogenic and well
the electrospun nanofibrous scaffolds may offer an optimal microenvi­
biodegradable. The main advantage of dECM is that biomolecules in
ronment that mimics native ECM for seeded cells [50]. Li et al. devel­
ECM are retained, supporting cell growth and viability. However, the
oped a novel nanoparticle-embedded electrospun nanofiber scaffold for
decellularization process presents challenges, particularly maximizing
controlled dual delivery of BMP-2 and DEX [51]. In vivo osteogenesis
cellular material removal while minimizing ECM damage. To evaluate
studies showed that controlled dual delivery of BMP-2 and DEX pro­
dECM’s potential for promoting the osteogenic process, Hashimoto et al.
motes calvarial bone defect repair; DEX effectively promotes early
compared MSC differentiation on three-dimensional dECM and two-
calcified bone formation, while BMP-2 facilitates long-term new bone
dimensional tissue culture polystyrene discs, observing significantly
formation. In conclusion, dual drug-loaded nanofiber scaffolds may be
higher alkaline phosphatase (ALP) activity in MSCs grown in decellu­
ideal candidates for bone tissue engineering.
larized bone matrix [42]. This finding suggests that the decellularized
bone matrix facilitates early osteogenic differentiation of MSCs.
Furthermore, when rabbit dECM from different age groups, such as 2.3. Limitations of traditional strategy mimicking ECM
neonates, children, and adolescents, was co-cultured with BMSCs,
BMSCs exhibited distinct cell morphology, roundness, and proliferation A primary focus of conventional bone tissue engineering is the
characteristics in vitro (Fig. 2C) [40]. To enhance the osteoinductivity of development of biomaterials that emulate the composition and structure
dECM, researchers have tried to incorporate different materials such as of the ECM to modulate bone regeneration. The method of mimicking
HA, glass-ceramics and Ti in dECM scaffolds, all of which proved to bone ECM is superior because it is simple and similar to bone ECM.
enhance osteogenic differentiation and bone repair [43,44]. These However, in many cases, the importance of the local cellular microen­
findings indicate that dECM, as a biomaterial, holds the potential for vironment in injury repair is often overlooked, and this simple approach
promoting bone regeneration. of mimicking bone-like structures may lack the physiological, chemical,
and physical cues that provide cells with the ability to form bone
2.2.2. Synthetic ECM scaffold [52–54]. It is well known that key components such as collagen and HA
Hydrogels, polymeric materials with water solubility, form three- provide the environment for osteogenic differentiation of stem cells in
dimensional network structures through cross-linking reactions be­ the bone structure, but natural ECM contains growth factors that are also
tween hydrophilic polymers. Owing to their biomimetic properties, necessary for normal bone formation. The dECM retains most of the
hydrogels can emulate the ECM internal structure and provide support original structure in biological tissues, yet some inactivation processes
for cells to perform physiological functions [45]. Many natural or syn­ during formation irreversibly destroy the active components, such as
thetic materials are used as hydrogel base units for bone repair. In the proteases and growth factors. It has been shown that different oxygen
last decade, hydrogels have been developed in various implantable concentrations, pH ranges, and appropriate stress stimulation can pro­
forms to address different types or locations of bone diseases [46]. mote osteoblast proliferation and matrix secretion. All these studies
Implantable hydrogels are widely used due to their mechanical strength suggest that we need to focus equally on the non-ECM part of the
and ease of shape adjustment at pre-designed sites. In-situ injectable microenvironment.
hydrogel scaffolds have excellent sol-gel properties allowing filling of Pericellular interactions in the microenvironment are dynamic, and
defective sites without traditional major surgery [47]. Thus, hydrogels their dynamics not only act as a reservoir for their signaling molecules
are considered promising candidates for bone tissue engineering. Due to but also mediate signals from other sources. These signals include a
the nanoscale size of natural tissues or organs, nanomaterials have variety of factors, including cell-cell, cell-growth factor, pericellular

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chemical changes, and physical stimuli [55]. These dynamic processes inflammatory phase to promote angiogenesis and stimulating osteo­
ultimately determine the equilibrium and potential aberrations of the clastogenesis during the repair phase to maintain bone homeostasis
tissue. In traditional strategy described above, researchers focus more on [66–68].
the simulation of the initial infrastructure, ignoring the signals that cells
must receive from the environment as they develop within or on the 3.1.3. Physical microenvironment
scaffold to achieve an ordered, specific genetic program that ultimately Except for the physiological and chemical cues mentioned above,
results in tissue/organ formation. Therefore, we believe that designing cells interact with and respond to physical stimuli (including mechani­
the cellular microenvironment requires more attention to the physio­ cal, photo, thermal, electrical, magnetic and acoustic stimuli) [69].
logical, chemical and physical cues in the microenvironment rather than Researchers have adopted this principle to achieve efficient bone repair
solely focusing on ECM features, which would be a revolutionary chance through the synergistic action of external field stimulation and respon­
for the tissue engineering field. sive scaffolds. Mechanical stimuli can affect cell behavior through
mechanotransduction. Photic stimuli (e.g., near-infrared light) can
3. Bone microenvironment upregulate osteogenic gene expression to enhance bone regeneration
[70]. Thermally responsive materials respond to temperature and serve
3.1. Composition of bone microenvironment to enhance cellular activity [71]. Electrical stimulation promotes
migration, proliferation and differentiation of osteoblasts [72]. One
The bone microenvironment consists of three key components: possible mechanism is that electrical stimulation upregulates intracel­
Physiological (e.g., neighboring cells such as macrophages), chemical (e. lular calcium concentration and subsequently regulates osteogenesis via
g., oxygen, pH), and physical factors (e.g., mechanics, acoustics). These the calmodulin pathway [73,74]. Magnetic stimulation therapy can be
components work in concert to provide functional support for bone classified into two types: static magnetic fields (SMF) or electromagnetic
growth and development [56]. fields (EMF), although the underlying biological mechanisms are still
elusive, in vitro studies have shown that it can significantly enhance
3.1.1. Physiological microenvironment osteoblast differentiation [75,76]. Acoustic fields induce material
The physiological well-being of bone is determined not only by the deformation through acoustic radiation forces [77,78]. Although
dynamic equilibrium between osteoblasts and osteoclasts but also by acoustic field stimulation has not been extensively utilized for engi­
other cells in the local microenvironment, such as immune and endo­ neering the bone microenvironment, it possesses considerable potential.
thelial cells. Immune cells, encompassing lymphocytes (B and T cells),
macrophages, and dendritic cells, have been demonstrated to secrete
active factors that impact bone formation [57,58]. For instance, mac­ 3.2. Importance of bone microenvironment
rophages promote osteoblastogenesis by releasing interleukin-18 (IL-18)
[59,60]. In the event of bone fractures, immune cells, particularly Over the past decade, comprehensive research has enhanced our
macrophages, are involved throughout the entire healing process, understanding of the effects of biochemical and biophysical cues on
providing defense against pathogens and releasing a diverse array of cellular behavior. The bone microenvironment, as a highly dynamic and
effectors to regulate bone remodeling. The immune system also con­ complex network, regulates the biological behavior of cells primarily
tributes to the development of pathological and chronic conditions in through the following mechanisms: 1) providing physiological and
osteoporosis [61]. Endothelial cells, which form the blood vessel linings, biochemical signals to cells; and 2) providing physical and stimulatory
along with pericytes, are crucial for bone tissue homeostasis by pro­ signals to cells. The principal challenge in comprehending the bone
ducing paracrine signaling molecules known as angiocrine factors [62]. microenvironment lies in adapting to its dynamic properties, where
Research has indicated that endothelial cells secrete several signaling cellular feedback plays a significant role. However, the spatial and
molecules via paracrine interactions, such as platelet-derived growth temporal variations of these cues, as well as their independent or col­
factor (PDGF)-BB, vascular endothelial growth factor (VEGF), and lective actions with cells in forming intricate microenvironmental net­
BMP-2, which play an active role in the regulation of bone homeostasis works, remain unclear. Gaining insight into the influence of these
[63]. dynamics on the regulation of cellular behavior is crucial for enhancing
the development of bionanomaterials that can be employed in designing
3.1.2. Chemical microenvironment cellular microenvironments and facilitating numerous biomedical ap­
The chemical microenvironment of bone contains numerous soluble plications. Below we describe how different cues guide changes in the
factors, such as nutrients (e.g., oxygen, pH) and signaling molecules (e. bone microenvironment and provide tools to interpret the
g., enzymes, cytokines). Oxygen is the most easily depleted nutrient, and microenvironment.
its insufficient supply has impeded the success of engineering intricate
and sizable tissue constructs. Among soluble signaling molecules, cyto­
4. Engineered bone microenvironment
kines have garnered significant attention in engineering biomimetic
cellular microenvironments. BMPs, a group of structurally similar,
The coordinated interplay of physiological, chemical, and physical
highly conserved functional proteins, belong to the TGF-β superfamily
signaling in the bone microenvironment is crucial for regulating cellular
[64]. BMP-2, a critical factor in osteogenesis, induces the differentiation
processes in both developing and mature skeletons [79]. Recent progress
of undifferentiated MSCs into chondrocytes and osteoblasts, which
in bone biology has resulted in a growing interest in using biomaterial
participate in bone and cartilage growth, development, and recon­
scaffolds and bioreactors to engineer microenvironments that mimic
struction processes [65]. Angiogenesis and osteogenesis are intimately
natural bone functions [80]. In the following sections, we systematically
connected, with VEGF performing distinct functions at various stages of
review how bone biology and tissue engineering have been integrated to
bone formation, such as recruiting macrophages during the
create controllable microenvironments at multiple levels (Table 1).

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Table 1
Microenvironment-targeted strategy.
Types Pathway Materials Function Ref.

Physiological Immunomodulation Titanium implant Micro-rough and hydrophilic surfaces promote the release of [81]
Microenvironment anti-inflammatory factors from macrophages
Polyethylene terephthalate Macrophages adhering to hydrophilic and anionic surfaces [82]
selectively produce anti-inflammatory cytokines
Magnesium containing microspheres Mg2+ release upregulates anti-inflammatory genes and triggers [83]
immune regulation
miR-181b exosomes Exo-181b activates the PRKCD/AKT signaling pathway to [84]
promote M2 polarization
Angiogenesis Sulfated chitosan scaffold Dual-module scaffold continuously releases rhBMP-2 and VEGF, [85]
synergistically promoting osteogenesis and angiogenesis
Nanofibrous gelatin-silica hybrid scaffold Vascular-mimicking microchannel scaffold promotes rapid [86]
vascularization and bone regeneration
Energy metabolism Bioenergetic-active material scaffold Scaffold degradation fragments can increase mitochondrial [87]
membrane potential to accelerate bone regeneration
GelMA hydrogel Mg2+ increases cellular bioenergy levels to promote [88]
osteogenesis induction
Citrate composite support Citrate-mediated elevation of cellular energy levels supports [89]
metabolic osteogenesis
Chemical Oxygen Liposomal/hydrogel complexes ROS-responsive hydrogel releases oxygen to promote bone [90]
Microenvironment regeneration
PCL/nHA/CaO2 scaffold The bionic scaffold releases oxygen continuously to promote [91]
bone defect repair
Bioactive glass/collagen–glycosaminoglycan Co2+ mimics hypoxic signaling to activate the HIF pathway to [92]
scaffold support osteogenesis
pH MOF@CaP nanoplatform Nanoplatform mimics low pH environment to enhance bone [93]
regeneration and capacity
Custom Titanium implant The alkaline microenvironment mediates the osteogenic [94]
differentiation of stem cells and promotes new bone formation
Enzymes and Chondroitin Sulfate/Polyethylene Glycol Matrix metalloproteinase-mediated degradation of hydrogels [95]
Cytokines hydrogel regulates stem cell differentiation
GelMA hydrogel Mineralized alkaline phosphatase enhances the osteogenic [96]
differentiation potential of BMSCs
Hyaluronic acid hydrogel Nanozymes mediate O2 production from endogenous H2O2 and [97]
provide a microenvironment for osteogenesis
Physical Mechanical forces Polydimethylsiloxane substrates Stiff materials have a higher osteogenic potential than soft [98]
Microenvironment materials
The flow loop apparatus Sustained low-velocity shear stress stimulates the expression of [99]
osteogenic markers in stem cells
Gelatin hydrogel Reversibly connected, highly elastic hydrogel adapts to [100]
dynamic stresses and supports bone regeneration processes
Temperature light-responsive poly (N-isopropylacrylamide- co Ultraviolet light stimulates the release of dexamethasone from [101]
-nitrobenzyl methacrylate) photosensitive materials to promote bone regeneration
Poly (vinyl alcohol) fibers Thermoresponsive fibers improve the toughness of calcium [102]
phosphate cement and enhance bone repair
Biphasic calcium phosphate scaffold Regulates drug release by changing the light source wavelength [103]
to promote bone repair
Electric field Whitlockite scaffold Scaffolds provide an endogenous electric field to the defect site [104]
and inhibit the activity of osteoclasts.
Triboelectric nanogenerator Mediated proliferation and differentiation of osteoblasts by [105]
electrical stimulation
Magnetic field Poly(lactide-co-glycolide) scaffold Magneto-thermal accelerated degradation behavior of magnetic [106]
scaffolds under alternating magnetic fields
Static magnetic field Magnetic fields can regulate the direction of osteoblast growth [107]
Acoustic Collagen sponge In situ recruitment of osteogenic factors by ultrasonically [108]
shocked microbubbles
Acoustically responsive scaffold/hydrogel Pulsed ultrasound recruits BMSCs for bone repair [109]
Programming design Poly(aryl-ether-ether-ketone) (PEEK) implant Programmed surface coating to release osteogenic drugs over [110]
time
GelMA hydrogel Programming a two-factor delivery system to match the bone [111]
repair healing process

4.1. Physiological microenvironment reciprocal relationship between bone cells and the immune system
[112]. Numerous factors typically categorized as immunological agents,
Maintaining the homeostasis of the physiological microenvironment such as interleukins (e.g., IL-6, IL-11, IL-17, and IL-23) [113–115],
of bone is paramount for preserving the vitality and functionality of tumor necrosis factor (TNF)-α [116], RANK and its ligand RANKL [117],
bone-related cells, and is a crucial aspect of bone regeneration [18]. This nuclear factor of activated T cells (NFATc1) [118] have been found to
review focuses on investigations concerning immune regulation, exert a significant influence on osteoclasts and osteoblasts. In particular,
angiogenesis, and energy metabolism within the bone physiological macrophages of the innate immune system undergo diverse polarization
microenvironment. states, with M1 macrophages exhibiting pro-inflammatory behavior and
M2 macrophages demonstrating anti-inflammatory characteristics
4.1.1. Immune regulation [119]. These macrophage phenotypes and their polarization are indis­
Osteoimmunology is a specialized area of research that examines the pensable for biomaterials to stimulate bone regeneration [120].

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Fig. 3. Biomaterials mimic the immune environment to promote bone repair. A) Sustained release of Zn2+ from bioactive ceramics induces M2 macrophages,
further promoting periosteal-derived progenitor cells-mediated bone regeneration. Reproduced and adapted with permission [126]. Copyright 2021, Springer Na­
ture. B) Blood-derived hybrid hydrogels promote bone healing by reprogramming the immune environment. Reproduced and adapted with permission [127].
Copyright 2022, American Chemical Society. C) Nanocomplexes deliver miR-21 and IL-4 in layers to promote macrophage polarization to the M2 phenotype.
Reproduced and adapted with permission [128]. Copyright 2021， Wiley-VCH GmbH. D) Mg–Al laminated double hydroxide coating induces macrophage polar­
ization to an M2 anti-inflammatory phenotype that exhibits good osteogenic and angiogenic potential. Reproduced and adapted with permission [129]. Copyright
2021, Elsevier. E) Dual-effect coated Ti screws modified with Zn2+ and BMP-2 co-regulate the bone immune microenvironment at the bone-implant interface.
Reproduced and adapted with permission [130]. Copyright 2022, Springer Nature.

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S. Hao et al. Materials Today Bio 22 (2023) 100741

Biomaterials have been recognized as a promising approach for properties to natural bone. For instance, natural collagen, fibrin gels,
regulating bone regeneration by altering surface morphology [81,121], and bone cement exhibit good osteoconductivity, but are fragile and
stiffness [122], porosity and pore size [123], hydrophilicity [124] and have poor mechanical properties. In contrast, materials such as bioactive
surface charge [82] through physical and chemical modifications, thus glass and polylactic acid have good degradation properties but limited
affecting macrophage polarization, phenotype and function and hydrophilicity and histocompatibility. Given these limitations, using a
contributing to the M1 to M2 phenotype transition. Biomaterials can single material for vascularized bone regeneration is challenging.
also provide bioactive molecules, including bioactive ions [83], drugs Tang et al. developed a dual modular scaffold that was designed to
[6], cytokines [125], and microRNAs(miRNAs) [84], which can activate release different growth factors with different characteristics while
anti-inflammatory signaling directly or indirectly. For instance, Zinc is maintaining their biological activity to promote angiogenesis and oste­
an essential trace element in bone formation and plays a critical role in ogenic capacity (Fig. 4A) [133]. Apart from the scaffold structure and
osteogenesis and immune processes. Therefore, it is frequently used to composition, cytokines and ionic components also play critical roles in
modify biological materials to improve bone immunomodulatory ca­ angiogenesis and bone regeneration. Therefore, scaffold properties can
pacity. Huang et al. investigated the role of Zn2+ in osteoinduction and be improved by incorporating these components to enhance vascular­
immunomodulation by combining it with a phosphate/poly(L-lactic ized bone tissue engineering. Angiogenesis is mediated by a complex
acid) scaffold (TCP/PLLA) [126]. Compared to a single scaffold, the interplay of molecular signals involving various cytokines and ions,
sustained-release scaffold of Zn2+ showed a stronger effect on bone including VEGF, Angiopoietin (ANG), Silicon (Si), Magnesium (Mg), and
differentiation while inducing macrophage polarization toward the M2 Calcium (Ca). BMP-2, which is the most potent osteogenic agent, was
phenotype, resulting in a favorable osteogenic microenvironment loaded into poly(lactic acid)-glycolic acid copolymer tubes by Bouyer
(Fig. 3A). et al., resulting in intact defect bridging and vascularized bone tissue
The incorporation of biomaterials with cytokines has emerged as a formation [134]. This indicates that BMP-2 has the potential to promote
direct and effective strategy for promoting bone regeneration. Human vascularized osteogenesis. Additionally, dimethyl oxalyl glycine
blood is naturally rich in various cytokines, and extensive bone injury is (DMOG) has gained considerable attention in vascularized osteogenesis.
often accompanied by a poor regenerative microenvironment, especially Bionic scaffolds loaded with DMOG have been shown to exhibit excel­
an unfavorable immune microenvironment. Studies have shown that lent angiogenesis and stable bone formation both in vitro and in vivo
autologous blood-derived hydrogels can create the right conditions for (Fig. 4B) [135]. Ha et al. co-loaded DMOG, an osteogenic factor, and a
osteogenesis by reprogramming the skeletal immune microenviron­ pro-angiogenic factor into nanofibrous scaffolds with an interconnected
ment, showing great promise in the field of personalized regenerative perfusable microchannel network (Fig. 4C) [86]. The microchannel
medicine (Fig. 3B) [127]. To take a more refined perspective, Deng et al. structure provided the necessary foundation for nutrient transport and
selected the key cytokines IL-4 and miR-21, which inhibit inflammation improved degradation, serving as a model for in vivo pre-vascularization.
and delivered them to the site of injury in a programmed manner to The primary benefit of revascularization is the ability to achieve
modulate the bone immune microenvironment (Fig. 3C) [128]. Surface perfusion immediately after implantation, accelerating the development
modifications of bone implants, including coating (Fig. 3D) [129] and of the entire capillary network. In a rabbit model, it was shown that
molecular click (Fig. 3E) [130] methods, have also been utilized to revascularized grafts enhanced the formation of new bone and capil­
achieve the same effect. Among the cells of the adaptive immune system, laries. Tissue engineering commonly employs 3D printing technology as
regulatory T cells may be promising candidates for a positive regulatory a strategy. 3D bioprinting technology enables precise localization of
effect on fracture healing. Chen et al. showed that regulatory T cell biomaterials, biochemistry, and living cells. Inspired by this technology,
exosomes can significantly enhance bone repair, demonstrating that Miao et al. designed a nano-dynamic hydrogel scaffold with the aid of
regulatory T cells are promising and effective therapeutic agents for 3D printing. VEGF-decorated black phosphorus nanosheets (BPNSs) and
bone reconstruction, but the exact mechanism still needs to be discov­ DNA impart functionality to the hydrogel, enhancing angiogenesis and
ered [131]. osteogenic activity (Fig. 4D) [136].
Despite the obvious progress in the field of bone immunology, many The greatest challenge for angiogenic strategies is that newly formed
questions remain. For example, the immune system shares a variety of capillaries after stent implantation are transient and require continuous
transcription factors, signaling molecules, and membrane receptors supplementation with exogenous nutrients [137]. Therefore, an
during bone repair, and the underlying molecular mechanisms by which in-depth understanding of cellular dynamics, cellular microenvironment
it promotes bone regeneration remain unclear. Therefore, it is crucial to and cell-cell interactions may guide the design of next-generation
identify the molecular mechanisms by which osteoblasts and the im­ angiogenic scaffolds.
mune system interact. In addition, bone injury patients with concomi­
tant autoimmune diseases are commonly seen in the clinic, and 4.1.3. Energy metabolism
autoimmunity may affect bone healing [132]. Future research advances Cellular energy homeostasis involves the regulation of energy pro­
could personalize microenvironmental therapies to address the need for duction and consumption in cells during normal physiological processes,
clinical treatment. achieved through nutrient uptake and biosynthesis (Fig. 5A) [138,139].
Two main metabolic pathways that convert nutrients to adenosine
4.1.2. Angiogenesis triphosphate (ATP) for energy to support biosynthetic activities are
Bone tissue is heavily reliant on the vascular system to receive oxy­ glycolysis and oxidative phosphorylation. The skeleton requires a sig­
gen and nutrients, as it is highly vascularized. The skeletal system is nificant amount of ATP to maintain its health, normal differentiation,
known to receive a substantial amount of blood output from the heart, and physiological functions. Cells follow a strict mechanism to regulate
estimated to be 10–15%. Thus, angiogenesis serves a key function in metabolic fluxes to maintain metabolic homeostasis [140]. This involves
driving the development of bone tissue. Multiple approaches have been the regulation of gene expression, mRNA transcription and translation,
suggested to improve angiogenesis in bone tissue regeneration, such as and the expression of transporter proteins and metabolic enzymes in
administering angiogenic growth factors like VEGF and FGF, selecting response to extracellular factors such as hormones and intertissued
appropriate seed cells like stem cells or mature vascular cells, and signals [141]. In this way, metabolic activities and pathways are regu­
designing three-dimensional (3D) bionic scaffolds. The effectiveness of lated to support desired physiological functions. For example, osteoblast
tissue engineering repair is closely related to the design of the bioma­ progenitors increase glucose uptake by upregulating the expression of
terial, which creates an environment suitable for cell growth, adhesion glucose transporter protein 1 (GLUT1) in response to osteogenic signals,
and differentiation. To promote vascularized osteogenesis, scaffold thereby meeting the energy requirements for osteogenic differentiation
materials used for tissue engineering must possess similar biological [136]. Additionally, in a hypoxic in vivo environment, undifferentiated

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Fig. 4. Biomaterials enhance angiogenesis to promote bone repair. A) 26SCS-functionalized bimodular scaffolds deliver BMP-2 and VEGF for synergistic
osteogenesis and angiogenesis. Reproduced and adapted with permission [133]. Copyright 2020, Elsevier. B) 3D-printed two-factor delivery scaffolds sequentially
release DMOG and Sr ions that match the angiogenic and osteogenic processes. Reproduced and adapted with permission [135]. Copyright 2023, American Chemical
Society. C) Nanofibrous gelatin-silica hybrid scaffold with the spatiotemporal release of DMOG and bone-forming peptide to enhance angiogenesis and improve bone
regeneration. Reproduced and adapted with permission [86]. Copyright 2022, Wiley-VCH GmbH. D) Dynamic DNA hydrogels loaded with black phosphorus
nanosheets continuously release VEGF and promote mature vessel growth to induce osteogenesis. Reproduced and adapted with permission [136]. Copyright
2022, Elsevier.

MSCs exhibit higher glycolytic activity and lower oxidative phosphor­ materials that induce material-derived cellular signaling. or instance, a
ylation activity, which suggests that cells may implement self-protective bioenergetically active scaffold was designed by Liu et al. to release
mechanisms to prevent aging caused by oxidative contingencies [142]. degradation debris in a controlled manner and produce metabolic in­
Recent evidence indicates that modulating cellular metabolism can termediates, which enter the mitochondria and enhance the tricarbox­
influence gene expression and signaling pathways to promote bone ylic acid (TCA) cycle, thereby increasing mitochondrial membrane
regeneration. One approach to achieve this is through the use of potential and promoting bone regeneration (Fig. 5B) [87]. Similarly,

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Fig. 5. Biomaterials improve energy metabolism for bone repair. A) Bioenergetic regulation and signaling during osteoblast differentiation. Reproduced and
adapted with permission [139]. Copyright 2022, Elsevier. B) Bioenergetically active material scaffolds that accelerate bone formation by degradation-mediated
increases in mitochondrial membrane potential. Reproduced and adapted with permission [87]. Copyright 2020, American Association for the Advancement of
Science. C) Mg2+ energy drive improved low-dose BMP-2-induced bone regeneration. Reproduced and adapted with permission [88]. Copyright 2022, Elsevier. D)
Citrate-based biomaterials support osteogenic differentiation by providing degradation products during degradation and regulating the metabolic pathway of energy
production. Reproduced and adapted with permission [89]. Copyright 2018, National Academy of Sciences.

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ion-doped biomaterials can effectively modulate metabolism to regulate significant roles in bone regeneration. This section outlines some of the
cellular function by controlling the release of metal ions that act as co­ essential factors, including nutrients such as oxygen and pH, as well as
factors for metabolic enzymes or indirectly affect enzyme activity. Lin signaling molecules like enzymes and cytokines.
et al. demonstrated that a Mg2+-based bioenergy-driven strategy
improved BMP-2-driven bone regeneration by increasing mitochondrial 4.2.1. Oxygen
membrane potential and upregulating metabolic enzyme activity Oxygen is an indispensable molecule for the maintenance of cell
through the Akt signaling pathway (Fig. 5C) [88]. In addition, the viability, growth, metabolism, differentiation, and intercellular
regulation of metabolites, cofactors, and key substrates can influence communication [143]. In healthy tissues, capillaries ensure the provi­
intracellular metabolic events, with citrate from citrate-based bio­ sion of sufficient oxygen to cells. Hypoxia arises when the distance be­
materials shown to promote osteogenic differentiation (Fig. 5D) [89]. tween cells and blood vessels surpasses 100–200 μm [144]. The primary
The regulation of cellular energy metabolism may be a determinant of cause of tissue hypoxia is disruption of the vascular network at the injury
cell survival, proliferation, differentiation, and specific functions. Thus, site, which results in delayed oxygen delivery to the adjacent cells.
understanding the specific energetic and biosynthetic requirements of Simultaneously, chronic hypoxia frequently contributes to widespread
different cell types is crucial for the design of effective regenerative cell death and tissue necrosis. Notably, various skeletal cells exhibiting
engineering strategies. high metabolic activity and oxygen demand exhibit heightened sensi­
Current research in bone tissue engineering in terms of cellular en­ tivity to hypoxic conditions. Consequently, it is imperative to ensure
ergy metabolism lags behind the neurological and cardiovascular fields. adequate oxygen supply to hypoxic tissues and regulate cellular meta­
One of the main difficulties is that osteogenic differentiation exhibits a bolism to adapt to the hypoxic environment [145].
high proliferation rate in the initial stages and a synthesis and deposition Tissue engineering facilitates in situ oxygen production by incorpo­
phase in the later stages mainly by mechanisms. Different cellular stages rating oxygen-generating components into biological materials. Oxygen
require different metabolic signals and energy allocation. This would be production based on hemoglobin and peroxides has achieved remark­
a great challenge to match the dynamic formation process with inactive able results, but emerging technologies such as oxygen microbubbles,
biomaterials. nanosponges and photosynthetic algae are equally worthy of in-depth
study (Fig. 6A). Sun et al. developed an innovative composite hydro­
4.2. Chemical microenvironment gel material capable of converting ROS to O2, with the capacity to
accelerate O2 production in response to excess ROS based on the re­
In the bone microenvironment, cellular interactions are critical, and quirements of the affected region [90]. This hydrogel demonstrates
there exists a plethora of nutrients and signaling molecules that play effective oxygen generation capacity, promoting angiogenesis,

Fig. 6. Biomaterials release oxygen to promote bone repair. A) Oxygen release mechanisms of various oxygen-producing biomaterials. Reproduced and adapted
with permission [147]. Copyright 2021, Elsevier. B) A novel composite hydrogel scavenges ROS and prolongs oxygen production to reverse the hypoxic microen­
vironment in areas of bone defects. Reproduced and adapted with permission [90]. Copyright 2022, Elsevier. C) 3D-printed bionic oxygen-containing scaffolds
enhance the expression of osteogenic regulatory transcription factors and accelerate osteogenesis. Reproduced and adapted with permission [91]. Copyright 2022,
American Chemical Society.

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inhibiting osteoclast differentiation, and enhancing osteoblast differ­ In the above study, different acid-base microenvironments showed
entiation under hypoxic conditions (Fig. 6B). Wang et al. incorporated different restorative effects in different pathological environments. It is
CaO2 in 3D printed scaffolds that exhibited good cytocompatibility and still difficult to determine the optimal pH range, and further in-depth
oxygen release, thus significantly improving cell survival and growth studies are needed in the future.
under hypoxic conditions (Fig. 6C) [91].
Hypoxia-inducible transcription factor (HIF) is among the most well- 4.2.3. Enzymes and cytokines
known transcription factors that mediate oxygen-sensitive signaling Enzymes are specific macromolecular biocatalysts, and all metabolic
pathways, stimulating the transcription of numerous genes and thereby processes lin the body require the participation of enzymes [152]. In
influencing angiogenesis, precursor cell recruitment, and differentiation recent years, enzyme-based biomaterials have received a lot of attention
[146]. The development of tissue-engineered scaffolds capable of [153]. For example, enzyme-responsive hydrogels typically utilize nat­
emulating local hypoxia within an environment exhibiting normal ox­ ural enzymes present in organisms or abnormally overexpressed at
ygen levels constitutes a rational approach, and this has emerged as a lesion sites, such as matrix metalloproteinases (MMP) [154], phospha­
contemporary research direction to incorporate key oxygen-dependent tases [155], and tyrosinases [156]. Anjum et al. prepared a natural
HIF signaling pathways in scaffold design and fabrication. Quinlan hydrogel based on an enzymatic reaction by grafting MMP in a hybrid
et al. incorporated cobalt ions into bioactive glass/collagen- system to maintain cell viability, proliferation, and migration through
glycosaminoglycan scaffolds, which mimic hypoxia and artificially sta­ MMP-mediated degradation of the hydrogel to regulate cell growth
bilize HIF-1α transcription factors [92]. The results demonstrated a factor delivery and stem cell differentiation [95]. However, MMP
significant enhancement of vascular endothelial growth factor expres­ overexpression in bone and chondrocytes can result in pathological
sion, highlighting the ability to activate the HIF pathway under nor­ changes in bone, such as osteoarthritis (OA), osteoporosis, and rheu­
moxic conditions. matoid arthritis (RA) [157]. In another study, mineralized ALP nano­
However, during tissue engineering development, the material particles were incorporated into hydrogels to assess their bone repair
cannot be considered an adequate oxygenation mechanism due to its effects in vivo. The osteogenic differentiation properties of BMSCs were
limited diffusion capacity and solubility in aqueous solutions. Therefore, significantly enhanced while retaining ALP enzyme activity (Fig. 8A)
the development of durable and homogeneous oxygen-releasing mate­ [96].
rials is critical for translation into clinical solutions. Enzymes can not only be used as cofactors to enhance scaffold
bioactivity but also be employed to create functionalized scaffolds via
4.2.2. pH 3D printing. Chen et al. designed an enzyme-functionalized scaffold that
The natural human microenvironment is mildly alkaline. It is releases glucose oxidase to alleviate the hyperglycemic environment and
established that systemic acidosis in humans results in bone loss, enhance bone regeneration in diabetic patients(Fig. 8B) [158]. Nano­
potentially due to the physicochemical dissolution of bone minerals zymes, a new generation of artificial enzymes, possess unique nano­
[148]. There are two common forms of acidosis, metabolic acidosis and material properties, such as high catalytic activity, good stability, and
respiratory acidosis. During metabolic acidosis, bones release buffered low cost. Introducing nanozymes into hydrogels can create highly
acids (protons) and calcium. During metabolic acidosis, bones release advanced bioactive platforms to address complex tissue-specific physi­
buffered acids (protons) and calcium. David et al. investigated the ef­ ological challenges. A recent study demonstrated that composite
fects of metabolic acidosis on bone in mice [149]. The results show that hydrogels loaded with nanozymes not only scavenged endogenous
metabolic acidosis can lead to the occurrence of bone resorption. Bone overexpressed ROS but also synergistically generated dissolved oxygen.
tissue pH can also be affected by inflammatory bone disease, tumor The effects of inhibiting local inflammatory cytokines and improving
environment, and local acidic microenvironments due to immune cell osseointegration were validated through in vivo and in vitro experiments
enrichment. The acidic microenvironment further decreases pH, pro­ (Fig. 8C) [97]. Despite these promising results, the question of how
moting increased bone resorption. Yuan et al. designed a composite enzymes with overlapping substrates can react more precisely remains
hydrogel with selective toxicity to osteosarcoma tissues [150]. The to be addressed.
loaded curcumin can be released in a pH-responsive manner at acidic Various growth factors play crucial roles in the bone repair process.
osteosarcoma sites through the breakage of subamine bonds in the Their effectiveness relies on the dose and release rate in vivo, as well as
hydrogel, achieving selective toxicity to osteosarcoma cells. This selec­ the drug delivery system, encompassing vectors, cells, and gene therapy.
tive toxicity and differentiation-promoting ability of pH-responsive BMP and VEGF are the most extensively studied growth factors in bone
hydrogels have been demonstrated in osteosarcoma cells and normal tissue engineering. Various BMP2-loaded scaffolds have been investi­
osteoblasts (Fig. 7A). The effective proton microenvironment boundary gated, including liposomes [159], gelatin sponges [160], hydrogels
of degradable biomaterials was recently found to be 400 ± 50 μm, [161], exosomes [162], immune complexes [163], 3D printed scaffolds
exceeding generally accepted value of 300 μm [151]. This further cor­ [164], etc., all of which demonstrate remarkable bone regeneration
roborates that biomaterials can significantly impact the cellular micro­ capabilities. Previous research on angiogenic factors has emphasized the
environment (Fig. 7B). Consequently, some researchers have leveraged role of VEGF in neovascularization and osteogenic recruitment,
these microenvironment characteristics to develop smart reactive bio­ revealing that VEGF delivery increases vascular density and stimulates
materials with therapeutic and regenerative functions. Zheng et al. minor bone regeneration in rabbits and rats with bone defects
aimed to design multifunctional nanoplatforms capable of releasing a [165–167]. Recent studies have indicated that the co-delivery of VEGF
low pH microenvironment [93]. These nanoplatforms can continuously with osteogenic growth factors synergistically enhances osteogenesis
release encapsulated bioactive factors at low pH conditions to actively (Fig. 8D) [168]. Bone-associated growth factors are diverse and
and precisely establish a reparative microenvironment for bone regen­ numerous, with dynamic concentrations and distributions under varying
eration. Moreover, slow degradation during nanoparticle healing pro­ physiological and pathological conditions, playing vital roles in pro­
vides sufficient in situ magnesium and silica for angiogenesis and moting systemic or local bone formation. Presently, the most significant
calcium and phosphate for osteogenesis (Fig. 7C). Alternatively, another challenge in tissue engineering is delivering appropriate growth factors
study focused on the impact of an alkaline microenvironment on bone in a temporally and tightly regulated sequence during the repair
regeneration. The researchers constructed a weakly alkaline inner layer cascade, holding immense potential for advancing bone repair medical
of Ca–O–Ti and a strongly alkaline outer membrane of MgO with Ti as interventions.
the substrate [94]. This customizable alkaline microenvironment sur­
face exhibited sustained resistance to infection and osseointegration,
offering novel insights into bone implant surface design (Fig. 7D).

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Fig. 7. pH-responsive biomaterials promote bone repair. A) pH-responsive hydrogel targets selective osteosarcoma cells to release pro-bone repair drugs.
Reproduced and adapted with permission [150]. Copyright 2022, American Chemical Society. B) Schematic diagram of the microenvironment of nearby cells affected
by the implanted biomaterial. Reproduced and adapted with permission [151]. Copyright 2019, American Chemical Society. C) A nanoscale drug delivery system
that reduces pH and can actively build a bone regenerative repair microenvironment. Reproduced and adapted with permission [93]. Copyright 2020, Elsevier. D)
Customizable alkaline surface to enhance the osteogenic properties of Ti implants. Reproduced and adapted with permission [94]. Copyright 2021, Elsevier.

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Fig. 8. Biomaterials release enzymes and cytokines to promote bone repair. A) Mineralase-based hydrogels promote BMSC osteogenic differentiation to induce
in situ mineralization. Reproduced and adapted with permission [96]. Copyright 2022, American Chemical Society. B) Enzymatic multifunctional scaffold loaded
with GOx and catalase alleviates hyperglycemic environment and promotes bone regeneration. Reproduced and adapted with permission [158]. Copyright 2021,
Wiley-VCH GmbH. C) Nanoenzyme-enhanced hydrogel improves the hypoxic environment for osseointegration at the RA prosthesis interface. Reproduced and
adapted with permission [97]. Copyright 2022, Springer Nature Limited. D) Multifunctional microcarriers with sequential delivery of BMP-2 and VEGF. Reproduced
and adapted with permission [168]. Copyright 2020, Springer Nature Limited.

4.3. Physical microenvironment withstand early mechanical loading, and guide new bone formation
[172]. Consequently, the efficacy of bone repair is strongly correlated
Physical stimuli can significantly influence bone formation and are with the mechanical properties of the scaffold, including stiffness, shear
typically categorized into two primary classifications: internal and stress, and dynamic stress, which can influence osteogenic effects.
external bone stimuli. Internal stimuli predominantly stem from me­ Current research has focused on investigating the impact of various
chanical alterations, while external stimuli encompass factors such as materials and scaffold stiffnesses on osteogenesis. In a study by Zhang
photothermal, electrical, magnetic, and acoustic. Moreover, by inte­ et al., polydimethylsiloxane substrates with different stiffnesses were
grating external stimuli with internal responses, a programmed spatio­ prepared to explore the potential mechanisms of mechanotransduction
temporally bone regeneration system can be designed and implemented. [98]. The results indicated that rat primary osteoblast differentiation
was more favorable on rigid substrates, with higher expression of ALP
4.3.1. Mechanical alterations and runt-related transcription factor 2 (Runx2) observed on substrates
Bone, a mechanosensitive tissue, responds to mechanical signals with a stiffness of 134 kPa. In another study, researchers compared the
from its environment through a process known as mechanotransduction orientation of collagen fibers in bone tissue microstructure and found
[169]. Extensive research demonstrates that mechanical stimulation that longitudinally aligned, dark-colored bone was more mineralized,
significantly impacts the development and remodeling of skeletal containing a higher ratio of inorganic to organic matrix components and
structures and offers a novel, drug-free approach to bone regeneration exhibiting increased stiffness and resistance to plastic deformation
[170,171]. In bone tissue engineering, biological scaffolds are under compression. In contrast, brighter-colored bone, containing a
frequently required as temporary structural supports to fill bone defects, higher proportion of collagen, provided enhanced ductility and energy

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Fig. 9. Mechanical properties of biomaterials promote bone regeneration. A) Effect of different arrangements of collagen fibers on bone stiffness. Reproduced
and adapted with permission [173]. Copyright 2021, American Chemical Society. B) Comparison of bone formation under different shear stresses. Reproduced and
adapted with permission [174]. Copyright 2019, Biomedical Engineering Society. C) Microfluidic chip generates constant shear stress to verify the effect of shear
force on osteogenic differentiation and shear stress distribution schematic. Reproduced and adapted with permission [178]. Copyright 2014, plos.org. D) Pressure
clouds and flow distribution of spiral structure brackets with the different modulus of elasticity. Reproduced and adapted with permission [183]. Copyright
2019, Elsevier.

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dissipation due to lower stiffness and rigidity (Fig. 9A) [173]. These irradiation of the photosensitive material g-C3N4/rGO generated
findings suggest that both intrinsic material properties and anisotropy photocurrent, rapidly inducing BMSCs into osteoblasts. The researchers
affect surface stiffness, warranting further in-depth comparative co-cultured photosensitive material with BMSC and the cell culture
research. dishes were simultaneously irradiated with blue light for 30 min per day.
Under physiological conditions, bone cells are constantly exposed to The spectroscopic results showed that photocurrent generation from
mechanical loads, such as shear stresses, which stimulate osteocytes and π-π* orbitals in visible light could provide a stronger driving force for
lead to changes in bone volume and structure to maintain an optimal osteogenesis [197].
skeletal structure (Fig. 9B) [174]. Fluid shear stresses are predicted to Temperature change also impacts bone formation. Thermally
range from 0.8 to 3 Pa [175,176], and they have been shown to initiate a responsive systems are powerful activation mechanisms for biomedical
series of osteogenic signaling events, including calcium release [177], and biomaterial applications, as body temperature typically ranges from
and nitric oxide [176] synthesis and release. Krekea et al. exposed planar 35 to 37◦ C, and temperature shifts induce functional changes [198].
cultures of BMSCs to shear flow [99]. The results demonstrated that Thermally responsive biomaterials play a significant role in bone
expression of late phenotypic markers of osteoblast differentiation regeneration, such as smart fibers and hydrogels. The researchers
increased with the duration of exposure to shear flow, with significant incorporated thermally responsive poly(N-isopropylacrylamide) fiber
enhancement of bone sialoprotein (BSP) and osteopontin (OPN) genes brushes with calcium phosphate [102]. This brush has a dual
observed at 30 and 120 min of shear flow. Similarly, Kim et al. subjected thermo-responsive transition, with the fibers dispersing in hydrophilic
MSCs to constant, very low shear stress generated by flow and observed calcium phosphate bone cement at 21◦ C and transforming to a hydro­
increased osteogenic differentiation of MSCs (Fig. 9C) [178]. These phobic state at 37◦ C to toughen this bone cement. Thermo-responsive
findings suggest that immature osteoblasts are mechanosensitive and hydrogels are a key biomaterial in that they exhibit
are associated with shear strength or shear patterns [179]. Therefore, temperature-dependent gel-sol transition in water [199]. Many ther­
shear stress is deemed an essential factor in bone scaffold development, mally responsive hydrogels based on synthetic and natural copolymers
contributing to osseointegration between the host and implant, a pre­ have been successfully prepared and further investigated [200,201].
requisite for implant stability. The synergistic effects of photo and thermo stimulation have proven
The dynamic stress microenvironment offers cells the capacity to efficacious in promoting bone regeneration. NIR light-generated pho­
influence behavior and fate through stress relaxation and remodeling tothermal effects facilitate osteogenesis, offering the advantages of non-
[180–182]. Traditional bone tissue engineering strategies aim to invasiveness and high spatial and temporal accuracy [202]. Photo­
develop a bone scaffold with an elastic modulus and yield strength thermal agents are capable of converting light energy into heat energy
comparable to human bone, employing helical structures and opti­ under NIR illumination, allowing for adjustments to photothermal
mizing cell inoculation efficiency by varying porosity and pore size hydrogels by modulating the concentration and ratio of the photo­
(Fig. 9D) [183]. In recent decades, adaptive hydrogels with reversible thermal agent, irradiation time, and laser intensity [203]. Gentle local
connections have garnered significant attention. These hydrogels are heating promotes cell proliferation, angiogenesis, wound healing, and
characterized by spatial dynamics of the matrix with reversible con­ bone regeneration [204], while moderate heat (45◦ C–50◦ C) causes
nections, providing plasticity and stress relaxation to adapt biophysical minimal damage to normal tissue cells but inflicts lethal damage to
signals during the repair process [184]. Supramolecular chemistry offers tumor cells [205]. For the healing of infected wounds, heat therapy
numerous non-covalent interactions for obtaining reversible connec­ (>50◦ C) is effective in inhibiting bacterial proliferation. Therefore, the
tions, including macrocyclic host-guest interactions [100], hydrogen photothermal effect can be controlled according to different tempera­
bonding [185], electrostatic interactions [186], and hydrophobic in­ tures for various applications [206]. In recent studies, upconversion
teractions [187]. Additionally, dynamic covalent chemistry presents nanoparticles have garnered attention as efficient photo-responsive
several options, such as reversible Diels-Alder reactions [188], hydra­ platforms. Yan [207] and Ye’s [208] teams employed NIR
zone bonding [189], thioester exchange [190], and borate bonding light-mediated photothermal reactions to release Epimedium(Fig. 10A)
[191]. Qian et al. prepared a supramolecular gelatin macromolecule and NO(Fig. 10B), respectively, as effective drugs against osteoporosis,
[100]. The resulting hydrogels can withstand excessive compressive and with both experiments exhibiting favorable osteogenic differentiation.
tensile strains and rapidly self-repair after mechanical damage. Hydro­ Photo-responsive systems can also achieve co-control of multiple tar­
gels with altered mechanical stress were shown to be promising carrier gets. Qin Zhao et al. developed a dual-targeting nanoscaffold (BCP-GNC)
materials for bone tissue repair. that modulates drug release by altering the light source wavelength,
thereby influencing scaffold temperature [103]. BCP-GNC releases IL-4
4.3.2. Photothermal effect at 690 nm and DEX at 808 nm, modulating innate and adaptive im­
Photo and thermal reactions serve as external stimulation treatments mune responses and promoting osteoinduction. Yang et al. designed a
and significantly contribute to bone regeneration promotion. Current multifunctional composite scaffold using 3D printing technology, uni­
research explores various types of light, including ultraviolet (UV), fying photothermal ablation of osteosarcoma, osteogenic differentiation
visible (Vis), near-infrared (NIR), and distinct wavelengths of laser light of progenitor stem cells, and enhanced angiogenesis through bioactive
[192]. Notably, NIR light, with its high tissue penetration depth and ions (Fig. 10C) [209].
photothermal effect, serves as an efficacious approach to foster osteo­ Another study reported that NIR-mediated photothermal responses
genesis [193,194]. inhibited osteolysis and promoted bone regeneration (Fig. 10D) [210].
UV light is frequently employed in photoresponsive biomaterial Accumulating evidence supports the efficacy of NIR-mediated photo­
systems, as its short wavelength facilitates drug release from bio­ thermal responses for targeted bone tumor treatment. Black phosphorus
materials [195]. In one study, a light-responsive microgel was synthe­ (BP), a cutting-edge two-dimensional material, exhibits exceptional
sized, which, under UV irradiation, promoted the release of DEX, photothermal properties, biocompatibility, and biodegradability [211].
inducing osteogenic differentiation of hMSC [101]. AlamarBlue assay Research has demonstrated that NIR-mediated photothermal heating of
and standardized ALP activity assay results indicated that DEX released BP induces its oxidation in the presence of oxygen and water, effectively
from microgels has the potential for inducing osteogenic differentiation degrading it into phosphate ions [212]. These phosphate ions subse­
of hMSC. By toggling the UV light source on and off, drug release can be quently attract nearby calcium ions to form HA, thereby achieving in situ
controlled, supporting clinical drug requirements. Furthermore, zirconia biomineralization [213]. Harnessing BP to enhance biomineralization
surfaces treated with UV light significantly enhanced osteogenesis, represents an innovative approach to fostering bone formation and
potentially due to accelerated cell attachment and spreading increased regeneration.
cytoskeleton development, and proliferation [196]. Blue light

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Fig. 10. Biomaterials respond to photo and thermal stimulation to promote bone repair. A) Upconversion nanoparticle loaded with epimedium promotes MSC
osteogenic differentiation. Reproduced and adapted with permission [207]. Copyright 2022, American Chemical Society. B) Targeted treatment of osteoporosis by
upconversion nanoparticles releasing NO. Reproduced and adapted with permission [208]. Copyright 2021, American Chemical Society. C) Wesselsite nanosheet
functionalized scaffold repairs tumor-induced bone defects. Reproduced and adapted with permission [209]. Copyright 2021, Wiley-VCH GmbH. D) Carboxy-capped
dendrimer-mediated photothermal response inhibits bone tumor growth and tumor-associated osteolysis. Reproduced and adapted with permission [210]. Copyright
2018, American Chemical Society.

4.3.3. Electricity and magnetism remodeling and repair [215]. Integrating smart materials with piezo­
Application of electrical or magnetic stimulation in bone tissue en­ electric properties into bone implants can enhance bone regeneration
gineering offers a promising strategy for bone regeneration [214]. Bone [216]. Piezoelectric materials facilitate bone regeneration by accumu­
inherently exhibits piezoelectric properties, generating electrical and lating electrical charge in response to mechanical stress, manifesting as a
biochemical signals in response to mechanical activity for bone voltage generated by mechanical stress (the positive piezoelectric effect)

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Fig. 11. Biomaterials respond to electrical and magnetic stimulation to promote bone repair. A) Schematic diagram of piezoelectric material surface me­
chanical strain induced charge generation triggering cell signaling pathway. Reproduced and adapted with permission [218]. Copyright 2020, WILEY-VCH Verlag
GmbH. B) Composite scaffold with piezoelectric properties provides an endogenous electric field to promote bone regeneration in bone defects. Reproduced and
adapted with permission [104]. Copyright 2022, Elsevier. C) Bionic piezoelectric bone membranes doped with polydopamine-modified hydroxyapatite (PHA) and
barium titanate (PBT) synergistically promote osteogenesis and immunity. Reproduced and adapted with permission [220]. Copyright 2023, American Chemical
Society. D) Schematic diagram of magnetron degradation in polymer implants. Reproduced and adapted with permission [106]. Copyright 2021, Wiley-VCH GmbH.
E) Electromagnetic co-stimulation of bionic 3D scaffolds synergistically promotes bone repair. Reproduced and adapted with permission [226]. Copyright 2019,
American Chemical Society.

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or a mechanical response to an applied voltage (the converse piezo­ [222]. A prevailing trend in magnetically responsive biomaterials is the
electric effect) (Fig. 11A) [217,218]. While both effects are crucial, the incorporation of iron oxide nanoparticles, as nanoparticles smaller than
positive piezoelectric effect has been predominantly investigated for 100 nm exhibit superparamagnetic properties that prevent particle
bone implant applications. Common piezoelectric biomaterials include agglomeration [223]. One study verified that magnetic nanoparticles
piezoelectric ceramics, piezoelectric polymers, and their composites generate magnetothermia in alternating magnetic fields, providing
[216,219]. crucial guidance for scaffold degradation (Fig. 11D) [106]. Magnetic
Recent research has focused on designing composite scaffolds with fields with varying parameters may differentially affect osteogenesis in
piezoelectric properties and sustained Mg2+ release using 3D printing the magnetic response regime [224]. The direction and strength of the
technology, which can restore the local endogenous electrical micro­ magnetic field influence bone regeneration. One study reported that
environment and promote osteogenic differentiation (Fig. 11B) [104]. cultured MC3T3-E1 cells aligned parallel to the static magnetic field
Additionally, multifunctional composite synergistic osteogenesis can be (SMF) after 60 h of exposure, marking the first evidence that the growth
achieved with piezoelectric materials, combining piezoelectric osteo­ direction of apposed cells can be regulated by the magnetic field [107].
genesis with immunomodulation for rapid in situ bone regeneration Moreover, the biological effect of the magnetic field became more pro­
(Fig. 11C) [220]. Nanogenerators can also convert environmental me­ nounced with increasing magnetic field strength within a specific range,
chanical energy into electrical energy [221]. One study proposed a but beyond that range, the effect diminished or even became inhibitory.
self-powered electrical system consisting of a triboelectric nano­ Yang et al. examined the induction of osteoblasts by SMFs at three
generator (TENG) and a flexible forked-finger electrode for in vitro different intensities (500 nT, 0.2 T, and 16 T) and found that iron con­
osteogenesis, significantly promoting osteogenesis and demonstrating centration and mRNA expression of transferrin receptor 1 were affected,
potential for clinical treatment of osteoporosis and related fractures suggesting iron involvement in the magnetic field’s effect on osteoblasts
[105]. The authors demonstrated that this electrical stimulator can [225].
clearly promote osteogenesis and has considerable potential for clinical Electrical and magnetic synergy can also benefit bone regeneration.
treatment. Magnetically active biomaterials exploit external magnetic Fernandes et al. demonstrated the feasibility of electromagnetic co-
fields or direct magnetic forces to enhance bone tissue regeneration stimulation by assembling piezoelectric polymers and magnetostrictive

Fig. 12. Biomaterials respond to ultrasound stimulation to promote bone repair. A) Schematic diagram of ultrasound-mediated targeted gene delivery to the
fracture site. Reproduced and adapted with permission [108]. Copyright 2017, American Association for the Advancement of Science. B) Schematic diagram of
endogenous cell recruitment by pulsed ultrasound remote stimulation. Reproduced and adapted with permission [109]. Copyright 2022, Elsevier. C) Schematic
diagram of LIPUS stimulation of periodontal stem cells to promote osteogenic differentiation. Reproduced and adapted with permission [238]. Copyright 2020,
Springer Nature. D) Schematic diagram of LIPUS stimulated micro-arc oxidized Ti implants for bone repair. Reproduced and adapted with permission [239].
Copyright 2019, American Chemical Society.

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S. Hao et al. Materials Today Bio 22 (2023) 100741

nanoparticles in response to magnetic stimulation, constructing a bionic programmable bio-design. By loading cells and bioactive cues directly
three-dimensional magnetically active scaffold for tissue recovery into bioink, bioprinting can create structures that mimic natural tissue
through co-stimulation (Fig. 11E) [226]. The applicability of pulsed while allowing for programmability (Fig. 13C) [241–244]. This
electromagnetic fields (PEMF) has been evaluated using a rat cranial advancement enables personalized treatment of bone-related diseases.
defect model [227]. In an 8 mm diameter rat cranial defect model, the Bioinks for bone tissue bioprinting are categorized into natural bioinks
experimental group receiving PEMF exhibited a significant effect on (collagen [245], chitosan [246], fibrin [247], gelatin [248], agarose
bone regeneration by applying a 12 μs width, a 60 Hz pulse frequency, [249] and alginate [250], etc.) and synthetic bioinks (Pluronic F-127
and a 10 G magnetic field strength. However, the complex biological [251], methylated gelatin [252], PEG [253], etc.). Numerous challenges
effects of electromagnetic fields and the underlying mechanisms of remain for clinical application of conventional 3D bioprinting in bone
PEMF pose challenges in defining treatment options, necessitating tissue engineering, such as reconstructing large and irregular bone tissue
extensive research to overcome this issue. for individualized needs, achieving vascularization and nerve regener­
ation when repairing extensive bone defects, and addressing mechanical
4.3.4. Acoustic properties [254,255]. Four-dimensional (4D) bioprinting, which in­
In vivo and in vitro studies have shown that ultrasound stimulation (e. tegrates the concept of time as a fourth dimension with 3D bioprinting,
g., low-intensity pulsed ultrasound (LIPUS), shock waves) is beneficial in permits printed objects to alter their shape or function in response to
promoting bone healing or reactivating failed healing processes [228]. external stimuli, cell fusion, or self-assembly after printing. This inno­
Ultrasound-responsive biomaterials can deliver signaling molecules vative approach offers a next-generation solution for tissue engineering,
directly or indirectly with the help of ultrasound stimulation [229,230]. providing the potential to construct complex functional structures
Functional fracture healing has been reported by ultrasound-mediated [256]. For instance, smart, renewable bioscaffolds synthesized using
delivery of target genes (Fig. 12A) [108]. At present, LIPUS is the PCL and crosslinkers with predetermined amounts of castor oil
most extensively studied and researched technique in the domain of demonstrated favorable shape memory effects and shape recovery at
ultrasound stimulation for bone repair [231]. The biological response to physiological temperatures [257]. Another study employed 4D printing
LIPUS is intricate, involving numerous cell types and multiple pathways. technology to dynamically regulate stem cell fate, enabling precise
Known mechanotransduction pathways implicated in cellular responses switching between proliferation and differentiation phases to better
include MAPK [232], other kinase signaling pathways, gap junction promote bone regeneration (Fig. 13D) [258]. The synthesized smart
intercellular communication [233], upregulation and aggregation of polymers exhibited satisfactory surface morphology, shape memory,
integrins, involvement of COX-2/PGE2 [234], iNOS/NO pathways mechanical properties, biocompatibility, and biodegradability. In recent
[235], and activation of ATI mechanoreceptors. A recent study discov­ years, bioprinting has garnered considerable attention in the biomedical
ered that by altering the radiation frequency of pulsed ultrasound, not field and clinical applications due to the emergence of
only could the release of bioactive molecules for recruiting endogenous stimulus-responsive biomaterials and a deeper understanding of tissue
BMSC be controlled, but also the capture of recruited BMSC into the regeneration. While various stimulus-responsive biomaterials and
stent could be facilitated through resonant gradient field-induced trap­ innovative strategies have been developed, 4D bioprinting remains in its
ping forces (Fig. 12B) [109]. Similarly, several other studies have re­ infancy, necessitating further research to address numerous challenges
ported some effects of LIPUS on cell differentiation and protein [259].
responses. Although clinical and experimental studies have shown Although responsive biomaterials have been developed for me­
enhanced effects of LIPUS on bone regeneration, the physiological chanical force, photothermal, electromagnetic and ultrasonic stimula­
mechanisms involved in the complex bone healing process remain un­ tion associated with the physical microenvironment, they are still in
clear and warrant further investigation (Fig. 12C–D) [236–239]. their infancy and have some common issues that need to be urgently
addressed. The first is that these novel biomaterials, their immune re­
4.3.5. Programmed spatiotemporally design sponses and metabolic pathways have not been systematically explored.
On-demand delivery of chemotactic and osteogenic biomolecules for Secondly, the manufacturing process of reactive materials is complex
bone regeneration is an appealing yet challenging endeavor, as it re­ and the specific mechanisms still need to be studied in detail. The
quires meeting the varying demands of distinct bone regeneration optimal parameters of external stimuli and changes in the internal
phases. Existing bone tissue engineering approaches face limitations in environment cannot be determined quickly in real-time and require the
coordinating appropriate biomolecule concentrations and treatment selection of suitable animal models for evaluation. Despite the enormous
time points on-demand due to insufficient spatial separation. Conse­ challenges, physical microenvironmental stimulation of biomaterials
quently, designing a programmable regulated delivery system remains a has far-reaching clinical applications in the future.
formidable task. In a recent study, researchers combined thermores­
ponsiveness with photothermal response to achieve rapid response 5. Bone microenvironment and bone organoids
concentrations in the initial burst release of the primary response, fol­
lowed by precise modulation of therapeutic effects using photo­ Organoids, defined as in vitro 3D cell clusters derived from induced
responsiveness [240]. The results suggest that a controlled and stable pluripotent stem cells, embryonic stem cells, or primitive tissues, rely on
promotion of osteogenic differentiation can be achieved (Fig. 13A). artificial microenvironmental matrices for self-renewal and self-
Other researchers have sought to integrate immunomodulation with organization. These formations emulate natural tissue structures and
osteogenic process programming. By coating the programmed surface exhibit organ functions akin to native tissues [260]. Despite the vari­
with poly(aryl ether ether ketone) (PEEK), IL-10 was released rapidly ability resulting from their self-organizing nature, organoids represent
within the first week, followed by slow release of DEX for up to four the closest in vitro model system to in vivo tissue conditions and offer a
weeks [110]. Suitable immunomodulatory activity sets the stage for promising avenue for personalized medicine. To date, organoids have
osteogenesis, while the stable release of DEX promotes subsequent bone been developed to simulate various human organs, including the brain
regeneration. Zhou et al. designed a hybrid dual growth factor delivery [261], lung [262], kidney [263], liver [264], pancreas [265], intestine
system with basic fibroblast growth factor (bFGF) and BMP-2 to further [266], and prostate [267]. However, the development of bone organoids
mimic the natural bone healing process and promote bone regeneration remains in its infancy, due to limited understanding of bone-related
by synergizing osteogenic and angiogenic functions. It provides a simple disease mechanisms and challenges in directing stem cell differentia­
and effective alternative method for bone defect treatment (Fig. 13B) tion [268]. Bone organoids combine stem cells with bioactive materials
[111]. to form three-dimensional bone-mimicking tissue with self-renewal and
Bioprinting technology presents a promising approach to achieving self-organizing properties [260]. The successful establishment of bone

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S. Hao et al. Materials Today Bio 22 (2023) 100741

Fig. 13. Programmable design of biomaterials for bone repair. A) Thermally responsive hydrogels are combined with light-responsive nanoparticles to construct
programmed delivery systems for on-demand growth factor release. Reproduced and adapted with permission [240]. Copyright 2022, Elsevier. B) Composite
hydrogel that mimics the natural cascade reaction of bone healing for spatiotemporally regulated release of cytokines. Reproduced and adapted with permission
[111]. Copyright 2021, Springer Nature. C) Multiple bioprinting technologies for bone repair. Reproduced and adapted with permission [244]. Copyright 2021,
Elsevier. D) Constructing multi-responsive bilayers using 4D printing technology to precisely regulate stem cell fate and optimize bone repair. Reproduced and
adapted with permission [258]. Copyright 2021, Wiley-VCH GmbH.

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S. Hao et al. Materials Today Bio 22 (2023) 100741

Fig. 14. Engineered bone microenvironments assist in building bone organoids. A) Schematic diagram of the process of building bone organoids. Reproduced
and adapted with permission [268]. Copyright 2022, Elsevier. B) The hydrogel-based construction of bone organoids bionically mimics the physiological micro­
environment and promotes cell adhesion, proliferation and differentiation. Reproduced and adapted with permission [270]. Copyright 2022, Elsevier. C) BMSC and
EC cells can form a pseudo-vascularized network within the mesenchymal compartment to generate bone marrow-like organs with in situ functional characteristics.
Reproduced and adapted with permission [272]. Copyright 2022, American Institute of Physics. D) Composite hydrogel sensory electrical stimulation reconstructs
the bone’s physical microenvironment to promote bone regeneration. Reproduced and adapted with permission [274]. Copyright 2022, Elsevier.

organoids depends not only on the selection of stem cells but, more organoid tissues. Despite its widespread use in organoid cultures,
importantly, on the capacity of introduced biomaterials to provide the Matrigel possesses certain drawbacks, such as heterogeneous origin,
requisite microenvironmental matrix for 3D cell model growth and variable composition, and complexity, which impede its clinical
differentiation (Fig. 14A). advancement. Consequently, alternative biomaterials are urgently
As aforementioned, the bone microenvironment can be classified required for bone organoids development. Bone tissue engineering
into physiological, chemical, and physical microenvironments, with techniques have identified hydrogels as a viable alternative to matrix
dynamic interactions between the physiological microenvironment and gels for bone organoids cultivation [270]. Hydrogels can provide a
bone organoids seed cells playing a critical role in regulating cellular three-dimensional aqueous microenvironment to activate cell adhesion
behavior and tissue regeneration [269]. This dynamic process involves and proliferation, enhance cell differentiation, more closely mimic the
the continuous generation, degradation, and remodeling of ECM com­ functionality of natural tissues, and allow modulation of their properties
ponents. Matrigel, a natural physiological microenvironmental compo­ through functional group modifications (Fig. 14B). It is anticipated that
nent derived from mouse tumors, is the most prevalent substrate for the biomimetic physiological microenvironment furnished by hydrogels
organoid cultures. It provides multiple physiological cues to induce stem will significantly advance overall regulation, thereby facilitating bone
cell differentiation, supports bone organoids growth, maintains extra­ organoids culture.
cellular and intercellular junctions, and facilitates self-organization into The chemical microenvironment, encompassing various soluble

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S. Hao et al. Materials Today Bio 22 (2023) 100741

factors, plays a crucial role in maintaining development and homeostasis time regulation has garnered substantial attention in biomedical
during bone organoids construction. Existing in vitro skeletal models are research and clinical applications due to the emergence of stimuli-
frequently limited to basic osteogenic functions. Nevertheless, by inte­ responsive biomaterials and enhanced comprehension of tissue regen­
grating engineered microenvironmental tissue engineering strategies, eration. Morrison et al. showcased the successful implementation of
organoids can present gradients of nutrients, gases, and signaling mol­ personalized 4D printed medical devices in the treatment of pediatric
ecules, thereby achieving a comprehensive integration of immune tracheobronchi [275]. 4D implants possess the capacity to
regulation, angiogenesis, and osteogenic functions [271]. Recent self-transform and self-mature over time, yielding significant benefits in
research has demonstrated that bone marrow-like organoids can be the management of adolescent patients with congenital malformations.
formed on a hydrogel microporous platform, aided by components of the The 4D concept holds immense potential for personalized treatment and
natural bone niche [272]. Morphologically, these organoids form precision medicine, emerging as a leading trend within the realm of
self-organized, vascular-like networks, which strongly support the bone tissue engineering.
further construction of vascularized bone organoids (Fig. 14C) [273]. To fully harness the potential of bone tissue engineering, it is
Constructing bone organoids with responsive physical microenvi­ imperative that researchers address these challenges, focusing on the
ronments enables rapid detection of and response to disease environ­ development of multifunctional integration and time-modulated 4D
ments and therapeutic effects. Various diseases exhibit distinct bone microenvironments. The successful incorporation of these ad­
pathological microenvironments, such as excess reactive oxygen species vances will not only contribute to a deeper understanding of the bone
and weak acidity in tumors, specific pH reductions and bacterially healing process but also propel the field toward more effective and
secreted enzymes in severe infections, and negative potential and spe­ personalized therapeutic solutions for bone tissue regeneration.
cific ion concentrations at bone defect sites [157]. Leveraging these
unique pathological characteristics, reversible or irreversible trans­
formations in physical properties or chemical structures of biomaterials Declaration of competing interest
can be induced by stimulating the surrounding physical microenviron­
ment, subsequently influencing cell fate and enhancing bone tissue We declare that we have no financial and personal relationships with
healing and regeneration. Recent studies have similarly shown that other people or organizations that can inappropriately influence our
re-establishing the physical microenvironment through electrical stim­ work, there is no professional or other personal interest of any nature or
ulation can significantly accelerate bone regeneration (Fig. 14D). [274]. kind in any product, service, and/or company that could be construed as
In summary, the bone microenvironment is inherently linked to the influencing the position presented in, or the review of, the manuscript
development of bone organoids. Engineered bone microenvironments entitled, “Microenvironment-Targeted Strategy Steers Advanced Bone
hold considerable potential for multifunctional co-regulation of bone Regeneration”.
organoids in simulating bone development and diseases. However, the
currently reported bone organoids can only represent a single function Data availability
of bone, such as bone formation, bone resorption, or hematopoiesis.
Achieving multifunctional integrated bone organoids remains a great No data was used for the research described in the article.
difficulty due to the different requirements of different types of stem
cells for co-culture microenvironments. Future research can be con­ Acknowledgments
ducted by constructing bone organoids with responsive microenviron­
ments and integrating engineered microenvironmental tissue S.Y.H., M.K.W., and Z.F.Y. contributed equally to this work. This
engineering strategies that can better mimic the complexity of bone work was financially supported by the National Natural Science Foun­
tissue, create more accurate in vitro models, and ultimately facilitate dation of China (82230071, 82172098), Shanghai Committee of Science
advances in bone tissue regeneration and personalized medicine. and Technology (23141900600, Laboratory Animal Research Project).

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