Temporally Controlled Therapeutic Strategies for Critical-Sized Bone Defects: Integrating Materials Engineering and Regenerative Medicine

Abstract

Critical-sized bone defects exceed the endogenous self-healing capacity of bone tissue and remain one of the most intractable clinical challenges in orthopedic reconstruction. Conventional therapeutic regimens, including autologous/allogeneic bone grafts and the Masquelet technique, are severely restricted by donor shortage, immune rejection, high infection risk, and insufficient functional regeneration efficacy. As an emerging paradigm, temporally controlled therapy precisely mimics the staged physiological demands of natural bone healing by programming the release timing, sequence, and dosage of therapeutic factors, thereby achieving dynamic regulation of bone regenerative microenvironments and bringing new breakthroughs for defect repair. This review systematically summarizes the biological basis, core material systems, key technological platforms, and molecular mechanisms of temporally controlled bone regeneration. We comprehensively discuss state-of-the-art delivery strategies based on smart biomaterials, including microenvironment-responsive hydrogels, multilayered gradient scaffolds, micro/nanoparticle carriers, and genetically engineered cell therapies. The spatiotemporal synergistic effects of core growth factors (BMPs, VEGF, PDGF) and innovative dynamic regulation technologies, such as 4D bioprinting and closed-loop feedback systems, are further highlighted. Distinct from conventional sustained-release systems that provide static drug supplementation, temporally controlled strategies enable stage-matched modulation of biological cascades covering inflammation, vascularization, osteogenesis, and bone remodeling. Notably, emerging rare-earth nanoparticle-integrated platforms offer unique advantages in responsive drug delivery, imaging-guided precise regulation, and adaptive tissue regeneration. Finally, we critically elaborate the current progress, translational bottlenecks, biosafety concerns, and future prospects of temporal therapeutic systems, aiming to provide comprehensive theoretical support and innovative perspectives for the design of advanced clinical repair strategies for critical-sized bone defects.

Graphical Abstract

Introduction

Definition, Classification, and Clinical Significance of Critical-Sized Bone Defects

A critical-sized bone defect is academically defined as “the smallest intraosseous defect that will not heal spontaneously during the lifetime of an experimental animal”. In clinical practice, this concept is typically specified as a segmental defect exceeding 2–3 cm in long bones, or a defect with a diameter more than 1.5 times that of the host bone in specific anatomical sites (eg, the jaw).¹ Based on their complex etiology, critical-sized bone defects can be systematically classified into four major categories: traumatic (eg, high-energy open fractures, combat injuries), pathological (eg, post-tumor resection, post-debridement for chronic osteomyelitis), congenital (eg, craniofacial developmental deformities), and iatrogenic (eg, periprosthetic osteolysis, osteoradionecrosis).²

The clinical dilemma is multi-dimensional and interwoven: Firstly, the loss of biomechanical stability renders the limb non-weight-bearing, severely impacting function and quality of life.³ Secondly, the biological microenvironment is catastrophically disrupted – interrupted local blood supply creates a “biological desert”, endogenous mesenchymal stem cells (MSCs) and growth factors are extremely scarce, while inflammatory and hypoxic conditions persist. Thirdly, the risk of infection remains high, especially in open injuries, where the formation of bacterial biofilms renders conventional antibiotic treatments ineffective, creating a vicious cycle of “infection-nonunion”.^4,5 Statistics indicate that millions of patients suffer from bone defects globally each year, with approximately 10–20% falling into the category of critical-sized defects, causing not only physical and psychological suffering but also imposing a heavy socio-economic burden. Therefore, developing novel therapeutic strategies capable of achieving perfect structural, functional, and biological regeneration has become an urgent need in orthopedics and regenerative medicine.⁶ Bone regeneration is a highly coordinated biological process involving three overlapping phases: inflammation, repair, and remodeling. Immediately after injury, the inflammatory phase regulates hematoma formation, immune activation, and recruitment of repair-associated cells. During the repair phase, angiogenesis, mesenchymal stem cell recruitment, osteogenic differentiation, and extracellular matrix deposition collectively contribute to new bone formation. Subsequently, the remodeling phase involves coordinated osteoblast and osteoclast activities, transforming immature woven bone into mature lamellar bone. Understanding this temporal sequence provides the biological foundation for designing stage-specific therapeutic interventions.⁷ Conventional sustained-release systems mainly aim to maintain therapeutic concentrations over an extended period, usually following predetermined release kinetics. In contrast, temporally controlled therapy emphasizes the coordination between therapeutic delivery and biological events, including inflammation regulation, vascularization, osteogenesis, and remodeling. Therefore, temporal control involves not only the duration of release but also the timing, sequence, dosage, and responsiveness of therapeutic signals.

Limitations of Traditional Treatment Methods

Currently, autologous bone grafting (often harvested from the iliac crest) is still considered the “gold standard” due to its triple advantages of osteoconduction, osteoinduction, and osteogenesis, along with the absence of immune rejection risk. However, its limitations are prominent: donor site morbidity rates are as high as 8%–39%, including chronic pain, hematoma, infection, and nerve injury; the obtainable quantity is limited, making it difficult to repair large defects; and shape matching is poor, complicating shaping.⁸ Allogeneic bone, while solving the source issue, carries risks of disease transmission, potential immune rejection, and slow “creeping substitution” with insufficient osseointegration strength due to the lack of living cells and growth factors.⁹ The Masquelet induced membrane technique (also known as the “biomembrane technique”), a two-stage procedure involving first inducing a vascular and growth factor-rich biological membrane using polymethylmethacrylate bone cement, followed by secondary bone grafting, provides a favorable “biological chamber” for bone regeneration. Although successful in treating large segmental defects, this technique involves a lengthy treatment period (often over 6 months), requires a second surgery, carries cumulative infection risks, and demands stringent requirements for membrane quality and grafting timing.^10,11 As for metallic implants (eg, intramedullary nails, locking plates, titanium meshes), they provide immediate mechanical stability but are essentially mechanical fillers, suffering from long-term complications such as stress-shielding-induced bone resorption, fatigue fracture, and aseptic loosening, and cannot achieve true biological integration with host bone.^12,13 The various shortcomings of these methods collectively drive the treatment paradigm towards regenerative medicine-based, temporally controlled strategies capable of dynamically regulating the healing process.

Different temporal delivery platforms exhibit distinct advantages depending on therapeutic requirements. PLGA microspheres provide controllable degradation kinetics and mature manufacturing processes, but their spatial regulation capability is relatively limited. Responsive hydrogels provide excellent biomimetic properties and environmentally triggered release but generally require improvement in mechanical stability. Multilayer electrospun scaffolds enable spatially organized sequential delivery and structural biomimicry, although fabrication complexity remains a challenge. Nanocomposite systems provide multifunctionality and external responsiveness but require further investigation regarding biosafety and clinical translation (Table 1).

Table 1.

Comparison of Typical Temporal Delivery Platforms for Bone Regeneration

Delivery Platform	Advantages	Limitations	Clinical Potential
PLGA microspheres	Tunable degradation and sustained release	Limited spatial control	High
Responsive hydrogels	Smart environmental response	Mechanical weakness	Moderate
Multilayer electrospun scaffolds	Sequential spatial delivery	Complex fabrication	Moderate-high
Nanocomposite systems	Multifunctional regulation	Biosafety concerns	Requires validation

The Rise and Core Philosophy of Temporally Controlled Therapeutic Strategies

The inspiration for temporally controlled strategies stems from a profound understanding of natural bone healing—a highly programmed, multi-stage, precisely orchestrated cascade. Physiological bone healing can be clearly divided into three overlapping yet functionally distinct phases: the inflammatory phase (days 0–7 post-injury), characterized by hematoma formation, inflammatory cell infiltration, and provisional extracellular matrix construction, aiming to clear necrotic tissue and initiate repair signals;^14,15 the reparative phase (days 7–28), centered on cartilaginous callus and woven bone formation, accompanied by vigorous angiogenesis and osteoblast differentiation; and the remodeling phase (lasting up to several years), involving the coordinated action of osteoclasts and osteoblasts to remodel mechanically weak woven bone into structurally robust lamellar bone.^16,17 Each stage is dominated by specific cell populations and precisely regulated by uniquely spatiotemporally encoded signaling networks.

The core philosophy of temporally controlled therapy is to deliver the right therapeutic factors, at the right dose and kinetics, to the right location at the right time. It aims to actively intervene in and optimize this natural process, with specific goals including: 1) Early-stage precision intervention: Rapidly controlling potential infection, modulating excessive inflammation, and recruiting endogenous stem cells during the inflammatory phase;^18,19 2) Mid-stage robust driving: Efficiently promoting functional vascular network formation and guiding/accelerating osteogenic differentiation during the reparative phase;^20,21 3) Late-stage fine-tuning: Providing appropriate osteogenic stimulation while balancing bone remodeling activity to promote the restoration of mechanical properties during the remodeling phase.²² This strategy fundamentally transcends traditional static models of “one-time filling” or “constant-rate release”, instead aiming to achieve dynamic, adaptive, and programmable control of the bone regeneration microenvironment through advanced smart material systems.

This review aims to construct a comprehensive knowledge framework from basic biological principles to cutting-edge engineering technologies, systematically elaborating on the key technological platforms, core material systems, underlying molecular mechanisms, current translational pathways, and future blueprint for achieving temporally controlled therapy for critical-sized bone defects.

Limitations of Existing Reviews and Scope of the Present Review

Previous reviews have comprehensively summarized biomaterial scaffolds, growth factor delivery systems, and bone tissue engineering approaches. However, most existing reviews mainly focus on material properties, biological functions, or sustained-release characteristics, while insufficient attention has been given to the dynamic relationship between therapeutic timing and the sequential biological events of bone regeneration. In particular, an integrated understanding connecting bone healing stages, temporal delivery strategies, advanced manufacturing technologies, and clinical translation challenges remains lacking. Therefore, this review aims to provide a systematic overview of temporally controlled therapeutic strategies for critical-sized bone defects by integrating biological principles with engineering innovations. This review is structured as follows. First, the biological basis of bone healing and the spatiotemporal regulation mechanisms underlying regeneration are introduced. Subsequently, advanced technologies for achieving temporal control, including smart biomaterial scaffolds, responsive hydrogels, nanoparticle-based delivery systems, genetically engineered cell therapies, and 4D bioprinting strategies, are discussed. The following sections summarize stage-specific therapeutic applications, preclinical validation, and challenges associated with clinical translation. Finally, future perspectives regarding intelligent, personalized, and clinically applicable temporal therapeutic systems are presented.

Scope and Methodology

This review focuses on temporally controlled therapeutic strategies for critical-sized bone defects, including biomaterial-based delivery systems, responsive scaffolds, nanoparticle-assisted regulation, engineered cell therapies, and advanced biofabrication technologies. Relevant literature was identified through searches of major databases including PubMed, Web of Science, and Scopus using combinations of keywords such as “critical-sized bone defects”, “temporal delivery”, “sequential release”, “smart scaffold”, “bone regeneration”, and “biofabrication”. Studies published mainly from 2000 to 2026 were considered, with particular emphasis on recent advances from 2020 onward. Original research articles, high-quality reviews, preclinical studies, and translational studies were included, whereas unrelated tissue engineering studies, studies without temporal regulation components, and publications lacking sufficient methodological information were excluded.

Theoretical Basis of Temporally Controlled Therapy: The Biological Program and Spatiotemporal Logic of Bone Healing

The Molecular Chronological Map of Natural Bone Healing

The molecular storm of the inflammatory phase (0–7 days): At the moment of fracture, activated platelets are not only central to hemostasis but also act as a potent “biologic factor depot”, explosively releasing Platelet-Derived Growth Factor (PDGF) and Transforming Growth Factor-β (TGF-β). The former strongly chemoattracts neutrophils and macrophages, while the latter initiates MSC recruitment.²³ Early-infiltrating macrophages predominantly exhibit a pro-inflammatory M1 phenotype, secreting Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), IL-6, etc, to clear debris and activate subsequent repair programs.²⁴ As the microenvironment changes, macrophages gradually polarize towards a reparative M2 phenotype, beginning to secrete Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Protein-2 (BMP-2), marking the transition from destruction to construction.^25,26 Concurrently, the hypoxic environment in the injury area stabilizes Hypoxia-Inducible Factor-1α (HIF-1α), which, as a key transcription factor, directly upregulates the expression of genes like VEGF, laying the molecular groundwork for the impending angiogenic surge.^27,28

The reparative phase (7–28 days): MSCs recruited to the injury site proliferate extensively under the drive of factors like BMP-2, BMP-7, and Fibroblast Growth Factor-2 (FGF-2), and initiate osteogenic differentiation programs regulated by core transcription factors such as Runx2 and Osterix.²⁹ Simultaneously, under the strong drive of VEGF, endothelial cells proliferate, migrate, and form new capillary networks, delivering oxygen and nutrients to the highly metabolically active repair tissue. By the end of this phase, woven bone is initially formed, but its structure is disorganized and mechanically weak.³⁰

The remodeling phase (beyond 28 days): This is the longest-lasting phase, whose core is the dynamic coupling of bone resorption and formation. Osteoclast differentiation and activity are mainly regulated by the balance between Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) and its decoy receptor Osteoprotegerin (OPG).³¹ Osteoblasts, through secreting ligands like those in the Wnt signaling pathway, maintain their own activity and influence osteoclasts. Guided by mechanical stress, osteoclasts resorb the disordered woven bone, and osteoblasts subsequently deposit structurally organized lamellar bone, ultimately restoring the original biomechanical properties and microstructure of the bone.^32,33

Spatiotemporal Synergy of Growth Factors and the “Therapeutic Window”

Growth factors do not act in isolation; their effects exhibit strict temporal dependency and synergy, constituting a complex regulatory network.

Research indicates that a brief but intense pulse stimulation of VEGF during the early reparative phase (approximately days 3–7) is most effective in inducing structurally intact and functionally mature new vascular networks.^34,35 Release that is too early (during the peak inflammatory period) may exacerbate vascular leakage and edema; release that is too late or sustained may lead to abnormal vascular structures or even hemangioma formation.³⁶

Different BMP members play distinct roles in the healing process. BMP-2 is a potent MSC chemoattractant and osteogenic differentiation inducer in the early stages,³⁷ whereas BMP-7 holds advantages in promoting bone matrix maturation and mineralization in later stages.³⁸ More noteworthy is the vascularization-osteogenesis coupling effect: Experiments demonstrate that temporally combining VEGF and BMP-2 (VEGF first, followed by BMP-2) results in far superior osteogenic outcomes compared to simultaneous or reverse-order administration.³⁹ This is because the initially established vascular network provides nutritional support and cellular transport channels for the subsequent large number of osteoblasts. Reported therapeutic windows for VEGF, BMP-2, and other signaling molecules are primarily derived from specific animal models and scaffold systems. These timeframes provide useful design principles but may vary depending on species, defect characteristics, material properties, and local biological conditions.

Each key therapeutic factor possesses an optimal biological window of action, known as the “therapeutic window”. The sophistication of temporally controlled therapy lies in engineering these therapeutic factors to precisely “hit” their respective windows, thereby maximizing efficacy and minimizing side effects.

The Dynamic Evolution of the Biomechanical Microenvironment

Bone healing is not only a biochemical event but also a biomechanical adaptation process. According to Wolff’s Law, the growth and remodeling of bone tissue follow the mechanical loads it bears.⁴⁰ In the early stages of healing, a certain degree of mechanical stability is crucial for hematoma organization and callus formation, but excessively rigid fixation (stress shielding) deprives the bone of physiological stress, thereby inhibiting callus growth, leading to bone resorption and delayed healing. Therefore, an ideal repair scaffold should possess “dynamic mechanical adaptability”: its initial mechanical strength must be sufficient to support the defect and maintain stability; as new bone tissue forms, the scaffold should gradually and controllably degrade, steadily and progressively transferring the mechanical load to the new bone, thereby stimulating its functional remodeling. This spatiotemporal matching of biodegradation and mechanical load transfer is the core embodiment of temporally controlled therapy at the macroscopic mechanical scale.⁴¹

Key Technological Platforms for Realizing Temporally Controlled Therapy

Translating the concept of temporally controlled therapy into reality is highly dependent on the cross-integration and technological innovation in materials science, drug delivery, and biofabrication.

Smart Biomaterial Scaffold Systems

Multi-Layer and Gradient Functional Scaffolds

Through biomimetic design, scaffolds with spatiotemporal heterogeneity are constructed by mimicking the gradient structure of natural bone from dense cortical bone to porous cancellous bone.^42,43 This multi-layered material system employs a stratified design and functionalization strategy. The outer/rapid-response layer, constructed from gelatin, alginate, or low-molecular-weight PLGA (eg, 50:50 ratio), can degrade rapidly within days to two weeks, and is used to load antibiotics (eg, vancomycin) for early-stage anti-infection or to carry VEGF to initiate vascularization.^44,45 The middle/core repair layer, using materials with a degradation period of 2–6 weeks such as collagen/nano-hydroxyapatite composites or medium-molecular-weight PLGA (eg, 75:25), serves as the main functional area for osteogenic differentiation, loading core osteogenic factors like BMP-2.⁴⁶ The inner/long-term sustained-release layer employs slowly degrading materials (over 6 weeks) such as polycaprolactone (PCL), bioactive glass (eg, 45S5), or high-molecular-weight PLGA (eg, 85:15), aiming to provide long-term structural support and continuously release bioactive ions like strontium (Sr²⁺) and silicon (Si⁴⁺), which possess dual osteogenic-promoting and osteoclast-inhibiting functions, achieving long-term regulation of the bone repair microenvironment.⁴⁷ (Figure 1).

Figure 1.

A biomimetic spatiotemporally heterogeneous scaffold recapitulates the gradient architecture of natural bone through a multilayered design with programmed degradation and sequential bioactive delivery. The rapidly degrading outer layer provides early antibacterial protection and promotes vascularization through antibiotic/VEGF release; the intermediate repair layer supports osteogenic differentiation via sustained delivery of osteogenic factors such as BMP-2; and the slowly degrading inner layer maintains long-term structural support while continuously releasing bioactive ions (eg, Sr²⁺ and Si⁴⁺). Together, this hierarchical system coordinates early infection control and angiogenesis, mid-stage osteogenesis, long-term bone remodeling, enabling stage-matched regulation throughout bone regeneration.

Precise manufacturing is achieved using techniques like layer-by-layer electrospinning, multi-nozzle 3D bioprinting, or sequential freeze-drying. To ensure strong interlayer bonding and prevent premature delamination of functional layers, interface engineering strategies are employed, such as selective chemical crosslinking using genipin or carbodiimide (EDC), or designing micro/nanoscale physical interlocking structures at the interfaces.^48,49

Microenvironment-Responsive Hydrogels

These “smart” materials can sense local pathophysiological changes in the bone defect and make specific responses, enabling “on-demand” release of factors. This smart responsive system achieves precise drug release through multiple environmental trigger mechanisms. In the acidic microenvironment (pH 6.5–7.0) caused by infection or ischemia, hydrogels based on chitosan, polyacrylic acid, or polyhistidine undergo swelling or degradation due to protonation or charge transitions, thereby releasing antibiotics or anti-inflammatory drugs.^50,51 Utilizing matrix metalloproteinases (MMPs), which are highly expressed during inflammation and remodeling, enzyme-responsive hydrogels can be constructed by using MMP-specific substrate peptides (eg, GPLGIAGQ) as crosslinking points. When MMP concentration increases, peptide bond cleavage leads to gel network dissociation, enabling the precise release of VEGF or the stem cell chemokine SDF-1α.^52,53 Simultaneously, targeting the high levels of reactive oxygen species (ROS) at inflammatory sites, redox-responsive systems containing disulfide bonds or thioketal linkages are designed. Such bonds break under ROS action, achieving targeted release of antioxidants like N-acetylcysteine (NAC) during oxidative stress peaks to protect nascent tissue from damage.^54–56 (Figure 2).

Figure 2.

Smart stimuli-responsive biomaterials enable on-demand therapeutic delivery by sensing pathophysiological changes within the bone defect microenvironment. In response to acidic conditions caused by infection or ischemia, pH-responsive hydrogels release antibiotics or anti-inflammatory agents through protonation-induced swelling or degradation. Under inflammatory and remodeling conditions, MMP-responsive hydrogels with cleavable peptide crosslinks precisely release pro-regenerative factors such as VEGF and SDF-1α. Meanwhile, ROS-responsive systems break redox-sensitive linkages to trigger antioxidant release, such as N-acetylcysteine (NAC), thereby reducing oxidative damage and protecting newly formed tissue. Collectively, these smart materials integrate infection control, inflammation modulation, vascularization, and tissue protection, achieving precise microenvironment-regulated bone regeneration.

Nanocomposite and Functional Hybrid Materials

Integrating functional nanomaterials as “components” into macroscopic scaffolds endows them with temporal functions surpassing those of the bulk materials. Mesoporous silica nanoparticles (MSNs), leveraging their huge specific surface area and tunable pore size (2–10 nm), enable ultra-high loading of growth factors and, through surface modification (eg, with polyethyleneimine), allow fine-tuning of the adsorption strength for different factors, thereby achieving sequential drug release.^57,58 Furthermore, bioactive glass nanofibers can not only continuously release ions like Ca²⁺ and Si⁴⁺, but their unique nanofiber morphology also provides contact guidance, directionally regulating cell adhesion, spreading, and migration.^59,60 Moreover, the introduction of photothermal nano-converters (eg, gold nanorods, copper sulfide nanodots, or 2D materials like MXene) grants the material external responsiveness.^61,62 Under near-infrared (NIR) light irradiation, they convert light energy into heat. This property can be used for photothermal antibacterial therapy (directly killing bacteria and disrupting biofilms via hyperthermia) and can serve as a “switch” to trigger the phase transition of thermosensitive materials (eg, pNIPAM), enabling on-demand, timed, and localized drug release.⁶³ Biodegradable magnesium-based materials represent a promising strategy for achieving dynamic mechanical adaptation because they can provide temporary mechanical support while gradually releasing bioactive magnesium ions. However, their clinical application remains limited by challenges including uncontrolled corrosion rates, hydrogen gas generation, localized alkalization, and mismatch between degradation behavior and new bone formation. Although temporally controlled delivery systems have demonstrated promising regenerative effects, inappropriate release timing, excessive therapeutic dosage, or uncontrolled degradation may compromise therapeutic outcomes. For example, prolonged exposure to high concentrations of growth factors may induce abnormal tissue formation, while certain nanomaterials may cause oxidative stress, inflammatory responses, or long-term accumulation concerns. These limitations highlight that successful temporal therapy requires precise control of dosage, release kinetics, degradation behavior, and host responses.^64,65

Temporally Controlled Release Technologies

Microsphere/Nanoparticle Delivery Systems

This is a classic and effective strategy for achieving different release kinetics. In temporally controlled release systems, multi-stage release microsphere systems and intelligent liposome technology demonstrate precise control capabilities. PLGA microspheres prepared via water/oil/water double emulsion-solvent evaporation can be used to build libraries of microspheres with degradation cycles ranging from days to months by precisely adjusting their molecular weight and monomer ratio (lactic acid/glycolic acid).^66,67 For example, physically mixing fast-degrading microspheres loaded with VEGF (PLGA 50:50, 10 kDa) with slow-degrading microspheres loaded with BMP-2 (PLGA 75:25, 50 kDa) can macroscopically simulate the physiological sequence of “promoting vascularization first, then osteogenesis”.^68,69 More ingeniously, core-shell structured microspheres can integrate burst release (shell) and sustained release (core) functions within a single particle. Concurrently, temperature-sensitive liposomes (eg, with a phase transition temperature of 39–42°C) offer an externally triggered pulsed release strategy: when exposed to external mild hyperthermia (eg, induced by photothermal nanomaterials) and reaching their phase transition temperature, their membrane permeability increases dramatically, enabling rapid, “burst” release of encapsulated drugs.^70,71

Genetic Engineering and Cell Programming

Recent advances in synthetic biology have enabled engineered cells to function as programmable therapeutic systems. Through inducible gene expression systems, biosensor-driven regulation, and environmental-responsive promoters, engineered cells can dynamically regulate therapeutic factor production according to local conditions. These approaches provide new opportunities for adaptive bone regeneration; however, challenges including gene delivery efficiency, immune compatibility, and long-term biosafety remain to be addressed. This involves modifying therapeutic cells to become “biological robots” that autonomously execute temporal instructions. At the genetic level, by integrating conditional gene expression switches and biosensor-driven systems, intelligent expression of therapeutic factors is achieved.^72,73 Conditional gene expression switches (eg, the tetracycline-inducible Tet-On/Off system or optogenetic tools) allow for remote, reversible, and dose-controlled spatiotemporal regulation of the expression of therapeutic factors like BMP-2 in transplanted cells through external stimuli such as oral antibiotics or surface light exposure. Complementing this are biosensor-driven autonomous expression systems, which place therapeutic factor genes under the control of endogenous biosensing promoters. For instance, using a hypoxia-response element (HRE) promoter to drive VEGF expression enables cells to automatically initiate angiogenesis programs upon sensing an ischemic/hypoxic microenvironment.⁷⁴ Alternatively, using osteoblast-specific promoters (eg, the osteocalcin OCN promoter) to drive BMP expression ensures that osteogenic factor expression is initiated only after cells differentiate into osteoblasts, achieving a fully automated, physiologically adapted therapeutic process.⁷⁵

Advanced Biofabrication Technologies: 4D Printing and Biomimetic Manufacturing

Compared with conventional three-dimensional fabrication, 4D bioprinting introduces time-dependent structural evolution into scaffold design. Stimulus-responsive biomaterials and shape-memory polymers enable implanted constructs to dynamically adapt to physiological environments through changes in morphology, mechanical properties, or drug release behavior. Recent advances in biofabrication and computational design have further promoted the development of adaptive regeneration systems. In the collaborative design of 4D bioprinting and biomimetic interface engineering, the introduction of “time” as a fourth dimension on top of three-dimensional structures enables the intelligent evolution of implants within the body.^76,77 4D bioprinting uses shape-memory polymers or stimulus-responsive hydrogels as bioinks. The initial structures built using this technology can undergo autonomous shape changes according to pre-programmed instructions under physiological stimuli such as body temperature or body fluids after implantation. Examples include self-rolling from a flat sheet into a tubular structure, or self-expanding from a compact state to conform to complex defects.^78,79 This dynamic shape-changing process can be further designed to expose new drug release sites or alter pore structure, thereby actively guiding directional cell migration and tissue regeneration.⁸⁰ Simultaneously, through biomimetic mineralization and interface engineering, the natural bone mineralization process is simulated inside the scaffold to construct gradients in chemical composition and crystal structure from amorphous calcium phosphate to crystalline hydroxyapatite.⁸¹ This gradient not only enables temporal ion release – rapid early release of amorphous calcium phosphate to activate cellular responses, followed by slow release of crystalline hydroxyapatite to enhance matrix mineralization – but its corresponding mechanical modulus gradient can also better match the host bone’s mechanical environment, optimizing stress distribution and synergistically promoting the functional regeneration of bone tissue.⁸² (Figure 3).

Figure 3.

4D bioprinting integrates time-dependent structural evolution with biomimetic interface engineering to create adaptive bone-regenerative implants. Using shape-memory polymers and stimulus-responsive hydrogels, printed constructs can autonomously self-roll, self-expand, or reshape after implantation in response to physiological cues, enabling defect conformability, dynamic pore regulation, and exposure of new drug-release sites to guide cell migration and tissue regeneration. Meanwhile, biomimetic mineralization establishes gradients from amorphous calcium phosphate to crystalline hydroxyapatite, enabling rapid early ion release for cellular activation followed by sustained mineral supply for matrix maturation, while the accompanying mechanical modulus gradient improves stress matching with host bone. Together, these spatiotemporally adaptive features promote coordinated structural adaptation, biochemical signaling, and functional bone regeneration.

Critical Comparison of Temporal Delivery Platforms and Translational Challenges

Although numerous temporal delivery systems have demonstrated promising outcomes in experimental models, their clinical feasibility varies considerably. PLGA-based microsphere systems possess relatively mature manufacturing processes and predictable degradation behavior, while responsive hydrogels provide adaptive release capabilities but require further improvement in mechanical stability. Multilayer scaffolds enable spatially organized sequential delivery but face challenges related to manufacturing complexity and reproducibility. Nanocomposite systems provide multifunctional properties, including imaging and external responsiveness; however, their long-term biosafety requires comprehensive evaluation.

Temporal Delivery Strategies for Core Therapeutic Factors

Based on the aforementioned technological platforms, precise temporally controlled therapeutic regimens can be designed for different stages of bone healing.

Early Stage (0–2 Weeks): Anti-Infection, Anti-Inflammation, and Laying the Vascular Network Foundation

To achieve multi-stage synergistic regulation during bone repair, a comprehensive strategy integrating intelligent anti-infection, immune microenvironment remodeling, and vascularization initiation has been designed.^83,84 Firstly, an intelligent anti-infection system is constructed by loading antibiotics like vancomycin or gentamicin into pH-responsive PLGA microspheres: This system accelerates degradation and releases high concentrations of antibiotics in a burst manner only when local infection causes a drop in microenvironmental pH, thereby achieving strong local antibacterial effects while simultaneously reducing systemic toxicity and the risk of antibiotic resistance.⁸⁵ Subsequently, to create a favorable repair environment, immune microenvironment remodeling is achieved by releasing cytokines like IL-4 and IL-10 from the scaffold, or by utilizing magnesium ions (Mg²⁺) and specific nanotopographies, to actively guide infiltrating macrophages to polarize from a pro-inflammatory M1 phenotype towards a pro-repair, pro-angiogenic M2 phenotype.^86,87 Concurrently, the vascularization initiation program is triggered: Using rapidly degrading hydrogel layers or ROS-responsive systems, a pulsed release of VEGF and SDF-1α is achieved in the early post-injury period. Here, SDF-1α strongly chemoattracts endothelial progenitor cells (EPCs) to home to the defect area, working synergistically with VEGF to rapidly construct a new vascular network, laying the foundation for subsequent substantial bone tissue regeneration.^88,89

Mid-Stage (2–8 Weeks): Driving Robust Osteogenesis and Vascular-Osteogenic Coupling

To achieve efficient, high-quality bone regeneration, core osteogenic factors are subjected to optimized delivery and synergistic design via temporal regulation strategies. Firstly, PLGA sustained-release microspheres or bioactive glass carriers are used to continuously release rhBMP-2, maintaining its local concentration within the optimal therapeutic window of 4–8 weeks.^90,91 This low-dose, long-cycle release mode significantly outperforms single high-dose administration in inducing the formation of structurally organized new bone and can effectively reduce side effects like heterotopic ossification. Building upon this, synergistic release of BMP-7 or intermittent administration of the parathyroid hormone analog PTH(1–34) during the mid-to-late repair phase (eg, around week 4) can further stimulate callus maturation and matrix mineralization, thereby enhancing the mechanical strength of the regenerated bone.^92,93 More crucially, the entire repair process follows the principle of “vascularization-osteogenesis sequential coupling”: Using bilayer scaffolds or sequential release systems ensures that the peak release of VEGF precedes that of BMP-2. The initially established rich vascular network not only provides nutrition for subsequent osteogenesis but its endothelial cells themselves can also secrete osteogenesis-promoting factors, forming a positive feedback loop that greatly enhances overall osteogenic efficiency and quality.^94,95

Late Stage (Beyond 8 Weeks): Guiding Bone Remodeling and Achieving Mechanical Compatibility

To achieve functional bone regeneration and ensure long-term stability of the repair, fine-tuning of the bone remodeling process is required in the late stages of bone repair. On one hand, during the remodeling phase, the material’s sustained release of low-dose RANKL or the provision of appropriate micro-strain stimulation via scaffold degradation can be used to moderately activate osteoclasts, initiating the necessary bone resorption and remodeling program.^16,96 Simultaneously, the synergistic release of OPG or strontium ions (Sr²⁺) inhibits excessive osteoclast activity, ensuring a slight predominance of bone formation, thereby achieving a net increase in bone mass within a dynamic balance.⁹⁷ On the other hand, this biochemical regulation process must be combined with biomechanical compatibility: The degradation rate of the scaffold needs to be dynamically synchronized with the formation rate of new bone to avoid mechanical collapse due to overly rapid degradation or stress shielding caused by overly slow degradation. Biodegradable magnesium alloys exemplify this concept. They corrode and degrade in vivo, initially providing mechanical support and releasing Mg²⁺, which has been proven to promote osteogenesis and angiogenesis.^98,99 As the new bone gradually matures and bears load, the magnesium scaffold degrades synchronously, achieving perfect mechanical load transfer and functional regeneration of bone tissue (Table 2).

Table 2.

Temporal Therapeutic Strategy and Delivery System Based on Bone Healing Stages

Healing Stage (Time)	Core Therapeutic Objectives	Key Strategies and Mechanisms	Main Delivery Carriers/Systems	Core Factors/Active Substances
Early Stage (0–2 weeks)	Anti-infection, Anti-inflammation, Establishing Vascular Network	1. Smart Anti-infection: pH-responsive drug release for localized high-concentration bactericidal effect, reducing systemic toxicity. 2. Immune Microenvironment Remodeling: Polarizing infiltrating macrophages from pro-inflammatory M1 to pro-repair M2 phenotype. 3. Vascularization Initiation: Pulsed release of cytokines to recruit endothelial progenitor cells and rapidly establish a nascent vascular network.	pH-responsive PLGA microspheres Fast-degrading hydrogel/ROS-responsive system	Antibiotics (Vancomycin/Gentamicin) IL-4, IL-10 VEGF, SDF-1α
Intermediate Stage (2–8 weeks)	Driving Robust Osteogenesis, Achieving Vascular-Osteogenic Coupling	1. Optimized Osteogenic Factor Delivery: Sustained release of rhBMP-2 via carriers to maintain an optimal therapeutic concentration window, minimizing side effects. 2. Growth Factor Synergy: Coordinated release of BMP-7 or intermittent PTH(1–34) in the mid-late phase to stimulate callus maturation and mineralization. 3. Sequential Vascular-Osteogenic Regulation: Ensuring VEGF release peaks precede BMP-2, creating a positive feedback loop to enhance efficiency.	PLGA sustained-release microspheres Bioactive glass Bilayered/sequential release scaffolds	rhBMP-2 BMP-7, PTH(1–34) VEGF
Late Stage (>8 weeks)	Guiding Bone Remodeling, Achieving Mechanical Compatibility	1. Regulating Bone Remodeling: Moderately activating osteoclasts via low-dose RANKL or microstrain from degradation to initiate necessary bone resorption/remodeling, while co-releasing OPG or strontium ions to inhibit excessive activity, favoring net bone formation. 2. Biomechanical Compatibility: Matching the scaffold degradation rate dynamically with the rate of new bone formation to enable smooth mechanical load transfer and avoid stress shielding or structural collapse.	Degradable materials (eg, Mg alloys) Sustained-release systems	RANKL, OPG Strontium ions (Sr2⁺) Magnesium ions (Mg2⁺)

Abbreviations: VEGF, vascular endothelial growth factor; BMP, bone morphogenetic protein; SDF-1α, stromal cell-derived factor-1α; PLGA, poly(lactic-co-glycolic acid); PTH, parathyroid hormone; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand; UCNPs, upconversion nanoparticles.

Preclinical Validation and Translational Status of Temporally Controlled Therapy

Confirmatory Efficacy in Large Animal Models

Large animal (sheep, goat, minipig) weight-bearing long bone (tibia, femur) critical-sized defect models serve as the “litmus test” for evaluating whether a therapeutic strategy can advance to the clinic. Research data are encouraging: Compared to simple implantation of BMP-2 in a collagen sponge, using a smart scaffold that temporally releases VEGF (week 1) and BMP-2 (weeks 2–8) in a 3-cm tibial defect model in sheep can shorten bone healing time by 30–40%. Micro-CT and biomechanical testing show that parameters of the new bone, such as bone volume fraction (BV/TV), trabecular thickness (Tb.Th), as well as torsional stiffness and maximum torque, are significantly superior to those in the control group and closer to normal bone levels.^100–102 In more complex infected bone defect models, systems integrating sequential antibiotic release with sequential growth factor release demonstrate powerful capabilities for simultaneous debridement and regeneration, offering innovative solutions for clinically challenging problems.

Real-World Challenges for Clinical Translation

Regulatory Maze: Temporally controlled therapy products are often “combination products” comprising biomaterials, active factors (drugs), and sometimes even cells. Their approval pathway is complex, requiring compliance with both medical device and pharmaceutical regulations, resulting in lengthy and costly processes.¹⁰³

Cost and Accessibility: The R&D, precision manufacturing, and quality control of smart materials increase product costs. How to reduce costs without sacrificing performance, making them accessible to healthcare systems and a wide range of patients, is a critical issue for industrialization.¹⁰⁴

Complexity of Personalized Protocols: Patient factors such as age, health status (eg, diabetes, osteoporosis), defect location, size, and infection status vary greatly, meaning that a “one-size-fits-all” temporal protocol may be ineffective. Developing personalized temporal protocol design platforms based on patient-specific parameters (eg, incorporating AI prediction) is a future direction.^105,106

Long-term Biosafety: Complex material systems and their degradation products may reside in the body for years. Their long-term biocompatibility, immunogenicity, and potential systemic effects require confirmation through longer-term follow-up studies.¹⁰⁷

Future Perspectives and Conclusion

Future Directions

Closed-Loop Intelligent Diagnosis and Treatment Systems: The ideal future implant would be a “smart organ” integrating biosensors (for real-time monitoring of local pH, pO₂, mechanical strain, specific biomarkers), microprocessors, and microfluidic actuators. It could sense the healing status in real-time and dynamically adjust the release profile of growth factors or drugs via AI algorithms, achieving fully automated, adaptive personalized treatment.

Fusion with “Living” Composite Materials: Deep integration of genetically edited MSCs or induced pluripotent stem cell (iPSC)-derived osteoblasts with smart scaffolds. These cells would not only serve as “factories” for factor production but also directly participate in constructing new bone tissue, forming a “living” repair system capable of self-renewal and self-regulation.

Multi-Omics and AI-Driven Design: Utilizing single-cell sequencing, spatial transcriptomics, and proteomics to analyze high-resolution spatiotemporal maps of the entire bone healing process at an unprecedented resolution. Combined with machine learning algorithms, new regulatory nodes and optimal temporal patterns can be mined from vast datasets, which can then inversely guide the design of new biomaterials and the formulation of personalized treatment plans.

Immune Engineering-Dominated Temporal Regulation: Moving beyond simple modulation of macrophage polarization to deeply explore how to temporally manipulate the specific functions of various immune cells (eg, T cells, neutrophils) in bone regeneration, reshaping the immune system from a passive “bystander” or “disruptor” in repair into an active “director” and “engineer” (Table 3).

Table 3.

Summary of Representative Preclinical Studies of Spatiotemporal Delivery Scaffolds for Bone Regeneration

Model	Defect Size	Material System	Release Strategy	Evaluation Indicators
Sheep long bone defect	Critical-sized segmental defect	Growth factor-loaded scaffold	VEGF/BMP sequential release	Micro-CT, mechanical testing
Rat femoral defect	Critical-sized defect	Responsive hydrogel	Stimulus-triggered release	Bone formation, angiogenesis
Large animal models	Weight-bearing defects	Multifunctional scaffolds	Sequential therapeutic regulation	Functional regeneration

Conclusion

From a clinical perspective, temporally controlled therapy provides a promising strategy to overcome limitations of conventional bone defect repair by coordinating infection control, vascularization, osteogenesis, and remodeling. From an industrial perspective, the development of standardized manufacturing processes and regulatory frameworks will determine the feasibility of clinical translation. From a basic scientific perspective, these systems provide new opportunities to investigate and manipulate the complex interactions between biomaterials, immune responses, and tissue regeneration. Temporally controlled therapy represents a paradigm shift from traditional concepts of bone defect repair. It is no longer satisfied with passive spatial filling but actively mimics and optimizes the natural spatiotemporal healing program. By integrating cutting-edge achievements in materials engineering, molecular biology, pharmaceutical sciences, and clinical medicine, it is dedicated to providing an ideal solution for critical-sized bone defects with higher biological integration, better functional recovery, and stronger individual adaptability. Although the journey towards widespread clinical application still faces multiple challenges from scientific, engineering, regulatory, and economic perspectives, the enormous potential and breakthrough progress demonstrated in this field are encouraging. Temporally controlled therapy represents a promising direction for improving critical-sized bone defect regeneration; however, extensive efforts are still required to overcome challenges related to biosafety, manufacturing, regulatory approval, and personalized treatment before broad clinical implementation. Ultimately, it aims to achieve the ultimate goal of transitioning from “repairing a defect” to “regenerating a living organ with complete biological function and mechanical performance”, bringing hope to millions of patients. Future research will undoubtedly focus on enhancing the intelligence, precision, and accessibility of these systems, propelling regenerative medicine to new heights. Future research should focus on several key challenges: (1) establishing personalized temporal treatment protocols according to patient-specific healing conditions; (2) developing real-time monitoring systems for in vivo release regulation; (3) improving long-term biosafety evaluation of multifunctional materials; and (4) establishing standardized criteria for clinical translation. Integration of artificial intelligence, biosensors, multi-omics analysis, and advanced manufacturing technologies may provide feasible solutions. Rare-earth nanoparticle-integrated sequential delivery systems represent an emerging direction by combining temporal regulation, multifunctional therapeutic effects, and imaging capabilities, although further studies are required to fully address biosafety and translational challenges. Among currently available strategies, sequential delivery systems based on biodegradable polymers, bioactive scaffolds, and growth factor regulation have demonstrated the strongest experimental evidence, particularly in promoting vascularization and osteogenesis in preclinical models. However, translation into clinical practice remains limited by challenges including manufacturing standardization, regulatory complexity, long-term biosafety evaluation, and patient-specific variability. Despite their promising multifunctional properties, rare-earth nanomaterials require careful safety evaluation before clinical translation. Potential concerns include ion release, long-term accumulation, systemic toxicity, and dose-dependent biological effects. For example, gadolinium-containing systems require particular attention because uncontrolled Gd release may raise concerns regarding biological retention and toxicity. In addition, combined use of multiple rare-earth elements may result in complex interactions affecting oxidative balance, immune responses, and degradation behavior. Therefore, comprehensive evaluation of pharmacokinetics, biodegradation, and long-term host responses is essential.

Data Sharing Statement

This is a review article, and all relevant information is provided in the article.

Ethical Approval and Consent to Participate

This is a review paper and does not involve direct research on humans or animals.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. All the authors listed meet the criteria for authorship as per the ICMJE guidelines, read the final manuscript and agree to publish this work.

Disclosure

The Authors declare that they have no competing interests financial or non-financial or any other interests that might be perceived to influence the results and/or discussion reported in this paper.