ZIF-8-Based Nanomedicine for Bone Regeneration: A Critical Appraisal of Preclinical Osteoimmunomodulatory Evidence and Translational Readiness

Abstract

Zeolitic imidazolate framework-8 (ZIF-8)-based biomaterials are increasingly investigated for bone repair because they combine cargo loading, environment-dependent degradation, surface engineering, and bioactive Zn²⁺ release. However, recent reviews already catalogue the multifunctional design and orthopedic applications of ZIF-8, while the directness of evidence supporting specific osteoimmunomodulatory claims remains less clear. This structured narrative review critically appraises preclinical ZIF-8 literature using a predefined evidence-classification framework that separates direct ZIF-8 evidence in bone-related models from indirect evidence derived from in vitro or non-bone studies, zinc biology, or other biomaterials, and from proposed mechanisms lacking direct validation. Macrophage-centered immunomodulation currently has the clearest direct support, including studies linking ZIF-8-containing systems to changes in inflammatory signaling and repair-associated macrophage phenotypes alongside bone regeneration. By contrast, direct evidence for local Th17/Treg regulation, neutrophil extracellular trap modulation, and broader immune-cell networks remains limited; these pathways should therefore be treated primarily as mechanistic hypotheses or future research directions. We also examine whether reported effects can be attributed to the ZIF-8 carrier itself, released Zn²⁺, loaded cargo, surface coatings, or composite matrices, and summarize study-level information on formulation, release conditions, dose, models, controls, immune endpoints, and regenerative outcomes. Particular attention is given to medium-dependent ZIF-8 stability, including phosphate and biological-fluid effects, dose-dependent cytotoxicity, the uncertain fate of Zn²⁺ and 2-methylimidazole, bone targeting versus local retention, and the scarcity of long-term pharmacokinetic, biodistribution, and large-animal data. Finally, we propose a translational-readiness framework spanning component attribution, standardized dose-release-toxicity testing, disease-specific validation, manufacturing reproducibility, sterilization, and regulatory planning. Most available evidence remains proof-of-concept and preclinical, indicating that mechanistic and safety validation should precede claims of clinical readiness.

Plain Language Summary

Large bone defects can be difficult to heal, especially when infection, poor blood supply, or prolonged inflammation disrupts the normal repair process. ZIF-8 is a tiny porous material made from zinc and an organic building block. It can carry medicines, release zinc ions, and be incorporated into hydrogels, coatings, membranes, or scaffolds for local treatment.

In this review, we examined preclinical studies of ZIF-8-based materials for bone repair. We focused not only on whether these materials improved bone formation, but also on how strongly the proposed biological mechanisms were supported. The clearest evidence indicates that selected ZIF-8-containing systems can regulate inflammation-related responses involving immune cells called macrophages while supporting bone regeneration. Evidence that ZIF-8 directly controls specific T-cell or neutrophil pathways remains limited and should be regarded as preliminary.

We also found that ZIF-8 may break down differently in acidic environments and in body fluids containing phosphate and proteins. Its benefits and risks depend on the dose, release rate, therapeutic cargo, and surrounding material. Before clinical use, researchers need to define safe exposure levels, track degradation products, study distribution and clearance, test relevant disease and larger-animal models, and establish reliable manufacturing and sterilization methods. ZIF-8 is therefore a promising preclinical platform, but it is not yet ready for routine clinical use.

Graphical Abstract

Introduction

Large and complex bone defects remain a major clinical challenge in orthopedic, dental, maxillofacial, and trauma surgery.^1,2 Defects caused by severe trauma, tumor resection, infection, metabolic bone disease, or implant failure often exceed the intrinsic regenerative capacity of bone and require grafting, scaffold implantation, or biomaterial-assisted reconstruction.^1,2 Although autologous bone grafting remains a clinical benchmark because it provides osteogenic cells, osteoinductive signals, and an osteoconductive matrix, its application is limited by donor-site morbidity, limited graft volume, prolonged operative time, and variable outcomes in patients with infection, diabetes, osteoporosis, vascular insufficiency, or chronic inflammation.^1–3 Allogeneic grafts and synthetic substitutes can partially overcome supply limitations, but they frequently show insufficient bioactivity, delayed integration, immune incompatibility, or unsatisfactory performance in pathological microenvironments.^2,4 These limitations indicate that effective bone repair requires more than structural filling; it requires active regulation of the local biological microenvironment.^4,5

The bone immune microenvironment has emerged as a critical determinant of regenerative outcomes.^4–6 After bone injury, immune cells participate in pathogen defense, debris clearance, cytokine secretion, angiogenic signaling, osteoprogenitor recruitment, and bone remodeling.^4,6 A transient inflammatory response is essential for initiating repair, whereas persistent or excessive inflammation can impair osteogenic differentiation, enhance osteoclastogenesis, disrupt vascularization, and delay tissue regeneration.^4,5 Macrophage polarization, Th17/Treg balance, neutrophil activation, neutrophil extracellular traps, reactive oxygen species, inflammatory cytokines, and RANKL/OPG-mediated osteoclast activity collectively influence the transition from inflammation to repair and remodeling.^4,6–8 Therefore, bone regeneration strategies should not simply suppress inflammation, but should modulate the timing, intensity, and cellular composition of immune responses.

Nanomedicine provides an attractive approach for spatially and temporally controlled regulation of pathological bone microenvironments.^5,9 Compared with free therapeutic agents or passive biomaterials, nanoplatforms can improve local retention, protect unstable bioactive molecules, enhance drug loading, enable stimuli-responsive release, and integrate multiple therapeutic functions within a single system.^9,10 In bone regeneration, nanomedicine platforms can be engineered to deliver antibacterial agents, anti-inflammatory compounds, antioxidants, osteogenic factors, angiogenic cues, proteins, peptides, or nucleic acids while responding to pathological stimuli such as acidity, oxidative stress, enzyme activity, or infection-associated signals.^5,9,10 These characteristics are particularly relevant for complex bone defects, where inflammation, infection, impaired vascularization, excessive bone resorption, and poor tissue integration often coexist.

Zeolitic imidazolate framework-8 (ZIF-8), a zinc-based metal–organic framework assembled from Zn²⁺ nodes and 2-methylimidazolate linkers, has attracted attention as a versatile carrier and bioactive material for bone repair.^10,11 Its framework can support multiple loading routes, surface engineering, and integration with hydrogels, membranes, coatings, and porous scaffolds.^9–11 Acidic conditions can accelerate framework disruption and cargo release, while released Zn²⁺ may contribute to osteogenic, antimicrobial, and immune-related effects within formulation- and exposure-dependent limits.^10,12,13 Importantly, ZIF-8 behavior in biological environments cannot be reduced to pH responsiveness alone; phosphate, proteins, amino acids, particle properties, and surface modification can substantially alter stability and release behavior.^14,15 These features make ZIF-8 an attractive platform for bone nanomedicine, but they also create important challenges in mechanistic attribution and safety evaluation.

Several recent reviews have comprehensively summarized ZIF-8-based systems for bone regeneration and orthopedic applications, covering material synthesis, drug delivery, scaffold integration, functionalization strategies, and biological performance.^16–19 For example, Rahaman and Mukherjee reviewed ZIF-8-based multifunctional biomaterials from the perspectives of design strategies, drug-delivery systems, scaffold integration, and biological functions, while Zhang et al focused on functionalized ZIF-8 platforms for orthopedic therapeutic applications and their associated challenges. Tahamtan et al specifically examined ZIF-8-based composites in bone scaffolds.^17–19 These contributions have substantially advanced understanding of how ZIF-8 can be engineered for bone-related applications; however, the rapidly expanding literature also raises a different question: how directly and convincingly are the proposed osteoimmunomodulatory mechanisms supported by the available experimental evidence?

Accordingly, the present review is not intended to provide another catalogue of ZIF-8 formulations or bone-regenerative applications. Instead, it adopts a structured narrative and critical-appraisal approach to distinguish direct ZIF-8 bone-model evidence, indirect evidence derived from zinc biology, non-bone models or related biomaterials, and proposed mechanisms that remain to be directly validated. Particular emphasis is placed on four unresolved issues: the relative strength of evidence across immune targets; the ability to distinguish the effects of the ZIF-8 carrier, released Zn²⁺, therapeutic cargo and other composite components; the quantitative relationship among formulation, degradation, release, dose and toxicity; and the experimental evidence required to judge translational readiness. The scope and analytical distinctions between representative recent reviews and the present review are summarized in Table 1.

Table 1.

Comparison of Representative Recent Reviews and the Distinctive Analytical Scope of the Present Review

Representative Review	Main Scope	Osteoimmunology Focus	Evidence Appraisal	Quantitative/Translational Emphasis	Distinctive Analytical Scope of the Present Review
Tahamtan et al, 202519	ZIF-8-based composites for bone scaffolds	Immune effects considered mainly within scaffold-mediated regeneration	No explicit direct/indirect/proposed evidence framework	Emphasis on scaffold design, material properties, and regenerative performance	Adds explicit evidence classification, component attribution, and translational-readiness analysis
Zhang et al, 202518	Functionalized ZIF-8 platforms for orthopedic applications	Immunomodulation considered within functionalized therapeutic systems	Not primarily organized according to mechanistic evidence directness	Strong emphasis on functionalization, targeting, delivery, and orthopedic challenges	Evaluates whether immune mechanisms are directly demonstrated in bone models and whether effects can be attributed to ZIF-8, Zn2+, cargo, or other components
Rahaman & Mukherjee, 202617	Multifunctional ZIF-8 biomaterials for bone regeneration	Immune regulation included as an important biological function	Broad functional synthesis rather than explicit direct/indirect/proposed classification	Drug delivery, scaffold integration, biological functions, biosafety, and translational limitations	Adds evidence hierarchy, study-level quantitative appraisal, component attribution, and translational-readiness assessment
Present review	Critical appraisal of ZIF-8-based bone nanomedicine	Central focus on the directness of osteoimmunomodulatory evidence	Direct / indirect / proposed or hypothetical evidence explicitly distinguished	Study-level formulation, dose, release, controls, safety, biodistribution, manufacturing, and model validity are critically assessed	Integrates evidence hierarchy, quantitative evidence audit, component attribution, disease-specific interpretation, and translational readiness

Review Methodology

Literature Search Strategy

This review was conducted as a structured narrative review with a predefined critical-appraisal framework rather than as a systematic review or meta-analysis. Literature searches were performed in PubMed, Web of Science Core Collection, and Scopus from database inception to August 15, 2026. Search terms combined ZIF-8-related terms (“ZIF-8” OR “zeolitic imidazolate framework-8” OR “zeolitic imidazolate framework 8”) with terms related to bone regeneration and orthopedic applications, including “bone regeneration”, “bone defect”, “osteogenesis”, “osseointegration”, “osteomyelitis”, “periodontal”, and “orthopedic”. Additional targeted searches combined ZIF-8-related terms with immune and translational concepts, including “macrophage”, “osteoimmunology”, “osteoclast”, “Th17”, “Treg”, “neutrophil extracellular traps”, “inflammation”, “toxicity”, “degradation”, “phosphate”, “biodistribution”, “pharmacokinetics”, “bone targeting”, “sterilization”, and “manufacturing”. Reference lists of highly relevant original studies and recent reviews were additionally examined to identify potentially relevant publications not retrieved through the initial keyword searches. Representative recent reviews identified through targeted searches were additionally evaluated to clarify overlap with previous literature and the distinct analytical scope of the present review.

Eligibility Criteria and Study Selection

Peer-reviewed original studies were prioritized when they directly evaluated ZIF-8 or ZIF-8-containing composites in bone regeneration, bone defects, orthopedic implants, bone infection, periodontal or craniofacial regeneration, tendon–bone interface repair, or other closely related musculoskeletal models. Studies were considered particularly relevant when they reported both regenerative outcomes and mechanistic, immune, inflammatory, degradation, release, or safety endpoints. Non-bone ZIF-8 studies, general Zn²⁺ biology studies, studies of other MOFs or zinc-based biomaterials, and broader osteoimmunology literature were included selectively when required to interpret biological plausibility, material behavior, safety, or an identified evidence gap. Reviews were used primarily to contextualize the field and identify additional original studies rather than as substitutes for primary evidence. Publications without sufficient relevance to ZIF-8 design, bone-related applications, immune mechanisms, safety, or translation were excluded from the core evidence synthesis.

Data Extraction and Critical Appraisal

For original studies included in the critical evidence synthesis, the following information was extracted when available: ZIF-8 formulation and modification, particle size, therapeutic cargo, loading or encapsulation characteristics, release medium and release conditions, ZIF-8 or Zn²⁺ exposure, cell or animal model, administration route, control groups, immune or inflammatory endpoints, osteogenic and regenerative outcomes, follow-up duration, and major experimental limitations. Quantitative information was reported only when explicitly provided in the original study; missing values were not inferred. Particular attention was given to whether the experimental design allowed the therapeutic contribution of individual platform components to be distinguished, including the ZIF-8 carrier, released Zn²⁺, loaded cargo, surface coating, scaffold, or hydrogel matrix.

Component attribution was considered adequately resolved when the experimental controls allowed the major active components to be independently interpreted, for example through comparisons involving unloaded ZIF-8, free cargo, matrix- or coating-only controls, or other appropriate component-specific groups. Attribution was considered partially resolved when only some components were isolated experimentally and unresolved when the reported effect could not be separated among ZIF-8, Zn²⁺, cargo, or other composite components. This classification was used to assess mechanistic interpretability rather than as a formal risk-of-bias tool.

Evidence Classification

Evidence was classified according to its directness to the proposed ZIF-8-mediated mechanism in bone regeneration, rather than by a formal clinical evidence-grading system. Direct evidence was defined as evidence from a ZIF-8 or ZIF-8-containing intervention evaluated in a bone-related in vivo model in which the relevant mechanistic or immune endpoint was directly measured, or from an integrated study in which mechanistic cellular experiments were linked to corresponding in vivo bone-regeneration outcomes. Indirect evidence included ZIF-8 studies limited to in vitro mechanistic observations without corresponding bone-model validation, ZIF-8 studies in non-bone disease models, general Zn²⁺ biology, studies of other MOFs or biomaterials, and bone-regeneration studies that reported therapeutic outcomes without directly evaluating the proposed mechanism. Proposed or hypothetical mechanisms were defined as biologically plausible pathways for which no direct ZIF-8 bone-model validation was identified.

Methodological Limitations

This review was designed to provide a structured critical synthesis across a heterogeneous and rapidly evolving field rather than a formal systematic review. Accordingly, no meta-analysis or formal risk-of-bias assessment was undertaken. The evidence-classification framework evaluates experimental directness and mechanistic interpretability rather than clinical certainty. Differences in ZIF-8 formulation, particle characteristics, cargo, dose, release medium, disease model, control design, analytical method, and follow-up duration limit direct quantitative comparison across studies. Furthermore, some mechanistic topics, particularly Th17/Treg regulation, NET modulation, and broader immune-cell networks, are supported mainly by indirect or extrapolated evidence. These limitations are explicitly considered when interpreting mechanistic claims and translational readiness.

ZIF-8 as an Environment-Responsive Nanomedicine Platform

The performance of ZIF-8 in bone nanomedicine is determined by the interplay among framework chemistry, cargo-loading route, particle properties, biological medium, surface modification, and delivery format. These variables influence cargo retention and release, Zn²⁺ exposure, degradation-product formation, cellular uptake, and potential toxicity. ZIF-8 should therefore be regarded as an environment-responsive platform whose behavior requires characterization under application-relevant conditions rather than being inferred from nominal pH responsiveness alone. Representative comparisons with alternative MOF and zinc-based systems are summarized in Table 2. The structural, cargo-related, degradation, functionalization, biological, and safety features of ZIF-8 relevant to bone nanomedicine are summarized in Figure 1.

Table 2.

Comparative Characteristics of ZIF-8 and Representative Alternative Metal-Organic or Zinc-Based Platforms Relevant to Bone Nanomedicine

Platform	Stability/Degradability	Metal-Related Considerations	Cargo Compatibility/Loading	Relevance to Bone and Osteoimmune Modulation	Main Trade-Off Relative to ZIF-8
ZIF-8	Environment-responsive and relatively degradable. Acidic conditions accelerate framework disruption, while phosphate-containing and protein-rich biological media can also promote transformation at near-neutral pH.	Released Zn2⁺ can contribute to osteogenic, antimicrobial, and immune-related effects, but excessive or prolonged exposure may be cytotoxic. The in vivo fate of 2-methylimidazole-related products remains incompletely defined.	Versatile loading through pore/surface adsorption, defect-associated incorporation, biomimetic mineralization, surface modification, and hybrid integration. Loading route should be matched to cargo size and chemistry.	Extensively investigated in preclinical bone regeneration. Macrophage-associated inflammatory remodeling currently has the clearest direct osteoimmune support.	Combines degradability, Zn2⁺ bioactivity, and cargo versatility, but medium-dependent instability, exposure-dependent Zn2⁺ toxicity, and uncertain degradation-product fate complicate translation.
ZIF-67	Structurally related to ZIF-8, but degradation and biological behavior remain formulation- and medium-dependent.	Co-based nodes provide distinct catalytic/redox properties but introduce cobalt-related exposure and toxicity concerns that differ from Zn-based ZIF-8.	The related zeolitic architecture supports cargo incorporation and surface engineering, although biocompatibility constraints may limit the usable exposure range.	Potential catalytic and redox functions are attractive, but bone-regenerative and osteoimmune evidence is less established than for ZIF-8.	May offer stronger catalytic/redox activity, but Co-related toxicological burden can be less favorable when bioactive Zn2⁺ release and broader bone-regeneration evidence are priorities.
UiO-series	Generally more structurally and hydrolytically stable than ZIF-8; degradation is slower and strongly dependent on framework defects, functionalization, and biological environment.	Common Zr-based systems avoid deliberate Zn2⁺ or Co2⁺ release, but metal-node and linker fate still require formulation-specific safety evaluation.	High porosity and tunable functional groups support drug loading; cargo compatibility depends strongly on pore dimensions, defects, and surface modification, especially for larger biomolecules.	Bone-oriented applications have been reported, but direct osteoimmune evidence is less extensive and platform-specific.	Greater stability may favor prolonged cargo retention, but can be less advantageous when predictable biodegradation and clearance are central design requirements.
MIL-series	Highly framework-dependent. Fe-, Al-, or other metal-based MIL materials differ markedly in stability, degradation, and ion release; family-level generalization is inappropriate.	Metal-related safety depends on the specific MIL framework and degradation products rather than on the MIL designation itself.	Broad diversity in pore architecture and chemistry enables varied loading strategies, but loading capacity and release behavior cannot be generalized across the family.	Regenerative and drug-delivery applications are reported for selected MIL systems, whereas bone-specific osteoimmune evidence remains heterogeneous.	Offers broad compositional and functional tunability, but substantial framework-to-framework heterogeneity reduces the value of simple family-level comparisons with ZIF-8.
Zn-polyphenol / metal-polyphenol systems	Dynamic coordination networks can be assembled under mild aqueous conditions and are often more structurally labile than crystalline MOFs; stability depends on metal/polyphenol chemistry and coordination conditions.	Zn-containing systems can retain Zn2⁺-related bioactivity while avoiding 2-methylimidazole. The polyphenol component is itself biologically active, which can complicate component attribution.	Cargo association generally relies on coordination, adsorption, coating, or matrix integration rather than a permanent crystalline microporous framework.	Bone-related coatings and hydrogels can provide osteogenic, antioxidant, anti-inflammatory, or antibacterial functions, although the evidence is not directly interchangeable with ZIF-8 studies.	Can provide simpler bioactive interfaces without 2-MIM, but less-defined network stoichiometry, composition-dependent stability, and batch reproducibility may complicate standardization.

Notes: This table provides a representative qualitative comparison rather than a universal ranking. Stability, cargo loading, biological activity, and safety remain formulation- and exposure-dependent.

Abbreviations: MOF, metal-organic framework; 2-MIM, 2-methylimidazole.

Figure 1.

Structural and biological features of ZIF-8 relevant to bone nanomedicine. ZIF-8 consists of Zn²⁺ nodes and 2-methylimidazolate linkers connected through Zn–N coordination. Representative cargos include small-molecule drugs, proteins and growth factors, and nucleic acids, which may be incorporated through adsorption, defect-associated loading, biomimetic mineralization, or hybrid-matrix integration. Surface functionalization and incorporation into hydrogels, membranes, implant coatings, or three-dimensional scaffolds can modify retention, degradation, release, and biological interactions. Degradation is environment dependent: acidic conditions accelerate framework disruption, while phosphate-containing and protein-rich media may also alter stability at near-neutral pH. The biological effects of released Zn²⁺ are dose- and exposure-dependent. Particle properties, administered dose, degradation products, biodistribution, immunocompatibility, and long-term fate remain important safety considerations. Icons schematically represent bone, endothelial, and immune cells, small-molecule drugs, proteins, and nucleic acids.

Structure, Loading Routes, and Cargo Compatibility

ZIF-8 is a zinc-based metal–organic framework composed of tetrahedrally coordinated Zn²⁺ centers and 2-methylimidazolate linkers, typically forming a sodalite-type framework.^10,20 Its internal cavities, external surface, crystal defects, and modifiable interfaces provide multiple opportunities for cargo association.^10,11 However, intrinsic microporosity should not be interpreted as evidence that all therapeutic cargos are physically accommodated within ideal framework pores. The dominant loading mechanism depends on cargo size and chemistry, synthesis route, crystal defects, solvent conditions, and post-synthetic modification.^9,10

For relatively small molecules, cargo association may involve pore-mediated adsorption, defect-associated incorporation, external-surface adsorption, and electrostatic, coordination, hydrogen-bonding, or hydrophobic interactions.^9,10 Experimentally measured loading should therefore not automatically be equated with occupation of the intrinsic microporous network unless pore accessibility or cargo localization has been directly demonstrated.

Biomacromolecules require a different interpretation. Proteins, enzymes, nucleic acids, and other cargos larger than the intrinsic ZIF-8 pore apertures can be incorporated through in situ biomimetic mineralization or one-pot co-precipitation, during which ZIF-8 nucleates and grows around the biomolecule rather than relying on diffusion through a preformed framework.^21,22 Liang et al, for example, demonstrated that BSA incorporation occurred through biomolecule-mediated nucleation and encapsulation rather than adsorption into the native ZIF-8 pore network.²¹

In hybrid systems, the therapeutic agent may also be associated partly or primarily with a surrounding polymer, hydrogel, membrane, implant coating, or porous scaffold.^9,10 Because these matrices can alter cargo retention, fluid access, degradation, and local exposure, studies should report the loading route and avoid describing ZIF-8 generically as a high-capacity porous carrier when cargo localization has not been established. Particle size, morphology, crystallinity, surface charge, and coating chemistry further influence uptake, degradation, and release and should be interpreted in the context of the final delivery format.^11,23

Environment-Dependent Degradation: Beyond Simple pH Responsiveness

Acid-sensitive degradation is an important property of ZIF-8. Under acidic conditions, protonation of the imidazolate linker weakens Zn–imidazolate coordination and promotes framework disruption, facilitating the release of Zn²⁺ and associated cargos.^10,11 However, pH alone does not determine ZIF-8 stability under biological conditions; the ionic and molecular composition of the surrounding medium can substantially alter framework integrity and degradation kinetics.^14,15

Phosphate is particularly important in this context. Velásquez-Hernández et al demonstrated substantial decomposition of ZIF-8 in 10 mM phosphate-buffered saline at pH 7.4 and 37°C, with phosphate shifting the Zn²⁺–imidazolate coordination equilibrium toward insoluble zinc-phosphate products.²⁴ Degradation was particle-size dependent, with nanoscale ZIF-8 losing crystallinity more rapidly than larger crystals under the tested conditions.²⁴ Neutral pH therefore does not guarantee ZIF-8 stability in the presence of strongly coordinating biological anions.²⁴

Biological media introduce further complexity. Luzuriaga et al showed that phosphate- and bicarbonate-containing buffers altered ZIF-8 morphology and composition, whereas serum produced more pronounced dissolution and premature cargo release.¹⁴ Cell-culture medium composition, serum proteins, amino acids, ionic strength, particle properties, and surface coatings can therefore modify ZIF-8 behavior.^14,15 Importantly, framework transformation and cargo release should not be assumed to proceed identically across different media.

These medium-dependent effects have direct implications for bone nanomedicine. Acidic conditions associated with infection, inflammation, or osteoclast activity may accelerate acid-mediated degradation, whereas phosphate-containing fluids and protein-rich environments can independently alter framework stability before the intended pathological niche is reached. Terms such as “pH-controlled”, “site-specific”, or “acid-triggered” release should therefore be used cautiously unless the final formulation has been evaluated under physiologically relevant conditions. Importantly, no single defect-site pH can be assumed across bone-regeneration settings, because local acidity varies with disease state, anatomical compartment, inflammatory burden, cellular activity, and time after injury. Disease-site pH should therefore be measured or experimentally justified for the intended application rather than treated as a universal trigger for ZIF-8 degradation.

Delivery matrices can further modify fluid access, diffusion, degradation, and release. Incorporation into a hydrogel, membrane, coating, or scaffold should therefore not be assumed to prolong release without experimental confirmation. Likewise, claims of sequential or stage-specific delivery should be supported by measured cargo and Zn²⁺ release profiles, preferably including phosphate- and protein-containing conditions in addition to simplified acidic buffers.

Zn²⁺ Release: Bioactivity, Exposure Metrics, and Dose-Dependent Risk

Zn²⁺ released during ZIF-8 degradation is biologically active rather than an inert degradation product. Zinc participates in skeletal homeostasis, extracellular-matrix synthesis, mineralization, immune regulation, and osteoblast–osteoclast coupling, providing a biological rationale for several regenerative effects reported for ZIF-8-containing systems.^12,13 At the platform level, nanoscale ZIF-8 has also been reported to promote osteogenic differentiation and bone repair through canonical MAPK signaling, although this mechanism should not be generalized across formulations without formulation-specific validation.²⁵ However, evidence derived from soluble zinc salts, other zinc-containing biomaterials, and ZIF-8 should not be treated as interchangeable because the magnitude and duration of zinc exposure depend on degradation, local retention, protein binding, particle uptake, and the surrounding biological environment.^12,13,23

A critical distinction is required among nominal ZIF-8 dose, extracellular Zn²⁺ concentration, cumulative zinc release, and intracellular zinc exposure. These parameters describe different aspects of biological exposure and cannot be substituted for one another. A nanoparticle mass concentration does not directly establish the concentration of bioavailable Zn²⁺ experienced by cells, while cumulative release measured in a simplified buffer may not reproduce exposure within a protein-rich defect, hydrogel, coating, or scaffold.

The biological window of Zn²⁺ is therefore context dependent rather than universal. Moderate exposure may support osteogenic or antimicrobial functions, whereas excessive or prolonged exposure can disturb ion homeostasis, increase oxidative stress, impair mitochondrial function, activate apoptosis-related pathways, and reduce cell viability.^23,26,27 The transition between beneficial and adverse effects varies with cell type, formulation, particle properties, degradation rate, medium, and exposure duration. Accordingly, a single universal “therapeutic” or “safe” Zn²⁺ concentration should not be extrapolated across ZIF-8-based bone-regeneration studies.

Representative studies illustrate that the biological response to Zn²⁺ depends on both concentration and exposure duration. In rBMSCs, Zn²⁺ concentrations of 2 and 5 μg/mL promoted adhesion, proliferation, and osteogenic differentiation, whereas 15 μg/mL produced adverse effects; in the same study, proliferation was evaluated over 1, 4, and 7 days, osteogenic outcomes over 3–14 days, and ROS generation and apoptosis after 24 h.²⁶ In another rBMSC study, 125 μM Zn²⁺ promoted survival, proliferation, migration, and osteogenic differentiation, whereas concentrations above 250 μM reduced viability and induced apoptosis; viability was assessed over 1, 3, and 5 days and apoptosis after 3 days.²⁷ Osteoblast-lineage responses are likewise time dependent: in SaOS-2 cells, 1 and 10 μM Zn²⁺ increased ALP activity after 4, 6, and 8 days, whereas 50 μM reduced it.²⁸ These study-specific differences reinforce that Zn²⁺ exposure cannot be interpreted independently of cell type, exposure duration, medium, and experimental context. Nominal ZIF-8-containing nanoparticle concentration should likewise not be interpreted as equivalent to bioavailable Zn²⁺ exposure. In one bone-regenerative ZIF-8/quercetin formulation, concentrations of 10–400 μg/mL were screened in BMSCs; concentrations above 100 μg/mL significantly inhibited cell growth, and 100 μg/mL was therefore selected for subsequent experiments.²⁹ This formulation-specific value should not be treated as a universal ZIF-8 safety threshold because cargo loading, particle properties, biological medium, and degradation collectively determine the resulting Zn²⁺ exposure. Quantitative reporting should distinguish the nominal ZIF-8 concentration or implanted dose from measured Zn²⁺ release and should specify release medium, exposure duration, and biological model. For locally implanted systems, the amount of ZIF-8 incorporated into the construct and, where available, local and systemic zinc exposure are also relevant. These study-level variables are summarized in Table 3 where reported; missing values should be treated as evidence gaps rather than estimated.

Table 3.

Study-Level Quantitative Evidence and Mechanistic Interpretability of Representative ZIF-8-Based Systems for Bone Regeneration

Study/Platform	Key Quantitative Formulation/Exposure	Model/Follow-Up	Key Controls	Mechanistic/Immune Evidence	Regenerative Outcomes	Evidence Appraisal
Si et al, 2025; MSC-Exos/ZIF-8@GelMA30	ZIF-8, 20 mg/mL; MSC-Exos, 1 × 1010 particles/mL; hydrogel mass retention, 81.3%, 62.5%, and 33.6% at 1, 2, and 4 wk; serum Zn2⁺, 18.81 μmol/L at 4 wk (control, 19.33 μmol/L; no significant difference)	Rat critical-size cranial defect; 8 wk	Control; Gel; ZIF-8@Gel; Exo@Gel; Z/E@Gel	Macrophage-associated phenotype; inflammatory cytokines; non-classical NF-κB	Micro-CT; histology; osteogenesis; angiogenesis	Direct osteoimmune evidence; attribution: partial-to-good. Zn2⁺ versus intact-ZIF effects remain incompletely separated.
Sun et al, 2025; ZIF-8/Que@GelMA29	ZIF-8/Que, 10–400 μg/mL screened; 100 μg/mL selected; DLE, 21.26%; DEE, 90.9%; release evaluated at pH 6.0 and 7.4	Rat cranial defect; 4 and 8 wk	Control (no hydrogel); GelMA; ZIF-8@GelMA; ZIF-8/Que@GelMA	Macrophage-associated response; inflammatory mediators	Micro-CT; histology; osteogenesis	Direct osteoimmune evidence; attribution: partial. Que and Zn2⁺ effects remain partly overlapping.
Yang et al, 2024; SFD/CS/ZIF-8@QCT31	QCT loading, 13.51%; sustained Zn2⁺/QCT delivery	Periodontitis-associated alveolar bone defect; 1, 4, and 8 wk	Blank; SFD/CS; SFD/CS/ZIF-8; SFD/CS/ZIF-8@QCT	Macrophage-associated immune remodeling (iNOS/CD206); inflammatory mediators	Alveolar bone regeneration; osteogenesis; angiogenesis	Direct disease-specific evidence; attribution: partial. Matrix, Zn2⁺, and QCT are all bioactive.
Niu et al, 2024; BMP-2@ZIF-8/PEG-NH2 + PDGF-BB hydrogel32	Sequential PDGF-BB/BMP-2 release; sustained Zn2⁺ release; administered doses NR	In vitro and in vivo bone-defect models	Multiple formulation controls	Primarily angiogenic/osteogenic rather than immune-specific	Vascularized bone regeneration; RNA-seq	Direct regenerative evidence, but not direct osteoimmune evidence; attribution: partial. Supports spatiotemporal delivery.
Liang et al, 2025; ZOL-Van@ZIF-8 (VZZ-8)33	Stable at pH 7.4; rapid degradation at pH 5.0; MRSA eradication, 93.84 ± 7.38%; biofilm inhibition, 95.36 ± 0.13%	Murine MRSA-associated PJI	Antibacterial/targeting comparators	TNF-α; IL-6	Reduced infection-associated osteolysis	Direct disease-model evidence; immune attribution: limited. Reduced inflammation may be secondary to bacterial clearance.
Qin et al, 2026; DEX@ZIF-8/ZnCu coating34	Controlled Zn2⁺/DEX release without marked burst; mechanical integrity maintained to 12 wk; ZIF-8/DEX loading NR	Rat inflammation-impaired femoral fracture; 12 wk	ZnCu and coating-related comparators	CD206/Arg-1; HIF-1α; PI3K/Akt/MAPK	Angiogenesis; osteogenesis; fracture healing	Direct osteoimmune evidence; attribution: partial/unresolved. ZnCu, Zn2⁺, DEX, and the coating are simultaneously bioactive.
GelMA@Sr-ZIF-8 study, 2025; GelMA@Sr-ZIF-835	5 wt% GelMA + 2 wt% Sr-ZIF-8; acidic conditions enhanced Zn/Sr release; STZ, 40 mg/kg	T2DM rat femoral defect; 2, 4, and 8 wk	Control; GelMA; GelMA@ZIF-8; GelMA@Sr-ZIF-8	Macrophage-associated inflammatory remodeling; redox regulation	Micro-CT; osteogenesis; angiogenesis; osteoclast-related outcomes	Direct disease-matched evidence; attribution: partial. Zn2⁺ and Sr2⁺ contributions overlap.
Jiang et al, 2025; ST/dECM/ZIF-8 bioadhesive36	At 8 wk, BV/TV 30.67 ± 4.04% vs 19.67 ± 5.03% in defect control; Tb.N, 1.69 ± 0.06 mm−1	Rat rotator-cuff tendon–bone repair; 4 and 8 wk	Defect; ST; ST/dECM; ST/dECM/ZIF-8	Local inflammatory response	Fibrocartilage formation; bone-tunnel regeneration; biomechanics	Direct tendon–bone evidence; attribution: partial. Findings should not be generalized to volumetric bone defects.
Li et al, 2025; miR-126@ZIF-837	ZIF-8, approximately 126 nm; miR-126@ZIF-8, approximately 173 nm; pH-responsive miRNA/Zn2⁺ release	In vitro only	ZIF-8/miRNA-related comparators	VEGF/HIF-1α and osteogenic signaling; no in vivo bone immune endpoint	Tube formation; mineralization; RUNX2/OCN	Indirect evidence. Supports nucleic-acid delivery feasibility but not an established in vivo osteoimmune mechanism.

Notes: Evidence classification follows the predefined framework described in Evidence Classification. Component attribution reflects the extent to which component-specific controls permit separation of the effects of ZIF-8, released Zn²⁺, therapeutic cargo, and other bioactive components. Quantitative values were included only when explicitly reported in the original studies and were not inferred from graphical data.

Abbreviations: Arg-1, arginase-1; BMP-2, bone morphogenetic protein-2; BMSC, bone marrow-derived mesenchymal stem cell; BV/TV, bone volume/total volume; CD206, cluster of differentiation 206; dECM, decellularized extracellular matrix; DEE, drug encapsulation efficiency; DEX, dexamethasone; DLE, drug loading efficiency; GelMA, gelatin methacryloyl; HIF-1α, hypoxia-inducible factor-1 alpha; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; micro-CT, micro-computed tomography; miR-126, microRNA-126; MRSA, methicillin-resistant Staphylococcus aureus; MSC-Exos, mesenchymal stem cell-derived exosomes; NF-κB, nuclear factor-κB; NR, not reported; OCN, osteocalcin; PDGF-BB, platelet-derived growth factor-BB; PEG-NH₂, amine-terminated polyethylene glycol; PI3K, phosphoinositide 3-kinase; PJI, periprosthetic joint infection; QCT, quercetin; Que, quercetin; RNA-seq, RNA sequencing; RUNX2, runt-related transcription factor 2; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; Tb.N, trabecular number; TNF-α, tumor necrosis factor-α; Van, vancomycin; VEGF, vascular endothelial growth factor; wt%, weight percentage; ZIF-8, zeolitic imidazolate framework-8; ZnCu, zinc–copper alloy; ZOL, zoledronic acid.

Because most bone-regenerative ZIF-8 systems are multicomponent constructs, biological effects should not automatically be attributed to Zn²⁺. Mechanistic interpretation therefore requires separation of carrier-, ion-, cargo-, and matrix-related contributions, while translational assessment requires formulation-specific dose–release–exposure relationships.

Surface Functionalization, Local Retention, and Hybrid Platform Design

Surface functionalization and hybrid integration can modify ZIF-8 behavior beyond that of the pristine framework. Polymer coatings, polydopamine, polysaccharides, peptides, cell-derived interfaces, hydrogels, membranes, and scaffold matrices may alter surface charge, colloidal stability, protein adsorption, cellular uptake, immune recognition, fluid accessibility, and degradation.^9,38,39 These modifications can improve cargo protection or local retention, but the resulting biological behavior should generally be attributed to the complete formulation unless individual contributions are experimentally resolved.

Surface modifications should be distinguished according to their intended function. Stabilizing modifications primarily alter colloidal behavior or degradation; biological-interface modifications regulate cellular interactions; cargo-retaining modifications affect loading and release; and targeting modifications are intended to increase association with a defined anatomical or cellular site.³⁹ A single modification may perform several functions, but these functions should not be inferred from material design alone.

Hybrid integration can also modify ZIF-8 degradation and exposure. Immobilization within a hydrogel, membrane, coating, or porous scaffold may restrict fluid access and alter diffusion, whereas matrix swelling, degradation, or ion exchange may accelerate or redistribute Zn²⁺ and cargo release.^9,40 Release and safety data obtained from free ZIF-8 nanoparticles should therefore not automatically be extrapolated to the corresponding composite formulation.

Targeting modifications should also be distinguished from strategies that merely improve local retention. This distinction is particularly important when interpreting claims of bone affinity or active bone targeting and is considered further in the disease- and delivery-specific context of Bone targeting and local retention: terminology should reflect the delivery mechanism.³³

Cross-Platform Comparison with Other MOFs and Analogous Zinc-Based Systems

ZIF-8 should not be considered a default or universally optimal MOF for bone nanomedicine. Platform selection depends on the desired degradation profile, cargo characteristics, treatment duration, route of administration, and acceptable exposure to metal- and linker-related products.^9,39 Comparison with alternative MOFs and non-MOF zinc-based systems is therefore necessary to define the conditions under which ZIF-8 offers a meaningful advantage.

ZIF-67 retains a related zeolitic imidazolate architecture but substitutes cobalt for zinc, providing distinct catalytic and redox-related functions while introducing different toxicological constraints.^39,41 UiO-series frameworks, commonly based on zirconium-containing nodes, generally provide greater structural and hydrolytic stability, which may favor prolonged cargo retention but be less desirable when biodegradation and predictable clearance are priorities.^39,42 MIL-series frameworks offer broad compositional and catalytic diversity, including widely investigated iron-based systems, but their degradation, ion release, and safety cannot be generalized across the family.^9,43 Comparisons should therefore be made at the level of the specific framework and formulation rather than by MOF family name alone.

Non-MOF zinc-based coordination systems, particularly metal–polyphenol networks and related zinc-containing coatings, provide another relevant comparison. Such systems can often be assembled under mild aqueous conditions and may offer adhesive, antioxidant, antibacterial, or bioactive interfaces without requiring a permanently crystalline porous framework.⁴⁴ ZIF-8 provides a more structurally defined architecture with established routes for encapsulation, biomimetic mineralization, and surface modification, whereas zinc–polyphenol systems may avoid some limitations associated with 2-methylimidazole but introduce uncertainties related to polyphenol composition, coordination stoichiometry, stability, and batch-to-batch reproducibility.^21,44

The value of ZIF-8 therefore lies in the combination of mild synthesis, versatile cargo incorporation, modifiable interfaces, environment-dependent degradation, and biologically active Zn²⁺ release.^10,38 These advantages are counterbalanced by phosphate- and biological-medium-dependent instability, exposure-dependent zinc toxicity, uncertainty regarding linker-product fate, and the possibility of premature transformation or cargo release.^14,15,23 Platform selection should therefore be function driven rather than material driven: the relevant question is not whether ZIF-8 is universally superior, but whether its degradation, loading, ion-release, and safety characteristics are appropriately matched to the intended application.

Biosafety, Degradation-Product Fate, and Long-Term Exposure

The biosafety of ZIF-8-based bone nanomedicine is formulation- and exposure-dependent, being influenced by particle properties, administered dose, degradation rate, exposure duration, delivery route, and surrounding matrices.^23,45 Framework degradation may generate intact or partially transformed particles, Zn²⁺, and 2-methylimidazole-related products with different local and systemic fates.^14,15 However, toxicological evidence for 2-methylimidazole is derived mainly from conventional exposure studies rather than from degradation-product studies of locally implanted ZIF-8, and its relevance to bone formulations therefore remains uncertain.⁴⁶ Although local implantation may reduce circulating exposure, it does not ensure confinement, and short-term cell-viability or histological findings cannot establish long-term safety.^47,48 Bone-regenerative formulations should therefore relate administered dose to degradation-product identity, time-dependent Zn²⁺ and cargo release, local and systemic exposure, persistence, clearance, and chronic tissue responses. Because no universal safety window applies across different formulations, safety must be established for the final biologically relevant product. The corresponding translational requirements are discussed in Translational Readiness: Safety, Pharmacokinetics, Manufacturing, and Regulation.

Bone Immune Microenvironment: Evidence Hierarchy for ZIF-8-Based Nanomedicine

Immune Dynamics During Bone Repair

Bone regeneration is governed by a dynamic immune–stromal–vascular network rather than by osteogenic cells alone. Immune cells, osteolineage cells, stromal and endothelial cells, extracellular-matrix components, cytokines, and chemokines interact throughout injury, repair, and remodeling. These interactions influence whether early inflammation resolves into coordinated vascularized bone formation or persists as a pathological environment that favors tissue injury and bone resorption.^4–6

Inflammatory, reparative, and remodeling processes overlap temporally rather than occurring as strictly separated stages. Early recruitment of neutrophils and monocyte/macrophage populations contributes to host defense, debris clearance, and recruitment of reparative cells, whereas persistent or dysregulated inflammation can impair osteogenesis, promote osteoclast activity, and disrupt vascular regeneration.^6,49 Osteoimmunomodulatory strategies should therefore regulate the magnitude, duration, and resolution of inflammation rather than simply suppress it.^4,5

For ZIF-8-based nanomedicine, however, the biological importance of an immune pathway must be distinguished from the strength of platform-specific evidence. Macrophage-associated inflammatory remodeling currently has the clearest direct support in ZIF-8-containing bone-regenerative systems, whereas evidence for Th17/Treg regulation, NET modulation, and broader immune-cell networks remains predominantly indirect or hypothesis-generating. This asymmetry forms the basis for the evidence hierarchy considered below. The overlapping phases of bone repair, representative immune–bone interactions, and the consequences of failed inflammatory resolution are summarized in Figure 2.

Figure 2.

Bone immune microenvironment and immune–bone coupling during bone repair. Bone healing involves overlapping inflammatory, reparative, and remodeling phases coordinated by immune, stromal, vascular, and bone-lineage cells. Timely inflammatory resolution supports repair-associated macrophage responses, osteogenesis, angiogenesis, and balanced remodeling, whereas failed resolution can lead to persistent inflammation, excessive osteoclast activity, impaired angiogenesis, and delayed regeneration. The lower panel summarizes representative interactions among immune cells, bone marrow mesenchymal stromal cells, endothelial cells, osteoblast-lineage cells, osteocytes, and osteoclasts. M1-like and M2-associated macrophages represent simplified, context-dependent states rather than fixed phenotypes. Black, green, and red arrows indicate cytokine/immune, pro-repair/angiogenic, and osteoclast-regulatory signaling, respectively; blunt-ended lines indicate inhibition. Purple, green, red, and blue dots denote pro-inflammatory, pro-repair, osteoclastogenic, and osteoprotective signals, respectively. The pathways shown represent general bone-repair biology and are not all directly regulated by ZIF-8.

Abbreviations: NETs, neutrophil extracellular traps; ROS, reactive oxygen species; BMSCs, bone marrow mesenchymal stromal cells; Th17, T helper 17 cells; Treg, regulatory T cells; RANKL, receptor activator of nuclear factor-κB ligand; OPG, osteoprotegerin.

Macrophage-Associated Inflammatory Remodeling: The Most Directly Supported Immune Axis

Macrophages are highly plastic regulators of inflammation, tissue repair, angiogenesis, and bone remodeling. Although the conventional M1/M2 terminology remains widely used in biomaterials research, macrophage activation in vivo is better regarded as a spectrum of context-dependent states rather than two discrete phenotypes. Expression of limited marker combinations such as iNOS or CD86 versus CD206 or Arg-1 should therefore not be interpreted as definitive evidence of distinct macrophage populations.^49,50

Macrophage function is also temporally dependent. Early inflammatory activity contributes to antimicrobial defense and clearance of damaged tissue, whereas persistent production of TNF-α, IL-1β, IL-6, ROS, and NF-κB-related signals can suppress osteogenic differentiation and promote osteoclastogenesis. Repair-associated macrophage states may subsequently support matrix remodeling, osteoprogenitor activity, and angiogenesis.^5,49 Mechanistic interpretation is therefore strengthened when phenotype-associated markers are combined with cytokine or signaling analyses, functional measurements, and temporal or spatial assessment rather than reduced to an “M2/M1 ratio”.

Among the immune mechanisms proposed for ZIF-8-based bone nanomedicine, macrophage-associated regulation has the strongest direct experimental support. Selected ZIF-8-containing platforms have simultaneously demonstrated altered inflammatory signaling or macrophage-associated phenotypes and improved bone-regenerative outcomes.^29,30 For example, MSC-Exos/ZIF-8@GelMA was associated with suppression of non-classical NF-κB signaling, reduced pro-inflammatory cytokine expression, a shift toward repair-associated macrophage characteristics, and improved bone formation and angiogenesis in a cranial-defect model.³⁰

These findings nevertheless support the activity of selected ZIF-8-containing formulations, rather than a universal intrinsic ability of ZIF-8 to induce a specific macrophage state. Therapeutic cargo, released Zn²⁺, surface modification, and surrounding matrices may all contribute to the observed response. The most defensible conclusion is therefore that selected ZIF-8-containing systems can modulate macrophage-associated inflammatory remodeling in parallel with improved bone repair, while the magnitude, timing, and component-specific basis of this effect remain formulation dependent.

Th17/Treg Regulation and Osteoclastogenesis: Biologically Relevant but Predominantly Indirect Evidence

CD4⁺ T-cell subsets influence bone remodeling partly through inflammatory signaling and the RANKL/OPG axis. Th17-associated responses can promote osteoclastogenesis through IL-17-related pathways, whereas regulatory T-cell responses may restrain excessive inflammation through IL-10, TGF-β, CTLA-4, and related signals.^7,51,52 However, Th17/Treg balance is not a simple binary switch, and measurements of isolated markers such as IL-17 or FOXP3 do not demonstrate a functional shift in local T-cell populations.

For ZIF-8-based bone nanomedicine, current evidence is mainly indirect. Zinc-related and general osteoimmunology studies suggest that Zn²⁺ exposure can influence T-cell responses,^7,53 but these findings do not establish that ZIF-8 restores Th17/Treg balance during bone regeneration. Direct validation requires a ZIF-8-containing intervention in a bone-related in vivo model, quantitative analysis of local T-cell populations, and corresponding assessment of RANKL/OPG signaling, osteoclast activity, and bone regeneration. Th17/Treg modulation should therefore be regarded as a biologically plausible research direction rather than an established ZIF-8 mechanism.

Neutrophils and NETs: Biological Relevance with Limited Direct ZIF-8 Evidence

Neutrophils support early antimicrobial defense after bone injury, whereas excessive or persistent activation can amplify inflammation and impair osteogenic and vascular repair.^6,54 Neutrophil extracellular traps (NETs) can immobilize microorganisms but may contribute to inflammatory tissue damage and osteoclast-related bone loss when excessively accumulated.^8,55,56

Direct evidence that ZIF-8 regulates NET formation during bone regeneration remains limited. In infected defects, bacterial clearance mediated by Zn²⁺, antimicrobial cargos, or other formulation components may secondarily reduce neutrophil recruitment and inflammatory signaling without demonstrating a NET-specific effect. Direct validation requires measurement of extracellular DNA together with NET-associated markers such as citrullinated histone H3, myeloperoxidase, or neutrophil elastase, ideally linked to bacterial burden, inflammation, osteoclast activity, and bone regeneration. NET modulation should therefore remain a hypothesis-generating mechanism rather than an established therapeutic pathway.

Immune–Vascular–Bone Coupling: A Systems-Level Objective with Heterogeneous Evidence

Successful bone regeneration requires coordinated immune regulation, vascularization, osteogenesis, and remodeling. Inflammatory signals influence osteoprogenitor and osteoclast activity, vascular ingrowth determines oxygen and nutrient supply, and reciprocal signaling between endothelial and bone-forming cells contributes to tissue reconstruction.^49,56 These processes are therefore biologically interconnected.

Demonstrating that they are mechanistically coupled by a ZIF-8 formulation, however, requires more than showing concurrent improvement in several endpoints. Changes in macrophage-associated signals, VEGF expression, vessel density, osteogenic markers, or bone volume may occur within the same study without establishing that one response causally mediates another. Similarly, enhanced angiogenesis does not by itself demonstrate osteoimmunomodulation.

Multifunctional ZIF-8 platforms are well suited to influence several compartments simultaneously because Zn²⁺ release can be combined with anti-inflammatory, antibacterial, angiogenic, or osteogenic cargos. Yet this multifunctionality also increases mechanistic ambiguity.^29,30 Current evidence therefore more strongly supports the conclusion that selected ZIF-8-containing systems can produce concurrent inflammatory, vascular, and osteogenic improvements than the stronger claim that ZIF-8 directly coordinates a unified immune–vascular–bone mechanism.

Immune–vascular–bone coupling should consequently be regarded as a systems-level therapeutic objective whose validation requires temporal integration of immune, vascular, osteogenic, and remodeling outcomes. Evidence that one biological compartment is necessary for improvement in another would provide substantially stronger mechanistic support than parallel endpoint changes alone.

Other Immune-Cell Populations: Emerging and Hypothesis-Generating Directions

B cells influence skeletal homeostasis through regulatory and osteoclastogenic pathways involving the RANKL/OPG axis.⁵⁷ Dendritic cells, mast cells, and natural killer cells have also been implicated in osteoimmune signaling and inflammatory bone remodeling.^58–60 However, direct bone-model evidence that ZIF-8-based nanomedicine specifically regulates these populations remains minimal. These pathways should therefore be considered emerging or hypothesis-generating rather than equivalent to macrophage-associated regulation.

Single-cell RNA sequencing, spatial transcriptomics, multiplex imaging, and related approaches may identify relevant cellular responses during fracture healing and biomaterial-associated tissue repair,^61,62 but exploratory associations require targeted functional validation before they can support mechanism-specific claims.

Overall, the current osteoimmune evidence hierarchy is markedly asymmetric: macrophage-associated inflammatory remodeling has the strongest direct support, whereas Th17/Treg regulation, NET modulation, and broader immune-cell responses remain predominantly indirect or hypothesis-generating. This distinction should guide both interpretation of existing studies and prioritization of future mechanistic work.

Therapeutic Strategies and Component Attribution in ZIF-8-Based Bone Nanomedicine

Therapeutic outcomes in ZIF-8-based bone nanomedicine commonly arise from interactions among the ZIF-8 framework, released Zn²⁺, therapeutic cargo, surface modifications, and the surrounding hydrogel, coating, membrane, or scaffold. The critical question is therefore not only whether a multifunctional formulation improves bone repair, but also whether the experimental design allows the contribution of individual components to be distinguished.

Mechanistic interpretation is strongest when the complete formulation is compared with relevant component controls. When several bioactive components are introduced simultaneously without such comparisons, therapeutic efficacy can be assigned to the composite platform, but specific effects cannot be confidently attributed to ZIF-8, Zn²⁺, the therapeutic cargo, or the surrounding matrix. Figure 3 integrates the pathological cues of bone defects, environment-responsive behavior of ZIF-8-based nanomedicine, evidence-weighted immune mechanisms, and regenerative outcomes.

Figure 3.

Mechanistic framework of ZIF-8-based nanomedicine in immune remodeling and bone regeneration. Pathological bone defects may involve acidity, infection or biofilm formation, oxidative stress, persistent inflammation, immune imbalance, excessive osteoclast activity, and impaired angiogenesis and osteogenesis. ZIF-8-based nanomedicine may respond through environment-dependent degradation, local Zn²⁺ and cargo release, and integration with hydrogels, coatings, membranes, or scaffolds. Macrophage-associated inflammatory remodeling currently has the strongest direct support in bone-related ZIF-8 studies. Anti-inflammatory, antioxidant, and antibacterial effects are frequently formulation- or cargo-dependent, whereas direct evidence for NET modulation and Th17/Treg regulation remains limited or indirect. Osteoclast regulation and osteogenesis–angiogenesis coupling represent additional regenerative outcomes, although the contributions of ZIF-8, Zn²⁺, cargo, and surrounding matrices are often incompletely resolved. Solid arrows indicate comparatively supported relationships; dashed arrows indicate emerging or predominantly indirect mechanisms.

Abbreviations: ROS, reactive oxygen species; NETs, neutrophil extracellular traps; BMSCs, bone marrow mesenchymal stromal cells; Th17, T helper 17 cells; Treg, regulatory T cells; RANKL, receptor activator of nuclear factor-κB ligand; OPG, osteoprotegerin; NF-κB, nuclear factor-κB; BMP-2, bone morphogenetic protein-2; PDGF-BB, platelet-derived growth factor-BB; RUNX2, runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; COL1A1, collagen type I alpha 1 chain; VEGF, vascular endothelial growth factor.

Component Attribution in Multifunctional ZIF-8 Platforms

The increasing complexity of ZIF-8-based regenerative systems creates a fundamental challenge for mechanistic interpretation. A typical composite may contain the ZIF-8 carrier, released Zn²⁺, one or more therapeutic cargos, a surface coating, and a hydrogel or scaffold matrix, each of which may influence inflammation, oxidative stress, cell survival, angiogenesis, osteogenesis, or antibacterial activity.^29,30,34 Improved regeneration by the complete formulation therefore does not establish that any single component is responsible for the observed response.

Component attribution should be considered at both therapeutic and mechanistic levels. At the therapeutic level, comparison of the complete formulation with individual components can determine whether combination provides additional benefit. At the mechanistic level, more stringent controls are required to establish whether a specific pathway is driven primarily by the ZIF-8 framework, released Zn²⁺, therapeutic cargo, or another component.

A useful minimum control structure includes the final composite together with unloaded-carrier, free-cargo, and matrix- or coating-only controls whenever experimentally feasible. Additional comparisons may be required for specific claims. For example, Zn²⁺-matched exposure can help distinguish soluble-zinc effects from intact-particle effects, whereas equivalent-dose free-cargo controls can determine whether ZIF-8 modifies efficacy through protection, localization, or controlled release.

Incomplete component controls do not invalidate demonstration of therapeutic efficacy, but they limit mechanistic inference. In such cases, conclusions should refer to the ZIF-8-containing composite platform rather than assigning the observed effect specifically to ZIF-8 or Zn²⁺.

Cargo-Specific Small-Molecule Delivery: Distinct Mechanisms and Exposure Requirements

Small-molecule cargos should be interpreted according to their individual pharmacological properties rather than grouped broadly as “anti-inflammatory” or “antioxidant”. Their biological effects depend on molecular target, local concentration, exposure duration, disease context, and the extent to which ZIF-8 modifies stability, retention, and release.⁶³

Quercetin-based systems illustrate overlapping carrier–cargo bioactivity. Quercetin has antioxidant and inflammation-modulating properties and may influence osteogenic and immune-associated responses, while ZIF-8 degradation simultaneously introduces biologically active Zn²⁺. Hierarchical systems combining these components can therefore improve the regenerative microenvironment, but therapeutic benefit may arise from quercetin, Zn²⁺, altered cargo exposure, or interactions among them rather than from a single component.²⁹

Curcumin requires a separate interpretation. As a pleiotropic molecule with antioxidant and inflammation-related activities, curcumin may influence several pathways relevant to inflammatory tissue injury and skeletal remodeling.⁶⁴ ZIF-8 incorporation may improve protection, dispersion, retention, or controlled exposure,^64,65 but reductions in ROS or inflammatory mediators should not automatically be assigned to the carrier. Antibacterial, inflammatory, osteoclast-related, and osteogenic outcomes should also be distinguished because improvement across these domains does not necessarily imply a common mechanism.

Dexamethasone raises a distinct dose–time problem. Its effects on inflammation and skeletal tissue depend strongly on concentration, duration, cellular context, and disease state. Controlled local delivery may reduce excessive inflammatory signaling under selected conditions, whereas prolonged or excessive glucocorticoid exposure can adversely affect bone formation and remodeling. The rationale for dexamethasone-loaded ZIF-8 should therefore focus on optimization of local exposure rather than assuming that glucocorticoid-mediated suppression of inflammation is inherently regenerative.³⁴ Other small-molecule systems, such as DMOG-loaded nanoscale ZIF-8, further illustrate how carrier-mediated exposure can be used to promote osteogenesis–angiogenesis coupling. Nevertheless, the effects of the cargo, released Zn²⁺, and altered delivery kinetics require separate interpretation.⁶⁶

Across these cargo classes, free-drug and unloaded-carrier comparisons are particularly important when the study seeks to demonstrate that ZIF-8 improves the therapeutic window through protection, localization, or controlled release. The relevant question is therefore not simply whether ZIF-8 can carry a bioactive molecule, but whether altered spatial or temporal exposure produces a measurable advantage over the free cargo.

Antibacterial Nanoplatforms: Separating Infection Control from Immunomodulation

ZIF-8-based antibacterial platforms may combine antimicrobial cargos, Zn²⁺ release, targeting modifications, and local delivery matrices.^33,67,68 Consequently, improved bacterial clearance may result from an individual component or the complete formulation, and antibacterial synergy should not be inferred without component-specific controls. Reduced bacterial burden can secondarily decrease inflammatory cytokines, neutrophil recruitment, oxidative stress, and tissue injury; however, these changes do not establish direct macrophage reprogramming or NET regulation unless the relevant immune endpoints are measured. Antibacterial efficacy, immune effects, host-cell compatibility, and bone regeneration should therefore be evaluated as related but mechanistically distinct outcomes. Short-term bacterial killing alone is insufficient to demonstrate a regenerative antibacterial platform.

Biomacromolecule, Extracellular-Vesicle, and Nucleic-Acid Delivery

ZIF-8 provides opportunities for delivering biologically fragile cargos, including proteins, growth factors, peptides, extracellular-vesicle-associated therapeutics, and nucleic acids. For these cargos, the principal potential advantages are protection from premature degradation, improved local retention, and controlled exposure rather than simple occupation of the intrinsic ZIF-8 micropores.^21,22

For proteins and growth factors, loading efficiency should be distinguished from retained biological activity. High cargo incorporation does not establish that a biomolecule remains structurally intact or therapeutically active after encapsulation, storage, degradation, and release. Evaluation should therefore connect cargo loading and release with retained functional activity and regenerative outcome.^21,32,69

For example, BMP-2-loaded ZIF-8 incorporated into PLGA/mesoporous bioactive-glass scaffolds produced sustained BMP-2 release and promoted osteogenic activity and in vivo bone formation, although the respective contributions of BMP-2, ZIF-8, and the scaffold remained partly overlapping.⁷⁰

Extracellular-vesicle-containing systems add further complexity because the vesicles themselves may regulate inflammatory, angiogenic, and osteogenic responses, while ZIF-8 and the surrounding matrix simultaneously alter retention, release, Zn²⁺ exposure, and cellular interactions.³⁰ Such formulations can provide convincing evidence of composite efficacy, but their mechanism cannot automatically be assigned to ZIF-8.

Nucleic-acid delivery is promising but remains less mature in bone-regenerative applications. A directly relevant example is miR-126-loaded ZIF-8, which has been investigated for vascularized bone regeneration.³⁷ This work supports the feasibility of delivering a biologically active microRNA through a ZIF-8-based system, but its principal evidence relates to angiogenic and osteogenic outcomes rather than broad osteoimmune-network regulation.³⁷

Successful nucleic-acid delivery also requires evidence beyond loading efficiency. Relevant considerations include protection from degradation, cellular uptake, intracellular trafficking, biologically effective dose, target-gene modulation, duration of activity, and off-target or immune effects. In vitro pathway modulation alone does not establish regenerative efficacy without corresponding bone-related in vivo validation.

Across biomacromolecule systems, four processes should therefore be distinguished: cargo loading, cargo protection, cargo release, and cargo function. Successful performance at one level does not guarantee success at the next, and regenerative interpretation should connect formulation properties with retained biological activity and tissue-level outcomes.

Sequential and Spatiotemporal Delivery: Matching Therapeutic Exposure to the Phases of Bone Repair

Bone regeneration is temporally heterogeneous. Early repair requires control of infection and excessive inflammation while preserving necessary host-defense responses, whereas later phases depend increasingly on vascular invasion, progenitor recruitment, matrix formation, osteogenic differentiation, mineralization, and remodeling.^6,56 This changing biological environment provides a rationale for sequential or spatiotemporally controlled delivery.

ZIF-8-based composites can generate different release profiles through environment-dependent degradation, cargo encapsulation, surface modification, and integration with hydrogels, coatings, membranes, or scaffolds.^29,32 However, different release rates do not by themselves demonstrate biologically effective sequential therapy.

A convincing sequential-delivery strategy should establish three progressively stronger levels of evidence: distinct material-release kinetics, temporally distinct biological responses, and a regenerative advantage of the programmed sequence over appropriate simultaneous or non-sequential controls. Only the final level demonstrates that temporal programming itself contributes to therapeutic benefit.

The same caution applies to microenvironment-responsive delivery. Acid-sensitive release demonstrated in a simplified buffer does not establish disease-specific temporal control in vivo because phosphate, proteins, matrix interactions, and local fluid exchange can modify degradation independently of pH.^14,15 Stage-specific delivery should therefore be supported by biologically relevant release measurements and, where possible, by evidence connecting material behavior with local therapeutic exposure.

Sequential designs may be particularly valuable when therapeutic functions have different or potentially conflicting temporal requirements. Potent antibacterial or anti-inflammatory activity may be advantageous early, whereas prolonged suppression of inflammatory signaling may interfere with later repair. Likewise, vascular stimulation and osteogenic maturation may require different temporal profiles. The objective is therefore not to maximize the number of therapeutic agents delivered, but to provide the appropriate exposure during the biological phase in which it is most useful.

Accordingly, sequential and spatiotemporal delivery should be interpreted according to the extent to which release timing, biological timing, and regenerative benefit are experimentally linked, rather than inferred from formulation architecture alone.

Disease-Specific Applications and Delivery Context

The suitability of a ZIF-8-based formulation for bone regeneration depends strongly on pathological and anatomical context. Infection, diabetes, osteoporosis, craniofacial disease, implant-associated defects, tendon–bone interfaces, and large osseous defects impose distinct biological, mechanical, and delivery constraints. Performance in one setting should therefore not be assumed to predict equivalent benefit in another.^71,72

Disease-specific interpretation requires more than matching a material property to a pathological feature. Acid-responsive degradation, antioxidant activity, angiogenic stimulation, antibacterial effects, or immune modulation provide a mechanistic rationale, but disease-specific efficacy requires validation in models that reproduce the relevant pathological barriers and clinically meaningful outcomes.^71,73,74

Infectious Bone Defects: Infection Control Must Translate into Durable Bone Repair

Infectious bone defects combine microbial persistence, biofilm formation, inflammatory tissue injury, and impaired osteogenesis. ZIF-8-containing systems have been investigated as antibiotic-loaded membranes, bone-targeting nanoparticles, responsive three-dimensional scaffolds, and infection-resistant implant interfaces.^{33,67,68,75,76} Recent studies have combined ZIF-8 with CuO-containing printed scaffolds or ZIF-sealed implant interfaces to integrate bacterial control with osteogenic, immunomodulatory, and osseointegration-related effects. However, these outcomes remain formulation specific because several antibacterial and bioactive components act simultaneously. Their evaluation should use disease- and anatomy-matched models and extend beyond planktonic bacterial counts or inhibition-zone measurements. Stronger evidence requires assessment of mature biofilm, infection recurrence, host-cell compatibility, inflammatory resolution, and structural bone regeneration. Decreases in inflammatory mediators after bacterial clearance should be interpreted primarily as secondary consequences of infection control unless macrophage-, neutrophil-, or NET-specific mechanisms are directly measured. The most relevant therapeutic endpoint is therefore durable infection control accompanied by restoration of bone structure and function.

Diabetic Bone Defects: Addressing Inflammation, Oxidative Stress, and Impaired Regeneration

Unlike infectious bone defects, where microbial eradication is the primary therapeutic goal, diabetic bone defects arise from a chronic metabolic and inflammatory milieu characterized by sustained inflammation, oxidative stress, microvascular dysfunction, impaired angiogenesis, and reduced osteogenesis.⁷³ ZIF-8-containing hydrogels and scaffolds may address these interacting barriers through local retention, environment-responsive release, and delivery of anti-inflammatory, antioxidant, angiogenic, or osteogenic agents.^35,77–79 Recent disease-matched studies have combined ZIF-8 with directional biomimetic scaffolds or photothermal hybrid hydrogels to regulate macrophage-associated inflammation, oxidative stress, vascularized osteogenesis, and bone homeostasis in diabetic defect models.^78,79 However, the contributions of ZIF-8, released Zn²⁺, therapeutic cargo, photothermal components, and surrounding matrices often remain unresolved.

Because systemic diabetes persists beyond the local defect, healthy bone-defect models or short-term osteogenic assays cannot establish disease-specific efficacy. Studies should therefore use validated diabetic models and integrate inflammatory, oxidative, vascular, osteogenic, resorptive, microarchitectural, and histological outcomes, together with appropriate component controls and adequate follow-up. These requirements distinguish diabetic repair from osteoporotic defects, where restoration of the formation–resorption balance is the central therapeutic concern.

Osteoporotic Bone Defects: Restoring Formation–Resorption Balance Requires Disease-Matched Evidence

Osteoporotic bone repair differs from regeneration in healthy bone because impaired bone formation occurs together with abnormal resorption and altered systemic skeletal metabolism. A formulation intended for this setting should therefore demonstrate benefit within an osteoporotic environment rather than relying solely on osteogenic activity in normal animals.

ZIF-8-based platforms may combine local drug delivery with Zn²⁺ exposure and osteogenic, anti-inflammatory, antioxidant, or anti-resorptive cargos.⁸⁰ However, osteogenic and anti-osteoclastogenic effects represent related but distinct therapeutic objectives. Increased osteoblast-associated markers do not demonstrate correction of excessive resorption, whereas reduced osteoclast activity alone does not establish restoration of structurally competent bone.

Selected ZIF-8-containing formulations have now been evaluated directly in osteoporotic bone-defect models, including a microenvironment-responsive hydrogel integrating EGCG and BMP-2@TA-ZIF-8 for staged anti-inflammatory and osteogenic regulation.⁸¹ An engineered simvastatin-loaded ZIF-8 hydrogel has also been evaluated directly in an osteoporotic environment and was reported to enhance vascularized bone regeneration. This strengthens disease-matched evidence but does not fully separate the contributions of simvastatin, Zn²⁺, ZIF-8, and the hydrogel matrix.⁸² The proposed responsiveness of ZIF-8 to osteoclast-associated acidity is also biologically plausible, but localized acidic resorption does not by itself demonstrate therapeutically advantageous osteoclast-specific degradation in vivo.

Disease-matched evaluation should therefore assess both formation and resorption, integrating osteoclast-related outcomes with bone microarchitecture, histological remodeling, and, where relevant, biomechanical performance. The strongest evidence will come from osteoporotic fracture or defect models demonstrating improvement in both abnormal remodeling and local regenerative outcome.

Periodontal, Periapical, and Craniofacial Defects: Anatomically Related but Biologically Distinct Applications

Periodontal, periapical, and craniofacial defects are anatomically related but biologically distinct. Periodontal regeneration requires restoration of alveolar bone together with periodontal ligament, cementum, and their functional interfaces; periapical disease is dominated by infection-associated inflammation and local bone resorption; and cranial defects primarily test osseous defect bridging, vascularization, and remodeling. These applications should therefore be considered separate evidence domains.

In periodontal regeneration, increased alveolar bone volume alone does not establish complete tissue restoration. Relevant outcomes include periodontal-ligament organization, cementum formation, alveolar-bone architecture, inflammatory control, and functional attachment,^83,84 particularly because the material operates within a saliva-exposed, polymicrobial, mechanically active environment. ZIF-8 has also been incorporated into guided-tissue-regeneration membranes to combine antibacterial and osteogenic functions.^85,86

Periapical disease has a different priority: durable suppression of the microbial source and resolution of inflammation-associated bone destruction. Antibacterial activity against organisms such as Enterococcus faecalis can provide relevant local evidence, but improvements in inflammation or radiographic lesions should be interpreted together with microbial clearance and tissue repair.⁸⁷

Cranial-defect models are valuable for assessing local retention, vascularized bone formation, defect bridging, and remodeling,^30,32 but they are generally non-load-bearing and do not reproduce the mechanical demands of many maxillofacial or orthopedic reconstructions. Positive cranial-defect results therefore provide valid proof-of-concept evidence for local bone regeneration without establishing suitability for load-bearing reconstruction.

Implant-Associated Osseointegration: Interface Stability and Functional Integration

Implant-associated regeneration depends on long-term stability of the material–implant–bone interface rather than bone formation alone. A therapeutic coating must remain integrated with the substrate, tolerate fluid exposure and mechanical loading, maintain controlled local release, and avoid persistent inflammation, fibrous encapsulation, corrosion-related complications, or excessive ion exposure.

ZIF-8-containing coatings have been investigated on titanium and other metallic substrates for local drug delivery, antibacterial protection, osteogenic stimulation, and modulation of peri-implant responses.^34,88,89 However, increased ALP activity, osteogenic-gene expression, or matrix mineralization does not establish functional osseointegration. More direct evidence includes bone–implant contact, peri-implant bone architecture, interfacial mechanical performance, and longer-term remodeling.

Coating integrity is equally important. Delamination, wear, corrosion, or rapid dissolution can alter the spatial and temporal exposure to ZIF-8, Zn²⁺, and therapeutic cargo. Release behavior should therefore be characterized using the final coated implant under conditions relevant to the intended environment rather than extrapolated from free nanoparticles.

Because substrate chemistry, surface topography, ZIF-8, released ions, and therapeutic cargos may all influence osseointegration, mechanistic interpretation should remain formulation specific. Disease-specific coatings should also be tested in the relevant setting—for example, infection-prone or osteoporotic implantation—rather than inferred from general osteogenic activity in healthy bone. The translational endpoint is durable, mechanically functional osseointegration with controlled local exposure.

Tendon–Bone Interface Repair: A Distinct Orthopedic Interface-Regeneration Problem

Tendon–bone healing should be considered separately from volumetric bone-defect regeneration because the therapeutic target is a mechanically graded soft-tissue–bone interface. Successful repair requires restoration of tendon attachment, fibrocartilage, collagen organization, mineralized interface formation, and mechanical load transfer rather than increased bone formation alone.

A ZIF-8-containing decellularized-matrix bioadhesive has been investigated in a rat rotator-cuff repair model and was associated with improved inflammatory responses, interface formation, and biomechanical performance.³⁶ This provides direct evidence for a ZIF-8-containing platform in tendon–bone healing, but the result should remain confined to the interface-regeneration context.

Relevant outcomes include collagen-fiber orientation, fibrocartilage maturation, continuity of the insertion, mineralized interface development, failure load, and stiffness. Material adhesion, degradation, retention, and release should likewise be evaluated under the tensile and shear conditions characteristic of the healing interface.

Accordingly, tendon–bone repair should be treated as a distinct orthopedic application whose success is defined by restoration of a structurally organized and mechanically functional attachment interface.

Large and Complex Bone Defects: Distinguishing Regenerative Delivery from Structural Reconstruction

Large and complex bone defects require more than local stimulation of osteogenesis. Depending on defect size and anatomical site, successful reconstruction may require vascularization, defect bridging, stable material retention, coordinated degradation, fixation, and sufficient mechanical competence to support healing.

In many composite systems, ZIF-8 primarily functions as a nanoscale carrier or bioactive component, whereas structural support is provided by a hydrogel, ceramic phase, polymer scaffold, metallic fixation device, or other matrix.^32,90 Increased bone formation in a non-load-bearing defect therefore demonstrates regenerative activity but does not establish suitability for mechanically demanding segmental reconstruction.

The anatomical model should be matched to the intended claim. Cranial defects provide useful proof-of-concept evidence for defect bridging and local regeneration, whereas claims of load-bearing reconstruction require models that also address fixation, mechanical stability, and functional recovery.

Evaluation should integrate complementary structural and functional outcomes. Imaging can quantify mineralized tissue and bridging, but residual high-density material may complicate interpretation; histology is important for tissue identity and host integration, while biomechanical testing is required when functional reconstruction is claimed. Long-term follow-up should further determine whether tissue maturation and remodeling remain coordinated with material degradation.

Evidence should therefore distinguish three progressively stronger outcomes: enhanced regenerative activity, successful defect bridging, and mechanically functional reconstruction. These levels should not be treated as equivalent.

Bone Targeting and Local Retention: Terminology Should Reflect the Delivery Mechanism

Bone targeting and local retention are related but distinct concepts. Direct implantation, local injection, scaffold incorporation, membrane confinement, or immobilization on an implant can all produce high local exposure without constituting active bone targeting. In such cases, localization is primarily determined by delivery route or physical confinement.

Active bone targeting is more appropriately reserved for systems in which a defined ligand, surface chemistry, or material feature increases preferential association with mineralized tissue, skeletal cells, or a disease-relevant molecular target. Bisphosphonate-derived ligands, zoledronic-acid modification, and selected bone-affinity peptides are examples of potential targeting strategies,^33,91 but the presence of such a moiety alone does not demonstrate successful targeting. Active cellular targeting has also been explored using erythrocyte-membrane-coated, aptamer-functionalized CDC20@ZIF-8 nanoparticles designed to recruit BMSCs and promote osteogenesis in bone-defect models.⁹² However, comparative biodistribution between targeted and otherwise matched non-targeted systems remains necessary to establish targeting efficiency independently of altered cellular uptake or formulation stability.

Evidence should ideally compare targeted and otherwise matched non-targeted formulations. Relevant measurements include local accumulation, retention, target-to-background or bone-to-organ distribution, and, where appropriate, therapeutic performance. For systemically administered nanoparticles, quantitative biodistribution is particularly important because increased skeletal localization must be interpreted alongside off-target exposure.

Bone affinity should also be distinguished from disease-specific targeting. Preferential association with hydroxyapatite or mineralized tissue does not establish selective localization to an infected lesion, osteoporotic region, inflammatory niche, or newly forming bone surface. Likewise, improved therapeutic efficacy alone cannot prove targeting because surface modification may also change colloidal stability, uptake, degradation, or release.

For many locally administered ZIF-8 systems, active targeting may not be necessary because clinically accessible defects, implant coatings, membranes, or hydrogels already provide effective localization. Terms such as local delivery, local retention, bone affinity, and active bone targeting should therefore be used according to the actual mechanism and comparative localization evidence.

Preclinical Evaluation Framework

Preclinical evaluation of ZIF-8-based bone nanomedicine should determine not only whether a formulation improves regeneration, but also whether the proposed sequence linking material properties, exposure, biological response, and tissue repair is experimentally supported. Physicochemical characterization defines what is administered; degradation and release determine the resulting exposure; mechanistic assays evaluate the proposed biological response; and imaging, histology, remodeling, and functional measurements determine whether these effects translate into meaningful tissue repair.^93,94

The required evidence should be claim dependent. An antibacterial platform, a macrophage-regulating formulation, an angiogenic delivery system, and a construct intended for load-bearing reconstruction require different confirmatory endpoints. Increasing the number of assays does not necessarily strengthen a study unless those assays are aligned with the therapeutic claim and connected within a coherent evidence chain.

Cytocompatibility and Exposure-Aware Safety Assessment

Cytocompatibility should be evaluated as a relationship between material exposure and cellular response rather than as acceptable viability at a single concentration or time point. Relevant variables include administered dose, particle characteristics, surface modification, degradation rate, biological medium, exposure duration, and final formulation composition, all of which can alter intact-particle exposure and the release of Zn²⁺, cargo, and other degradation products.^14,15,23

Initial viability assays provide useful screening information but are insufficient as stand-alone evidence of safety. Depending on the intended application, complementary assessment may include membrane damage, apoptosis, mitochondrial dysfunction, oxidative stress, intracellular zinc accumulation, proliferation, or relevant cell-specific functional outcomes. The objective is to identify the exposure conditions under which cellular dysfunction emerges rather than to accumulate multiple toxicity assays without a defined exposure framework.^23,26,27

Dose reporting should distinguish the administered ZIF-8-containing formulation from measured Zn²⁺ exposure and, where relevant, cargo exposure. Nominal nanoparticle concentration cannot be assumed to produce equivalent biologically available zinc across different particle sizes, coatings, matrices, or media. Cytotoxicity comparisons across studies should therefore be interpreted cautiously when these variables are incompletely reported.

Cell selection should also reflect the intended application. Osteoblast-lineage cells or mesenchymal stromal cells may be appropriate for osteogenic compatibility, whereas endothelial, immune, periodontal, tendon-derived, or other cell populations may be required when their responses are central to the therapeutic claim. For locally implanted systems, the tested concentration range should reflect plausible local exposure rather than relying exclusively on low-dose systemic-equivalent conditions.

Thus, preclinical cytocompatibility is more appropriately interpreted as a dose–exposure–response relationship than as a binary “biocompatible/non-biocompatible” classification.

Immune Endpoints Should Match the Mechanistic Claim

Immune evaluation should be selected according to the mechanism being claimed rather than applied as a uniform marker panel to every ZIF-8 formulation. A change in a limited number of markers may demonstrate an altered inflammatory environment, whereas claims of immune-cell reprogramming or pathway-specific regulation require more direct and functionally informative evidence.⁵⁰

For macrophage-associated claims, conventional marker pairs should be complemented by cytokine, signaling, functional, temporal, or spatial evidence when stronger mechanistic conclusions are intended.^49,50 Th17/Treg claims require direct assessment of relevant T-cell populations rather than inference from general inflammatory cytokines or soluble-zinc studies.⁷ NET-related claims require NET-associated measurements rather than antibacterial efficacy or neutrophil abundance alone,^8,54,55 while anti-osteoclastogenic claims should connect osteoclast number or activity with downstream remodeling and structural outcomes.

Across these mechanisms, three levels of inference should be distinguished: descriptive evidence, in which an immune endpoint changes after treatment; mechanistic association, in which that change is linked to relevant regenerative outcomes; and causal evidence, in which experimental manipulation demonstrates that the immune pathway contributes to the therapeutic effect. Most current ZIF-8 bone-regeneration studies remain within the first two levels.

Immune profiling should therefore be sufficiently deep to support the stated mechanism, but additional markers should not be included solely for descriptive completeness. The most informative experiments are those in which immune endpoints are prespecified by the therapeutic hypothesis and connected to material exposure and tissue-level repair.

Regenerative Endpoints: Distinguishing Molecular Activity from Tissue-Level Repair

Regenerative endpoints represent different levels of evidence and should not be treated as interchangeable. Osteogenic, angiogenic, osteoclastic, histological, structural, and functional measurements answer related but distinct questions.^94,95

Osteogenic markers such as ALP, RUNX2, COL1A1, OCN, OPN, and mineralization assays can demonstrate activation of osteoblast-associated programs, particularly in vitro. They do not, however, establish formation of mature bone in vivo. Likewise, increased VEGF expression provides molecular evidence of angiogenic signaling, whereas endothelial migration or tube formation adds functional in vitro support; stronger tissue-level evidence requires assessment of vessel formation, vascular architecture, or perfusion within the regenerating defect.^56,95

Similar caution applies to bone resorption. Reductions in TRAP-positive cells, RANKL/OPG-related signaling, or osteoclast-associated proteins may indicate decreased resorptive activity, but suppression of resorption alone does not demonstrate complete regenerative recovery without corresponding evidence of bone formation, maturation, and remodeling.

Regenerative evidence can therefore be organized into three progressively stronger levels: molecular or cellular activity, tissue formation and organization, and structural or functional restoration. Increased RUNX2 expression represents osteogenic activity; histologically verified new bone and defect bridging provide tissue-level evidence; and restoration of mechanical competence provides stronger evidence of functional reconstruction. Claims should be calibrated to the highest level directly demonstrated.

Temporal assessment is also important because early molecular changes may not predict durable repair. Multi-time-point or longitudinal designs are particularly valuable when a study proposes a sequence linking material degradation, biological modulation, and mature tissue regeneration.

Imaging and Biomechanical Assessment: Avoiding Overinterpretation of Mineralized Signals

Imaging provides essential structural information in bone-regeneration studies, but its interpretation depends on the composition of the implanted material and the limitations of the imaging modality. Micro-computed tomography (micro-CT) is widely used to quantify bone volume fraction, bone mineral density, trabecular parameters, residual defect area, and defect bridging. These measurements are highly informative but should not automatically be interpreted as direct measurements of newly formed bone.^96,97

A particular challenge arises when a scaffold, ceramic phase, metallic component, or other high-attenuation material generates an X-ray signal that overlaps with mineralized tissue. Under these conditions, threshold-based segmentation may misclassify residual material as bone or obscure the interface between new tissue and the implanted construct.^97,98 The magnitude of this problem depends on material composition, degradation state, acquisition parameters, reconstruction settings, and segmentation strategy.

Micro-CT analysis should therefore distinguish new mineralized tissue from residual material whenever technically feasible. Depending on the experimental system, useful approaches may include material-only controls, baseline imaging, phantom calibration, longitudinal scanning, or appropriately validated multi-threshold segmentation. No single segmentation strategy, however, can be assumed to perform reliably across all ZIF-8-containing composites.

Histology or histomorphometry provides important complementary evidence when tissue identity is uncertain. Histological assessment can determine whether radiopaque regions correspond to mature or woven bone, residual scaffold, fibrous tissue, or mixed material–tissue interfaces and can provide information on vascularization, inflammation, degradation, and host integration.^97,99

Structural imaging should also be distinguished from functional reconstruction. Increased BV/TV or apparent defect filling does not establish that the regenerated region can withstand physiologically relevant loading. When mechanical restoration forms part of the therapeutic claim, biomechanical testing should be matched to the application.^94,95 Pull-out or push-out testing may be appropriate for implant osseointegration; tensile failure load and stiffness are relevant to tendon–bone repair; and bending, torsional, or compressive testing may be required for load-bearing bone reconstruction.

Thus, imaging, histology, and biomechanics provide complementary levels of evidence: micro-CT characterizes mineralized structure, histology establishes tissue composition and organization, and biomechanical testing determines whether structural repair translates into functional competence. Concordance among these modalities provides stronger evidence than any one measurement alone.

Limitations of Current Preclinical Models and Reporting Practices

The preclinical evidence base for ZIF-8-based bone nanomedicine remains heterogeneous in formulation, model selection, treatment exposure, follow-up duration, and outcome definition. Such heterogeneity is expected across platforms designed for different therapeutic purposes, but cross-study interpretation becomes difficult when key variables are incompletely reported or when conclusions extend beyond the capabilities of the experimental model.

Model validity is a central limitation. Healthy bone-defect models are appropriate for evaluating biocompatibility, local retention, osteogenic activity, and proof-of-concept regeneration, but they do not directly establish efficacy in infected, diabetic, osteoporotic, or otherwise pathological environments. Likewise, non-load-bearing cranial defects can provide valid evidence of local regeneration and defect bridging without reproducing the mechanical requirements of segmental long-bone reconstruction.^71,94 Models should therefore reproduce the biological, anatomical, and mechanical barriers relevant to the intended claim.

Follow-up duration should similarly reflect the process being evaluated. Early mineralization may be sufficient to demonstrate initial regenerative activity but not complete material degradation, mature bone remodeling, infection recurrence, durable osseointegration, chronic inflammatory responses, or mechanical maturation.^94,95 Observation periods should therefore be selected according to both biological healing and material behavior.

Cross-study comparison is further limited by inconsistent quantitative reporting. Particle size and dispersity, loading method and efficiency, administered ZIF-8 and cargo dose, release conditions, Zn²⁺ exposure, delivery route, surrounding matrix, control groups, and follow-up time are frequently reported unevenly. Apparently conflicting biological results may therefore reflect differences in exposure or formulation rather than true differences in therapeutic efficacy. These study-level variables should be reported explicitly and form an important basis for quantitative evidence comparison in Table 3.⁹³

Experimental design determines the level of inference that can be supported. A comparison between an untreated control and a multifunctional ZIF-8 composite may demonstrate efficacy of the complete formulation but provides limited component-specific information. Similarly, a mechanistic endpoint measured only at the terminal time point may not establish the temporal sequence connecting material exposure, immune regulation, and tissue regeneration.

Finally, most evidence remains concentrated in vitro and in small-animal models. These systems are appropriate for mechanistic investigation and early proof-of-concept testing but provide limited information on clinical-scale material volume, surgical handling, mechanical loading, systemic exposure, and long-term functional integration. Larger-animal studies become particularly relevant when the claim involves load-bearing reconstruction, implant-scale performance, clinically relevant delivery dimensions, or durable functional restoration;^71,94,95 they should be selected according to the translational question rather than treated as a universal requirement.

Taken together, the strength of preclinical evidence depends on four interconnected factors: model validity, exposure characterization, claim-matched endpoints, and adequate follow-up. Proof-of-concept regeneration establishes feasibility, whereas translational confidence requires convergence of these domains with durable tissue-level and, where relevant, functional outcomes.

Translational Readiness: Safety, Pharmacokinetics, Manufacturing, and Regulation

Translation of ZIF-8-based bone nanomedicine requires progression from proof-of-concept efficacy toward formulation-specific evidence of reproducible exposure, safety, disposition, manufacturability, and clinically relevant benefit. Improved bone formation in a small-animal model is an important early result, but it does not establish that the same formulation can be manufactured consistently, administered at a clinically relevant dose, degraded predictably, or cleared without unacceptable local or systemic effects.¹⁰⁰

Translational readiness should therefore be considered as a connected evidence chain. The final product must be reproducibly defined; its dose, degradation, and release must be linked to biological exposure; therapeutic effects must occur within an acceptable safety margin; and these properties must remain stable after scale-up, sterilization, storage, and final-product processing.^100,101 The required evidence package will depend on the delivery format, route of administration, intended duration of exposure, primary mode of action, and clinical indication rather than on ZIF-8 as a material class alone.

Dose–Exposure–Toxicity Relationships and the Zn²⁺ Therapeutic Window

Translation requires a formulation-specific relationship among administered dose, degradation, Zn²⁺ and cargo exposure, therapeutic activity, and toxicity. Nominal ZIF-8 concentration, soluble Zn²⁺ concentration, cumulative release, and intracellular zinc exposure are not interchangeable because they are influenced by particle properties, biological medium, surface modification, local retention, and delivery matrices.^14,15,23 Studies should therefore combine time-resolved release testing under physiologically relevant conditions with measurements of local and systemic exposure and corresponding efficacy and toxicity endpoints. Rather than extrapolating a universal safe concentration, investigators should define a therapeutic window for the final formulation while accounting for cell type, exposure duration, administration route, and disease context.^26,27 Component-specific controls, including unloaded ZIF-8, soluble Zn²⁺, free cargo, and matrix-only groups, should be included when feasible. A translationally credible formulation should reproducibly maintain therapeutic exposure below levels associated with unacceptable local or systemic injury.

Degradation-Product Fate and Clearance

Disappearance of the original ZIF-8 framework does not demonstrate biological clearance. Degradation may generate Zn²⁺, 2-methylimidazole-related species, partially transformed particles, and medium-dependent products.^14,15 Available in vivo studies provide formulation- and route-specific information on ZIF-8 biodistribution or tolerability,^47,48 but they do not establish the complete fate of degradation products released from locally implanted bone constructs. Studies should therefore identify relevant degradation products and determine their time-dependent retention at the treatment site, entry into systemic or lymphatic pathways, accumulation in clearance organs, further transformation, and elimination.

Longer follow-up is particularly important for locally implanted, repeatedly administered, slowly degrading, or highly loaded formulations. These assessments should be performed using the final composite product because coatings, cargos, hydrogels, and scaffolds can alter degradation and exposure. Biodegradability should therefore not be treated as synonymous with biological safety.

Local and Systemic Disposition: Pharmacokinetics, Biodistribution, and Retention

Pharmacokinetic assessment should reflect the route and delivery format of the final formulation. Conventional plasma pharmacokinetics may be informative for intravenously administered nanoparticles, whereas locally implanted hydrogels, coatings, membranes, or scaffolds are better characterized through the relationship among local retention, release, systemic leakage, and duration of exposure.^47,48

For local applications, studies should determine how long the formulation or its biologically relevant components remain at the treatment site and whether Zn-related species, cargo, or other degradation products enter the systemic circulation. Local administration should not be assumed to ensure confinement because fluid exchange, degradation, cellular uptake, and mechanical forces can redistribute material-derived species.

Systemically administered or actively targeted formulations require quantitative biodistribution. Relevant measures include circulating exposure, skeletal accumulation, target-to-nontarget distribution, uptake by major clearance organs, persistence, and elimination.^33,47 Comparative localization is particularly important when active bone targeting is claimed.

Carrier, cargo, and degradation-product disposition may also differ substantially. A therapeutic molecule may be released and cleared while carrier-derived products persist, or the carrier may disappear while the cargo remains locally active. Tracking only one component can therefore provide an incomplete description of formulation exposure.

The translational objective is to define where the formulation and its major products go, how long biologically relevant exposure persists, and whether that distribution is compatible with both efficacy and safety.

Immunotoxicity and Chronic Inflammatory Risk

Immunotoxicity represents a distinct concern because ZIF-8-based systems may be intentionally designed to modify inflammatory responses while also producing unintended or persistent immune effects. Short-term anti-inflammatory activity should therefore not be interpreted as evidence of long-term immune safety.

Immune safety should be evaluated in relation to the magnitude, duration, and reversibility of host responses. Transient inflammation may be compatible with normal tissue repair, whereas persistent inflammatory-cell infiltration, foreign-body reactions, fibrosis, or prolonged immune dysfunction may indicate an unfavorable response.^102,103 Conversely, excessive or prolonged immune suppression may be undesirable when antimicrobial defense remains important.

The relevant safety profile depends on the final formulation, including particle properties, degradation behavior, Zn²⁺ exposure, cargo, coatings, and matrices. Long-term immune safety therefore cannot be inferred from favorable expression of a small number of macrophage-associated markers.

Assessment should instead determine whether immune modulation remains controlled and resolves appropriately during tissue repair. Depending on formulation and indication, relevant outcomes may include persistent inflammatory-cell infiltration, foreign-body response, fibrosis, systemic inflammatory changes, and other product- or mechanism-specific endpoints. Specialized pathways such as Th17/Treg or NETs need not be measured indiscriminately unless they are central to the therapeutic or safety hypothesis.

Manufacturing, Sterilization, and Storage Stability

Manufacturing readiness requires reproducible control of the material attributes that determine biological exposure and therapeutic performance. For ZIF-8-based products, relevant critical quality attributes may include particle size and dispersity, morphology, crystallinity, composition, surface chemistry, cargo identity and loading, residual reagents, degradation behavior, release kinetics, and endotoxin burden. Composite systems may additionally require control of matrix composition, crosslinking, coating properties, pore architecture, mechanical characteristics, and spatial distribution of ZIF-8.^100,101

Scale-up should demonstrate that these critical attributes remain within predefined ranges. A larger production volume cannot be assumed to generate a biologically equivalent product if changes in particle or composite properties alter degradation, Zn²⁺ exposure, cargo release, or cellular interactions.^100,101

Sterilization should likewise be considered part of product development rather than a final processing step. ZIF-8-containing products may incorporate crystalline framework material together with drugs, proteins, polymers, extracellular vesicles, hydrogels, or implant coatings, each of which may respond differently to sterilization.

Gamma irradiation warrants specific consideration because ionizing radiation can modify both the crystalline framework and other formulation components. In an experimental radiation-stability study of archetypal MOFs, ZIF-8 exhibited framework degradation under high cumulative γ-irradiation doses; however, these experiments extended to the MGy range and were not designed to validate a medical-device sterilization process.¹⁰⁴ They therefore demonstrate the potential radiation sensitivity of ZIF-8 but should not be extrapolated directly to sterilization-relevant conditions.

A direct head-to-head comparison of γ-irradiation and ethylene oxide (EtO) sterilization for drug-loaded ZIF-8 bone-regenerative products was not identified. EtO is non-ionizing and presents a different compatibility profile, including potential interactions with therapeutic cargos, polymers, coatings, and other composite components as well as the need to control residual sterilant. Consequently, current evidence does not support a general conclusion that either γ-irradiation or EtO is intrinsically preferable for ZIF-8-based bone products.

Importantly, sterilization can modify therapeutic release even when gross material integrity appears acceptable in non-ZIF controlled-release systems, γ-irradiation over 4–33 kGy significantly accelerated the initial release phase of 5-fluorouracil from PLGA microparticles, whereas irradiation at 25 kGy produced a modestly faster release of an encapsulated peptide from PLGA microspheres.^105,106 These findings are indirect to ZIF-8 but demonstrate why sterilization compatibility should be assessed using the final formulation rather than inferred from preservation of crystallinity alone.

Sterilization studies should therefore compare the final product before and after processing with respect to framework integrity, particle or surface properties, cargo activity, degradation and release kinetics, mechanical performance where applicable, and biological function. Storage stability should be evaluated using the same principle to ensure that predefined product attributes remain acceptable throughout the intended shelf life.^100,101

Regulatory Considerations and a Product-Specific Translational-Readiness Framework

Regulatory development should be defined by the final therapeutic product, not by ZIF-8 as an isolated material class. A ZIF-8 formulation may function as a drug-delivery nanoparticle, implant coating, injectable hydrogel, membrane, scaffold component, or multifunctional combination product, each with different exposure profiles, mechanical roles, modes of action, and regulatory requirements.^100,107

At present, no ISO standard specifically dedicated to MOF- or ZIF-8-based implants was identified; regulatory evaluation should instead follow standards applicable to the final product category, tissue contact, sterilization method, and identified risks.

Relevant examples include ISO 10993–1:2025 for biological safety evaluation within a risk-management process,¹⁰² ISO 10993–18:2020 for chemical characterization of medical-device materials,¹⁰⁸ and ISO 10993–6:2026 for assessment of local effects after implantation.¹⁰³ Radiation sterilization can be developed and validated under ISO 11137–1:2025,¹⁰⁹ whereas EtO sterilization is currently addressed by ISO 11135:2014,¹¹⁰ and residual ethylene oxide and ethylene chlorohydrin are specifically addressed by ISO 10993–7:2026.¹¹¹ Device-level risk management is further guided by ISO 14971:2019.¹⁰⁷

These standards do not establish the safety or efficacy of ZIF-8 itself; rather, they define product-specific evidence domains that may become applicable to a ZIF-8-containing implant, coating, scaffold, hydrogel, drug-delivery system, or combination product.

Regulatory evidence should then connect product identity with exposure and intended clinical performance. Local implants may require particular emphasis on degradation-product exposure, tissue response, durability, and systemic leakage, whereas systemically administered or actively targeted formulations require greater attention to quantitative biodistribution, clearance, off-target exposure, and targeting selectivity. Biological endpoints should similarly match the intended indication: antibacterial coatings require durable infection control and implant integration, load-bearing constructs require structural and mechanical evidence, and immunomodulatory products require evidence that intended immune effects remain compatible with longer-term immune safety.

Existing standards and regulatory frameworks should therefore be selected according to the final product category rather than assuming a single MOF-specific pathway. Biological evaluation, chemical characterization, sterilization, implant performance, manufacturing quality, drug-delivery requirements, or combination-product considerations may become relevant depending on the formulation and intended use.

A practical translational-readiness framework can be organized as a progression from product definition → physiologically relevant degradation and exposure → mechanism and efficacy → local and systemic safety → disease- and application-matched functional validation → reproducible manufacturing and product stability. Progression should be claim dependent rather than platform dependent: not every formulation requires the same animal model, pharmacokinetic analysis, mechanical test, or immune assay.

Critically, the strength of the translational claim should not exceed the weakest essential component of this evidence chain. Strong proof-of-concept bone formation cannot compensate for undefined systemic exposure, unresolved degradation-product fate, inadequate long-term safety, or irreproducible manufacturing. Most ZIF-8-based bone-regenerative systems therefore remain within early preclinical development, and translational progress should be judged by convergence of product definition, exposure, mechanism, disease-specific efficacy, safety, and reproducibility rather than by formulation complexity alone.

Conclusions

ZIF-8 is a versatile platform for bone nanomedicine because it supports multiple cargo-loading strategies, environment-dependent degradation, surface modification, and biologically active Zn²⁺ release. However, the strength of evidence is uneven across the proposed osteoimmunomodulatory mechanisms. The clearest direct evidence indicates that selected ZIF-8-containing formulations can regulate macrophage-associated inflammatory responses while supporting bone regeneration. Antibacterial activity, local retention, osteogenic support, and angiogenic enhancement have also been demonstrated in preclinical models, although the respective contributions of ZIF-8, released Zn²⁺, therapeutic cargo, surface modifications, and surrounding matrices are often incompletely resolved.

By contrast, evidence for ZIF-8-mediated Th17/Treg regulation, NET modulation, and broader immune-cell networks remains predominantly indirect or hypothesis-generating. Zinc biology provides mechanistic plausibility, but such evidence should not be interpreted as direct demonstration of a ZIF-8-specific mechanism. Nucleic-acid delivery is similarly promising but remains insufficiently validated for broad osteoimmune claims.

Translation is further limited by the influence of phosphate-containing and protein-rich media on ZIF-8 degradation, the absence of a universal Zn²⁺ safety window, and the uncertain fate of linker-related and other degradation products. Long-term pharmacokinetics, biodistribution, immunotoxicity, disease-specific efficacy, sterilization compatibility, storage stability, and manufacturing reproducibility remain inadequately defined for most formulations.

Five priorities should guide future research: (1) establish formulation-specific dose–release–exposure–toxicity relationships under physiologically relevant conditions; (2) use component-specific controls to distinguish the contributions of ZIF-8, Zn²⁺, cargo, coatings, and matrices; (3) characterize long-term degradation-product fate, biodistribution, clearance, and immune safety; (4) validate structural and functional outcomes in disease- and application-matched models, including larger animals when justified; and (5) develop scalable manufacturing, sterilization, storage, and quality-control processes that preserve critical product attributes.

Overall, ZIF-8-based bone-regeneration systems remain at the preclinical proof-of-concept stage, with evidence dominated by in vitro and small-animal studies. Progress toward clinical use will depend more on reproducible product definition, mechanistic attribution, exposure control, long-term safety, and functional validation than on increasing formulation complexity.

Funding Statement

This work was supported by the National Natural Science Foundation of China for Young Scientists Project (82405109), the Tianjin Education Commission Scientific Research Program (2024ZXZD001), the Tianjin Science and Technology Program Project (25JCZDJC01350), and the Tianjin Public Health Science and Technology Major Special Project (25ZXWZSY00010).

Declaration of Generative AI and AI-Assisted Technologies in the Writing Process

During the preparation of this manuscript, the authors used ChatGPT (OpenAI, GPT-5) solely for English-language editing to improve grammar, clarity, and readability. The authors subsequently reviewed and verified the entire manuscript, including all scientific statements, citations, and references, and take full responsibility for the originality, accuracy, and integrity of the published work. The authors confirmed that the use of this tool complied with the applicable terms of use. No generative AI tool was used to generate scientific data, results, analyses, or figures.

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Disclosure

The authors report no conflicts of interest in this work.