Metabolic Node-Directed Biomaterials for Bone Repair
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Abstract
Glucose metabolism connects the nutrient demands of repair cells with the design of bone biomaterials.
Material architecture, interfacial chemistry and local delivery can influence substrate availability, enzyme
regulation and metabolite handling, creating opportunities to coordinate osteogenic, vascular and immune
responses. This Review examines representative studies published online from 2018 through September
2026, organized around four strategies: glucose acquisition, enzymatic regulation, lactate transport,
production and removal, and immune-cell metabolic reprogramming. Within each strategy, materials are
compared by their metabolic targets, responding cells, intervention conditions and bone outcomes. The
comparisons distinguish substrate supply from extracellular consumption, enzyme expression from activity,
and lactate release from transport inhibition or catalytic conversion. They also connect metabolic responses
with anti-inflammatory, antimicrobial, antioxidant and angiogenic functions. Across these approaches, the
direction and timing of intervention depend on the cellular and repair context. Relating controllable material
properties to local exposure, metabolic function and tissue recovery provides a basis for selecting delivery
systems and identifying informative mechanistic tests. This organization places metabolic nodes within the
material design process and highlights the experimental connections needed to advance functional bone
regeneration.
Keywords: bone biomaterials; glucose metabolism; glycolysis; lactate metabolism; immunometabolism;
bone regeneration
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1. Introduction
Bone repair depends on coordinated interactions among osteogenic, immune and vascular cells within the
extracellular matrix. These interactions provide targets for biomaterial intervention. [1] In mesenchymal
stromal cells (MSCs), glucose metabolism supplies energy and biosynthetic substrates, with requirements
that vary according to oxygen availability and cellular state. [2] Defining these requirements helps identify
the metabolic process, responding cell population and repair stage that a material should target.
Substrate availability is particularly important in constructs carrying living cells. Glucose supplementation
sustained human MSC survival in three-dimensional fibrin under near-anoxic, glucose-limited conditions. [3]
For glucose-consuming materials, an alginate/glucose oxidase/calcium phosphate-catalase scaffold consumed
glucose in a cell-free, high-glucose system, whereas the alginate/glucose oxidase formulation alone impaired
cell proliferation readouts. [4] These studies address different aspects of the same design problem:
maintaining glucose access in nutrient-limited constructs while controlling extracellular glucose consumption
in environments where depletion is the intended intervention.
The desired response also depends on the recipient cell. In separate high-glucose experiments, free myricitrin
(MYT) increased medium glucose consumption by bone-marrow MSCs, whereas the PN@MHV composite
hydrogel reduced consumption by RAW264.7 macrophages. [5] Such responses support directing material
activity toward the metabolic requirements of specific cell populations. At the tissue level, the enzymefunctionalized scaffold improved bone formation in diabetic calvarial defects. [4] Comparing these cellular
and tissue responses with material composition can reveal which intervention sites are useful in a given
repair environment.
Existing reviews have organized diabetic bone biomaterials by therapeutic function, examined nanomaterial
interactions with the bone microenvironment, and cataloged interventions in macrophage
immunometabolism. [1, 6, 7] MSC-focused reviews have also connected glucose requirements with
postimplantation function and engineering approaches. [2] A metabolic-node framework provides a shared
basis for comparing scaffolds, coatings and delivery systems that influence the same substrate pool or
reaction. It also allows differences in material architecture and cellular mechanism to be examined alongside
bone outcomes.
This review examines how specific glucose-metabolic nodes can inform biomaterial design for bone repair.
We relate extracellular reactions, molecular responses and functional metabolic measurements to material
properties and tissue outcomes, using mechanistic perturbations to evaluate the proposed connections.
Comparisons retain the responding cell, disease model, exposure conditions and experimental comparator.
We also consider how metabolic regulation accompanies anti-inflammatory, antimicrobial, antioxidant and
angiogenic functions, and examine the treatment comparisons used to assess therapeutic synergy.
The review focuses on representative bone biomaterial studies published online from 2018 through
September 2026. Dental-specific applications are excluded, and mechanistic background studies are
identified separately. We first discuss the roles of glucose metabolism in biomaterial-mediated repair, then
examine four strategies: glucose acquisition, enzymatic regulation, lactate production, transport and
clearance, and immune-cell metabolic reprogramming. Figure 1 maps these strategies to the cellular and
experimental settings represented in the literature. The final discussion compares their design implications,
identifies unresolved mechanisms and considers the experimental steps needed to connect metabolic
intervention with functional bone regeneration.
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2. Roles of glucose-metabolic regulation in biomaterial-mediated bone repair
2.1. Cellular energy requirements and repair functions
2.1.1. Energy supply and osteogenic fate
Early cell survival establishes the population available for subsequent repair, making glucose availability a
basic consideration in cell-bearing constructs. At 0.1% O₂, human mesenchymal stromal cells (hMSCs)
supplied with 5 g/L glucose in serum-free minimal medium retained more than 60% of the day-1 viable-cell
number after 14 days without medium renewal. In a separate three-dimensional fibrin experiment at the same
oxygen level, 1 g/L glucose preserved approximately 77% of the initial viable-cell number after seven days.
[3] Sustaining substrate access during this period of restricted exchange provides the foundation for
evaluating later osteogenic function.
The metabolic support offered by a construct also depends on how the intended cells respond to the material.
Laponite-engineered apoptotic vesicles (L@Apo) increased oxygen consumption and extracellular
acidification in rat bone-marrow stromal cells (BMSCs), together with stronger osteogenic-marker responses
at seven days and mineral staining at 21 days. [8] Cell history introduces another source of variation. In
osteoblasts from pooled diabetic Charcot donors cultured on printed titanium, the authors proposed metabolic
inflexibility as an explanation for the observed responses. [9] Construct design therefore requires attention to
both nutrient provision and the capacity of the intended cell population to use that support during
differentiation.
2.1.2. Immune and vascular contributions
Endothelial metabolism connects implant surfaces with vascular functions that support regenerating tissue. A
titanium-based TiO₂/Bi₂O₃ semiconductor network increased extracellular acidification rate (ECAR) and
PFKP immunofluorescence in human umbilical-vein endothelial cells (HUVECs). The glycolytic inhibitor 2deoxyglucose weakened the surface-associated improvements in tube formation and migration. [10] These
experiments place the functional metabolic response in endothelial cells and suggest a vascular route through
which surface engineering may support bone repair. Figure 1 situates this route alongside osteogenic and
immune responses.
A material may need to elicit different metabolic responses in neighboring cell populations. During mouse
BMSC osteogenic induction at 40 mM glucose, free MYT increased medium glucose consumption. In a
separate experiment, the PN@MHV composite reduced consumption by high-glucose-exposed RAW264.7
macrophages. [5] The distinction between free cargo and complete formulation is relevant to this
comparison, as is the difference in cell identity. Together with the endothelial findings, these results support
evaluating whether a metabolic intervention accommodates osteogenic demand, immune regulation and
vascular function within the shared repair environment. [10]
2.2. Metabolic constraints in the repair microenvironment
2.2.1. Local glucose and oxygen availability
The value of glucose provision depends on the oxygen environment experienced by the cells. In
subcutaneous fibrin constructs containing pooled hMSCs in nude mice, core oxygen tension was 0.13 ±
0.06% on day 1 and 0.14 ± 0.04% on day 3, substantially below the surrounding tissue values (mean ± SD, n
= 3). [3] Combined with the near-anoxic survival experiments, these measurements show how nutrient access
can influence cell persistence in poorly oxygenated constructs. This ectopic implantation model provides a
basis for examining the balance among glucose supply, oxygen availability and cellular demand during early
implantation.
Glucose-depleting materials bring the balance between substrate removal and host-cell nutrition into
formulation design. In a GOx-containing bone-cement study, BMSCs cultured in low-glucose medium
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showed a reduced CCK-8 metabolic-activity signal even at the lowest tested GOx condition. The endothelial
experiments used high-glucose medium, introducing a difference in substrate availability as well as cell
identity. [11] Oxygen consumption can further constrain the response: dissolved oxygen decreased in both
GOx and GOx/catalase systems, despite the oxygen-generating reaction catalyzed by catalase. [4] Selecting a
catalytic formulation thus requires simultaneous consideration of residual glucose, net oxygen balance and
host-cell function.
2.2.2. Inflammatory stage, disease context and exposure
The timing of metabolic intervention influences how an inflammatory response contributes to repair. Lactateloaded collagen sponges increased day-1 CD86-positive cells and early vascular readouts, and Nodinitib-1
attenuated these responses. In a separate arm of the same study, lactate delivered with Bio-Oss Collagen
increased calvarial bone volume fraction and mineral density at eight and 12 weeks. Delaying implantation
until day 3 or 7 produced no significant vascular benefit at the shared day-14 endpoint, while also shortening
treatment exposure. [12] The results connect early lactate-responsive inflammation with vascular support and
establish treatment duration as part of the timing question.
Composition and cellular activation state also shape the response. Dexamethasone-containing manganese
nanogels reduced iNOS/CD86 and TNFα while increasing Arg1/CD206 and selected repair-associated
factors in the supernatant of LPS-stimulated macrophages. In direct BMSC experiments, Alg-MD favored
early ALP/RUNX2 responses, whereas Alg-MD@CS favored later OPN expression and mineral staining.
Bone formation was assessed separately in conventional femoral-condyle defects. [13] Calcium-citrate
loading produced another exposure-dependent pattern: the highest loading reduced both ECAR and oxygen
consumption under IL-4 stimulation. [14] These findings make inflammatory stimulus, treatment duration
and the selected functional endpoint integral to material comparison.
2.3. Connecting metabolic measurements with bone outcomes
2.3.1. From metabolic response to mechanism
The choice of metabolic measurement determines which aspect of material activity can be compared. A
ROS-responsive Qu-Cr titanium coating increased the intracellular glucose pool in MSCs after seven days of
H₂O₂-induced oxidative stress, whereas a curcumin-loaded composite scaffold increased glucose
consumption estimated from culture medium. [15, 16] Intracellular glucose reflects the balance between
uptake and utilization; medium depletion also depends on cell abundance and non-cellular losses. At the
enzyme level, calcium-phosphate cement/PCL scaffolds with CaCit10/15 loadings reduced
phosphofructokinase activity in LPS-stimulated RAW264.7 cells. [14] In a BMP2/aFGF membrane system,
UK5099 reduced ALP and mineral staining, demonstrating pharmacological sensitivity to mitochondrial
pyruvate-carrier inhibition. [17]
The material itself can also influence the assay. Bioactive-glass particles and their dissolution products
altered LDH and ALP signals, supporting the use of material blanks and independent measurements when
interpreting enzyme-based readouts. [18] Targeted perturbations can then test the proposed intermediary. For
example, the authors of a macrophage-directed system identified mannose-receptor knockdown and in vivo
macrophage depletion as the next steps in mechanistic validation. [19] Table 1 relates expression
measurements, metabolite pools, functional assays and perturbations to the questions they address. This table
helps select experiments that connect a material property with its proposed metabolic effect.
2.3.2. From cellular responses to bone repair
Bone-related endpoints span differentiation, tissue preservation and mechanical recovery. Time-dependent
supernatant ALP in MG63 cells on titanium-alloy lattices describes an in vitro osteoblastic response. [20]
Bone-targeted AZD3965 nanoparticles improved trabecular structure in ovariectomized mice, while
osteoblast number and RUNX2 positivity showed no significant increase over ovariectomized controls; the
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outcome therefore favors bone preservation through reduced resorption. [21] Ti-VN₄ fixation implants were
evaluated by maximum load, stiffness and histological healing at three and five weeks in rabbit tibial
fractures, providing endpoints directly relevant to fracture function. [22]
The comparator and observation period determine the benefit captured by each endpoint. At eight weeks, a
metformin-loaded hydrogel improved bone mineral density, trabecular thickness and trabecular number
relative to unloaded gel, whereas the BV/TV difference was not significant. [23] Model identity is equally
important: blood-lactate measurements during forced swimming and bone protection in a separate model
describe different aspects of material performance. [24] Table 1 brings these distinctions together so that
each material can be evaluated through its measured metabolic node, responding cell and bone outcome. The
following sections apply this approach, beginning with glucose acquisition.
3. Glucose availability and acquisition
3.1. Supply, diffusion and mass transport
3.1.1. Scaffold architecture and effective diffusion
Scaffold architecture influences the glucose available to embedded cells by changing transport through the
material. In acellular experiments at 37 °C, electrospun polycaprolactone (PCL) scaffolds had lower effective
glucose diffusivity in culture medium than in water, and larger interfiber spaces supported faster diffusion.
Because the geometric characteristics varied together, the comparison reflects the combined pore
architecture. [25] Pore organization and fluid composition therefore need to be considered together when
specifying transport through a cell-bearing scaffold, with diffusivity measured in the intended culture
medium.
Perfusion introduces spatial variation that bulk-medium measurements can overlook. A radial-flow
bioreactor simulation predicted heterogeneous pericellular glucose concentrations, with poorly perfused
regions falling below the bulk-medium concentration. The model assumed uniform cell seeding and oxygenindependent glucose consumption; an alginate-gelatin bead model also omitted oxygen transport. [26, 27]
These assumptions become consequential when cell density, bead size or flow changes. Combining diffusion
measurements with local glucose and oxygen profiles would allow material geometry and perfusion to be
evaluated against the exposure actually experienced by the cells. [25–27]
3.1.2. Metabolic responses to cell-sheet organization and geometry
Gelatin hydrogel-fragmented fibers changed both cell-sheet organization and metabolic readouts. MC3T3-E1
sheets containing these fibers consumed more glucose, contained more ATP and had a lower lactate/glucose
molar ratio after 24 h of reculture. Under osteogenic induction that included BMP-2, they also showed
greater mineralization and calcium content. The authors attributed the pattern to improved oxygen
availability, although local oxygen, diffusion and oxygen-consumption rates were unmeasured. [28] The
study relates sheet organization to substrate use and induced mineralization in an osteoblastic cell line; the
lower lactate/glucose ratio describes the measured metabolic balance without assigning a lactate-clearance
mechanism.
Titanium lattices illustrate the importance of matching metabolic and differentiation measurements in time.
In MG63 cultures, medium glucose declined between days 4 and 8 on nominal 900-µm cubic Ti-6Al-4V
lattices, whereas the highest mean supernatant ALP at day 14 occurred on the 400-µm body-centered cubic
geometry. These were descriptive group means, and cell counts were available only on the final day. [20] A
study published online in 2017 provides methodological context: day-14 DNA-normalized glucose
consumption was lower in osteogenically induced rat MSC constructs on PLLA than in basal-medium
constructs. [29] Comparing geometry-dependent consumption alongside maturation stage and
contemporaneous cell abundance makes the result more useful for construct design.
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3.2. GLUT-associated glucose acquisition
3.2.1. GLUT1 and membrane microdomains
Cur@MS combines curcumin-loaded gelatin microspheres with a printed PCL/hydroxyapatite/β-tricalciumphosphate scaffold. After 24 h, BMSC cocultures showed greater medium-based glucose consumption than
scaffold controls. Unloaded scaffolds also increased consumption, making the microsphere-bearing control
important for assessing the additional contribution of curcumin. GLUT1 staining became denser and
clustered near nuclei, while cholesterol depletion dispersed overlapping GLUT1 and lipid-raft signals. Total
cellular cholesterol did not differ significantly among the untreated scaffold groups. [16] The proposed
mechanism consequently centers on the spatial organization of GLUT1 and membrane microdomains
alongside the consumption response.
BAY-876 and methyl-β-cyclodextrin reduced glucose-consumption readouts to low levels across groups,
eliminating significant differences between them. These interventions probe GLUT1-directed inhibition and
broader cholesterol-dependent membrane organization, respectively. In 5-mm rat calvarial defects, Cur@MS
improved bone parameters at day 28. A separate inhibitor experiment examined day-3 GLUT1/RUNX2
staining and favored local delivery over oral curcumin. [16] The combined findings identify delivery route
and membrane organization as candidate design variables, supported by cellular pharmacology and separate
repair experiments.
3.2.2. GLUT4 signaling and mechanically coupled glucose availability
ROS-responsive coatings regulate glucose handling through cargo release and associated signaling. CaCO₃quercetin-chromium nanoparticle coatings increased normalized intracellular glucose after seven days in
H₂O₂-exposed MSCs from osteoporotic rats. PI3K-related signals and IRS2/Akt phosphorylation increased,
and GLUT4 immunolocalization was consistent with redistribution toward the membrane. Molecular
docking supported a proposed interaction between Qu-Cr and IRS2. The coatings also improved peri-implant
bone at 30 days in ovariectomized rats. [15] Together, the signaling and localization data help interpret a
glucose-pool measurement that integrates both transport and utilization.
Dynamic hyaluronan (HA) networks approach glucose regulation through mechanical cues. RGDfunctionalized HA-ADA and HA-CA differed in host-guest chemistry and relaxation behavior, while the
authors reported similar elastic moduli. After three days of osteogenic culture, human MSCs in HA-ADA
showed greater GLUT1 transcription and immunofluorescence, accompanied by higher protein-normalized
glucose content in cell extracts. HA-ADA carrying osteogenically preconditioned rat MSCs improved bone
formation at eight weeks in bilateral rat calvarial defects. [30] The coating and hydrogel address implant
integration in osteoporosis and cell-assisted defect repair, respectively. Their findings identify signaling and
network relaxation as routes to altered glucose availability, with transporter-specific perturbation needed to
resolve the contribution of GLUT regulation. [15, 30]
3.3. Extracellular glucose depletion
3.3.1. GOx-mediated and enzyme-mimetic glucose consumption
GOx-bearing materials consume glucose before cellular uptake. An injectable hydroxyapatite/GelMAHAMA bone cement containing GOx lowered glucose over 72 h in a cell-free solution initially containing 2
mg/mL glucose; GOx-free formulations had little effect. [11] Printed Alg/GOx/CaP@CAT scaffolds, which
combine GOx with catalase-bearing calcium phosphate, lowered glucose from 11.2 to 6.5 mmol/L over 72 h
and outperformed Alg/GOx in the same assay. [4] In db/db mouse tibial osteotomies, the complete bone
cement produced little separation among groups at four weeks, followed by BMD and BV/TV values
approximately 1.69 and 1.84 times those of untreated controls at eight weeks. [11] The printed composite
improved eight-week bone formation in 5-mm calvarial defects in type 2 diabetic rats. [4] Both systems
combine extracellular catalysis with repair benefits obtained from the complete formulation.
6
Enzyme-mimetic platforms broaden the range of coupled reactions. MoSe₂/PtCu exhibited GOx-like and
CAT-like activities, while a polydopamine layer immobilized native lactate oxidase. A coupled TMB assay
supported glucose oxidation, and a separate peroxide-decomposition assay measured oxygen production. [31]
These measurements characterize individual cell-free reactions; the lactate branch is considered in Section
5.2.2. Across these platforms, catalyst composition, residual glucose and reaction products are
interdependent formulation variables. Local measurements of these formulation variables would connect
catalytic performance with the repair outcomes of the mineral, polymer and enzyme components.
3.3.2. Oxygen balance, peroxide and combined treatment
Catalyst loading must be matched to both substrate supply and host-cell requirements. In the bone-cement
study, BMSCs in low-glucose medium showed a reduced CCK-8 metabolic-activity signal at the lowest
tested GOx concentration, 2.5 µg/mL, whereas HUVEC experiments began with a higher glucose supply.
[11] Adding CaP@CAT to printed scaffolds limited peroxide accumulation and improved attachment and
CCK-8 responses in rat BMSCs and HUVECs relative to Alg/GOx. Both formulations nevertheless
consumed dissolved oxygen overall. [4] Because the comparison introduces calcium phosphate and catalase
together, it supports selection of the composite on its overall cellular response. Residual glucose, net oxygen
change and peroxide accumulation provide a common chemical basis for refining the formulation under
matched starting conditions.
Antibacterial systems add ionic or thermal functions to glucose oxidation. With near-infrared irradiation, Cupolydopamine/GOx-coated sulfonated PEEK improved peri-implant BV/TV and interface bone formation at
four and eight weeks in infected diabetic rat femoral-condyle models. The study compared irradiation
conditions within the composite treatment but did not include Cu-only or GOx-only implants. [32] In another
platform, BPQD@Cu-MOF, the GOx-like assay was performed at pH 9, making activity at physiological pH
a relevant formulation question. [33] Component controls and local measurements of temperature, pH and
peroxide would clarify how these functions contribute to repair. Figure 2 compares their intervention sites
and environmental consequences, while Table 2 places their cellular and bone outcomes alongside those of
the other metabolic strategies.
4. Enzymatic regulation and metabolic cascades
4.1. HK/PFK responses and cellular attribution
4.1.1. Enzyme expression and catalytic activity
Physical cues alter glycolytic gene expression over time. Human MSCs on fibronectin-coated
polyacrylamide substrates expressed more PFKM, PKM2, LDHA and SLC2A1 on stiff than on soft matrices
at day 1, with stiffnesses of 40 and 4.47 kPa, respectively. By day 7, HK2 was the only tested glycolysisrelated gene that remained upregulated. [34] Fibronectin-modified porous polylactic-acid microparticles also
increased HK2 protein in rat BMSCs after seven days relative to solid particles. [35] These physical
environments share an HK2 expression response, while the transient PFKM response makes culture duration
central to their comparison. Transcript and protein measurements describe related but distinct levels of this
adaptation. [34, 35]
Macrophage studies provide direct activity measurements at PFK. Calcium-citrate-loaded PCL coatings on
calcium-phosphate cement reduced protein-normalized PFK activity in LPS-stimulated RAW264.7 cells after
48 h at CaCit10/15 loadings; CaCit0/5 had no significant effect. [14] In experiments accompanying DMOGloaded autologous blood clots, the two higher DMOG pretreatments increased erythrophagocytosisassociated Fe²⁺ fluorescence and lowered PFK activity. The lowest treatment produced no significant change
in either measurement relative to zero-DMOG controls. The authors proposed erythrophagocytosis-derived
iron as a mediator of the metabolic response. [36] These assays measured PFK activity without resolving the
isoform, whereas the MSC study identified PFKM transcription. [14, 34, 36] The design objective is
7
consequently cell specific: supporting MSC adaptation and lowering macrophage PFK activity require
different exposure conditions and functional endpoints.
4.1.2. Metabolic nodes in donor and recipient cells
Cell-derived materials require the metabolic state of the donor to be distinguished from the response to the
implanted product. After 24 h of oscillating fluid flow, MLO-Y4 osteocytes increased PFKP transcripts and
culture-supernatant lactate relative to static controls; HK1, LDHA, PFKM, PGAM1 and PGK1 comparisons
were nonsignificant. Whole-cell lysate from these donors was incorporated into an alginate hydrogel
scaffold, HSOOL, which increased eight-week bone volume fraction in calvarial defects in male BALB/c
nude mice relative to defect-only and blank-scaffold controls. [37] Here, PFKP describes the donor-cell
response to preconditioning, while bone formation measures the performance of the lysate-containing
construct.
Surface-engineering studies assess metabolism in cells directly contacting the material. The TiO₂/Bi₂O₃
heterojunction network, 3D-NTBH, increased ECAR and PFKP immunofluorescence in HUVECs relative to
titanium and TiO₂-network controls; PFKP was assessed after 48 h. Corresponding implants improved new
bone formation and OCN staining at four and eight weeks in rats. The authors related the endothelial
response to fibronectin-mediated adhesion, angiogenesis and bone integration. [10] These two approaches
offer different routes to metabolic design: controlling the state of cells that supply biological cargo, or
presenting a surface that influences metabolism in local endothelial cells. [10, 37]
4.2. ALDOA, GAPDH and downstream reactions
4.2.1. Ligand dynamics and ALDOA availability
Dynamic ligand presentation can alter the subcellular availability of a glycolytic enzyme. In an RGD-bearing
gold-nanoparticle platform on glass, aniline-catalyzed hydrazone exchange generated the dynamic CAT
interface, which was compared with an uncatalyzed NOC interface. Human MSCs on CAT yielded more
digitonin-releasable ALDOA and showed higher GLUT4 protein and transcript abundance, together with
increased PKM, TCA-cycle and OXPHOS-related transcripts. [38] Immunoblotting after digitonin
permeabilization quantified the releasable ALDOA pool. The experiment links interface dynamics to enzyme
partitioning and metabolic gene expression.
Rac1 knockdown reduced GLUT4, PKM and related metabolic transcripts in CAT-cultured cells. The
authors proposed that cytoskeletal rearrangement releases actin-associated ALDOA, coupling ligand motion
to metabolic adaptation. After one day of adhesion and seven days of osteogenic induction, CAT increased
ALP and the osteogenic proteins COL I, OCN and RUNX2. [38] This in vitro system suggests that ligand
mobility can be tuned alongside enzyme accessibility during stem-cell differentiation. A useful next
comparison pairs ALDOA partitioning with catalytic-activity measurements to clarify how the releasable
fraction contributes to the osteogenic response.
4.2.2. GAPDH, PK and enzyme-directed delivery
Drug delivery provides another means of influencing enzyme activity. Dimethyl fumarate (DMF)-loaded
liposomes in collagenase-responsive CM@GM microspheres reduced macrophage GAPDH activity,
measured kinetically in lysates and normalized to total protein. In bone-marrow-derived macrophages from
diabetic rats, the loaded system lowered ECAR and increased OCR. At four weeks, it also reduced bacterial
counts and histological inflammation in MRSA-infected femoral defects in STZ-diabetic rats. [39] The
formulation thus combines changes in host bioenergetics with infection control. Because DMF acts on
multiple targets, GAPDH activity is one component of the pharmacological response produced within this
delivery environment.
Calcium-citrate scaffolds show how enzyme suppression varies with loading and activation state. CaCit10/15
in CPC/PCL scaffolds reduced protein-normalized PK activity in LPS-stimulated RAW264.7 cells after 48 h.
8
At the highest loading, IL-4-stimulated cultures showed lower ECAR and OCR. [14] Non-material
mechanistic evidence provides additional context: during human MSC adipogenesis, TRAF4 overexpression
reduced PKM2 Y105 phosphorylation and increased total cellular PK activity, while knockdown produced
the opposite effects. PKM2-IN-1 and DASA-58 reversed the respective adipogenic responses. [40] This
bidirectional perturbation illustrates how catalytic activity, a specific regulatory modification and lineage
outcome can be examined together. The same comparison strategy can relate enzyme activity to lineage
outcome in material studies while distinguishing total PK activity from PKM2-specific regulation.
4.3. Pyruvate handling and energetic coupling
4.3.1. PDK, PDH and mitochondrial pyruvate entry
Pyruvate handling can be influenced through regulatory expression, enzyme activity or mitochondrial
transport. In high-glucose BMSCs, free MYT, the cargo used in PN@MHV hydrogels, restored PDK1
expression together with HKII, PFK1 and LDHA. [5] Calcium-citrate-functionalized CPC/PCL scaffolds
reduced PDH activity in LPS-stimulated RAW264.7 macrophages at CaCit10/15 loadings. [14] PDK1
expression and PDH activity describe different aspects of the junction between glycolysis and mitochondrial
oxidation. Their interpretation depends on the responding cell and the metabolic program that the
intervention is intended to support.
Mitochondrial entry was tested pharmacologically in a Janus membrane that delivered BMP-2 and aFGF
from its inner layer. UK5099, an inhibitor of the mitochondrial pyruvate carrier (MPC), reduced ALP and
mineralization staining in treated BMSCs. In a separate rat calvarial-defect experiment, the dual-factor
membrane produced greater eight-week BV/TV than either single-factor membrane or controls. [17] These
findings identify MPC-sensitive differentiation as a mechanism worth examining within growth-factor
delivery. Combining MPC perturbation with PDH activity, phosphorylation and glucose tracing would
establish how substrate routing contributes to the response and distinguish glucose-derived pyruvate from
other respiratory fuels.
4.3.2. Parallel activation and metabolic redistribution
Respiration and glycolytic readouts can increase together during osteogenic treatment. After seven days of
exposure to BFGP+PZ Janus-membrane extracts, BMSCs showed higher OCR and ECAR than cells exposed
to growth-factor-free membrane extracts. [17] Free L@Apo vesicles produced a similar paired increase. [8]
The carrier comparison adds a separate question: L@Apo-G/P hydrogels showed no significant four-week
BV/TV advantage over Apo-G/P in rat femoral-condyle defects. [8] Matched cargo and carrier controls are
therefore needed to relate vesicle-induced metabolic changes to the added benefit of local delivery.
Electrical stimulation produced a different response. Bio-SIG, generated by a self-sustained system coupled
to a gold-coated fixation plate, increased basal and compensatory glycoPER while reducing basal, maximal
and ATP-linked OCR in MC3T3-E1 cells after two weeks. GlycoPER estimates glycolytic proton efflux,
whereas ATP-linked OCR describes respiration coupled to ATP synthesis. The authors proposed calcium
signaling as part of the mechanism, and the one-month BV/TV comparison between stimulated and control
rat femoral defects was nonsignificant. [41] These results make cell identity and stimulation duration
important when comparing metabolic redistribution with the parallel activation observed in BMSCs.
Enzyme-focused design therefore includes extracellular substrate conversion (Figure 2) and cellular
regulation of enzyme abundance, accessibility and activity (Table 2).
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5. Lactate transport, production and removal
5.1. Endogenous production and material-derived lactate
5.1.1. Cellular regulation and external supply
Lactate-directed materials act at several locations: intracellular production, external supply, membrane
transport and extracellular conversion, followed by signaling or protein-modification responses (Figure 3).
Under oxidative stress, L-arginine-loaded HKUST-1, LA@HK, increased Ldha transcription and
intracellular lactate in mouse BMSCs relative to HKUST-1. ABAT overexpression lowered lactate in
LA@HK-treated cells, whereas GCLM depletion produced no significant change, distinguishing the lactate
response from the parallel antioxidant branch. Incorporation of LA@HK into GelMA increased bone volume
in rat calvarial defects at four and eight weeks relative to HKUST-1/GelMA. [42] Transcription, metabolite
abundance and ABAT perturbation thus describe complementary aspects of the intracellular response.
Degradable polymers and lactate salts supply lactate through different exposure profiles. A PLGA coating on
porous Ti6Al4V released both lactic and glycolic acids. [43] An SDF1-bearing, calcium-lactate-coated
PCL/regenerated-cellulose scaffold released approximately 20% of its measured lactate within 24 h and 75%
by three weeks in cell-free PBS. [44] Sodium-lactate-loaded PCL/nHA scaffolds sustained lactate availability
in BMSC culture medium, where release, cellular production and utilization jointly determine concentration.
[45] Cumulative release in PBS and concentrations measured during cell culture therefore describe different
processes. Buffering also requires separate consideration: β-TCP moderated PLGA degradation-associated
acidification, introducing pH control without directly specifying a change in lactate mass. [46]
5.1.2. LDH-like catalysis and reaction direction
LDH-mimetic surfaces are designed to generate lactate through interfacial catalysis, whereas lactate-bearing
scaffolds supply a stored substrate. [22, 44] V-N₄ or Rh-N₄ single-atom coatings on titanium alloy or
stainless steel were tested in a pyruvate/NADH system by following the decline in NADH absorbance at 340
nm. The authors assigned the reaction to pyruvate reduction toward lactate and also reported NOX-like
NADH oxidation in the catalyst platform. [22] NADH turnover is the direct measurement in this assay;
lactate and pyruvate quantification is a next step for resolving product formation in the presence of the
competing cofactor-consuming reaction.
The coating format combines catalytic chemistry with fracture fixation. In rabbit tibial fractures, Ti-VN₄
plates improved load-bearing capacity and stiffness at three and five weeks relative to unmodified controls,
while bone area showed no significant difference at week 3. The authors interpreted repair through combined
enzyme-mimetic activity and Piezo1/Ca²⁺/Nrf2 antioxidant signaling. [22] The difference between early
bone-area and mechanical outcomes suggests that interfacial function should be evaluated through tissue
maturation as well as mineralized area. Formulation refinement can use coatings that separate catalytic and
mechanical contributions to identify which functions to retain.
5.2. Transport, utilization and extracellular removal
5.2.1. MCT inhibition and control of bone resorption
Bone-targeted delivery can direct lactate-transport inhibition toward excessive resorption. PH/DPA@A
nanoparticles combine a ROS-responsive hyaluronic-acid derivative, an Asp8-bearing component and
AZD3965. In the accompanying pharmacology experiments, free AZD3965 lowered extracellular lactate and
increased intracellular lactate after five days of RANKL stimulation in bone-marrow macrophage cultures.
[21] The paired response supports impaired export in RANKL-differentiated osteoclast cultures. Measuring
both compartments gives the extracellular decrease its mechanistic context and separates transport blockade
from extracellular enzymatic removal.
10
After six weeks in ovariectomized mice, PH/DPA@A improved bone volume fraction, trabecular number
and trabecular separation relative to free drug. Osteoclast surface, osteoclast number and MMP9-positive
area declined, whereas osteoblast number and RUNX2 positivity showed no significant increase over
ovariectomized controls. [21] The outcome pattern supports an antiresorptive explanation for bone
preservation. Its design implication is cell specific: restricting lactate export in resorbing cells addresses a
different biological task from supplying or processing lactate for osteogenic cells within a defect.
5.2.2. LOx-mediated conversion and respiratory support
The MPL hybrid immobilizes native lactate oxidase (LOx) on a MoSe₂/PtCu platform that also exhibits
GOx-like and CAT-like activities. MPL retained lactate-oxidizing activity in cell-free assays. [31] LOx
converts lactate to pyruvate and hydrogen peroxide, coupling substrate removal to changes in local redox
chemistry. In BMSCs exposed to 30 mM glucose and 5 mM lactate, the composite restored basal and
maximal respiration, spare respiratory capacity and ATP-linked oxygen consumption. The authors proposed
that LOx-derived pyruvate contributes to TCA-cycle metabolism, based on metabolite pools and respiratory
measurements. [31] Lactate-derived carbon tracing offers a direct test of that proposed connection.
Active MPL produced stronger ATP and mineralization responses than formulations containing inactivated
LOx, relating retained enzyme activity to the cellular response. In diabetic rats with infected calvarial
defects, local MPL-containing gel combined with early near-infrared irradiation improved micro-CT repair
outcomes at four, eight and 12 weeks. Tissue coexpression of SIRT3, GPX1 and MFN2 was consistent with
the authors’ mitochondrial interpretation. [31] This formulation combines extracellular substrate conversion
with respiratory support in defect repair, complementing the transport-inhibition strategy used for
antiresorption. Optimizing this formulation requires joint consideration of the catalytic reaction, redox
products and recipient-cell metabolism.
5.3. Lactate signaling and lactylation
5.3.1. Receptor-associated and endothelial responses
Lactate-releasing materials can influence bone-forming cells directly or support them through the vascular
environment. A PCL/regenerated-cellulose membrane bearing calcium lactate (CaL), with optional SDF1
functionalization, illustrates the first route. In CaL-exposed human BMSCs, OR5AN1 knockdown reduced
intracellular calcium fluorescence and RUNX2, BMP2 and OCN expression. Both CaL-containing
membranes improved new bone formation in rat femoral defects relative to the CaL-free membrane, while
mean BV/TV remained comparable with and without SDF1. [44] These results support OR5AN1
participation in the cellular response and allow receptor-associated signaling and added recruitment functions
to be evaluated separately.
A reticular PLGA coating on porous titanium supported an endothelial route under diabetic conditions. The
coating produced no significant improvement in proliferation or ALP in rat osteoblast monoculture, but both
increased after non-contact HUVEC coculture. Lactic acid at 112.3 µM reproduced endothelial protection,
whereas glycolic acid at 166.3 µM had little effect. The coating and lactic acid lowered CML, pentosidine
and RAGE; aminoguanidine reproduced part of the protection, and PLGA attenuated AGE-induced injury.
Dose influenced the response: 100 and 500 µM lactic acid increased endothelial proliferation, while 2500
µM mildly inhibited it. [43] These findings favor a degradation profile that maintains endothelial function
and is evaluated through the resulting support for osteogenesis.
5.3.2. Histone lactylation and osteoclast regulation
Magnesium-containing intramedullary nails connect an ionic material cue with osteoclast-associated
lactylation. In rat femoral fractures, Mg-IMNs increased four-week BV/TV and reduced trabecular separation
relative to Ti-IMNs. In RANKL-induced RAW264.7 cultures, magnesium reduced NFATc1 expression and
osteoclast maturation while increasing lysine lactylation and H3K18la. The implanted Mg group also showed
11
greater lactylation in ATP6V0D2-associated regions. [47] The concurrent reduction in multinucleated TRAPpositive cells places this response within regulation of the resorptive component of fracture healing.
Separate pharmacological experiments examined lactate and oxamate. Lactate suppressed osteoclast
maturation and increased H3K18la enrichment at the Nfatc1 promoter in CUT&RUN-qPCR, whereas
oxamate produced opposite responses. The animal pharmacology used Ti-IMNs throughout. [47] Material,
lactate-response and chromatin experiments therefore converge on an osteoclast-associated pathway while
retaining distinct intervention settings. H3K18-specific perturbation and Nfatc1 rescue would connect
promoter enrichment with differentiation more directly. Implant release kinetics can then be evaluated
against osteoclast activity and fracture progression to identify when this response is most useful during
remodeling.
5.3.3. STAT1 lactylation and osteogenic responses
Non-histone lactylation introduces protein localization and function as material-responsive variables. In
human BMSCs cultured on a printed PCL/nHA scaffold containing 1% w/w sodium lactate, anti-K-Lacenriched proteomics and MS/MS identified STAT1-K193 as a lactylation site. Increased STAT1 lactylation
accompanied cytoplasmic accumulation. Wild-type and K584R STAT1 were predominantly cytoplasmic,
whereas K193R remained nuclear. After endogenous STAT1 knockdown and 14 days of osteogenic
induction, wild-type reconstitution retained ALP and mineralization responses, while K193R reduced these
endpoints and COL1A1, OCN and OPN expression. [45] The reconstitution experiment connects K193
function with the osteogenic response, although lysine substitution can affect properties beyond lactylation.
The sodium-lactate scaffold increased 12-week BV/TV and trabecular indices in 5-mm rat calvarial defects
relative to the unmodified scaffold. [45] To connect this material outcome with the cell-based mechanism,
K193 dependence needs to be tested in vivo. A related strategy uses HLP2 liposome-hydrogel delivery of
DR8, a peptide designed to mimic non-lactylated ENTR1. Its proposed GLUT1 connection rests on docking
and colocalization, and the authors identified downstream glycolytic reprogramming as future mechanistic
work. [48] DR8 mimics a protein modification state, whereas sodium lactate aims to promote endogenous
modification. Both approaches make the identity and function of the affected protein central to formulation
design. Comparing these materials requires the target cell to be specified because osteoclasts and BMSCs
responded differently (Table 2).
6. Immune-cell metabolic reprogramming
6.1. Glucose-metabolic nodes in immune cells
6.1.1. Transporters and glycolytic enzymes
Figure 4 organizes the experimental routes connecting immune-cell metabolism with communication and
bone repair. Silver nanoparticle-loaded TiO₂ nanotubes illustrate a surface-based route. After 24 h,
RAW264.7 macrophages on these surfaces expressed less GLUT1 than cells on titanium or unloaded
nanotubes. STF31 further reduced GLUT1 and iNOS while increasing Arg1; PI3K activation with 740Y-P
restored p-Akt and GLUT1 expression and reduced LC3. Conditioned medium from the silver-bearing
surfaces increased MC3T3-E1 osteogenic gene expression, day-7 ALP staining and day-21 mineralization
relative to medium from the two silver-free surfaces. [49] These experiments connect a PI3K/Akt-associated
transporter response with an osteogenesis-supporting conditioned environment, which contains macrophage
products and any material carried through the transfer protocol.
Other carriers act at enzyme activity or coordinated transcription. DMF-loaded liposomes in chondroitinsulfate/gelatin-methacrylate microspheres reduced macrophage GAPDH activity. [39] Calcium-citratefunctionalized calcium-phosphate scaffolds lowered PFK activity in LPS-stimulated RAW264.7 cells after
48 h at the two higher loadings, while the lower-loading groups showed no significant reduction. [14] A
Ce/Zn-doped hydroxyapatite-gelatin microsphere/short-fiber scaffold, FM@CeZnHA, lowered HK2, GLUT
12
and PKM2 transcripts in LPS-stimulated macrophages. [50] These carriers can be compared at their
measured regulatory levels: transporter abundance, enzyme activity and transcript profiles describe distinct
steps between material exposure and immune function.
6.1.2. Functional metabolism across activation states
Several materials increased respiratory activity while lowering glycolytic readouts in inflammatory
macrophage models. FM@CeZnHA reduced ECAR and increased basal and maximal respiration in LPSstimulated macrophages; several respiratory comparisons with unstimulated controls were nonsignificant.
[50] Methacrylated-alginate hydrogels containing dexamethasone-loaded hollow MnO₂, with or without
chitosan coating, similarly reduced ECAR-derived glycolytic parameters and increased basal, maximal and
ATP-linked respiration in LPS-treated RAW264.7 cells. [13] SDSSD-functionalized, zinc-sulfide-loaded
zeolitic imidazolate framework nanoparticles produced the same broad response in LPS/IFN-γ-stimulated
bone-marrow macrophages, including increased spare respiratory capacity. [51] Paired acidification and
respiration measurements therefore offer functional criteria for comparing immune-directed formulations.
Glucose demand varies with the initial activation state. In RAW264.7 cells without LPS stimulation,
glucomannan-modified nano-hydroxyapatite (GHANPs) increased 24-h glucose utilization, lowered
supernatant lactate and increased OCR relative to unmodified particles and medium-only controls. Utilization
was estimated from medium measurements normalized to cell number. [19] The pattern is compatible with
increased respiration alongside sustained glucose demand; substrate tracing would determine how much of
that respiration is fueled by glucose. By contrast, the highest calcium-citrate loading reduced both ECAR and
OCR after 48 h in IL-4-treated RAW264.7 cells. [14] Material exposure should therefore be selected against
the activation context, with respiratory function, nutrient demand and inflammatory activity evaluated
together. [50]
6.2. Connecting immune responses with bone repair
6.2.1. Conditioned media and osteogenic recipients
Macrophage-conditioned media can convey the effects of different material platforms to osteogenic cells.
Ce/Zn-containing hydroxyapatite in short-fiber microsphere scaffolds and MnO₂-dexamethasone nanogels
generated conditioned media that increased ALP and mineralization in rat BMSCs. [13, 50] Within the
nanogel series, Alg-MD outperformed its chitosan-coated counterpart in several osteogenic readouts. [13]
These experiments used RAW264.7 donors, whereas the bone-targeted ZIF-H₂S-SDSSD system conditioned
bone-marrow macrophages under LPS/IFN-γ stimulation. Its medium increased BMSC ALP at seven days
and calcium deposition at 14 days. [51] Across these delivery formats, recipient-cell differentiation provides
a functional link between material exposure during macrophage conditioning and osteogenesis.
The recipient lineage can change the outcome. ZIF-H₂S-SDSSD-conditioned medium produced no
significant reduction in osteoclast F-actin rings or TRAP staining, despite improving BMSC differentiation.
Low-speed centrifugation preceded mixing with induction medium, leaving residual particles and soluble
material components as possible contributors. [51] Cleared-medium experiments, measured carryover
controls and cytokine neutralization provide a test strategy for distinguishing altered macrophage output from
direct effects on the recipient. This distinction is particularly relevant when the intended treatment must
promote bone formation and regulate resorption simultaneously.
6.2.2. Direct cellular effects and in vivo repair
Direct material exposure adds a parallel contribution. ZIF-H₂S and ZIF-H₂S-SDSSD increased BMSC ALP,
OCN and calcium deposition, while directly treated osteoclast cultures showed reduced F-actin rings and
TRAP-positive area. In ovariectomized mice with femoral fractures, the targeted formulation increased callus
indices at days 7 and 14, reduced the index at day 28, and increased callus BV/TV at all three assessments.
[51] The temporal pattern is consistent with earlier callus maturation. Component comparisons involving
13
zinc and sulfide could clarify how the direct and conditioned-medium responses contribute to that
progression.
Scaffold studies extend the comparison to defect repair. GHANPs in GelMA increased bone volume and
bone surface in bilateral rat calvarial defects at eight weeks, with accompanying structural and histological
improvements. [19] FM@CeZnHA improved selected bone indices in noncritical rat femoral-condylar
defects at two and four weeks. [50] Anatomical site, intrinsic healing capacity and follow-up differ between
these models. Together, the cellular and tissue findings support treating immune conditioning, direct
osteogenesis and resorption control as adjustable contributions, with cell-specific perturbations at defined
repair stages providing a way to test their relative importance.
6.3. Cell-specific and staged intervention
6.3.1. Different metabolic directions in neighboring cells
PN@MHV combines a MYT-containing HA-PBA-PVA hydrogel with folate-modified, PX478-loaded
liposomes to support osteogenic metabolism while restraining inflammatory glycolysis. In the BMSC
experiments, free MYT restored glucose consumption and lactate production during osteogenic induction
under 40 mM glucose, with responses followed at days 3, 7 and 14. Separately, PN@MHV reduced both
measurements in high-glucose RAW264.7 cultures after 48 h. The authors interpreted these opposing
responses through cell-dependent regulation of HIF-1α and glycolysis. [5] The comparison makes the
intended metabolic direction, delivery component and exposure duration specific to each cell population.
The formulation rankings also differed between cell culture and fracture repair. MHV alone produced more
mineralized area than PN@MHV in BMSC cultures at day 21, whereas PN@MHV achieved higher BV/TV
in diabetic mouse femoral fractures at days 14 and 21. [5] This reversal supports evaluating a composite
across the multicellular repair environment, with macrophage regulation remaining a proposed explanation
for its tissue-level advantage. A staged test can measure uptake and metabolic responses separately in
BMSCs and macrophages, then use cell-resolved glucose tracing within the defect to test whether the
opposing responses coexist during improved healing.
6.3.2. Timing, release and dose
The timing of lactate exposure can favor an early immune and vascular response. Lactate-loaded collagen
sponges increased day-1 CD86-positive cells and vascular indices from days 3 to 14 in mouse calvarial
defects; Nodinitib-1 attenuated these responses. Starting treatment on day 3 or 7 produced no significant
angiogenic improvement at day 14 and also shortened exposure. In the separate regeneration arm, lactate
delivered with Bio-Oss Collagen increased BV/TV and BMD at eight and 12 weeks. [12] Matching carrier
and cumulative exposure would clarify how the early vascular response contributes to later bone formation.
Release kinetics and dose provide further control over treatment timing. Chitosan coating delayed
dexamethasone release from MnO₂-based AlgMA nanogels. In directly exposed, H₂O₂-challenged BMSCs,
Alg-MD favored early ALP/RUNX2 responses, whereas Alg-MD@CS performed better for later OPN
expression and mineralization. The macrophage assays used LPS stimulation, while the femoral-condyle
repair model had no infection challenge. [13] Dose effects were also evident in the CaCit system, where 15%
loading suppressed IL-4-induced CD206/Arg1, and in DMOG-loaded clots, where four-week new-bone
volume did not differ significantly between 0.2 and 0.4 mM groups. [14, 36] These comparisons favor
selecting local dose and release duration against phase-specific immune and osteogenic outcomes. Table 2
places the immune-directed systems within the broader comparison of metabolic intervention sites.
14
7. Synthesis and discussion
7.1. Comparing strategies and explaining divergent findings
7.1.1. Starting conditions and intervention location
The choice between supplying and consuming glucose depends on the local constraint and the cell population
intended to benefit. Glucose sustained hMSC survival in near-anoxic minimal medium, while GOx/catalase
scaffolds consumed glucose in a glucose-rich, cell-free system. [3, 4] The catalytic system also consumed
oxygen overall, and GOx-containing bone cement reduced BMSC CCK-8 metabolic activity at the lowest
tested enzyme loading in low-glucose culture. [4, 11] Reaction rate must therefore be considered against
substrate availability, oxygen supply and the requirements of repair cells.
Lactate strategies illustrate the importance of intervention location. Calcium-lactate-coated scaffolds release
substrate into surrounding fluid, whereas immobilized LOx on MoSe₂/PtCu showed lactate-oxidizing activity
in cell-free spectral assays. [31, 44] Free AZD3965, investigated as the payload of a bone-targeted carrier,
lowered extracellular lactate while increasing the intracellular pool in osteoclast cultures. [21] The same
extracellular measurement thus has different implications for release, oxidation and transport inhibition.
Compartment-resolved measurements therefore guide selection of the intended material function. In
multicomponent systems, cell-resolved delivery and metabolism would further establish whether the
proposed interventions occur together within the repair site. [5]
7.1.2. Context-dependent responses and incremental benefit
Divergent respiratory responses can reflect different biological tasks. Laponite-primed apoptotic vesicles
increased BMSC OCR, although maximal respiration did not differ significantly from unprimed vesicles.
Bio-SIG lowered OCR in MC3T3-E1 cells after prolonged electrical stimulation. [8, 41] Likewise, the
highest tested lactic-acid dose lost the proliferative benefit seen at lower doses in HUVECs. [43] Whether a
metabolic change is favorable depends on cell state, exposure duration and the functional outcome.
Matched comparators identify the benefit gained from additional formulation complexity. At four weeks,
laponite-primed-vesicle hydrogels did not significantly exceed unprimed-vesicle hydrogels in BV/TV; SDF1
addition to calcium-lactate scaffolds produced comparable mean BV/TV in rat femoral defects. [8, 44] For a
metformin-loaded PEG-peptide/clay hydrogel, the eight-week advantage over unloaded carrier appeared in
BMD and trabecular measures, while BV/TV remained nonsignificant. [23] These endpoint-specific findings
help identify which material functions contribute to measurable repair improvements. Nonsignificant
comparisons leave the incremental benefit unresolved and should be retained when selecting payload
combinations.
7.2. Mechanistic validation and measurement quality
7.2.1. Substrate fate and causal intervention
Substrate tracing can connect a controllable material function with the metabolic process it is intended to
change. For MPL, time-resolved tracing of labeled lactate into extracellular pyruvate and intracellular TCA
intermediates would test the proposed connection between LOx activity and respiratory recovery. [31]
Active-enzyme, inactivated-enzyme and cell-free groups should share substrate concentrations, oxygen
conditions and sampling times. Dynamic HA hydrogels require a complementary approach: tracer uptake
combined with GLUT1 perturbation and re-expression across matched networks. [30] Following glucosederived carbon downstream, together with viability measurements, would relate transporter regulation to
metabolic function while separating metabolic dependence from general cellular injury. Table 3 links these
questions to material variables and suitable validation experiments.
Mediator testing is most informative when the material background is held constant. For Mg intramedullary
nails, a factorial comparison of implant composition and lactate-pathway intervention would extend the
15
pharmacology performed with titanium nails. [47] In DMOG-loaded clots, iron-chelation and PFKrestoration experiments, accompanied by erythrophagocytosis measurements, would distinguish the proposed
iron/PFK mechanism from changes in erythrocyte clearance. [36] STAT1 studies could extend wildtype/K193R complementation through matched expression, site-resolved lactylation analysis and lineagedefined implantation experiments. [45] Demonstrating that a mediator changes both the intended cellular
response and the corresponding bone outcome within the same treatment would provide a stronger basis for
choosing dose, delivery location and exposure window.
7.2.2. Assay recovery and reporting
Composition-specific interference can alter the interpretation of metabolic assays. Adding Ca-containing
mesoporous bioactive glass only during SaOS-2 lysis reduced LDH readings, whereas Mg-containing glass
produced no significant LDH change. Material-conditioned lysis buffer also produced an upward ALP trend
with Mg-containing glass. [18] These results make extraction efficiency and assay chemistry part of material
evaluation. Comparing particles, particle-free eluates and material-free controls, with enzyme standards
introduced before extraction and after separation, would locate signal loss or enhancement. Calibrated DNA
measurements or imaging-based cell counts can provide an independent reference.
Sampling and replication should match the level of the proposed mechanism. Repeated cell-number estimates
and validated assay recovery across titanium-lattice geometries would improve normalization of medium
glucose changes. [20] Donor identity should also be retained through culture and analysis; the
healthy/diabetic Charcot osteoblast study pooled three donors per group before testing. [9] Reports should
distinguish donor, construct and animal replication and specify medium exchange, exposure duration and
normalization denominators to support comparison across studies. Accessible calibration curves,
supplementary data and correction histories would further support reuse of the evidence. Table 1 summarizes
the relationship between each measurement and its interpretation.
7.3. Material design and translation
7.3.1. Combined treatment and functional recovery
Combined functions are most useful when they address a defined obstacle to repair. Under near-infrared
irradiation, Cu/PDA/GOx-coated PEEK showed antibacterial activity in vitro and improved peri-implant
bone formation in infected diabetic rat femoral-condyle models, accompanied by lower inflammatory
markers. [32] Other systems addressed peroxide accumulation or vascular support: catalase limited GOxgenerated H₂O₂ in enzyme-functionalized scaffolds, while TiO₂/Bi₂O₃ heterojunctions supported more
perfused vascular structures around rat femoral implants. [4, 10] In these systems, antimicrobial action,
peroxide control and vascular support address different repair constraints. Matched component groups and
interaction analysis provide a test of quantitative synergy, including in multifunctional Zn/H₂S-delivering
systems. [51]
Translation also requires endpoints that reflect the expected function of regenerated bone. Se-MBG increased
bone volume fraction and trabecular thickness in rat calvarial defects, with histological evidence of new bone
formation, but the study did not assess mechanics. [52] Ti-VN₄ plate-screw fixation improved maximum load
and stiffness at three and five weeks in rabbit tibiae. [22] Following repair from initial mineralized filling
through remodeling would relate early structural gains to sustained function. For load-bearing applications,
local metabolic exposure should be assessed alongside tissue maturation, mechanical recovery and
degradation or coating integrity throughout the intended healing period. Manufacturing evaluation should use
the same functional criteria to establish reproducible loading, retained catalytic activity after processing and
consistent release between batches.
16
7.3.2. Design principles
Glucose acquisition, enzymatic regulation, lactate handling and immune-cell metabolic reprogramming
organize bone biomaterials according to the processes they are intended to influence. Across these strategies,
the central design task is to match intervention location, direction and duration to the target cells and stage of
repair. Scaffold transport and mechanics, catalytic surfaces and delivered agents can then be compared
through their effects on a defined metabolic node and a relevant cellular function.
A material-mechanism-outcome comparison is strongest when local exposure, metabolic response and bone
repair are examined within the same experimental setting. Bone formation, reduced resorption, vascular
support and mechanical recovery contribute different information to that comparison. Targeted perturbations
can identify which metabolic changes support the desired outcome and guide subsequent formulation
choices. Metabolic measurements can guide material selection according to the biological requirements of
bone regeneration.
Declarations
Acknowledgments and funding
____________________________________________________________
Conflict of interest
____________________________________________________________
Data availability
____________________________________________________________
Author contributions
____________________________________________________________
Declaration of generative AI use
____________________________________________________________
17
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21
Figure 1
22
Figure 1. Four glucose-metabolic intervention sites in bone biomaterials. (A) Cellular context defines the metabolic
task. Glucose supplementation supports human MSC survival in near-anoxic, glucose-limited 3D fibrin. Under high
glucose, free myricitrin and PN@MHV show different glucose-use responses in BMSCs and RAW264.7 cells,
respectively. (B) Glucose acquisition encompasses pore-dependent diffusion, cellular uptake-related responses and
extracellular consumption. The examples comprise cell-free scaffold transport, pharmacologically sensitive medium
glucose depletion with Cur@MS, and GOx/CAT-containing scaffolds. (C) Enzyme-level interventions include
increased digitonin-releasable ALDOA on a dynamic ligand interface, reduced PFK/PK/PDH activities in CaCitexposed LPS-stimulated RAW264.7 cells, and parallel ECAR/OCR increases with free L@Apo in BMSCs. PDH links
pyruvate to mitochondrial oxidation. (D) Lactate handling encompasses lactate availability in SL-scaffold cultures,
altered intracellular/extracellular lactate pools with free AZD3965, a cell-free spectral lactate-oxidation readout with an
LOx-bearing material, and OR5AN1-associated calcium responses under CaL exposure. These assays describe different
compartments and measurement types. (E) Alg-MD/Alg-MD@CS connect macrophage respiratory responses with
conditioned-medium effects on recipient BMSCs and a separate femoral-defect outcome. The 3D-NTBH example
represents a parallel endothelial branch, with HUVEC metabolic and vascular responses alongside peri-implant bone
formation. Solid arrows indicate the stated biochemical step, measured comparison or experimental transfer; dashed
arrows identify a proposed connection between evidence levels. Up/down arrows indicate within-study directions. The
diagram synthesizes separate experiments; molecular specificity and in vivo mediation follow the evidence available
for each branch.
Sources: [3–5, 8, 10, 13, 14, 16, 21, 25, 31, 38, 44, 45].
23
Figure 2
24
Figure 2. Material interventions at different levels of glucose acquisition. (A) Cell-free PCL experiments measure
scaffold diffusion, with lower diffusivity in culture medium than in water; a separate radial-flow model predicts
pericellular gradients. (B) Cur@MS increases medium glucose depletion in BMSCs, and BAY-876 or MβCD removes
the between-group difference. Qu–Cr coating experiments assess GLUT4 localization and intracellular glucose pools.
(C) GOx consumes glucose and oxygen while producing peroxide. CAT reduces peroxide accumulation, with a net
decline in dissolved oxygen in the tested combined system. Diabetic calvarial repair is a separate outcome of the
Alg/GOx/CaP@CAT scaffold. (D) Cell identity, basal medium and formulation jointly define GOx compatibility; MgMOF pairing improves the tested cellular readouts. (E) Free myricitrin increases glucose use in high-glucose BMSCs,
whereas PN@MHV reduces it in high-glucose RAW264.7 cells. PN@MHV also improves BV/TV in the separate
diabetic-fracture experiment. Arrows organize observations within their experimental conditions, and ↑/↓ indicate
direction rather than effect size. Cellular glucose pools, medium depletion, scaffold diffusion and animal repair retain
their distinct measurement meanings.
Sources: [4, 5, 11, 15, 16, 25, 26, 53].
25
Figure 3
26
Figure 3. Lactate-directed biomaterials organized by source, reaction and signal. (A) PLGA degradation, CaL-coating
release and lactate availability in SL-scaffold cultures represent different source and compartment measurements. (B)
Single-atom implant assays follow NADH absorbance in the presence of pyruvate, testing the direction assigned to
pyruvate reduction; direct product analysis remains relevant because NOX-like activity also consumes NADH. Fracture
outcomes are presented separately. (C) Free AZD3965 changes lactate pools in RANKL-induced BMMs; PH/DPA@A
delivery preserves bone in OVX mice, with osteoblast numbers remaining nonsignificant. (D) MPL combines cell-free
lactate-related spectral evidence with improved BMSC respiratory readouts. Its gel formulation with NIR is assessed in
infected diabetic defects. Lactate-carbon tracing would resolve the proposed connection to TCA-cycle utilization. (E)
OR5AN1 knockdown reduces CaL-associated calcium fluorescence and selected osteogenic transcripts in human
BMMSCs; this supports a receptor-associated cellular response alongside the scaffold's femoral-repair observations. (F)
Mg increases H3K18la in osteoclast-lineage cells. Independent lactate/oxamate comparisons alter osteoclast responses
and Nfatc1-promoter H3K18la enrichment; the animal drug comparisons use Ti implants. (G) STAT1-K193
identification and WT/K193R add-back provide non-histone, site-associated functional evidence in vitro. Calvarial
repair is evaluated in a separate scaffold experiment. Orange text specifies interpretive scope. Horizontal dashed rules
separate evidence summaries from qualifications, and ↑/↓ denote the reported directions in the stated comparisons.
Sources: [21, 22, 31, 43–45, 47].
27
Figure 4
28
Figure 4. Connecting immune-cell metabolism with bone repair. (A) Independent materials act at GLUT1 expression,
total PFK activity or GAPDH activity. The DMF-system metabolic direction follows the plotted results. (B) ZIF–H₂S–
SDSSD lowers glycolytic ECAR and increases OCR parameters in activated BMMs. GHANPs increase the glucose-use
proxy and OCR while lowering extracellular lactate in RAW264.7 cells. (C) Conditioned medium from treated
macrophages enhances recipient BMSC osteogenesis. This transfer assay measures the activity of the complete
conditioned milieu. (D) Direct BMSC mitochondrial/osteogenic effects and direct osteoclast inhibition constitute
additional treatment branches. The source text reports a nonsignificant conditioned-medium anti-osteoclast comparison.
(E) OVX-fracture, femoral-defect and diabetic-fracture studies assess repair in their respective disease and material
contexts. (F) Early lactate/collagen-sponge treatment increases CD86-associated and vascular responses, and NOD1
inhibition attenuates the vascular response. Delayed treatment gives no significant vascular benefit at the shared
endpoint. Later bone improvement is tested using lactate with Bio-Oss Collagen, a different carrier, while start time and
exposure duration covary. ↑/↓ denote within-study directions; CM arrows denote experimental transfer. Dashed rules
separate interpretive qualifications. The branches organize cell-level, transfer and animal evidence for evaluating how
immune metabolic responses contribute to repair.
Sources: [5, 12–14, 19, 39, 49, 51].
29
Table 1
Metabolic and bone-repair readouts and their interpretation. Examples cover therapeutic biomaterials, pathological material exposure, mechanism studies and transport models. The validation
approaches are proposed by the review authors.
Sources: [8, 13, 15, 16, 18, 19, 21, 22, 25, 27, 28, 39–41, 45, 47–49, 51, 54, 55].
Readout
What it measures
Interpretive scope
Complementary validation
References
GLUT expression /
colocalization
Protein abundance, localization or spatial
proximity.
Uptake flux, direct molecular binding
or in vivo necessity.
Pair functional uptake with binding assays and
isoform-specific perturbation/rescue; verify cell
identity and imaging resolution.
[15, 48, 49]
Medium glucose depletion /
net use
Net change within a specified culture
interval and normalization scheme.
A GLUT-isoform rate or the fate of all Control cell number, viability and acellular
consumed carbon.
adsorption; add uptake kinetics, isotope tracing and
mass balance.
ECAR / glycoPER
Acidification or calibrated glycolysisrelated function under the stated injection
protocol.
Uncorrected absolute carbon flux or
MCT-specific activity.
OCR / ATP-linked OCR
Respiration and pharmacologically derived
ATP-associated oxygen consumption.
Exclusive glucose fueling or a directly Resolve mitochondrial/non-mitochondrial oxygen use; [8, 51]
measured ATP-production rate.
add substrate tracing and an independent ATPproduction assay.
ATP / lactate / succinate
pools
Amount or concentration at the sampled
endpoint.
Production rate, carbon flux or a
uniquely determined metabolic
direction.
[16, 19]
Document injection order, non-glycolytic acidification [41, 51, 55]
and normalization; corroborate with lactate and tracer
measurements.
Match sampling time and units; normalize to viable
cells/protein and measure turnover or isotope-labeled
flux.
[28, 51, 54]
30
Table 1 (continued, part 2/3)
Readout
What it measures
Interpretive scope
Complementary validation
Paired intra-/extracellular
lactate
A distribution consistent with perturbed
transport or metabolism.
Exclusive MCT1 action, extracellular Measure volumes, pH and viability; combine transport [21]
enzymatic clearance or causal order in kinetics with isoform-specific rescue and lactate addvivo.
back.
Lysate LDH / ALP activity
An enzyme-assay response under the tested
lysis and material conditions.
LDHA regulation, altered lactate
production or improved osteogenesis
by itself.
Enzyme abundance / activity
/ modification
A change at the specifically measured
regulatory layer.
Interchangeability of these layers or a Measure abundance, PTM and activity separately with [39, 40]
proven change in whole-pathway flux. stated substrate and normalization; add selective
rescue and tracing.
Lactylation enrichment / site
variants
Modification association or site-related
functional dependence.
Equivalence of histone and nonhistone effects, or absolute lactylation
specificity of K→R.
Validate antibodies and sites; use endogenous editing
and rescue, control other lysine modifications, and
retain conflicting source findings.
[45, 47]
Conditioned-medium
recipient osteogenesis
Osteogenic activity in the post-treatment
environment.
Exclusive mediation by macrophagesecreted factors or macrophage
metabolism.
Quantify residual drug/particles; compare direct
exposure, depletion/separation and factor
neutralization.
[13, 51]
Use material blanks, lysis-stage spiking, particle
removal/recovery and orthogonal assays; distinguish
trends from significant differences.
References
[18]
31
Table 1 (continued, part 3/3)
Readout
What it measures
Interpretive scope
Complementary validation
References
Micro-CT /
histomorphometry
Structural differences in the stated model,
endpoint and comparator.
Direct osteogenesis, mechanical
recovery or superiority at every time
and parameter.
Separate bone volume from area, resorption from
defect repair, and significant from null comparisons;
test mechanics independently.
[8, 21, 22]
GLUT modulation, independent
external validation or bone
regeneration in vivo.
Use independent validation data; include oxygen and
[25, 27]
cellular consumption, resolve parameter/unit conflicts,
and test biological endpoints.
Physical diffusion / numerical Transport or predictions under explicit
modeling
geometric and biological boundary
conditions.
32
Table 2
Representative bone biomaterials: metabolic nodes, cellular evidence and bone outcomes. Each entry pairs within-study metabolic observations with the tested cells, interventions and bone
models; null comparisons and the scope of mechanistic interpretation are stated alongside.
Sources: [4, 5, 8, 13–16, 19, 21, 22, 30, 31, 38, 39, 41, 43–45, 47, 51].
Material
Node and cells
Metabolic evidence
Bone model and outcome
Interpretive scope
References
Glucose acquisition
GLUT1-associated glucose
Curcumin-loaded gelatin handling; BMSCs
microspheres in a 3Dprinted
PCL/hydroxyapatite/βTCP scaffold
Medium glucose depletion increased;
BAY876 or MβCD reduced the
response.
Rat calvarial defect, 28 d: improved
bone parameters versus scaffold
controls.
Net consumption is not isoform-specific
uptake. Pharmacology and early staining do
not establish metabolic necessity for late
repair.
[16]
Glucose acquisition
CaCO3–quercetin–Cr
nanoparticles in a ROSresponsive
gelatin/carboxymethylchitosan Ti coating
GLUT4 localization and
glucose pool; MSCs
Qu–Cr treatment increased intracellular OVX rat femoral Ti implant, 30 d:
glucose and altered GLUT4 localization improved peri-implant bone.
under oxidative stress.
A glucose pool is not uptake flux.
Component effects and GLUT4 necessity
remain unresolved.
[15]
Glucose acquisition
RGD-functionalized
hyaluronan host–guest
hydrogels with dynamic
HA-ADA/HA-CA
networks
GLUT1 expression and
AMPK-associated energy
response; hMSCs
Dynamic networks increased GLUT1
Rat calvarial defect with rMSC-loaded
expression and glucose pools; AMPKα1 hydrogels, 8 wk: improved bone
knockdown attenuated an ATP-related
formation.
staining response.
hMSC assays and rMSC implants are
[30]
distinct. AMPK perturbation does not prove
GLUT1-specific uptake or in vivo
mediation.
Glucose acquisition
3D-printed
alginate/GOx/CATassisted calciumphosphate nanosheet
scaffold
Extracellular glucose
oxidation and peroxide
handling; acellular assays
Glucose and H2O2 decreased; the
complete catalytic system still
consumed oxygen overall.
T2D rat calvarial defect, 8 wk: improved External catalysis is not host GLUT/HK
[4]
repair.
regulation; CAT coupling does not establish
net oxygen supply.
Enzyme-linked
regulation
Aniline-catalyzed RGD–
AuNP interface tethered
to glass through PEGhydrazide/benzaldehyde
hydrazone bonds
Digitonin-releasable
ALDOA and Rac1associated response;
hMSCs
More ALDOA was released by
digitonin; Rac1 knockdown attenuated
associated metabolic transcripts.
In vitro osteogenic-marker/ALP
response; no animal bone-repair
endpoint.
Released enzyme fraction is not catalytic
activity or carbon flux. CAT denotes the
catalyst condition here, not catalase.
[38]
33
Table 2 (continued, part 2/4)
Material
Node and cells
Enzyme-linked
HK2/PFK1/PDK1
regulation
expression and glycolytic
Self-powered Bio-SIG:
function; MC3T3-E1 cells
PLLA/PCL nanofiber–
PDMS generator
coupled to a gold-coated
PEEK fixation plate
Metabolic evidence
Bone model and outcome
Interpretive scope
References
Glucose uptake and glycoPER
increased, OCR decreased; 2-DG
attenuated osteogenic readouts.
Rat femoral defect: improved later bone
formation; BV/TV at 1 month was not
significantly different.
Gene expression is not enzyme activity.
Broad 2-DG inhibition does not identify a
unique enzyme or establish in vivo
necessity.
[41]
PDH is not a glycolytic enzyme. No PFK
selectivity; the 10% versus 15% difference
is not established as significant, and
supporting SI is unavailable.
[14]
Enzyme-linked
regulation
Calcium-phosphate
cement with a
PCL/calcium-citrate
coating
PFK, PK and PDH activity; Multiple enzyme activities decreased
LPS- or IL-4-conditioned
under LPS; high citrate loading also
RAW264.7 cells
suppressed IL-4-associated
CD206/Arg1.
OVX rat femoral-condyle defect, 8 wk:
10% loading had the highest reported
bone-formation proportion.
Enzyme-linked
regulation
Laponite-primed BMSC
apoptotic vesicles
(L@Apo) delivered in a
GelMA/PEG hydrogel
PFKM expression and
bioenergetic readouts;
BMSCs
PFKM immunofluorescence, ECAR
and OCR increased; 2-DG attenuated
osteogenic responses.
Rat femoral-condyle defect: 4- and 8No PFKM-specific rescue. Assay
[8]
week assessment; 4-week BV/TV versus timing/injection labels and PEG end-group
Apo hydrogel was not significant.
descriptions conflict; no uniform superiority
across endpoints.
Lactate handling
Lactate release and
Electrospun
OR5AN1-associated Ca2+
PCL/regeneratedsignaling; hBMMSCs
cellulose/calcium-lactate
membranes, with or
without immobilized
SDF1
Lactate was released; OR5AN1 siRNA
reduced Ca2+ fluorescence and
osteogenic transcripts under soluble
CaL treatment.
Rat femoral defect, 2/4 wk: CaLcontaining groups improved repair; extra
SDF1 bone-volume benefit was not
shown.
No direct ligand-binding or in vivo receptor
necessity. Calcium versus lactate
contributions and conflicting soluble CaL
doses remain unresolved.
[44]
Lactate handling
Reticular PLGA (50:50)
coating on electronbeam-melted porous
Ti6Al4V
LA release and endothelial protection
were observed; LA and GA controls
used different concentrations.
Osteoblast monoculture responses were
not significant.
Diabetic sheep iliac implants, 12 wk:
improved bone ingrowth (BV/PV).
No MCT/LDH necessity or carbon tracing;
do not label the endpoint BV/TV or infer
direct osteoblast stimulation.
[43]
Polymer-derived lactic
acid; endothelial cells, with
osteoblasts tested
separately
34
Table 2 (continued, part 3/4)
Material
Node and cells
Metabolic evidence
Bone model and outcome
Interpretive scope
References
Lactate handling
LOx immobilized on
PDA-coated
MoSe2/PtCu (MPL),
delivered in Alg–PVA–
PBA hydrogel with NIR
Extracellular lactate
oxidation; acellular
chemistry and BMSC
respiration
A spectral assay was interpreted as
lactate depletion; BMSC OCR
recovered under high-glucose/highlactate conditions.
Diabetic infected rat calvarial defect,
4/8/12 wk: improved bone repair.
The spectral response is not an absolute
lactate time course. No lactate-carbon
tracing into the TCA cycle; conflicting
Fig.6 values are excluded.
[31]
Lactate handling
VN4/RhN4 single-atom
carbon catalyst coatings
on titanium or stainlesssteel fixation devices
Exogenous LDH-like
pyruvate-to-lactate
reaction; acellular assay
NADH loss served as the catalytic
proxy; lactate product was not directly
quantified.
Osteoporotic mouse tibial fracture:
This is a proposed lactate-generation
improved repair; rabbit Ti–VN4 fixation direction, not clearance. NOX-like NADH
improved load and stiffness at 3/5 wk.
consumption and the catalytic-to-cellular
causal link remain unresolved.
Lactate handling
Perforated
intramedullary nail
containing a magnesium
rod, compared with a
titanium-rod nail
H3K18 lactylation in
osteoclast precursors;
RANKL-treated
RAW264.7 cells
Mg + RANKL: H3K18la increased.
Separate lactate/oxamate arms showed
Nfatc1-promoter enrichment changes
and osteoclast responses.
Rat femoral fracture, 4 wk: BV/TV
Lactate/oxamate animal arms used Ti
[47]
increased and Tb.Sp decreased versus Ti implants, not Mg rescue. Promoter
control.
enrichment does not establish
transcriptional suppression or site necessity.
Lactate handling
3D-printed
PCL/nanohydroxyapatit
e scaffold containing
sodium lactate
Non-histone STAT1-K193
lactylation; hBMSCs
Site-resolved mass spectrometry and
WT/K193R reconstitution linked the
site to osteogenic readouts.
Rat calvarial defect, 12 wk: improved
bone formation.
K→R is not uniquely lactylation-specific;
no in vivo site-necessity test. Conflicting
ChIP and protein-half-life claims are
excluded.
[45]
Lactate handling
AZD3965-loaded PH
(PBAP–
hyaluronan)/DPA
(DSPE–PEG–Asp8)
nanoparticles
MCT1/2-associated lactate
export; RANKL-treated
BMMs
Free AZD3965 increased intracellular
and decreased extracellular lactate;
these assays are distinct from
nanoparticle treatment in vivo.
OVX mice: nanoparticles improved
bone preservation versus free drug;
osteoblast number/RUNX2 did not
significantly increase.
Antiresorption is not defect regeneration.
No exclusive MCT1 assignment or
demonstrated direct osteogenesis; free-drug
results are not nanoparticle-specific flux
evidence.
[21]
[22]
35
Table 2 (continued, part 4/4)
Material
Node and cells
Immune-cell metabolic
reprogramming
Dexamethasone-loaded
hollow MnO2/chitosan
particles in
photocrosslinked
methacrylated alginate
Metabolic evidence
Bone model and outcome
Interpretive scope
References
ERK/HIF-1α/GLUT1ECAR decreased and OCR increased;
associated response; LPSERK inhibition produced a samestimulated RAW264.7 cells direction response. Conditioned
medium promoted BMSC osteogenic
readouts.
Rat femoral-condyle defect, 2/4 wk:
improved repair in a separate, non-LPS
injury model.
Same-direction inhibition is not rescue.
Drug/particle carryover and direct action
were not fully separated from macrophagemediated effects.
[13]
Immune-cell metabolic
reprogramming
Dimethyl-fumarate
liposomes in
chondroitin-sulfatemethacrylate/gelatinmethacrylate
microspheres
GAPDH activity and
metabolic function;
diabetic macrophages
GAPDH activity and ECAR decreased;
OCR increased according to the
inherited figure check.
Diabetic infected femoral defect, 4 wk:
increased new-bone area.
DMF is pleiotropic; no node-specific
rescue. ECAR/OCR text/figure
inconsistencies remain flagged; new-bone
area is not BV/TV.
[39]
Immune-cell metabolic
reprogramming
SDSSD-functionalized,
zinc-sulfide-loaded ZIF
nanoparticles
Glycolytic and respiratory
function; activated BMMs
ECAR decreased and OCR increased.
OVX femoral fracture, 7/14/28 d:
Conditioned-medium and direct-particle improved callus BV/TV and maturation
BMSC osteogenic responses were
pattern.
tested separately.
Immune-cell metabolic
reprogramming
Cationic-glucomannanfunctionalized
nanohydroxyapatite
delivered in GelMA
GLUT1/HK2/PGK
expression and net glucose
use; RAW264.7 cells
Medium glucose use and OCR
increased while supernatant lactate
decreased; gene expression was
measured separately.
Rat calvarial defect, 8 wk: bone volume No GLUT1-specific transport or glucose[19]
(BV) and bone surface (BS) increased as carbon tracing. Lactate-pool reduction is not
separate endpoints.
proven enzymatic clearance; SI-derived
ECAR statistics are excluded.
Immune-cell metabolic
reprogramming
MYT–HA–PBA–PVA
hydrogel carrying folatemodified PX478
liposomes (PN@MHV)
Cell-dependent glucose
consumption; BMSCs and
RAW264.7 cells assayed
separately
Free MYT increased BMSC glucose
consumption; PN@MHV reduced
RAW264.7 glucose consumption under
high glucose.
T2D mouse femoral fracture, 14/21 d:
PN@MHV improved BV/TV versus
MHV.
No in vivo metabolic rescue. Residual
[51]
particles may contribute to conditionedmedium effects; the 5 versus 10 mg/kg dose
conflict remains unresolved.
At 21 d, isolated BMSC mineralization
favored MHV over PN@MHV. No in vivo
dual-cell flux test; free MYT is not the
complete hydrogel.
[5]
36
Table 3
Design priorities and proposed validation paths derived from published studies. The priorities and experiments are review-author proposals, organized around the observations and unresolved
questions in the cited studies.
Sources: [3–5, 8, 11, 18, 21, 24, 25, 27, 31, 36, 39, 40, 43–45, 47, 48, 53, 56].
Design principle
Evidence and question
Proposed validation
Proposal scope
References
Define glucose–oxygen
windows for the target cell
and repair stage
Glucose manipulation is context-dependent; Cross initial glucose and oxygen
No universal glucose threshold; culture survival or
local exposure and the timing of depletion
conditions; monitor local
systemic glycemia does not establish local defect
versus replenishment remain unresolved.
glucose/O2/H2O2, viable-cell
rescue.
function and later bone endpoints with
inactive-enzyme, component and
glucose-add-back controls.
[3, 4, 11]
Separate lactate source,
reaction direction and pH
effects
Release and oxidation address different
contexts; cell-specific sources and whether
benefit reflects carbon use or detoxification
remain uncertain.
Match pH, ions, osmolality and
exposure; track lactate, pyruvate and
H2O2 with labeled lactate, inactive
catalysts and substrate/product addback.
Do not assume more or less lactate is always
beneficial, or reverse LDH-like pyruvate reduction
into clearance.
[31, 43, 45]
Distinguish antiresorption,
defect repair and transporter
isoforms
Osteoclast export inhibition and osteoblast
lactate-associated signaling address
different tasks; lineage-specific
MCT1/MCT2 roles remain incompletely
tested.
Combine lineage-resolved transport
and isoform-specific rescue with
lactate add-back; assess formation,
resorption, coupling and mechanics in
fracture/defect models.
OVX bone preservation is not direct osteogenesis; SIonly genetic claims do not supply completed causal
verification.
[21, 44, 45]
Link enzyme state to
selective intervention and
flux
Expression, activity and modification are
distinct; multienzyme effects and cell/stage
dependence prevent assignment of a unique
optimal target.
Measure abundance, PTM, activity
and labeled-carbon flux under
material exposure; use node-specific
rescue and separate osteogenic,
immune and osteoclast dose/stage
responses.
Broad 2-DG or multi-enzyme inhibition is not enzyme [36, 39, 40]
selectivity; non-material mechanism studies are
background evidence.
Resolve component
contributions and cross-scale
discrepancies
In vitro component rankings and in vivo
repair can differ; existing multi-component
data do not quantify interaction or synergy.
Use factorial component controls and
prespecified interaction tests; connect
matched exposures in multicellular
assays to cell-resolved in vivo
measurements and long-term
mechanics.
Do not overwrite a null comparison or in vitro ranking [5, 8]
with the in vivo result; lack of significance is not
equivalence.
37
Table 3 (continued, part 2/2)
Design principle
Evidence and question
Proposed validation
Proposal scope
Manage oxygen and catalytic
by-products together with
substrate
Peroxide removal can coexist with net
oxygen consumption; activity retention,
oxygen limitation and peroxide leakage in
implants are unresolved.
Measure glucose/lactate/O2/H2O2/pH A CAT-like label does not prove sustained oxygen
[4, 53]
trajectories and retained activity; build delivery; improved bone does not prove elimination of
reaction–transport balances with
peroxide toxicity.
inactive-catalyst, ion/pH and
perfusion controls.
DR8 colocalization is not proven endogenous
delactylation; promoter enrichment and K→R
experiments do not by themselves establish a
complete mechanism.
References
Advance modification
Histone H3K18la, non-histone STAT1associations to cell-specific in K193 and DR8-related hypotheses address
vivo tests
different levels; endogenous occupancy,
specificity and in vivo necessity remain
open.
Combine site-resolved MS,
endogenous editing/rescue and
matched release exposures; connect
cell-specific lactate, modification,
gene function and
formation/resorption endpoints.
Control assay interference
and retain source uncertainty
Material–assay interactions and source
inconsistencies can mimic metabolic
regulation; the magnitude and mechanism
of assay bias are often unknown.
Use acellular blanks, lysis-stage
Lower LDH assay signal is not LDHA inhibition;
spiking, particle removal/recovery and distant fatigue blood-lactate effects cannot establish a
independent assays; separate rawlocal bone mechanism.
material, released and cell-generated
lactate across time and location.
[18, 24]
Connect transport models to
viable cells and bone
outcomes
Predictions depend on calibration and
omitted boundaries; low residual glucose
can indicate either use or substrate
limitation.
Validate independently and include
oxygen, lactate/pH and viable-cell
consumption; measure concentration
fields, per-cell metabolism,
matrix/mineralization and mechanics
in matched geometries.
[25, 27, 56]
Fitted trends are not external validation; modeled cell
number is not bone formation, and unresolved units
preclude universal design thresholds.
[45, 47, 48]
38