Evaluation of a hydroxyapatite (HAp) material commonly begins with Ca/P ratio, XRD, FTIR, particle size, and SEM. Together, these methods provide information on elemental composition, crystalline phase, chemical groups, particle-size distribution, and particle morphology. They do not fully describe what happens at the outermost interface after the material enters an aqueous environment containing ions and proteins.
A 2026 study in Biomaterials Research primarily investigated the mechanism in vitro and also included a preliminary rat subcutaneous implantation experiment. The authors placed porcine bone-derived biogenic HAp (BHA) particles in culture medium and in a RAW 264.7 murine macrophage system, examining liquid-phase Ca²⁺, particle zeta potential, direct cell contact, and TLR4-related signaling. This article focuses on the in vitro mechanism data. The study extends the analysis from bulk composition to the solid-liquid interface: materials with similar routine bulk characterization may still behave differently in a defined liquid environment.
Ca/P and Interface Data Describe Different Levels
The theoretical molar Ca/P ratio of stoichiometric HAp is approximately 1.67. That value describes the overall numerical relationship between calcium and phosphorus in a sample. It does not identify the phase on its own or reveal the immediate state of calcium sites, phosphate-related sites, and the hydration layer in a particular liquid environment. Our earlier article on whether a Ca/P ratio near 1.67 can confirm HAp covers this distinction.
After contact with liquid, an HAp surface can change with pH and ionic composition through protonation and deprotonation, ion adsorption and exchange, and, under certain conditions, dissolution and reprecipitation [3,5]. A 2005 surface-chemistry study showed that external Ca²⁺ can adsorb to HAp and alter surface-charge behavior depending on pH, electrolyte concentration, and available sites [3]. Ca/P and interface testing are therefore complementary: one describes bulk composition, while the other follows a material's response in a defined environment.
How the Study Built Its Case for Surface Calcium Enrichment
The study used porcine-derived BHA particles measuring approximately 0.25 to 1 mm. They were incubated in complete culture medium at a material-to-liquid ratio of 180 mg/mL for 24 hours. ICP-OES showed a lower Ca²⁺ concentration in the supernatant after contact with BHA. The authors therefore distinguished a relatively calcium-enriched particle interface from a surrounding liquid phase with lower Ca²⁺ [1].
The paper's “surface calcium enrichment” was not a directly imaged calcium-rich layer. The interpretation was supported by depletion of Ca²⁺ from the liquid phase, reduced enrichment capacity after calcium pre-adsorption, and changes in zeta potential. This evidence is consistent with transfer of Ca²⁺ from the liquid to the material interface, but it is not equivalent to a higher surface Ca/P ratio measured by routine EDS.
Direct Contact and Conditioned Medium Produced Different Results
The researchers compared BHA-conditioned medium with direct macrophage contact on BHA particles. The conditioned medium represented the liquid environment after its Ca²⁺ concentration had fallen. The direct system exposed cells to the particle surface. Tnf-α, Il-18, Ccl3, and Ccl4 expression increased in the direct-contact group, as did TNF-α and MIP-1α protein levels. Inflammatory activation was substantially weaker with conditioned medium alone [1].
Under these conditions, a lower Ca²⁺ concentration in the surrounding medium did not explain the full response; direct interaction with the material interface was more important. The experimental design did not isolate protein adsorption, surface microstructure, or other ionic processes individually, so the observed response cannot be attributed to Ca²⁺ alone.
TLR4 Was Involved, but It Was Not the Only Pathway
Transcriptomic and RT-qPCR results showed increased TLR4-related signaling after direct BHA contact, together with elevated intracellular Ca²⁺. Pretreatment with the TLR4 inhibitor TAK-242 reduced Tlr4, Cd14, Ly96, several inflammatory markers, and intracellular Ca²⁺ [1]. These results support a role for TLR4 in the macrophage response to this BHA system.
The blockade experiment did not show that surface Ca²⁺ directly binds TLR4, nor does it justify the claim that HAp invariably activates TLR4. Some signals remained above control levels after inhibition, and Il-6 and Il-18 did not decline in parallel. The authors therefore left room for additional regulatory pathways. A related 2024 study of porcine-derived HAp prepared at different sintering temperatures also linked surface calcium enrichment, cellular calcium influx, and inflammatory activity, providing earlier evidence for the 2026 work [2].
Pre-Adsorption Changed Interface Behavior Without an Obvious Routine Signature
The authors repeatedly exposed BHA to DMEM to pre-adsorb Ca²⁺. After three and six cycles, the particles had less capacity to remove additional Ca²⁺ from fresh medium. Zeta potential shifted from −11.14 mV for untreated BHA to −8.60 mV and −7.51 mV. This indicates a change in electrokinetic behavior near the slipping plane. Zeta potential is related to interfacial charge, but it is not a direct substitute for true surface charge density.
SEM, XRD, and EDS detected no changes in morphology, crystallinity, or elemental composition, while TLR4-related gene expression declined [1]. This does not prove that “only the interface changed and everything else remained identical.” It means no corresponding difference was detected within the scope and sensitivity of those methods. Routine phase and morphology measurements remain important, but they cannot capture every dynamic ion-exchange process.
| Evidence | What it mainly supports | What it cannot establish alone |
|---|---|---|
| Ca²⁺ in the supernatant | Transfer of Ca²⁺ between liquid and material | Thickness and spatial distribution of an enriched layer |
| Zeta potential | A change in electrokinetic behavior near the particle interface | The complete distribution of true surface charge density |
| SEM, EDS, and XRD | Detectable morphology, local elemental composition, and crystallinity | All dynamic adsorption and exchange processes |
| Cell and blockade assays | Participation of TLR4-related responses in a defined system | All HAp materials or in vivo responses |
Interface Testing Should Follow the Application Question
This article focuses on the in vitro system comprising a specific porcine-derived BHA, millimeter-scale particles, and RAW 264.7 cells. The paper also implanted BHA pretreated with whole blood or serum in a rat subcutaneous model and assessed inflammatory-cell infiltration on day 3; with only three animals per group, this was a preliminary validation. Neither the in vitro mechanism data nor this short-term animal result can be transferred directly to synthetic HAp powders, CaHA microspheres, coatings, complete medical devices, or patients. Particle origin, thermal history, phase and crystallinity, particle size, and specific surface area can all alter solid-liquid interface behavior.
Whether interface testing is warranted should be determined by the downstream application and research question. A generic “surface calcium state” should not automatically become a release item on every certificate of analysis. Basic release testing should continue to address phase, Ca/P ratio, particle-size distribution, morphology, impurities, and batch consistency. When downstream development raises questions about ion exchange, protein adsorption, or immune-cell contact, the test plan may then add time-resolved liquid-phase ion analysis, zeta potential, surface-sensitive methods, and an appropriate direct-contact model.
This follows the same principle discussed in our article on why D50 and SEM should not be expected to give the same particle-size answer: a method must match the question. Ca/P describes bulk composition; XRD identifies crystalline structure; particle-size analysis and SEM describe particle state; interface experiments examine how the material changes in a defined environment. Distinguishing these measurement levels helps prevent a single metric from being used to explain phenomena beyond its detection scope.
This article focuses on the in vitro mechanism section of the study; the paper also included a preliminary rat subcutaneous implantation experiment. “Surface calcium enrichment” refers to the authors' interpretation based on liquid-phase ion changes, pre-adsorption, and interfacial electrokinetic results. The findings come from a defined porcine-derived BHA system, are not a standardized quality attribute, and cannot be directly extrapolated to other HAp materials, medical products, or clinical outcomes.
References
- Mai M, Su M, Deng S, et al. Surface Calcium Enrichment of Biogenic Hydroxyapatite Regulates Macrophage Inflammation via Toll-Like Receptor 4 Signaling. Biomaterials Research. 2026;30:0409. DOI: 10.34133/bmr.0409.
- Li C, Su M, Mai M, et al. Calcium Enrichment Activity Initiates Extracellular Calcium Influx-Dependent Inflammatory Response of Biologically-Derived Hydroxyapatite. Materials Today Bio. 2024;28:101231. DOI: 10.1016/j.mtbio.2024.101231.
- Harding IS, Rashid N, Hing KA. Surface Charge and the Effect of Excess Calcium Ions on the Hydroxyapatite Surface. Biomaterials. 2005;26(34):6818–6826. DOI: 10.1016/j.biomaterials.2005.04.060.
- Kieswetter K, Bauer TW, Brown SA, et al. Characterization of Calcium Phosphate Powders by ESCA and EDXA. Biomaterials. 1994;15(3):183–188. DOI: 10.1016/0142-9612(94)90065-5.
- Gómez-Morales J, Iafisco M, Delgado-López JM, et al. Progress on the Preparation of Nanocrystalline Apatites and Surface Characterization: Overview of Fundamental and Applied Aspects. Progress in Crystal Growth and Characterization of Materials. 2013;59(1):1–46. DOI: 10.1016/j.pcrysgrow.2012.11.001.