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REVIEW 4 major objections 6 minor 42 references

Single-step method for the immobilization of hydroxyapatite on 3D-printed porous polyetherketoneketone implants

T0 review · 4 major / 6 minor · reviewed 2026-08-12 · deepseek-v4-flash

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Pith's one-line read A single-step hydroxyapatite dip nearly doubles stem-cell adhesion on 3D-printed porous PEKK implants while preserving pore geometry and compressive strength.

desk verdict Useful single-step coating method with solid materials characterization, but the osteogenic differentiation claim is undermined by Alizarin Red staining the HAp coating itself. read the letter →

arxiv 2501.08133 v1 pith:4XVKCWEB submitted 2024-11-29 physics.med-ph cond-mat.mtrl-sciphysics.bio-ph

Semen Goreninskii , Igor Akimchenko , Alexander Vorobyev , Mikhail Konoplyannikov , Yuri Efremov , Evgeniy Sudarev , Andrei Zvyagin , Evgeny Bolbasov
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Sergei Tverdokhlebov
This is my paper · ORCID
classification physics.med-phcond-mat.mtrl-sciphysics.bio-ph
keywords hydroxyapatitepolyetherketoneketonesurfacemodificationboneimplant3Dprintingstemcelladhesionosteogenicdifferentiationfuseddepositionmodeling
verification ladder T0 review T1 audit T2 compute T3 formal

The pith

A machine-rendered reading of the paper's core claim, the machinery that carries it, and where it could break.

The reading

The paper proposes a one-step surface treatment that immobilizes hydroxyapatite (HAp), the mineral of bone, onto 3D-printed porous polyetherketoneketone (PEKK) bone implants. The treatment is a 3-minute immersion of the implant in a suspension of HAp particles in a hexafluoropropanol/water mixture, followed by vacuum drying. The authors report that this preserves the implant's pore structure and compressive strength while rendering the surface fully wettable (0° water contact angle) and coating up to about 35% of it with HAp. Stem-cell adhesion rises from about 121 to 234 cells/mm², and the HAp-coated implants induce osteogenic differentiation, so the method could make bioinert PEKK implants more bone-friendly without sacrificing mechanical design.

What carries the argument

The load-bearing mechanism is solvent-induced surface swelling: HFP (1,1,1,3,3,3-hexafluoropropan-2-ol) swells the PEKK surface when mixed with water, and HAp particles suspended in that mixture become immobilized on the softened polymer during a 3-minute vortexing step. The authors link the uneven distribution of HAp to the semi-crystalline structure of PEKK, where crystalline and amorphous regions swell differently. The immobilized particles then change three surface properties relevant to cells: roughness rises from 5.0 to 13.4 µm, the surface becomes fully hydrophilic, and calcium and phosphate are presented at the interface.

What would settle it

Expose PEKK samples to the same HFP/water mixture without HAp, dry them by the same vacuum protocol, and seed MSCs; if this control reproduces any of the viability loss, adhesion increase, or Alizarin Red staining, the paper's attribution of the biological effects to HAp would be falsified. Quantifying residual HFP on the actual coated implants would independently settle the confound.

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Extended reading notes

Core claim

The central claim is that porous FDM-printed PEKK implants can be bioactivated in a single stage: immersion in a HAp suspension in an HFP/water mixture causes the polymer surface to swell and trap HAp particles. The coating, at 5–20 wt% HAp in the suspension, is confined to the surface and covers up to 35.0±14.0% of it, raising calcium and phosphorus surface content to 17.4±4.1 and 8.0±1.7 wt% respectively and bringing the water contact angle from 102±3° to 0°. Morphology (pore diameter ~460–480 µm, line thickness ~0.42–0.46 µm) and compressive properties (Young modulus ~5.2–5.6 MPa, yield strength ~28–31 MPa) remain statistically unchanged. In vitro, the modified surface roughly doubles stem-cell adhesion (121±40 to 234±8 cells/mm²) and, unlike unmodified PEKK, produces Alizarin Red-positive osteogenic differentiation.

Load-bearing premise

The biological conclusions depend on the assumption that vacuum drying removes HFP to trace levels, so the improved adhesion and differentiation reflect the hydroxyapatite coating rather than residual solvent effects.

Editorial extensions

If this is right

  • One-step HAp immobilization can be added to existing FDM-printed PEKK implants without changing the printed geometry, so pore size and infill can be optimized for mechanics and cell infiltration separately from bioactivation.
  • Because the 5 wt% suspension already yields a significant adhesion increase, the practical coating step can be short (3 minutes) and uses no vacuum chambers or magnetrons, lowering cost and equipment barriers.
  • The treated surfaces induce osteogenic differentiation of MSCs in vitro, pointing toward improved osseointegration if the effect carries into animal models.
  • Preserved compressive strength means the coating does not introduce the mechanical penalties seen with bulk HAp-filled filaments.

Reading between the lines

Editorial extensions of the paper, not claims the author makes directly.

  • The attribution of the 10–15% viability decrease to trace residual HFP is plausible but unverified; a direct measurement of residual solvent (or an HFP-only control) would separate coating effects from solvent toxicity.
  • The adhesion plateau between 5% and 20% HAp suggests that surface coverage beyond ~10% adds little for attachment; any additional benefit of higher Ca/P may show up only in longer-term differentiation assays.
  • Because the differentiation readout is Alizarin Red staining at a single 14-day time point, the claim of induced osteogenic differentiation would be strengthened by expression analysis of osteogenic markers such as ALP, Runx2, or osteocalcin.
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Editorial analysis

A structured set of objections, weighed in public.

Desk editor's note, referee report, and a circularity audit.

Referee Report

4 major / 6 minor

Summary. The manuscript reports a single-step method for immobilizing hydroxyapatite (HAp) particles on the surface of 3D-printed porous PEKK implants using an HFP/water suspension. The authors characterize morphology, elemental composition, wettability, compressive mechanical properties, and in vitro stem cell responses. They report that the treatment preserves pore diameter and compressive strength, increases surface hydrophilicity to a 0° contact angle, and improves MSC adhesion (from 121±40 to 234±8 cells/mm²). They further claim that the HAp-modified implants induce osteogenic differentiation of MSCs. The method is presented as an improvement over the authors' earlier two-step approach.

Significance. If the claims are substantiated, the method is attractive: it is simple, rapid (3 min), equipment-light, and preserves the scaffold's mechanical properties, which matters for load-bearing bone implants. The paper provides quantitative EDS confirmation of HAp presence, quantitative adhesion counts with statistics, and ISO-based compression testing. However, the biological claim of osteogenic differentiation is currently not supported by the evidence presented, because Alizarin Red S stains calcium phosphate and will directly stain the immobilized HAp coating. This is a load-bearing claim in the abstract and conclusions. The morphology data also contain an internally inconsistent unit error and a contradiction between the text and Table 1 for surface roughness. These issues need to be resolved before the manuscript can be accepted.

major comments (4)
  1. [Table 1 and Section 3] Table 1 reports the thickness of the printed line as 0.42±0.02 µm for the control sample, which is physically implausible for FDM printing with a 0.4 mm nozzle. The text repeats this value. This is not a cosmetic typo: the morphology-preservation claim rests on these measurements, and the reported roughness values (5.0–13.4 µm) are an order of magnitude larger than the reported line thickness, making the data internally inconsistent. The unit is almost certainly meant to be millimeters, or the variable is the line width rather than thickness. Please correct the units and re-verify all morphological measurements and their reporting.
  2. [Section 3, roughness paragraph] The text states that 'The immobilization of HAp particles resulted in the decrease of the roughness up to 13.4±1.3 µm', but Table 1 shows that Ra increased from 5.0±0.7 µm (control) to 13.4±1.3 µm (20% HAp). The word 'decrease' contradicts the data. This is not merely a wording issue, because increased roughness is subsequently invoked as a mechanism for improved cell adhesion. Please correct the statement to match the data, or provide the actual measurements if the trend is opposite.
  3. [Section 2.7 and Figure 2 (bottom row)] The osteogenic differentiation claim is not supported by the presented Alizarin Red S data. Alizarin Red S is a calcium-binding dye that will stain the immobilized HAp particles on the modified scaffolds; the red signal on 5%, 10%, and 20% HAp samples, which intensifies with increasing HAp content, is exactly the pattern expected from direct staining of the calcium phosphate coating rather than from mineralized extracellular matrix produced by differentiated MSCs. The control sample lacks HAp and therefore shows no red signal, so it does not control for this artifact. The paper provides no cell-free stained scaffold control, no decalcification step, no quantification of stained area, and no molecular markers (e.g., ALP, Runx2, OCN). The abstract and conclusions state that HAp immobilization induces osteogenic differentiation, but this is not established. Please either add appropriate controls and quantitative differentiation assays, or remove/qualify the differentiation claim throughout the manuscript.
  4. [Section 3, cell viability paragraph] The paper attributes a 10–15% decrease in cell viability to 'trace amount of residual HFP' without measuring residual HFP or including a solvent-only control. The same samples are used for the adhesion and differentiation experiments, so residual solvent could confound all biological outcomes. This is acknowledged in the text, but the current data do not rule out the possibility that the improved adhesion is partly a cellular response to residual HFP rather than to HAp. Please quantify residual HFP (e.g., by GC-MS or similar) or include a solvent-treated control without HAp in the biological experiments.
minor comments (6)
  1. [Section 2.7] The text states that '100 ml of DMSO was added', but the context (dissolving formazan crystals in a 96-well plate) indicates this should be microliters (µl), not milliliters.
  2. [Section 2.8] The phrase 'non-parametric ANOVA test' is imprecise; please state the specific test (e.g., Kruskal-Wallis with post hoc comparison) used for the in vitro data.
  3. [Section 3, compressive properties paragraph] The sentence 'The control PEKK samples demonstrated extremely low Young modulus and yield strength (5.4±0.4 and 30±2 MPa, respectively) (Table 1)' cites Table 1, but these values appear in Table 3. Please correct the cross-reference.
  4. [Section 2.4] The method for measuring 'HAp-coated area of the sample, %' in Table 1 is not described. Please specify the image-analysis procedure used to determine the percentage of coated surface.
  5. [Table 2] The Ca/P ratio of about 2.1 is noticeably higher than the stoichiometric HAp value of 1.67; this discrepancy is not discussed. A brief comment on possible causes (e.g., non-apatite calcium phosphate phases, EDS measurement uncertainty, or surface contamination) would help the reader.
  6. [Section 2.3] There is a typo: 'dryed with filter paper' should be 'dried with filter paper'.

Circularity Check

1 steps flagged · score 6.0 of 10

The osteogenic differentiation claim is circular: Alizarin Red S stains the HAp coating itself, so the red signal is guaranteed by the deposited calcium phosphate rather than by new cellular mineralization.

  1. self definitional [Section 2.7 (Alizarin Red staining protocol) and Section 3, final paragraph (osteogenic differentiation results), Figure 2 (bottom row)]
    "Next, the cells were dyed with Alizarin Red solution for 1 hour. ... In turn, red round coloring typical for the osteogenically differentiated cells [41] was observed for the cells incubated with the samples with immobilized HAp. The observed effect was intensified with the increasing amount of HAp from the results of elemental composition (Table 2)."

    Alizarin Red S is a calcium-binding dye, and the applied modification is hydroxyapatite, a calcium phosphate. On modified samples the red signal is therefore expected from the immobilized HAp particles themselves; the control has no HAp and consequently shows no red. The reported intensification with HAp content (Table 2) is precisely the trend expected if the dye is staining the coating, not newly formed mineralized matrix from differentiated MSCs. The paper provides no cell-free stained control and no decalcification step to separate coating-derived stain from cellular calcium deposits, so the 'osteogenic differentiation' observation is not independent of the input material; the assay signal is produced by the very substance whose biological effect is claimed.

full rationale

Apart from the differentiation assay, the paper's measurements are direct and self-contained: pore/line dimensions, EDS composition, water contact angle, compression testing, and cell counting are all new experimental observations with no fitted parameters and no equations that map inputs to claimed outputs by construction. The self-citation to the authors' prior two-step HFP method [20] is motivational and explanatory rather than load-bearing; the present results are not consequences of that citation. The main circular step is the use of Alizarin Red S as evidence for HAp-induced osteogenic differentiation. Because the dye binds calcium and the samples are coated with calcium phosphate, the red signal, and its increase with HAp content, is guaranteed by the coating itself in the absence of any cell-specific mineralization assay. This makes the differentiation claim partially circular, but it does not affect the independent validity of the method-development, morphology, strength, or adhesion claims.

Assumptions & free parameters 0 free parameters · 4 assumptions · 0 invented entities

No free parameters or invented entities. The central claims rest on four domain assumptions about the swelling mechanism, residual solvent, WCA interpretation, and EDS phase identification.

assumptions (4)
  • domain assumption HFP swells the PEKK surface, enabling entrapment of HAp particles during the 3-minute immersion.
    Central mechanism of the proposed method, based on the authors' prior work (ref [20]) and not independently verified here.
  • domain assumption Residual HFP after vacuum drying is low enough that the in vitro cell responses reflect the HAp coating rather than solvent effects.
    The paper reports a 10-15% viability decrease in coated samples and attributes it to trace residual HFP (Section 3, cell viability paragraph).
  • domain assumption A water contact angle of 0° measured immediately on a porous sample reflects surface chemistry rather than capillary wicking into the 400-600 µm pores.
    WCA is reported as a surface property, but the sample is highly porous; the measurement is taken immediately after drop placement (Section 2.5).
  • domain assumption The Ca/P ratio of approximately 2.1 indicates the presence of hydroxyapatite; the deviation from stoichiometric HAp (1.67) is not explained.
    EDS data in Table 2 report Ca/P around 2.0-2.2, which is used as evidence of HAp identity without discussion of possible other calcium phosphate phases.

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Pith. "Pith review of Single-step method for the immobilization of hydroxyapatite on 3D-printed porous polyetherketoneketone implants." pith.science (2026). https://pith.science/paper/4XVKCWEB

@misc{pith2026250108133,
  author       = {Pith},
  title        = {Pith review of: Single-step method for the immobilization of hydroxyapatite on 3D-printed porous polyetherketoneketone implants},
  year         = {2026},
  howpublished = {\url{https://pith.science/paper/4XVKCWEB}},
  note         = {Machine review of arXiv:2501.08133}
}
read the original abstract

The development of tissue engineering structures (scaffolds) for the reconstruction of bone tissue defects is the relevant task of modern biomedical materials science. Compared to metal-based structures, polymer constructs provide numerous advantages, among them - better processibility and metallosis avoidance. Owing to its high mechanical performance and biocompatibility, polyetherketoneketone (PEKK) became a promising material for the development of such structures. Previously, a method for the immobilization of hydroxyapatite (HAp) on PEKK surface was proposed by our group for the enhancement of stem cell adhesion. In the present study, we propose a single-step method of HAp immobilization on the surface of 3D-printed porous PEKK implants. The proposed approach allowed to preserve the morphology (pore diameter, width of the printed lines) of the pristine implants. With that, up to 35.0+-14.0 % of the sample surface were coated with HAp particles, which resulted in improved hydrophilicity (0 degrees water contact angle). The calcium and phosphorus content on the surface of the modified samples was up to 17.4+-4.1 and 8.0+-1.7 wt. %, respectively. Importantly, the proposed modification preserved compressive strength of the 3D-printed porous PEKK implants. HAp immobilization provided better adhesion of stem cells (from 121+-40 cells/mm2 to 234+-8 cells/mm2) and induce their osteogenic differentiation.

Figures

Figures reproduced from arXiv: 2501.08133 by the authors.

Figure 1
Figure 1. SEM images of the samples (magnification ×50, magnification ×2000 in the inserts) 2 µm 2 µm 2 µm 2 µm 50 µm 50 µm 50 µm Control 5 % HAp 10 % HAp 20 % HAp [PITH_FULL_IMAGE:figures/full_fig_p007_1.png] view at source ↗
Figure 2
Figure 2. Fluorescent microscopy of the cells adhered to the surface of the samples (top row) and optical micrographs of the cells dyed with Alizarin Red after the incubation with the samples (bottom row) (magnification of ✕40) Thus, the deposition of HAp particles using the proposed single-step method, improved the adhesion of stem cells to the surface of the FDM-printed porous PEKK-based implants. The observed effect may be… view at source ↗

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Pith tools

Reviewed August 12, 2026 · model on record in the stance chip above.