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Eggshell derived scaffold of hydroxyapatite-ammonium bicarbonate nano-composite: Bioactivity and cytotoxicity studies · 10.1016/j.heliyon.2024.e36493

Name: Verified Rigor Record — Eggshell derived scaffold of hydroxyapatite-ammonium bicarbonate nano-composite: Bioactivity and cytotoxicity studies · 10.1016/j.heliyon.2024.e36493
Item: Eggshell derived scaffold of hydroxyapatite-ammonium bicarbonate nano-composite: Bioactivity and cytotoxicity studies · 10.1016/j.heliyon.2024.e36493
Rating: 73
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Other Study Type

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: moderateReviewer agreement: lowEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.
No data or code availability links were detected to verify.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper synthesizes and characterizes a hydroxyapatite scaffold from eggshell waste with descriptive in vitro biocompatibility assays. Its scientific premise is well-founded and reporting transparency is adequate (limitations discussed, funding/COI declared). However, key rigor gaps exist: no randomization/blinding/power analysis for the cell-based assays, key resources lack catalog numbers and versions, and the data availability statement is vague ('on request'). The paper would benefit from more complete reporting of experimental design and resource identification.

Evaluated as an in vitro materials characterization study; biological variables and ethical approvals were not applicable (commercial cell line, no animals/humans); statistical analysis was not applicable (no inferential tests). One reviewer classified the study as interventional, but the primary methodology is materials synthesis and characterization. The citation check found 16 references not found in registry (no retractions); these should be verified.

12 major claims checked against the paper's own evidence: all adequately supported.

5 integrity concerns flagged (0 high).

Rigor

Eight Dimensions2 pass · 2 warn · 1 fail

12 major claims3 partial9 supported

Prior work on HAp synthesis methods, biological HAp sources, and eggshell-derived calcium phosphate are cited (paragraphs 1–7 of Introduction). The rationale—using eggshell waste to fabricate a porous scaffold and study its biocompatibility—is clearly linked to gaps in prior research ('the fabrication of porous scaffold nano-composite to enhance the cell diffusion... and the detailed study of their toxicity has not been well explored'). A limitation of the present study is also explicitly discussed in a dedicated section (3.8).

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The paper establishes a clear scientific foundation by citing prior work on hydroxyapatite synthesis from waste materials and identifies a gap in the literature regarding detailed toxicity studies of porous scaffolds.

Evidence

paraphrase[Introduction, paragraph 3]

“The introduction references several prior studies on HAp synthesis from eggshells and other biological sources.”

direct quote[Introduction, final paragraph]

“Herein, eggshells waste which is often discarded as useless materials will be utilized as source of calcium in the production of hydroxyapatite biomaterial that can be deployed in biomedical application, thus, adding values to agricultural wastes.”

direct quote[Introduction, paragraph 4]

“the fabrication of porous scaffold nano-composite to enhance the cell diffusion during implant and the detailed study of their toxicity has not been well explored.”

PASSReviewer 2· 90% conf

The paper adequately establishes its scientific foundation by citing prior work, providing a logical rationale, and addressing limitations of previous research.

Evidence

direct quote[Introduction, paragraphs 1-3]

“Though, there have been reports on the synthesis of HAp powder from waste ago-materials as mentioned above, the fabrication of porous scaffold nano-composite to enhance the cell diffusion during implant and the detailed study of their toxicity has not been well explored.”

direct quote[Introduction, paragraph 4]

“In this present study, our aim was to fabricate HAp powder from waste eggshells which serve as calcium source and reacted with ammonium dihydrogen phosphate {(NH4)H2(PO4)} solution via wet chemical method to form a scaffold nano-composite.”

direct quote[Introduction, paragraph 3]

“the fabrication of porous scaffold nano-composite to enhance the cell diffusion during implant and the detailed study of their toxicity has not been well explored.”

PASSReviewer 3· 95% conf

The paper adequately establishes its scientific foundation by citing prior work on hydroxyapatite synthesis, eggshell-derived materials, and the gap in porous scaffold toxicity studies.

Evidence

paraphrase[Introduction, paragraphs 2–5]

“Multiple prior studies on HAp synthesis routes, biological HAp from bone/shell, and eggshell-derived calcium phosphate are cited (e.g., references [12]–[20] in the text).”

direct quote[Introduction, paragraph 6]

“Thus, this work tends to utilize agro-waste of eggshell for the production of porous scaffold hydroxyapatite-ammonium bicarbonate nano-composite.”

direct quote[Section 3.8 Limitation of the study]

“This study focused on the use of eggshell as the main source material for the synthesis of the prepared hydroxylapatite.”

The study is an in vitro experiment on synthesized scaffold materials. Randomization, blinding, and a priori power analysis are not reported. Inclusion/exclusion criteria for the cell-based assays are not stated. Biological vs technical replicates are not distinguished. Outlier handling is not mentioned. Controls are partially adequate (0% AMB sample is used as a control, and cells without scaffold as a 100% viability reference). Independent replication is not reported. The scoring rule for in vitro experiments requires ≥60% of applicable criteria to pass; here, only 1 of 9 applicable criteria (controls) can be considered adequate, yielding ~11%.

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsNOT REPORTED

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodNOT REPORTED

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The study design lacks several key rigor elements for in vitro experiments: no randomization, blinding, power analysis, or pre-specified inclusion/exclusion criteria are reported, and replicate distinction and outlier handling are not addressed.

Evidence

direct quote[Section 2.8, paragraph 1]

“ESTHAp with 0 % AMB was considered as the control sample, while cell without any samples which determines the viability of control cells, was set as 100 %.”

absence[Methods, Sections 2.1–2.11]

Randomization, blinding, power analysis, and other key design elements are absent.

absence[Methods, Sections 2.8–2.10]

Replicate distinction and outlier handling are not addressed.

N/AReviewer 2· 90% conf

The study is a materials synthesis and characterization with in vitro cell tests; no formal experimental design with randomization, blinding, or power analysis is applicable.

Evidence

absence[Methods, sections 2.1-2.11]

No randomization, blinding, or power analysis described.

PASSReviewer 3· 95% conf

The experimental design is clearly described as a bench-scale materials synthesis and characterization study; all applicable sub-criteria are adequately reported.

Evidence

direct quote[Section 2.8, Assessment of biocompatibility and cell growth]

“ESTHAp with 0 % AMB was considered as the control sample, while cell without any samples which determines the viability of control cells, was set as 100 %.”

paraphrase[Sections 2.1–2.11]

“Procedures for eggshell calcination, HAp synthesis, scaffold preparation with AMB, sintering, characterization (XRD, FT-IR, SEM, TEM, TGA), bioactivity, MTT assay, and cell adhesion are all described in step-by-step detail.”

absence[Methods]

No mention of outlier handling.

The paper uses MG63 osteosarcoma cells obtained from Sigma, India, and explicitly states no cells were extracted from animal or human. No biological subjects with organismal variables are used. Thus, all sub-criteria are not applicable, making the dimension itself not_applicable.

Sub-criteria

demographicsN/A

sex reportedN/A

sex justifiedN/A

age weight healthN/A

housing conditionsN/A

species strain sourceN/A

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 90% conf

The study is a materials characterization and in vitro cytotoxicity experiment using a commercial cell line (MG63); no animal or human subjects are involved, and the cell line is not a biological variable that requires reporting of sex, age, housing, etc.

Evidence

direct quote[Ethics statement, after Conclusion]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India. We didn't use cells extracted from either animal or human.”

N/AReviewer 2· 85% conf

The study uses a cell line (MG63), not animal or human subjects; biological variables like sex, age, housing are not applicable.

Evidence

direct quote[Ethics statement]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India. We didn't use cells extracted from either animal or human.”

PASSReviewer 3· 95% conf

Biological variables relevant to this materials study are reported: species/strain of the cell line, and source.

Evidence

direct quote[Section 2.8]

“MTT assay performed using MG63 osteosarcoma cell lines”

direct quote[Ethics statement]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India.”

The paper explicitly states that cells were simulated/obtained from Sigma and no animal or human material was used. The study does not involve non-public subject-level data, so ethical approvals are not applicable.

Sub-criteria

iacuc statementN/A

informed consentN/A

irb ethics statementN/A

regulatory complianceN/A

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 90% conf

The study uses a commercial cell line (MG63) and does not involve human participants, animals, or non-public identifiable data; hence, ethics approval is not required.

Evidence

direct quote[Ethics statement]

“We didn't use cells extracted from either animal or human.”

direct quote[Ethics statement]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India.”

N/AReviewer 2· 95% conf

No human or animal subjects were used; ethics approval is not required.

Evidence

direct quote[Ethics statement]

“We didn't use cells extracted from either animal or human.”

PASSReviewer 3· 95% conf

The study uses commercially obtained, simulated cell lines; no human or animal subjects were involved, so ethics approval is not applicable.

Evidence

direct quote[Ethics statement]

“We didn't use cells extracted from either animal or human.”

Antibodies are named with vendor only, no catalog numbers; cell line source is given but no authentication; reagents are listed with vendor but no catalog numbers; software tools are named but versions not provided; mycoplasma testing is not reported. All applicable sub-criteria are scored as inadequate or not reported, leading to an overall fail.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedINADEQUATE

organisms identifiedN/A

antibodies identifiedINADEQUATE

cell line authenticationINADEQUATE

software tools identifiedINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Key resources are partially identified: the investigational scaffold material is described, but several reagents and equipment lack vendor/catalog numbers and software versions are not fully reported.

Evidence

direct quote[Section 2.1, paragraph 1]

“The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Dimethyl sulfoxide (DMSO)... Phosphate-buffered saline (PBS), Ca(OH)2, Ammonia, ammonium bicarbonate, simulated body fluid, (NH4)H2PO4, and ELISA plate used were purchased from Sigma, India”

direct quote[Section 2.10, paragraph 1]

“The Becton Dickinson CellquestPro software was used to evaluate the data.”

direct quote[Ethics statement]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India.”

FAILReviewer 2· 70% conf

Key resources (antibodies, cell line, reagents, software) are identified but inadequately detailed; mycoplasma testing is absent.

Evidence

direct quote[Methods, section 2.10]

“the cells were stained with antihuman CD29-PE, CD73-PE, and CD44-FITC antibodies (all from BD Pharmingen)”

direct quote[Ethics statement]

“The cells used in this experiment were simulated cells to mimic those of living components which were obtained from Sigma, India.”

absence[Methods, section 2.8-2.10]

Mycoplasma testing not reported.

PASSReviewer 3· 95% conf

Key resources are adequately identified: reagents, software, and investigational product (the scaffold) are named with sources.

Evidence

direct quote[Section 2.1]

“The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Dimethyl sulfoxide (DMSO), Phosphate-buffered saline (PBS), Ca(OH)2, Ammonia, ammonium bicarbonate, simulated body fluid, (NH4)H2PO4, and ELISA plate used were purchased from Sigma, India”

paraphrase[Sections 2.5, 2.10, 2.11]

“Bruker D8 Advance XRD, Bruker TENSOR 27 FT-IR, Carl Zeiss Supra 55VP SEM, FEI Tecnai G220 TEM, SDT Q600 TGA/DSC, Schimadzu balance, Shimadzu UTM, Leica TCS SP2 confocal microscope, BD FACS Calibur with CellquestPro software.”

absence[Methods]

Mycoplasma testing is not reported.

The paper reports viability percentages, compressive strength values, and crystallite sizes without any hypothesis tests, p-values, or confidence intervals. Therefore, the sub-criteria for statistical analysis (tests, assumptions, p-values, effect sizes) are not applicable.

Sub-criteria

tests namedN/A

exact p valuesN/A

effect sizes ciN/A

data presentationN/A

software identifiedN/A

assumptions verifiedN/A

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

N/AReviewer 1· 80% conf

The study presents descriptive results and a bar chart of cell viability percentages but performs no inferential statistics (no p-values, confidence intervals, or statistical tests), so statistical analysis criteria are not applicable.

Evidence

absence[Results, Section 3.7]

No inferential statistics are reported.

direct quote[Section 3.7, paragraph 1]

“It was observed that on comparing the sample of ESTHAp containing 0 % AMB with other samples in the range of 0.01–1 mM, viability of above 85 % MG63 cells was achieved except for ESTHAp with 40 % AMB which showed some level of toxicity.”

N/AReviewer 2· 90% conf

No inferential statistical tests were performed; only descriptive statistics (percentages, means) are reported.

Evidence

absence[Results, sections 3.1-3.8]

No statistical tests or p-values reported.

WARNReviewer 3· 90% conf

Statistical analysis is minimal; no inferential tests are reported for the quantitative comparisons, so assumptions and exact p-values are not reported; data presentation is descriptive.

Evidence

absence[Results, sections 3.5–3.7]

No inferential statistical tests are named or performed.

paraphrase[Section 3.7, Figure 11]

“MTT assay results are reported as percentages but without error bars, individual data points, or per-group n in the text (the figure may show them, but the image is not accessible in the text).”

absence[Methods]

No statistical software is identified.

The paper includes a data availability statement: 'Data will be made available on request.' This is 'reported_but_inadequate' because it fails to specify a repository, platform, or concrete access conditions and timeframe. No raw data are deposited in a public repository. No custom code was generated (standard commercial instruments were used), so code sharing is not applicable.

Sub-criteria

code sharingN/A

accession numbersN/A

repository depositNOT REPORTED

data availability statementINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

A data availability statement is present but vague ('Data will be made available on request'), without specifying a repository or access mechanism, and no code is shared.

Evidence

direct quote[Data and code availability section]

“Data will be made available on request.”

absence[Data and code availability section]

No repository deposit or accession numbers are provided.

absence[Data and code availability section]

No code sharing is mentioned.

FAILReviewer 2· 85% conf

Data availability statement is vague ('on request'); no repository deposit, accession numbers, or code sharing.

Evidence

direct quote[Data and code availability]

“Data will be made available on request.”

absence[Data and code availability]

No repository deposit or accession numbers.

WARNReviewer 3· 95% conf

A data availability statement is present but vague ('on request' with no mechanism); no code is shared.

Evidence

direct quote[Data and code availability]

“Data will be made available on request.”

absence[Data and code availability]

No raw data repository deposit is mentioned.

Methods (sections 2.1–2.11) are detailed enough for independent replication. A dedicated limitations section (3.8) is present, though it only discusses one limitation (single biological source). The conclusions in the abstract and section 4 are proportional to the descriptive results (no causal or clinical claims beyond the in vitro evidence). Funding sources (Taif University, project TU-DSPP-2024-13) and a conflict-of-interest statement (none declared) are provided. Trial registration is not applicable. No reporting guideline is referenced.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The paper has transparent reporting in some areas (limitations discussed, funding/COI stated, conclusions generally proportional) but lacks trial registration (n/a), a reporting guideline checklist, and fails to report all pre-specified outcomes (the cell adhesion study showed unstable samples but was still included, and negative results are mentioned).

Evidence

direct quote[Section 3.8, paragraph 1]

“There is one major limitation in this study that could be addressed in future research work.”

direct quote[Funding and Declaration of competing interest sections]

“This research was funded by Taif University, Saudi Arabia, project No.(TU-DSPP-2024-13)... The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.”

absence[Methods]

No reporting guideline is mentioned.

PASSReviewer 2· 85% conf

Methods are detailed, limitations are discussed, funding and COI are declared, conclusions are proportional.

Evidence

direct quote[Results, section 3.8]

“There is one major limitation in this study that could be addressed in future research work.”

direct quote[Funding, Declaration of competing interest]

“This research was funded by Taif University, Saudi Arabia, project No.(TU-DSPP-2024-13).”

direct quote[Results, section 3.7]

“except for ESTHAp with 40 % AMB which showed some level of toxicity.”

PASSReviewer 3· 95% conf

Methods are described in sufficient detail to be reproducible; limitations are discussed; conclusions are proportional; funding and COI are disclosed.

Evidence

paraphrase[Sections 2.2–2.11]

“Detailed step-by-step protocols for synthesis, scaffold preparation, sintering, characterization, bioactivity, MTT assay, and cell adhesion studies are provided.”

direct quote[Funding and Declaration of competing interest]

“This research was funded by Taif University, Saudi Arabia, project No.(TU-DSPP-2024-13). ... The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.”

direct quote[Section 3.8]

“There is one major limitation in this study that could be addressed in future research work. This study focused on the use of eggshell as the main source material for the synthesis of the prepared hydroxylapatite.”

30 copyedit issues flagged (2 major): mostly typo, grammar, clarity.

Checked 40 references: 23 verified — 16 unresolved, 1 not checked.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

The paper is already published. An informed reader should weigh the lack of randomization, blinding, and power analysis for the cell-based assays, the absence of reagent catalog numbers and software versions, and the vague data availability statement — these gaps reduce reproducibility but do not invalidate the descriptive findings. The internal contradiction between MTT viability (>85%) and cell adhesion failure (no cells near heat-treated samples) and the incorrect hydroxyapatite formula are issues that warrant correction or clarification.

1.
HIGHreporting
Clarify the internal contradiction between MTT viability results (>85% viability for up to 30% AMB) and the cell adhesion study (no cells observed near heat-treated samples); explain whether the sample conditions differ (e.g. sintered vs. unsintered) and reconcile the apparent inconsistency. [Section 3.7]
The apparent contradiction undermines the biocompatibility conclusion and requires explicit clarification for informed readers.
2.
HIGHreporting
Correct the incorrect chemical formula of hydroxyapatite in the Introduction from Ca10(PO4)(OH)2 to Ca10(PO4)6(OH)2; ensure consistency with the correct formula used in equation (3). [Introduction, paragraph 2]
The incorrect formula is a factual error that may confuse readers and affect trust in the chemical understanding.
3.
HIGHreporting
Revise the Ethics statement: MG63 cells are a human osteosarcoma cell line, so the statement 'We didn't use cells extracted from either animal or human' is misleading; clarify that the cells are of human origin but were obtained commercially as a cell line. [Ethics statement]
The current statement is factually inaccurate and could be flagged as a misrepresentation by journal editors or readers.
4.
HIGHdata code
Replace the vague data availability statement ('Data will be made available on request') with a deposit of raw data (XRD, FTIR, TGA, cell viability measurements) in a public repository (e.g., Zenodo, Figshare) with a DOI, or provide a detailed managed-access procedure with contact information and timeline. [Data and code availability section]
The current 'on request' statement does not meet current journal standards for data sharing and reduces reproducibility; many journals require a concrete data access plan.
5.
HIGHrigor
Provide vendor catalog numbers for all key reagents (MTT, DMSO, PBS, Ca(OH)2, ammonium bicarbonate, (NH4)H2PO4, ELISA plate) and the MG63 cell line (at a minimum the Sigma product number and RRID). [Section 2.1, Ethics statement]
Catalog numbers are essential for reproducibility of chemical and biological reagents; their absence was flagged by two reviewers.
6.
HIGHrigor
Provide software versions for all analysis software used (CellquestPro, Shimadzu UTM software, Leica software, Bruker software) in the relevant methods sections. [Sections 2.5, 2.10, 2.11]
Software versions are essential for reproducibility of data processing and analysis.
7.
HIGHrigor
Add a statement on mycoplasma testing for the MG63 cell line, including the method and result. [Section 2.8]
Mycoplasma contamination can affect cell viability and assay results; its absence is a standard reviewer concern for cell-based studies.
8.
MEDIUMreporting
Report the number of biological replicates (independent experiments) and technical replicates (wells per condition) for the MTT assay; distinguish between them and report per-group sample sizes. [Section 2.8]
Without replicate distinction, the precision and reliability of the viability data cannot be assessed.
9.
MEDIUMreporting
Add a statement on outlier handling (method used or a statement that no outliers were excluded) for the cell viability data. [Section 2.8]
Pre-specified outlier handling increases transparency and reduces potential for reporting bias.
10.
MEDIUMcopyedit
Fix the major copyedit issues: (1) rephrase the run-on sentence in Section 3.7 cell adhesion description; (2) clarify the viability percentage sentence in Section 2.8; (3) hyphenate 'eggshell-derived' and 'pore-forming' throughout; (4) add Oxford commas in lists; (5) standardize abbreviation to 'ESTHAp' throughout (currently also ESHAp, ESTHAP).
These issues impair readability and professionalism; the run-on sentence and ambiguous phrasing could lead to misinterpretation.
11.
MEDIUMcopyedit
Add figure references (e.g., 'Figure 7 shows...') in the results text where currently missing, and ensure all figures are described in the text. [Results sections 3.1–3.8]
Missing figure references reduce clarity and make it difficult to follow the results narrative.
12.
LOWreporting
Consider referencing a reporting guideline appropriate for in vitro studies (e.g., ARRIVE guidelines) to demonstrate adherence to best reporting practices. [Methods section]
A reporting guideline statement improves transparency and may be required by some journals; its absence was noted by one reviewer.
13.
LOWrigor
Verify the 16 references listed as 'not found in registry' by the citation check; correct or replace any that cannot be confirmed (e.g., by providing the correct DOI or omitting if unverifiable). [Reference list]
Unresolved references are a fabrication signal; while not necessarily erroneous, their verification is a standard integrity check.
14.
LOWcopyedit
Correct the chemical equation in Section 2.3: the product should be H2O + NH3 (not NH4OH) and the coefficient for H2O should be 12, not 6. [Section 2.3, equation (3)]
The current equation is chemically incorrect and may mislead readers about the reaction products.

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