Verified Rigor RecordRigor Reviewed

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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: 72
Author: Αlpha¹

Paper source

Αlpha¹ Editorial

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

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Computational Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: ModerateReviewer agreement: moderateEngine: 7.5.0Source: 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.

12 major claims checked against the paper's own evidence: 1 not fully backed by the presented evidence (unsupported or overstated).

8 integrity concerns flagged (1 high).

Rigor

Eight Dimensions1 pass · 5 warn

12 major claims1 overstated5 partial6 supported

Prior work on natural-sourced HAp is cited (Shih-Ching et al., Aseel and Nihad, Leyla et al., Sandile et al.) with mention of their limitations. The logical rationale from waste utilization to scaffold fabrication is clearly explained. The study acknowledges that porous scaffold nanocomposite fabrication and toxicity studies have 'not been well explored,' addressing limitations of prior research.

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 90% conf

The scientific premise is adequately established: prior work on HAp synthesis from biogenic sources is cited, the rationale for using eggshell waste and ammonium bicarbonate as a pore former is clearly stated, and a gap (limited study of porous scaffold nanocomposite toxicity) is identified.

Evidence

direct quote[Introduction, paragraphs 2-3]

“For instance, Shih-Ching et al. [] reported the formation of HAp phase via ball milling followed by calcinations... Also, Aseel and Nihad [] used calcium oxide from eggshell...”

direct quote[Introduction, paragraph 3]

“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 2]

“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.”

PASSReviewer 2· 90% conf

The paper clearly establishes the scientific need for cost-effective biomaterials from waste eggshells and cites relevant prior work on hydroxyapatite synthesis, though the limitations of prior research are only partially addressed.

Evidence

paraphrase[Introduction, paragraph 2]

“The introduction cites Shih-Ching et al., Aseel and Nihad, Leyla et al., Sandile et al., and Nongnuch et al. for HAp from eggshell and other sources.”

direct quote[Introduction, last paragraph]

“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.”

absence[Introduction]

Limitations of prior work are only cursorily addressed.

PASSReviewer 3· 95% conf

The scientific premise is well established, with prior work cited and a clear rationale linking the use of eggshell-derived hydroxyapatite scaffolds to tissue engineering applications.

Evidence

direct quote[Introduction, paragraph 4]

direct quote[Introduction, paragraph 5]

direct quote[Introduction, final paragraph]

“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”

The study involves preparing different weight percent of AMB (0% control) and sintering at various temperatures; this serves as a design with multiple groups and a control. No randomization, blinding, or power analysis is reported; these are typical for in vitro studies but still count as not_reported. Inclusion/exclusion criteria for cells are not stated. Outlier handling is not described. Distinction between biological and technical replicates is not made. Independent replication is not mentioned. The use of 0% AMB as a control sample is adequate. For this in vitro study, 6 sub-criteria (randomization_method, randomization_unit, blinding_levels, power_analysis, replicate_distinction, independent_replication) are considered not_applicable per the guidelines for interventional (preclinical/in vitro) studies, but the scoring rule requires at least 2 applicable sub-criteria; here inclusion_exclusion, outlier_handling, and controls are scored. Controls is pass, inclusion_exclusion and outlier_handling are not_reported, resulting in a warn status.

Sub-criteria

controlsADEQUATE

power analysisN/A

blinding levelsN/A

outlier handlingNOT REPORTED

randomization unitN/A

inclusion exclusionNOT REPORTED

randomization methodN/A

replicate distinctionNOT REPORTED

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

The study design for this materials science paper lacks several standard methodological elements: no randomization, blinding, power analysis, or pre-specified inclusion/exclusion criteria. The reporting of replicates, controls, and independent replication is inadequate or missing.

Evidence

direct quote[Section 3.7, 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[Section 2.8, 2.9]

Number of experimental replicates not reported for MTT assay or cell adhesion.

absence[Methods, sections 2.8-2.10]

No pre-specified inclusion/exclusion criteria or outlier handling reported.

WARNReviewer 2· 85% conf

The study describes materials synthesis and characterization in detail but lacks essential design elements such as randomization, blinding, a priori power analysis, distinction of biological versus technical replicates, explicit inclusion/exclusion criteria, and outlier handling, and does not report independent replication.

Evidence

absence[Methods]

No randomization method or blinding described.

direct quote[Section 2.11]

“These molded cylindrical samples were brought forward for a compression test, which was conducted on a minimum of five samples per group”

paraphrase[Section 2.8]

“The vitality percentage of cells cultured with 0% AMB ESTHAp pellets and various AMB modified scaffolds was determined by comparing its values to 100% of control cells cultured without ESTHAp pellets.”

WARNReviewer 3· 90% conf

The study design has several strengths, including the use of multiple scaffold formulations and controls, but lacks randomization, blinding, power analysis, and explicit inclusion/exclusion criteria, which are not applicable for an in vitro study; however, other elements like replicate distinction and independent replication are also not addressed.

Evidence

direct quote[Section 3.7, Figure 11a]

“ESTHAp with 0 % AMB was considered as the control sample”

absence[Section 2.8 Assessment of biocompatibility and cell growth]

No explicit inclusion/exclusion criteria for cells are reported.

absence[Section 2.8 and 3.7]

No distinction between biological and technical replicates is provided.

The paper uses MG63 osteosarcoma cell lines obtained commercially. No animal or human subjects are used. The 'Ethics statement' explicitly states: '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.' Therefore, none of the biological variable sub-criteria (sex, age/weight/health, species/strain/source, housing, demographics) are applicable because the study is entirely in vitro with a cell line. Even the cell line source is reported (Sigma, India), but that falls under key_resources, not biological_variables.

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· 95% conf

The study uses an established MG63 osteosarcoma cell line (purchased from Sigma, India) and does not involve animal or human subjects. No biological variables such as age, sex, weight, or health status are relevant for this in vitro/computational study.

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.”

WARNReviewer 2· 85% conf

The study uses a human osteosarcoma cell line (MG63) and reports the cell line source (Sigma, India) but does not report sex of the cell line (which is male-derived MG63 is known, but not stated), nor age/weight/health status (not applicable for a cell line), and no species/strain information beyond the cell line name. Housing conditions and demographics are not applicable.

Evidence

direct quote[Section 2.2 and Section 2.8]

“Uncrushed eggshells were sourced locally from Abeokuta, Ogun State of Nigeria”

direct quote[Section 2.8]

“MG63 osteosarcoma cell lines”

N/AReviewer 3· 95% conf

The study uses a cell line (MG63) and does not involve animals or human subjects with biological variables such as sex, age, or health status; therefore, this dimension is not applicable.

Evidence

direct quote[Section 2.8]

“MTT assay was performed using MG63 osteosarcoma cell lines”

The ethics statement says: '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.' Since the cell line is a commercially available, established line and no new human/animal subjects were used, the dimension is not_applicable. However, the eggshells are a waste product, not animal subjects.

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 only commercially purchased cell lines (MG63 osteosarcoma) and does not involve human participants, animals, or non-public identifiable data. Therefore, no ethics approval is required.

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.”

N/AReviewer 2· 95% conf

The study uses MG63 osteosarcoma cell lines obtained from Sigma, India, and the authors explicitly state they did not use cells from animals or humans. The eggshells are agricultural waste. Thus no human or animal subjects are involved, making ethics approvals 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.”

WARNReviewer 3· 90% conf

The ethics statement mentions simulated cells from Sigma, India, and states no human or animal cells were used, but the study involves a human-derived cell line (MG63), which typically requires ethical oversight; the provided statement is inadequate.

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.”

absence[Ethics statement section]

No IRB or ethics committee approval is mentioned.

Applicability: This is an in vitro study using chemicals, cell lines, and software. Antibodies_identified: not_applicable because antibodies are used only in cell adhesion studies (α-tubulin and Cy3 secondary antibodies) but are mentioned without vendor/catalog (Sigma only for α-tubulin, no RRID). Actually it is applicable: antibodies are used (anti-α-tubulin). The text says 'immunostaining with an antibody against the cytoskeletal marker αtubulin (Sigma)' — vendor only, no catalog, no RRID => reported_but_inadequate. Cell_line_authentication: MG63 cells are from Sigma but not authenticated (no STR) => reported_but_inadequate. Mycoplasma_testing: not reported. Organisms_identified: eggshells from chicken (not explicitly stated as Gallus gallus), source 'locally' => reported_but_inadequate. Reagents_identified: many chemicals (Ca(OH)2, ammonium bicarbonate, (NH4)H2PO4, etc.) are named but not with catalog numbers. The MTT reagent is described with full name but also no catalog. => reported_but_inadequate. Software_tools_identified: Bruker software for XRD, Leica TCS SP2 software for confocal, BD CellquestPro for flow cytometry, but versions not given => reported_but_inadequate. Only one sub-criterion (reagents_identified) might be argued as adequate for simple chemicals, but overall most are inadequate. Pass threshold requires >=60% adequate; none are adequate, so warn.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedINADEQUATE

organisms identifiedINADEQUATE

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 cell line (MG63) and some reagents are named with suppliers, but many reagents lack catalog numbers, RRIDs, or complete details (e.g., antibody clones, primer sequences). Software tools are identified with versions.

Evidence

paraphrase[Section 2.1 (implicitly) and 3.7]

“Paraphrase: MG63 osteosarcoma cell lines were obtained from Sigma, India, but no catalog number or STR authentication is reported.”

direct quote[Section 2.10]

“the cells were stained with antihuman CD29-PE, CD73-PE, and CD44-FITC antibodies (all from BD Pharmingen) or their corresponding isotype-matched controls (Biolegend).”

direct quote[Section 2.5]

“The XRD patterns of all the samples were measured using Bruker D8 Advance X-ray Diffractometer with Cu Kɑ (λ = 1.5405 Å) radiation...”

WARNReviewer 2· 85% conf

The study identifies many key chemicals and reagents by name but lacks vendor/catalog numbers for most (except MTT assay reagents from Sigma, India). The cell line is stated as MG63 from Sigma but without catalog number or authentication. Software tools are mentioned but not with full versioning. No antibodies are identified with RRIDs.

Evidence

direct quote[Section 2.9]

“immunostaining with an antibody against the cytoskeletal marker αtubulin (Sigma)”

absence[Section 2.8]

Cell line MG63 from Sigma but no authentication mentioned.

absence[Methods]

Mycoplasma testing not reported.

WARNReviewer 3· 85% conf

Key chemicals and reagents are mostly identified, but the cell line source and authentication are not detailed, and software tools are identified without versions or RRIDs; the investigational product (HAp-AMB composite) is adequately identified.

Evidence

direct quote[Section 2.8]

“MTT assay was performed using MG63 osteosarcoma cell lines”

direct quote[Section 2.10]

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

direct quote[Section 2.10]

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

No statistical tests are named in the paper. For the MTT assay, only viability percentages (e.g., 'above 85%') are reported without any comparison test (e.g., t-test, ANOVA). For compressive strength, only mean values are given without error bars or statistical comparison. No p-values, effect sizes, or confidence intervals are reported. The statistical software used is not mentioned (BD CellquestPro is used for flow cytometry data, but that is not a general statistical package). Data presentation: figures are included (Fig. 11a shows MTT data, presumably as a bar graph) but individual data points are not shown, and error bars are not defined (SD/SEM). Normality assumptions are not mentioned. The mathematical plausibility of reported means is not checkable because Ns for MTT are not reported per concentration; the compressive strength means are reported without SD. Thus, among the 7 sub-criteria: tests_named (not_reported), assumptions_verified (not_reported), exact_p_values (not_reported), effect_sizes_ci (not_reported), software_identified (not_reported), data_presentation (reported_but_inadequate: figures exist but no individual data points, error bars undefined), mathematical_plausibility (not_applicable: no numerical summary statistics to verify). Out of 7 applicable criteria, 0 adequate, 1 inadequate, 6 not_reported → <30% → fail. However, given that this is a hypothesis-generating materials paper without formal hypothesis testing, a fail is too harsh. I'll treat the dimension as 'not_applicable' because no inferential statistics are performed. The paper only reports descriptive summaries. The scoring rule says: 'n/a no inferential statistics'. The paper uses no inferential statistics, so set status to not_applicable.

Sub-criteria

tests namedNOT REPORTED

exact p valuesNOT REPORTED

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedNOT REPORTED

assumptions verifiedNOT REPORTED

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

The paper reports some statistics (compressive strength, MTT viability percentages) but does not name any inferential statistical tests, verify assumptions, or report exact p-values, effect sizes, or confidence intervals. Software for statistical analysis is not identified. Data presentation uses bar graphs (from figures) but individual data points are not shown.

Evidence

absence[Results and Methods]

No statistical tests are named in the manuscript.

direct quote[Section 3.7]

“viability of above 85 % MG63 cells was achieved except for ESTHAp with 40 % AMB which showed some level of toxicity.”

absence[Results section 3.6, 3.7]

No p-values, effect sizes, or confidence intervals are reported.

WARNReviewer 2· 80% conf

The paper reports some quantitative results (compressive stress values, MTT viability percentages) but does not name any statistical tests performed, does not report p-values, effect sizes, confidence intervals, or test assumptions. The software for statistical analysis is not identified. Data presentation uses bar graphs and textual descriptions but without error bars defined or individual data points shown for most results.

Evidence

absence[Methods, Results]

No statistical tests named.

direct quote[Section 3.6]

“The ESTHAp samples containing 10, 20, 25 and 30 % of AMB have withstood a compressive stress up to 5, 20, 30 and 42 N/mm2 respectively.”

absence[Results]

No p-values or effect sizes reported.

FAILReviewer 3· 80% conf

The statistical analysis is severely lacking: no statistical tests are named, no p-values are reported, no effect sizes or confidence intervals are provided, and the data presentation uses bar graphs from an MTT assay without individual data points or defined error bars.

Evidence

absence[Section 3.7]

No statistical tests are named for any comparison.

direct quote[Section 3.7]

“viability of above 85 % MG63 cells was achieved except for ESTHAp with 40 % AMB which showed some level of toxicity.”

absence[Section 3.7]

No individual data points or error bars are described.

The data availability statement says 'Data will be made available on request' without specifying a repository, platform, or conditions. This is a vague statement, rated as reported_but_inadequate. No repository deposit, accession numbers, or code sharing are mentioned. For this type of in vitro study, data availability is applicable, and code sharing is not applicable as no custom code is reported. The inadequate statement leads to a warn status.

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'), lacking a concrete mechanism or repository. No code sharing is applicable. No repository deposit or accession numbers are reported.

Evidence

direct quote[Data and code availability section]

“Data will be made available on request.”

absence[Data and code availability section]

No raw data repository deposit or accession numbers are provided.

WARNReviewer 2· 90% conf

The paper includes a data availability statement that is vague ('Data will be made available on request') without specifying a repository or access mechanism. No data or code is deposited. No accession numbers are provided.

Evidence

direct quote[Data and code availability section]

“Data will be made available on request.”

absence[Data and code availability section]

No repository deposit mentioned.

WARNReviewer 3· 95% conf

A data availability statement is present but vague ('Data will be made available on request'), which is inadequate; no code sharing statement or repository deposit is provided.

Evidence

direct quote[Data and code availability section]

“Data will be made available on request.”

Methods for synthesis, characterization, and cell assays are detailed enough for replication. No trial registration is applicable. No reporting guideline (e.g., ARRIVE) is mentioned. The limitations are noted but superficial (only one limitation mentioned). The conclusion is fairly proportional but overclaims on biocompatibility without statistical support. Funding and COI are reported. All outcomes mentioned in methods (MTT, adhesion) are reported, but results are presented without statistical analysis. This results in 2 of 5 applicable sub-criteria adequate (methods_completeness, funding_coi), giving a warn status.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedINADEQUATE

limitations discussedINADEQUATE

conclusions proportionalINADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

The paper is generally transparent in methods and limitations, but lacks trial registration (n/a), a reporting guideline check, and does not report all outcomes (cell adhesion data described as unstable with no cells observed, yet conclusion emphasizes biocompatibility). Funding and COI are reported.

Evidence

direct quote[Section 3.7, paragraph 2]

“the inverted microscopic images of MG 63 cells incubated with ESTHAp samples and treated with heat at 1000 °C appears to be unstable in the media used with particle leaching observed, and no cells observed near to the samples.”

direct quote[Conclusion]

“The (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay results showed that samples of scaffold eggshell derived hydroxyapatite showed higher degree of viability which suggests the biocompatibility of the samples even up to a weight percent of ammonium bicarbonate of 30 %.”

direct quote[Section 3.8]

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

WARNReviewer 2· 85% conf

The methods are quite detailed for synthesis and characterization, but not for some aspects (e.g., no statistical analysis plan, no trial registration needed, no reporting guideline referenced). Limitations are discussed briefly (only one limitation). Conclusions are generally proportional. Funding and conflict of interest are reported.

Evidence

direct quote[Section 3.8]

“This study focused on the use of eggshell as the main source material for the synthesis of the prepared hydroxylapatite... future study could focused on the use of some other biological origins”

absence[Methods]

No reporting guideline referenced.

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”

WARNReviewer 3· 90% conf

Methods are described in sufficient detail for replication, limitations are briefly discussed, and funding/COIs are reported; however, no trial registration or reporting guideline is used, and not all outcomes are transparently reported (e.g., cell adhesion results are negative).

Evidence

direct quote[Section 3.8 Limitation of the study]

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

direct quote[Funding and Declaration of competing interest]

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

absence[Entire paper]

No reporting guideline is referenced.

23 copyedit issues flagged (1 major): mostly typo, grammar, clarity.

Checked 40 references: 23 verified — 17 unresolved.

Full text (36,404 chars)Analyzed: Jul 6, 2026

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