[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
^1Department of Nephrology, Jiangnan University Medical Center, Wuxi, China ^2Department of Nephrology, Shanghai General Hospital of Nanjing Medical University, Shanghai, China ^3Department of Neonatology, The Affiliated Wuxi Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, China ^4Department of Pediatric Laboratory, The Affiliated Wuxi Children's Hospital of Jiangnan University (Wuxi Children's Hospital), Wuxi, China ^5Department of Cardiology, Jiangnan University Medical Center, Wuxi, China ^6Department of Laboratory Medicine, Wuxi No. 5 People's Hospital, Wuxi, China ^7Institute of Metabolism and Regenerative Medicine, Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China ^8Department of Medicine, University of Verona, Verona, Italy ^9IRCCS Sacro Cuore – Don Calabria Hospital, Negrar di Valpolicella, Italy ^10Southampton National Institute for Health and Care Research Biomedical Research Centre, University Hospital Southampton, Southampton General Hospital, Southampton, UK ^11MAFLD Research Center, Department of Hepatology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China ^12Institute of Hepatology, Wenzhou Medical University, Wenzhou, China ^13Key Laboratory of Diagnosis and Treatment for The Development of Chronic Liver Disease in Zhejiang Province, Wenzhou, China

Experimental evidence suggests that dyslipidemia may induce lipotoxicity, low-grade inflammation, and increased oxidative stress in renal tissues [1–4], thereby contributing to the development of both glomerulosclerosis and tubulo-interstitial fibrosis [5]. Dyslipidemia is

Results: Mice were fed by a 60% kcal fat diet for 20 weeks developed the typical traits of metabolic syndrome with subsequent renal lipid accumulation and renal injury. Treatment with the FXR agonist CDCA or GW4064 decreased body weight, renal lipid accumulation, as well as renal injury. Moreover, renal β-catenin signaling was activated and improved with FXR-agonist treatment in HFD-fed mice. To examine whether FXR affected β-catenin signaling, and was involved in tubulo-interstitial fibrosis, we explored the FXR expression and function in ox-LDL induced-renal tubular injury. In rat proximal tubular epithelial cells (NRK-52E) stimulated by ox-LDL, FXR protein was decreased compared to control group, and phosphorylated (Ser675) β-catenin was activated by ox-LDL in a dose- and time-dependent manner. Ox-LDL enhanced α-SMA and fibronectin expressions and reduced E-cadherin levels, whereas FXR agonism or FXR overexpression inhibited fibronectin and α-SMA expressions and restored E-cadherin. Moreover, FXR agonist treatment also decreased phosphorylated (Ser675) β-catenin, nuclear translocation and β-catenin-mediated transcription induced by ox-LDL in NRK-52E cells. We showed that FXR could bind with β-catenin via the AF1 domain, and disrupt the assembly of the core β-catenin/TCF4 complex.

not only a cause of kidney injury, but also a consequence of chronic kidney disease (CKD), especially in nephrotic syndrome [6]. Consequently, treatment of lipid-mediated kidney injury is clinically important.

Conclusion: These experimental data suggest that FXR activation, via modulating β-catenin signaling, may contribute to attenuating the development of lipid-mediated tubulo-interstitial fibrosis.

Methods: We investigated the effects of HFD on mouse kidney and renal tubular epithelial cells both in vivo and in vitro, and observed the changes of FXR and β-catenin pathway. FXR agonist was also used to alleviate this HFD-induced effect, and the interaction between FXR and β-catenin was further verified.

CKD causes major alterations in plasma lipoprotein profile, by inducing high levels of triglycerides, very low-density lipoprotein (VLDL) and

Objective: Activation of farnesoid X receptor (FXR), a bile acid nuclear receptor, may be implicated in the pathophysiology of diabetic nephropathy. We explored a possible role for FXR activation in preventing renal fibrosis in high fat diet (HFD)-fed mice.

Keywords: Bile acid; FXR; β-Catenin; Tubular epithelial cells; Renal fibrosis

Received September 19, 2023 • Revision received October 18, 2023 • Accepted November 22, 2023 • Available online 28 November 2023

Dan-Qin Sun^1,2,14, Fuqiang Yuan^3,4,14, Meng-Zhu Fu^5, Meng-Yang Zhong^1, Shi-Liang Zhang^6, Yan Lu^7, Giovanni Targher^8,9, Christopher D. Byrne^10, Ming-Hua Zheng^11,12,13,**, Wei-Jie Yuan^2,*

https://doi.org/10.1016/j.molmet.2023.101841

Dan-Qin Sun

© 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

oxidized low-density lipoprotein (ox-LDL), as well as low levels of high-density lipoprotein cholesterol [7]. Increased ox-LDL levels have been shown to cause renal injury through multiple mechanisms. Previous experimental studies have focused not only on the role of macrophage cells and endothelial dysfunction, but also on the role of ox-LDL in promoting glomerular sclerosis and tubulo-interstitial fibrosis [8,9]. It has been reported that ox-LDL may induce tubulo-interstitial fibrosis possibly through lysyl oxidase (LOX)-1-related oxidative stress via the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways [10].

Renal tubulo-interstitial fibrosis is typically characterized by accumulation of extracellular matrix (ECM) in the interstitial space, thus leading to destruction of renal parenchyma [11,12]. Studies showed that the tubular epithelial-to-mesenchymal transition is a fundamental process for driving renal fibrosis development [11]. Previous studies also showed that the β-catenin pathway is an evolutionarily conserved, developmental signaling pathway that regulates multiple biological processes. Among these, the activation of β-catenin signaling may induce tubular epithelial–mesenchymal transition and kidney fibrosis after injury [12].

Specifically, once initiated by its upstream activator, β-catenin is stabilized and binds to the T cell factor/lymphoid enhancer-binding factor to trigger transcription of β-catenin target genes, which are involved in regulating cell proliferation, stem cell maintenance, or targeting genes associated tubular epithelial–mesenchymal transition and fibrogenesis, such as fibronectin, E-cadherin and Snail [13]. In addition, it is also possible to hypothesize that inhibiting the activity of β-catenin signaling could become a new therapeutic option to delay CKD progression. However, to our knowledge, there is currently limited research on the β-catenin signaling pathway and the mechanisms of ox-LDL-induced injury in renal tubular epithelial cells, and it remains still unclear whether treatment can reduce this ox-LDL-induced kidney injury.

The farnesoid X receptor (FXR) is a nuclear bile acid receptor and transcription factor playing a key role in bile acid homeostasis, and lipid and glucose metabolism, with chenodeoxycholic acid (CDCA) being the most potent endogenous ligand. In addition, GW4064 is a highly effective and selective nonsteroidal agonist of FXR, which is usually used as a tool compound for investigating the physiological functions of FXR. Furthermore, obeticholic acid (OCA) is a semisynthetic derivative of CDCA, also known as INT-747, which was developed for the treatment of various liver diseases, including biliary atresia and non-alcoholic steatohepatitis [14,15]. FXR is mostly expressed in the liver and intestine, as well as widely in non-classical bile acid target tissues such as the kidneys [16]. However, it is becoming increasingly clear that FXR can also play a role in renal physiology and renal diseases [17]. FXR may be involved in the progression of many kidney diseases, including acute kidney injury and diabetic nephropathy, by regulating fatty acid oxidation, inflammation, apoptosis, fibrosis and other signaling pathways [18–20]. Our previous study also reported a possible molecular mechanism of FXR's involvement in lipid-induced kidney injury [4]. Experimentally, it has been reported that FXR activation reduces markers of extracellular matrix, i.e., TGF-β, Smad3, but the precise mechanisms for these effects are poorly known [21,22]. Furthermore, it has been reported that FXR is associated with β-catenin and may inhibit its activity in hepatocellular carcinoma [23]. Apart from its role in hepatocellular carcinoma development, whether FXR activation may also improve ox-LDL-induced kidney fibrosis, possibly through the β-catenin signaling pathway, is currently not known.

To test this experimental hypothesis, different strategies of FXR activation (either via GW4064, chenodeoxycholic acid [CDCA] and INT-747 [OCA], or via a FXR plasmid) were used both in proximal tubular epithelial cells (NRK-52E) and in high-fat diet (HFD)-fed mice.

The NRK-52E cells were purchased from the China Center for Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone; GE healthcare) with 5% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 100 U/mL penicillin/streptomycin (NCM Biotech, Suzhou, China) in a 5% CO2 atmosphere at 37°C. GW4064 (Cat. No. S2782, Selleck Chemicals LLC, Houston, USA) and CDCA (Cat. No. HY-76847, MedChemExpress, Monmouth Junction, USA) were dissolved in dimethyl sulfoxide (DMSO) and kept the final DMSO concentration below 0.1%. For lipid treatment, the NRK-52E cells were seeded at ~70% confluence in complete medium with 5% FBS and then serum-starved for 12 h, followed by ox-LDL treatment (Yiyuan Biotechnology, Guangzhou, China) for various times. For some experiments, after serum starvation, cells were either treated with FXR agonists or ICG-001 (β-catenin inhibitor, Cat. No. S2662, Selleck Chemicals LLC, Houston, USA) for another 24 h.

The construct pCMV3-C-flag-β-catenin (pβ-catenin) was purchased from SinoBiological (Beijing, China). The plasmids of pcDNA3 and pcDNA3-flag-FXR (pFXR) were purchased from Zoonbio Biotechnology (Nanjing, China). The coding region of Rattus FXR (GI:11120689) gene was amplified and cloned into pcDNA3-flag vector. All plasmids were identified by sequencing. We also constructed the negative control siRNA, siRNA oligo duplexes targeting FXR and β-catenin (Tsingke Biotechnology Co., Ltd) for transient knocking down gene expression using Lipofectamine 8000.

For transfection, NRK-52E cells were transfected with pFXR and pβ-catenin construct or FXR siRNA with or without β-catenin siRNA using Lipofectamine 8000 (Invitrogen; Thermo Fisher Scientific, Inc.) and Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.). The pcDNA3 construct (Invitrogen) or control siRNA was transfected as a vehicle control. Dual luciferase reporter assay was performed using a dual-luciferase reporter assay system kit (Promega) according to the instruction manuals. The β-catenin transcriptional activity was determined using the TOPflash reporter system, which contains the thymidine kinase (TK) minimal promoter and two sets of triplicate TCF binding sites upstream of the luciferase open reading frame (Millipore). NRK-52E cells were co-transfected with the TOPflash plasmid (1 μg) and the FXR-expression vector or the empty vector pcDNA3 or pβ-catenin or FXR siRNA or β-catenin siRNA, and together with the internal control reporter plasmid (Renilla reniformis luciferase, 0.1 μg). After 6 h, ox-LDL was treated for 24 h. Then, the cells were lysed for luciferase assay.

RIPA lysis buffer (Beyotime Institute of Biotechnology) containing PMSF (Beyotime Institute of Biotechnology) and phosphatase inhibitors (NCM Biotech) was used for cell lysis and protein extraction. Protein was determined by NanoDrop (Thermo Fisher Scientific, Inc.) and mixed with SDS-PAGE sample buffer and separated by SDS-PAGE. The following antibodies were used in the study: anti-E-cadherin (Cat. No. 14472; Cell Signaling), anti-phospho-β-catenin (Ser675) (Cat. No. 9567; Cell Signaling), anti-phospho-β-catenin (Ser33/37/Thr41) (Cat. No. 9561; Cell Signaling), anti-β-catenin (Cat. No. 610154; BD Transduction Laboratories, San Jose, CA), anti-TATA-binding protein

(TBP) (AB181-100, Abcam), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (abs132004; Absin Bioscience), anti-FXR (sc-25309, Santa Cruz; Cat No: 25055-1-AP, Proteintech), anti-Snail (ab17732, Abcam), anti-Fibronectin (ab2413, Abcam), and anti-α-SMA (A5228, Sigma Aldrich), respectively.

NRK-52E cells were washed twice with pre-chilled PBS, and nuclear proteins were extracted using nuclear and cytoplasmic extraction reagents according to the manufacturer’s procedures (Thermo Scientific, Rockford, IL, USA). After centrifugation, the cell pellets were re-suspended in buffer A and lysed with homogenizer. The cell nuclei were collected by centrifugation at 5000 rpm for 15 min and washed with buffer B. The nuclei were lysed in SDS sample buffer. TBP was used as loading control.

NRK-52E cells were washed twice with pre-chilled PBS and then lysed on ice by adding 1 mL of native lysis buffer (1% Triton X-100, 1% protease inhibitors cocktail and 1% phosphatase inhibitors cocktail I and II (NCM Biotech)). After pre-washing with IgG, cell lysates were incubated with 4 μg anti-β-catenin or anti-FXR for 2 h at room temperature and then precipitated with 30 μL protein A/G plus agarose beads. Precipitated complexes were analyzed by Western blotting for FXR and β-catenin detection.

Twenty-four male C57 BL/6J mice (5 weeks, weight 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Company. These mice were maintained in the animal center of Shanghai General Hospital under standard conditions (temperature: 24 °C; humidity: 40–70%; 12/12 h of light/dark cycle and air exchange). After 3 weeks, the mice were randomly divided into 4 groups: a 10% fat diet group (Control group, n = 6) (Cat. MD12031, medicience. Ltd); a 60% high fat diet (HFD) group (Cat. MD12033, medicience. Ltd) (n = 6); a HFD group with 0.1% CDCA (n = 6) (Cat. No. HY-76847, MedChemExpress) (Cat. MDDZ515, medicience. Ltd); and a 30 mg/kg INT-747 group (n = 6) (Dalian Meilun Biotechnology Co., Ltd) (Cat. MDDZ516, medicience. Ltd). A high fat diet (HFD) usually includes 60 kcal % fat, 20 kcal % protein, and 20 kcal % carbohydrate, respectively [24]. At 15 weeks of age, one mouse in the HFD group was found dead in a cage. The cause of death was uncertain, but the hair on the head and back of several mice in the cage was gone. The dead mouse was more seriously affected. We speculated that the mouse in the cage had been involved in a violent incident.

After a 12-week treatment, all mice were euthanized by intraperitoneal injection of sodium pentobarbital (100 mg/kg) and samples were harvested for further determinations, including Oil Red O staining, immunohistochemistry and Western blot analyses. Urine specimens were collected on Monday morning of week 20. Urinary albumin-to-creatinine ratio (ACR) was measured using a fully automatic biochemical analyzer (Rayto Life and Analytical Sciences Co. Ltd). Biochemical measurements including blood urea nitrogen (BUN), serum creatinine, albumin, alanine aminotransferase (ALT), total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-c) were measured using an automated analyzer (Abbott AxSYM), according to the standard laboratory methods. All mice were treated in accordance with animal protection guidelines and experiment procedures were approved by the guidelines of the ethics committee of the Shanghai General Hospital.

Kidney cryosections (3 μM) were fixed with 4% paraformaldehyde for 15 min, then blocked with blocking buffer for 1 h at room temperature followed by β-catenin (#37447S, Cell Signaling) incubation for 10–12 h at 4 °C.

For the immunocytofluorescence, NRK-52E cells were seeded on coverslips, fixed with methanol and acetone (1:1) at –20 °C for 10 min, blocked with 20% normal donkey serum for 30 min at room temperature, and then mixed with anti-E-cadherin. Primary antibody incubation was made for fibronectin, α-SMA, FXR or β-catenin. Afterwards, NRK-52E cells were stained with Cy3- or Cy2-conjugated secondary antibodies for 30 min. Cells were also double-stained with 4′,6-diamino-2-phenylindole-hydrochloric acid (DAPI, P0131, Beyotime, Shanghai, China) to visualize nuclei. Finally, cells were observed under a Leica fluorescence microscope.

Renal specimens were fixed overnight with 4% paraformaldehyde at 4 °C, then paraffin-embedded and 3-micron sections were prepared. Masson staining and Sirius red staining were performed according to manufacturer’s instructions. In each group, each slice was randomly selected with at least three 100-fold visual fields to be photographed, and calculated the positive areas of Masson staining and Sirius red staining. For immunohistochemical staining, antigen retrieval was performed at 96 °C for 10 min in nitrate solution. Then, sections were blocked with 3% bovine serum albumin (Cat. No. A8020; Solarbio Science & Technology) for 30 min at room temperature, followed by anti-β-catenin, α-SMA or fibronectin Connexin primary antibody incubation overnight at 4 °C. Finally, slides were incubated with secondary antibody, washed and developed with DAB (Cat. No. K5007; Dako; Agilent Technologies).

All statistical analyses were performed using SPSS statistics version 22.0 (IBM SPSS Statistics. Armonk, NY, USA). One-way analysis of variance (1-way ANOVA) was used for multiple group comparisons. The Student-Newman-Keuls post-hoc test was used for post-hoc analyses. A p-value <0.05 was deemed to indicate statistical significance.

FXR protein expression was decreased in NRK-52E cells after 50 μg/mL ox-LDL treatment, as confirmed by Western blot analyses (Figure 1A and B). Immunofluorescence staining showed nuclear localization of FXR in basal conditions, whereas the staining for FXR stimulated by ox-LDL decreased after 6 h (Figure 1C). Immunofluorescence staining also showed nuclear localization of β-catenin incubated with ox-LDL (50 μg/mL) for 6 h, whereas β-catenin was located in cell–cell junction and cytoplasm under basal circumstance (Figure 1D). It has been reported that phosphorylation β-catenin at Ser33/37 promotes its degradation while phosphorylation at Ser675 induces β-catenin accumulation in the nucleus and increases its transcriptional activity [25]. Figure 1A and B showed that an ox-LDL-stimulated β-catenin activation in NRK-52E cells, as revealed by upregulation of phosphorylated (Ser675) β-catenin and down-regulation of phosphorylated (Ser33/37) β-catenin in a dose and time dependent manner.

Figure 2A and B showed that ox-LDL (50 μg/mL) induced the biomarkers of myofibroblasts (such as fibronectin, α-SMA and Snail), and suppressed the expression of E-cadherin after 24 h. Then, treatment with FXR agonists (either GW4064 or CDCA) attenuated the ox-LDL-induced expression of α-SMA and Snail at different dosages. Quantitative determination revealed a 1.3-fold induction of fibronectin protein stimulated by ox-LDL, then treatment with CDCA attenuated the induction in different dosages (Figure 2D). Moreover, for determining the function of FXR induction, FXR was overexpressed by transient transfection with the expression vector of flag-tagged FXR in NRK-52E cells. We found that FXR overexpression inhibited fibronectin expression and improved E-cadherin expression induced by ox-LDL (Figure 2C and E). Immunofluorescence staining showed that ox-LDL led to a weakening of E-cadherin expression in plasma membranes and an increase in fibronectin and α-SMA expression (Figure 2F–H). Treatment with FXR agonists (either CDCA or GW4064) also improved the ox-LDL-induced tubular fibrosis. These observations suggest that ox-LDL was able to trigger tubulo-interstitial fibrosis, but activation of FXR expression ameliorated the fibrosis in tubular epithelial cells in vitro.

In order to assess the requirement for inhibition of β-catenin signaling to decrease fibronectin induction by ox-LDL, we inhibited β-catenin function with the antagonist ICG-001. The NRK-52E cells were treated with ICG-001 (10 μM), followed by ox-LDL incubation. As shown in Figure 3A and B, ICG-001 induced inhibition of β-catenin signaling and attenuated 1.6-fold fibronectin production by ox-LDL, which was similar to the effect of inhibition of this pathway by FXR activation.

To determine the possibility that activation of FXR expression repressed ox-LDL-induced fibrosis in NRK-52E via inhibiting β-catenin signaling, we first investigated whether FXR activation inhibited the phosphorylation (Ser 675) of β-catenin. In these experiments, we found that FXR agonist inhibited the phosphorylation (Ser 675) of β-catenin in a dose-dependent manner (Figure 3C). Overexpression of FXR also inhibited the expression of phosphorylation (Ser 675) of β-catenin (Figure 3D). Moreover, we also investigated the intracellular distribution of β-catenin in tubular epithelial cells, by scrutinizing β-catenin subcellular localization incubated with ox-LDL after treatment with FXR agonists. The alteration of FXR activation did not affect cellular β-catenin levels, but ox-LDL promoted β-catenin translocation into the nuclei and GW4064 decreased β-catenin expression in the nuclei (Figure 3E and F). We also explored the β-catenin-mediated transcriptional activity in a TOPflash luciferase reporter system, which demonstrated that exogenous FXR inhibited the 1.7-fold transcriptional activity induced by ox-LDL (Figure 3G). Then, NRK-52E cells were co-transfected with TOPflash reporter plasmid and either FXR siRNA with or without β-catenin siRNA, which confirmed that FXR siRNA could increase 1.3-fold β-catenin transcriptional activity; then, co-transfected with β-catenin siRNA, FXR siRNA did not increase the β-catenin transcriptional activity (Figure 3H–J). These data suggest that

FXR is a key factor for the stabilization and nuclear translocation of β-catenin mainly via modulating β-catenin phosphorylation. Since the nuclear translocation of β-catenin is a fundamental process for controlling its target gene transcription in the nucleus that causes tubular fibrosis, our results also suggest that activation of FXR expression can repress ox-LDL-mediated fibrosis in tubular epithelial cells mainly via inhibiting β-catenin signaling.

Using immunofluorescent staining, we showed that FXR and β-catenin were co-localized in the nucleus in ox-LDL-stimulated NRK-52E cells

(Figure 4A). The interaction between FXR and β-catenin was confirmed by co-immunoprecipitation. In NRK-52E cells, we examined the interaction of FXR with β-catenin in the basal state, after treatment with GW4064, and also after treatment with GW4064 incubated with ox-LDL (Figure 4B and C). Expression of FXR, pDEL-FXR and pβ-catenin were confirmed by Western blotting with anti-flag (Figure 4D and E).

To explore the role of FXR interaction with β-catenin in ox-LDL-mediated β-catenin transcription activity, the indicated constructs were transfected and the TOPflash signal was determined (Figure 4F). Indeed, β-catenin overexpression restores the decrease in signaling caused by FXR under ox-LDL treatment. Notably, FXR overexpression is sufficient to reduce the TOPflash signal while this could be partially

## A

| ox-LDL (μg/ml) | -           | + | + | + |
| -------------- | ----------- | - | - | - |
| GW4064 (μM)    | -           | - | - | + |
| ICG-001 (μM)   | -           | - | + | - |
| 285-           | Fibronectin |   |   |   |
| 36-            | GAPDH       |   |   |   |

## B

```mermaid
graph TD
A[DMSO] --> B[1.0]
C[DMSO+ox-LDL] --> D[2.0]
E[ICG-001+ox-LDL] --> F[1.25]
G[GW4064+ox-LDL] --> H[1.25]
```

## C

| ox-LDL (μg/ml) | -                    | + | + | + | + |
| -------------- | -------------------- | - | - | - | - |
| GW4064 (μM)    | -                    | - | 1 | 2 | 5 |
| 100-           | p-β-catenin (Ser675) |   |   |   |   |
| 100-           | β-catenin            |   |   |   |   |
| 36-            | GAPDH                |   |   |   |   |

## D

|        | pcDNA3               |   | pFXR |   |
| ------ | -------------------- | - | ---- | - |
| ox-LDL | -                    | + | -    | + |
| 100-   | p-β-catenin (Ser675) |   |      |   |
| 100-   | β-catenin            |   |      |   |
| 36-    | GAPDH                |   |      |   |

## E

| ox-LDL (μg/ml) | -         | + | + |
| -------------- | --------- | - | - |
| GW4064 (μM)    | -         | - | + |
| 100-           | β-catenin |   |   |
| 36-            | GAPDH     |   |   |

## F

| ox-LDL (μg/ml) | -         | + | + |
| -------------- | --------- | - | - |
| GW4064 (μM)    | -         | - | + |
| 100-           | β-catenin |   |   |
| 45-            | TBP       |   |   |

## G

```mermaid
graph TD
A[pcDNA3] --> B[1.0]
C[pcDNA3+ox-LDL] --> D[2.0]
E[pFXR+ox-LDL] --> F[1.25]
```

## H

|     | control siRNA | FXR siRNA |
| --- | ------------- | --------- |
| 50- | FXR           |           |
| 36- | GAPDH         |           |

## I

|      | control siRNA | β-catenin siRNA |
| ---- | ------------- | --------------- |
| 100- | β-catenin     |                 |
| 36-  | GAPDH         |                 |

## J

```mermaid
graph TD
A[control siRNA] --> B[1.0]
C[FXR siRNA] --> D[1.4]
E[β-catenin siRNA] --> F[1.0]
G[FXR siRNA + β-catenin siRNA] --> H[1.0]
```

Figure 3: Overexpression of FXR inhibits activation of β-catenin signaling in tubular epithelial cells. (A–B) ICG-001 and GW4064 inhibit fibronectin expression induced by ox-LDL. NRK-52E cells were pretreated with ICG-001 10 μM or GW4064 5 μM for 30 min, then incubated with ox-LDL (50 μg/mL) for 24 h. (C) After pretreatment with various concentrations of GW4064 for 30 min, NRK-52E cells were incubated with 50 μg/mL ox-LDL for 24 h. The protein level of phospho-β-catenin (Ser675) was examined by Western blot analysis. (D) Ectopic expression of FXR decreases phospho-β-catenin (Ser675) induced by ox-LDL. (E–F) Ox-LDL promotes β-catenin translocation into the nuclei and GW4064 decreases β-catenin expression in the nuclei. (G) NRK-52E cells were co-transfected with TOPflash reporter plasmid and either pcDNA3 or pFXR vector, followed by incubation with or without 50 μg/mL ox-LDL. (H–I) Specific knockdown of FXR and β-catenin with siRNA oligo duplexes was confirmed by western blot in NRK-52E. (J) NRK-52E cells were co-transfected with TOPflash reporter plasmid and either control siRNA, FXR siRNA or β-catenin siRNA. Down-regulation of FXR in NRK-52E cells promotes β-catenin mediated transcriptional activity. Relative luciferase is reported (n = 6) and values shown are means ± S.E.M, *p < 0.05.

MOLECULAR METABOLISM 79 (2024) 101841 © 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com

Figure 4: FXR/β-catenin interaction is induced in ox-LDL stimulated tubular epithelial cells. (A) Co-localization of FXR and β-catenin in NRK-52E cells. NRK-52E cells were double stained with anti-FXR antibody and β-catenin antibody. (B–C) Association of FXR with β-catenin was confirmed by in vitro co-immunoprecipitation. Immunoprecipitation was carried out in NRK-52E cell lysates using anti-FXR antibody or anti-β-catenin, followed by IgG agarose incubation. After washing, bound β-catenin or FXR was detected by Western blot. (D–E) Overexpression of pFXR, pDEL-FXR and pβ-catenin in NRK-52E cells were confirmed by Western blotting with anti-Flag antibody. (F) NRK-52E cells were co-transfected with TOPflash reporter plasmid and either pcDNA3, pFXR vector or pβ-catenin, followed by incubation with or without 50 μg/mL ox-LDL. (G) Diagram shows FXR functional domains, including the AF-1 domain, the DNA binding domain (DBD), the hinge domain, the ligand binding domain (LBD), and the AF-2 domain. (H) The AF1 domain of FXR was required for suppression of β-catenin mediated transcriptional activity. NRK-52E cells were transfected with TOPflash reporter, β-catenin and pFXR, pDEL-FXR. Luciferase activity was measured using cell lysates 24 h after transfection. Relative luciferase is reported (n = 3) and values shown are means ± S.E.M, *p < 0.05.

MOLECULAR METABOLISM 79 (2024) 101841 © 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).  7
www.molecularmetabolism.com

The FXR structure is characterized by a N-terminal AF1 domain, a DNA binding domain (DBD), a hinge domain, a ligand binding domain (LBD), and a C-terminal AF2 domain, respectively (Figure 4G). To identify the specific domain, which could mediate the interaction with β-catenin and is potentially involved in its inhibitory effect on β-catenin transcriptional activity, we constructed a Flag tagged full length FXR, a AF1 domain deletion mutant defined as pDEL-FXR (124–469), and β-catenin pFXR and FXR deletion mutants were co-transfected with pβ-catenin and TOPflash reporter in NRK-52E cells. We found that the full length FXR could inhibit β-catenin mediated transcription activity, while the pDEL-FXR without the N-terminal AF1 domain did not have any effect on β-catenin mediated TOPflash reporter activity (Figure 4H). These results indicate that the N-terminal AF1 domain in FXR is required for its inhibitory activity.

Compared to mice treated with control diet, HFD-fed mice had higher body weight (49.01 ± 1.89 g vs. 27.59 ± 1.62 g), higher total cholesterol (5.73 ± 0.65 vs. 2.55 ± 0.64 mmol/L), higher LDL-cholesterol (0.46 ± 0.11 vs. 0.19 ± 0.07 mmol/L), and had more severe renal injury, with a remarkable increase in albuminuria (37.1 ± 3.79 vs. 23.24 ± 4.77 mg/g), serum creatinine (4.8 ± 1.3 vs. 1.11 μmol/L) and BUN levels (10.7 ± 1.81 vs. 7.5 ± 1.39 mmol/L). Treatment with CDCA or INT-747 improved albuminuria and decreased levels of body weight, serum ALT, BUN, creatinine and lipids (Figure 5). In addition, Oil red O staining showed that HFD-fed mice also had greater kidney lipid accumulation in the glomeruli and tubulo-interstitium, which was improved by treatment with either CDCA or INT-747 (Figure 6A).

Furthermore, we also examined the expression of β-catenin, fibronectin and α-SMA in the kidneys of HFD-fed mice treated by either CDCA or INT-747 in order to examine the FXR-mediated inhibition of β-catenin in renal fibrosis (Figure 6B–C). The Masson’s staining and Sirius red staining revealed that treatment with either CDCA or INT-747 decreased tissue fibrosis in HFD-fed mice (Figure 6D–F). As shown in Figure 7A–C, we examined β-catenin protein expression and localization in 20-week mice. Immunohistochemical staining analysis showed that positive β-catenin staining was markedly increased in renal tubular cells of HFD-fed mice compared to the control mice, and β-catenin was predominantly localized in the nuclei of tubular cells in HFD-fed mice (Figure 7D–E, arrowheads). Western blot analysis also showed an increase in phosphorylated β-catenin (Ser675) expression (Figure 8A). Treatment with either CDCA or INT-747 decreased positive β-catenin levels in immunohistochemical staining, and the expression of phosphorylated β-catenin (Ser675) was decreased in CDCA or INT-747 treatment groups. In addition, immunohistochemical staining and Western blot analyses showed that FXR activation led to a reduction in fibronectin and α-SMA expressions. Compared to control mice, quantitative determination revealed a ~5-fold induction of fibronectin protein, ~4-fold induction of the relative abundance of phosphorylated β-catenin (Ser 675) protein, and 1.3-fold induction of α-SMA protein in HFD mice (Figure 8B–D). Moreover, treatment with FXR agonists attenuated these protein inductions. These data indicated that FXR activation may protect against renal fibrosis in HFD-fed mice possibly by inhibiting the β-catenin pathway.

Our experimental data show that ox-LDL represses the expression of the epithelia marker E-cadherin, and induces the expression of fibronectin and α-SMA, as markers of myofibroblasts. Conversely, FXR activation leads to induction of E-cadherin and repression of fibronectin and α-SMA levels stimulated by ox-LDL, thereby confirming that ox-LDL is able to trigger tubulo-interstitial fibrosis, but activation of FXR expression ameliorates the fibrosis in tubular epithelial cells in vitro. In addition, ox-LDL decreases FXR abundance in the nuclei and increases β-catenin phosphorylation and nuclear localization, thus activating the β-catenin signaling pathway. FXR activation inhibits the β-catenin phosphorylation and nuclear localization, thereby reducing the activation of β-catenin signaling and its target gene expression (Figure 8E–F). In a mouse model of HFD-induced nephropathy, we have also shown that treatment with FXR agonists reduces body weight, improves plasma lipid profile and albuminuria, and represses the expression of fibronectin, α-SMA and β-catenin phosphorylation. Overall, these results suggest that FXR may interact with β-catenin, regulating β-catenin signaling activity, and thereby participating in the development of renal fibrosis.

To date, growing evidence supports a role of dysregulated lipid metabolism in the pathogenesis of renal disease [30–32]. Increased lipid metabolites may induce the release of multiple pro-inflammatory and pro-fibrotic cytokines and chemokines, and promote the expression of renal extracellular matrix, inflammation and fibrosis, thereby contributing to the development and progression of CKD [33]. Lipotoxicity may also adversely affect renal lipid metabolism, leading to increased production of proteins involved in lipid synthesis, while decreasing those involved in lipid oxidation. When excess free fatty acids are not oxidized, they are esterified with glycerol and become intra-renal lipid droplets, which may induce inflammation and cell apoptosis, thus further aggravating CKD [34].

FXR is a ligand activated transcription factor that is involved in the regulation of bile acid metabolism [26]. It has been reported that FXR agonists may also improve proteinuria, as well as podocyte injury, mesangial expansion and tubulo-interstitial fibrosis in both diabetic DBA/2J and db/db mice [27]. However, the precise mechanisms by which FXR activation may improve renal fibrosis require better understanding. In our experimental study, we showed that FXR expression was downregulated in ox-LDL-mediated injury of tubular cells, and that FXR over-expression inhibited ox-LDL-mediated renal injury. Consistently, a previous study reported that FXR activation suppressed renal fibrosis in a unilateral ureteral occlusion mouse model via regulating Smad3 [28]. Another experimental study also reported that FXR activation suppressed the β-catenin-mediated activation of Wnt target genes, which are involved in spontaneous hepatocellular carcinoma [23]. Recently, one study also reported that FXR may exert a tumor-suppressor role in colorectal cancer by antagonizing β-catenin signaling [29].

Although FXR activation can exert important hepato-protective effects against various stress conditions [35,36], very few studies have examined the possible renal protective effects of FXR activation in HFD-fed mice. FXR activation has been reported to improve metabolic derangement and insulin resistance, all of which may reduce the risk of developing diabetic nephropathy [27]. In our experimental study, the FXR agonists CDCA or INT-747, both ameliorated lipid dysfunction and albuminuria in HFD-fed mice, associated with regulation of β-catenin

restored by β-catenin overexpression. These data indicate that FXR interacts with β-catenin, being implicated in the regulation of β-catenin transcription activity in ox-LDL treated conditions.

against renal fibrosis in HFD-fed mice possibly by inhibiting the β-catenin pathway.

| A                                                         | B                                                            |
| --------------------------------------------------------- | ------------------------------------------------------------ |
| Graph A: Body weight (g)                                  | Graph B: Urinary ACR (mg/g)                                  |

| C                                                        | D                                                      |
| -------------------------------------------------------- | ------------------------------------------------------ |
| Graph C: Total cholesterol (mmol/L)                      | Graph D: LDL-c (mmol/L)                                 |

| E                                                        | F                                                        |
| -------------------------------------------------------- | ------------------------------------------------------ |
| Graph E: Triglycerides (mmol/L)                          | Graph F: Serum ALT (U/L)                                |

| G                                                        | H                                                              |
| -------------------------------------------------------- | ------------------------------------------------------------- |
| Graph G: Serum BUN (mmol/L)                              | Graph H: Serum Creatinine (mmol/L)                           |

Figure 5: Characteristics of mice in the different groups after treatment. FXR agonist (CDCA or INT-747) treatment improves body weight (A), albuminuria, expressed as urinary albumin to creatinine ratio (ACR) (B), serum total cholesterol (C), LDL-c (D), triglycerides (E), ALT levels (F), BUN (G) and creatinine (H) at 20 weeks in 60% HFD-treated mice. Values shown are means ± S.E.M, *p < 0.05.

MOLECULAR METABOLISM 79 (2024) 101841 © 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

## A
Oil red O
staining

## B
Fibronectin

## C
α-SMA

## D
Masson

## E
Sirius red

## F

Masson staining
(Relative level %)
```
0    10    20    30
CTL ┤
HFD ████████████████████
CDCA ████████
INT-747 ██████
```

Sirius red staining
(Relative level %)
```
0    5    10   15
CTL ┤
HFD ████████████
CDCA ██████
INT-747 ████
```

Figure 6: FXR activation prevents renal lipid accumulation and protects against renal fibrosis in HFD mice. (A) Oil red O staining of kidney sections. (B-C) Representative micrographs display the induction of localization of fibronectin and α-SMA in HFD mice. Kidneys from control, HFD, CDCA and INT-747 group for 20 weeks were stained immunohistochemically for fibronectin and α-SMA protein. Representative images of Masson staining (D) and Sirius red staining (E). The positive area of Masson and Sirius red were calculated (F) (n = 5). Values shown are means ± S.E.M. *p < 0.05.

MOLECULAR METABOLISM 79 (2024) 101841 © 2023 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
www.molecularmetabolism.com

One of the main findings in our study is that the possible beneficial effects of FXR activation on lipid-induced tubulo-interstitial fibrosis might be, at least partly, mediated by modulation of the β-catenin signaling activity. Our study shows that FXR activation suppresses the β-catenin-mediated activation of the Wnt target genes, which are involved in tubulo-interstitial fibrogenesis. First, after ox-LDL treatment, the FXR abundance was decreased in the nuclei, and FXR interacted with β-catenin, especially in the nuclei. Second, FXR inhibition induced β-catenin signaling activation, whereas increased expression/activity of FXR decreased β-catenin signaling activity. Third, after ox-LDL stimulation, the abundance of β-catenin translocated into the nucleus, β-catenin transcriptional activity and its target gene expression were increased (whilst the total β-catenin abundance did not change). A recent study showed that in HEK293 cells, the full length FXR and its mutants containing the N-terminal AF1 domain were able to bind with β-catenin; conversely, other FXR mutants, which did not have the N-terminal AF1 domain, lost their β-catenin binding ability [23]. Moreover, to identify the possible underlying mechanisms, in our study we showed that FXR interacted with β-catenin signaling using the co-immunoprecipitation assay, and tested the effects of FXR on β-catenin mediated transcriptional activity using a cell-based TOPflash reporter assay in ox-LDL-exposed NRK-52E cells. We also showed that the AF1 domain of FXR was required for the suppression of β-catenin-mediated transcriptional activity.

activity. Collectively, these data support a possible renal protective effect of FXR activation to inhibit tubulo-interstitial fibrogenesis in lipid-induced nephropathy.

In conclusion, our experimental study shows that FXR activation may be implicated in decreasing β-catenin signaling activation and protecting against renal fibrosis development. Our study also shows that through interaction with β-catenin, FXR may act as a mediator for ox-LDL regulation β-catenin signaling in tubular epithelial cells. However, further investigation of the regulation and function of the FXR-β-catenin interaction in the development and amelioration of renal fibrosis in vivo in man is required to better elucidate whether FXR agonists can be used to treat or protect against tubulo-interstitial fibrosis.

This work was supported by grants from the National Natural Science Foundation of China (81970624, 82000690), China Postdoctoral Science Foundation (2023M732681), Top-notch Talents from Young and Middle-Age Health Care in Wuxi (BJ2020026), Youth Research Project Fund from Wuxi Municipal Health Commission (Q201932) and Technology Development Fund in Wuxi (N20202001). GT was supported in part by grants from the School of Medicine, University of Verona, Italy. CDB was supported in part by the Southampton NIHR Biomedical Research Centre (NIHR 203319), UK.

Shi-Liang Zhang and Meng-Yang Zhong: Resources.
Giovanni Targher and Christopher D. Byrne: Writing – Review & Editing.

Fuqiang Yuan and Yan Lu: Resources.
Wei-Jie Yuan: Supervision.

Figure 8: FXR activation protects against β-catenin signaling in HFD mice. Representative Western blot analysis (A) and quantitative data (B–D) show the activation of β-catenin and induction of fibronectin, α-SMA and reduction of FXR protein in HFD mice. (E–F) Schematic Figure illustrating the molecular mechanisms by which FXR and β-catenin may act to regulate the development of renal tubulo-interstitial fibrosis. Ox-LDL-exposed tubular epithelial cells, activates tubular markers of extracellular matrix (fibronectin, α-SMA) and promotes the development of tubulo-interstitial fibrosis via activity of the FXR/β-catenin complex (E). In contrast, up-regulation of FXR activity negatively regulates the FXR/β-catenin complex and inhibits tubular markers of extracellular matrix towards the ox-LDL-induced tubular interstitial fibrosis (F).

The authors would like to thank Dr Wei-Chun He (Center for kidney disease, Second Affiliated Hospital, Nanjing Medical University) for providing TOPflash plasmid and pcDNA3 plasmid. The authors would like to thank Yi Du and Shuang Xu for providing technical guideline, then thank Gang Tang for experimental assistance.

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.

Data will be made available on request.

farnesoid X receptor
NRK-52E
rat proximal tubular epithelial cells
chronic kidney disease
oxidized low-density lipoprotein
lysyl oxidase
extracellular signal-regulated kinase
mitogen-activated protein kinase
extracellular matrix
chenodeoxycholic acid
high-fat diet

[31] Kume S, Uzu T, Araki S, Sugimoto T, Isshiki K, Chin-Kanasaki M, et al. Role of altered renal lipid metabolism in the development of renal injury induced by a high-fat diet. J Am Soc Nephrol 2007;18(10):2715–23.

[32] Sun DQ, Ye FZ, Kani HT, Yang JR, Zheng KI, Zhang HY, et al. Higher liver stiffness scores are associated with early kidney dysfunction in patients with histologically proven non-cirrhotic NAFLD. Diabetes Metab 2020;46(4):288–95.

[33] Abrass CK. Cellular lipid metabolism and the role of lipids in progressive renal disease. Am J Nephrol 2004;24(1):46–53.

[34] Wahl P, Ducasa GM, Fornoni A. Systemic and renal lipids in kidney disease development and progression. Am J Physiol Renal Physiol 2016;310(6):F433–45.

[35] Zhou J, Cui S, He Q, Guo Y, Pan X, Zhang P, et al. SUMOylation inhibitors synergize with FXR agonists in combating liver fibrosis. Nat Commun 2020;11(1):240.

[36] Han CY, Rho HS, Kim A, Kim TH, Jang K, Jun DW, et al. FXR inhibits endoplasmic reticulum stress-induced NLRP3 inflammasome in hepatocytes and ameliorates liver injury. Cell Rep 2018;24(11):2985–99.


[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
Extraction and characterization of biocompatible hydroxyapatite (HAp) from red big eye fish bone: Potential for biomedical applications and reducing biowastes

Over the past decade, India has seen remarkable growth in fish production, solidifying its position as the world’s second-largest producer. Approximately 6.4 % of the global fish mass production and aquaculture originate from India. According to research by the National Fisheries Development Board (NFDB), India produced around 12 million metric tons of fish in 2017–2018, contributing significantly to inland sector production, accounting for about 65 % [1,2]. Additionally, cultured fisheries contribute nearly half of the world’s total fishery production. Despite this success, there’s a substantial issue with fish discards by-products such as heads, scales, fins, trimmings, viscera, and skin constituting 20–70 % of the overall fish weight. India alone generates approximately 4 million metric tons of fish waste annually, with only a small fraction being repurposed into fish meals, fertilizers, pet food, or animal feed [3,4]. The majority is irresponsibly discarded into oceans, seas, and landfills, posing environmental hazards. However, these waste materials hold potential as sources of biomolecules and other valuable elements. Implementing stricter ecological standards and regulations for waste disposal, as well as exploring the possibility of retrieving quality by-products, is imperative [5,6]. Unfortunately, most domestic markets lack efficient waste management systems and fail to

In this study, nanostructured hydroxyapatite (HAp) was successfully extracted from the bones of Priacanthus macracanthus (Red Big Eye), a species commonly processed in the fish industry, generating significant waste. The extraction process utilized an alkaline hydrolysis method optimized with 2 M sodium hydroxide at 250 °C for 5 h, producing high-purity HAp. Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of characteristic phosphate peaks at 1044 cm⁻¹ and 963 cm⁻¹, and hydroxyl peaks at 632 cm⁻¹. Powder X-ray diffraction (XRD) analysis showed prominent peaks at 2θ values of 25.9°, 32.2°, 39.8°, and 46.7°, corresponding to the crystalline planes of HAp. Field-emission scanning electron microscopy (FESEM) revealed spherical HAp particles with sizes ranging from 50 to 80 nm. Biocompatibility was assessed using human osteoblast-like MG-63 cells, showing a proliferation rate of 92 % compared to the control. Cytotoxicity tests indicated no significant adverse effects, supporting the potential use of this HAp in biomedical applications. Importantly, this method offers a sustainable solution for managing fish bone waste, contributing to pollution control by reducing environmental burdens associated with discarded bone wastes. Future research will focus on in vivo biocompatibility studies and exploring applications in pollution mitigation and tissue engineering. This study highlights the dual benefits of utilizing biowaste for valuable HAp production and addressing environmental pollution challenges, making it a promising approach for sustainable material synthesis and environmental management. The cost-effective and environmentally friendly process further underscores the feasibility of scaling up this method for industrial applications, providing a greener alternative to conventional HAp synthesis.

journal homepage: www.editorialmanager.com/scenv

Keywords:
Fish bone
Hydroxyapatite
MG-63 cell line
Polymer
Human osteoblast
FESEM

E-mail address: yuvarajdinakarkumar@gmail.com (D. Yuvaraj).

2949-8392/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. recognize the value in what they discard, exacerbating the problem. Even industries equipped with waste management facilities face challenges such as machinery malfunctions, odour issues, and high production costs [7]. Consequently, there’s an urgent need to address the improper utilization of fish discards and develop eco-friendly, advanced technologies to process these wastes from both marine and inland sectors effectively, ensuring their responsible and accurate utilization [8,9].

2. The status of fish bone discards and its environmental issues are a complex and multifaceted problem. The significant increase in fish processing by-products and the need for eco-friendly management practices are the need in the present situation [10,11]. These discards create adverse ecological impacts of fishery discards and the need for sustainable management [12,13]. The potential for extracting bioactive substances from fish waste [10] and the applications of treated fish waste [11] offer promising solutions, but the challenge lies in their implementation and the need for responsible and sustainable management practices.

3. Bones, provide support, protection, and mobility to our bodies, are intricate compositions of organic and inorganic elements. Comprising roughly 70% minerals and 30% organic matter, bones are essential for maintaining the structural integrity of the body and facilitating various physiological functions. Among the mineral components that contribute significantly to bone composition, hydroxyapatite (HA) stands out as a key player.

4. Hydroxyapatite, a calcium phosphate mineral with the chemical formula Ca₁₀(PO₄)₆(OH)₂, is the primary mineral phase found in bone tissue. It forms the rigid matrix that gives bones their strength and hardness, essential for withstanding mechanical stresses and supporting bodily movements. In fact, approximately 95% of the mineral content in bone consists of hydroxyapatite crystals [14]. This remarkable abundance underscores the critical role that hydroxyapatite plays in maintaining skeletal health and function.

5. The importance of hydroxyapatite extends beyond its structural role in bone tissue. In biomedical applications, hydroxyapatite has garnered significant attention due to its biocompatibility, bioactivity, and osteoconductive properties. These attributes make it an attractive material for a wide range of medical uses, including bone grafts, orthopedic implants, dental restorations, and drug delivery systems [15]. Its ability to integrate seamlessly with surrounding tissue without triggering adverse reactions or toxicity makes hydroxyapatite an invaluable asset in regenerative medicine and tissue engineering.

6. The quest for synthetic biomaterials capable of mimicking the properties of natural bone has been ongoing for decades. The development of the first generation of biomaterials intended for use inside the human body, dating back to the 1960s and 1970s, marked a significant milestone in the field of biomedical engineering [16]. Since then, researchers have been exploring various approaches to fabricate biomimetic materials that can interact harmoniously with the body’s biological environment.

7. Calcium phosphate ceramics, including hydroxyapatite, have emerged as promising candidates for biomedical applications. The calcium/phosphorus atomic ratio (Ca/P) serves as a fundamental descriptor for these ceramics, with hydroxyapatite (Ca/P = 1.667) and β-tricalcium phosphate (β-TCP, Ca/P = 1.5) being among the most extensively studied compounds [4]. Understanding the physicochemical properties and biological behaviors of these materials is crucial for their successful implementation in medical settings.

8. One of the key advantages of hydroxyapatite lies in its ability to bind tightly to bone tissue, promoting osseointegration and facilitating the regeneration of damaged or diseased bone. Unlike some synthetic biomaterials that may elicit inflammatory responses or immune reactions, hydroxyapatite exhibits excellent biocompatibility, making it an ideal choice for medical implants and devices [17]. Moreover, hydroxyapatite can be tailored to meet specific clinical requirements by adjusting its composition, morphology, and surface characteristics.

9. Despite the widespread use of hydroxyapatite in biomedical

10. applications, challenges remain in the synthesis and fabrication of HA-based materials with desired properties. Various synthesis methods have been developed to produce hydroxyapatite, including chemical precipitation, hydrolysis, sol-gel processing, hydrothermal synthesis, emulsion techniques, sonochemical methods, solid-state reactions, and mechanochemical approaches [18]. Each of these methods offers unique advantages and challenges, influencing the final properties of the synthesized hydroxyapatite.

11. In addition to synthetic routes, hydroxyapatite can also be obtained from natural sources, such as bone tissue or marine organisms. Natural hydroxyapatite exhibits non-stoichiometry and may contain trace elements that mimic the composition of human bone, including sodium, zinc, magnesium, potassium, silicon, barium, fluoride, and carbonate ions [19]. These trace elements can influence the properties and biological behavior of hydroxyapatite, highlighting the importance of understanding the complexities of natural mineral sources.

12. The synthesis of hydroxyapatite often involves high-temperature processes, such as calcination, to achieve the desired crystalline structure and phase purity. Calcination, typically conducted at temperatures ranging from 800 to 1100 °C, plays a critical role in controlling the physicochemical properties of hydroxyapatite and ensuring its suitability for biomedical applications [20]. The impact of calcination temperature on the crystallinity, phase composition, and surface properties of hydroxyapatite has been extensively studied, with researchers striving to optimize synthesis conditions for specific medical applications.

13. In recent years, researchers have explored alternative synthesis approaches for hydroxyapatite, including environmentally friendly methods that minimize the use of hazardous chemicals and energy-intensive processes. One such approach is alkaline hydrolysis, which offers several advantages, including the potential to produce nanostructured hydroxyapatite and the ability to incorporate carbonate ions into the crystal lattice [21]. Alkaline hydrolysis has emerged as a promising technique for the synthesis of hydroxyapatite with tailored properties, opening up new possibilities for biomedical applications.

14. In our current scientific study, the synthesis of hydroxyapatite from discarded Priacanthus bone was done. By employing both thermal calcination and alkaline hydrolysis techniques, hydroxyapatite was extracted and characterized for implying its biomedical applications. Chemical analyses, spectroscopic studies, diffraction measurements, and microscopic observations will be conducted to evaluate the composition, structure, and morphology of the synthesized hydroxyapatite.

15. The outcomes of our research could have significant implications for the field of biomaterials and tissue engineering. By elucidating the synthesis pathways and properties of hydroxyapatite derived from natural sources, we aim to contribute to the development of advanced materials for bone regeneration, implant coatings, and drug delivery systems. Ultimately, our goal is to harness the remarkable properties of hydroxyapatite to improve patient outcomes and advance the field of regenerative medicine.

16. 2.1. Preparation of hydroxyapatite by alkaline (A-HAp) and thermal (T-HAp) calcination

17. To remove any remaining traces of meat and skin, the bone of Priacanthus underwent boiling water washing for three days. To eliminate contaminants from the cleaned bones, a solution containing 1% NaOH and C₃H₆O was utilized (with a bone to sodium hydroxide solid/liquid ratio of 1:50). The bones were then rinsed, pulverized, and subjected to baking at 50–60 °C for a period of twenty-four hours. Subsequently, 2 g of pulverized Priacanthus bone was treated with 2 M sodium hydroxide (Sigma Aldrich) at 250 °C for 5 h (with a solid-liquid ratio of 1:30). A 2 M sodium hydroxide solution was prepared for water-soluble extraction, and this process was repeated to remove organic components. The

resulting liquid was filtered and cleaned using a suction pump to achieve a neutral pH. The product was then dried in an oven at 100 °C. For thermal calcination, 3 g of Priacanthus bone was heated to 900 °C in a silicon chamber for five hours using an electrical muffle furnace (Sisco India) [22].

A FTIR, XRD, and SEM analysis was conducted on the isolated Hydroxyapatite (HAp) powder to determine its structure and chemical composition. The phase and purity of crystalline HAp were determined by X-ray diffractometers (PANalytical, 207756, Netherlands). To confirm the structure of calcined HAp powder, FTIR spectrometers (Bruker Tensor 27 Instruments, Billerica, MA, Germany) were used. SEM (JEOL JSM-5610LV Field Emission Gun) was used to determine the average particle size of powder.

MG-63 human osteosarcoma cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 2 millimolar glutamine, and 100 μg ml⁻¹ penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere with 5% carbon dioxide. To assess the biological compatibility of HAp nano- and micro-crystals, MG-63 human osteoblastic cells were utilized. The cells were seeded at a density of 2 × 10³ cells/well in 96-well plates. After 24 h, the cells were exposed to freshly prepared media containing A-HAp and T-HAp at concentrations of 12.5 and 25 μg ml⁻¹, respectively. HAp-free cells served as the blank control. Following a one-day incubation period, the media was removed, and 100 μl of MTT (5 mg ml⁻¹) was added to each well. The cells were incubated with MTT for four hours, after which 100 μl of (CH₃)₂SO was added to dissolve the formazan salt. The optical density (OD) at 540 nm was measured using a GENios R microplate reader (Tecan Austria GmbH, Austria) to quantify the formazan formation, which indicates cell viability. The percentage of cell viability and proliferation were calculated by comparing the absorbance values to those of the control group.

Previous research revealed that HAp obtained at a lower temperature had larger surface area nanocrystals than HAp produced by the thermal calcination process at a higher temperature [23,24]. Bovine bone was thermally calcined using the vibro-milling technique to split the HAp [25] into nanocrystalline and microstructure forms [26]. Since the thermal calcination method was also used to separate HAp from chicken femur, sheep femur, sheep skull flat bone, and bovine femur bone, there is no degradation at a high temperature (1100 C) [21]. CO₂, CaO, and (CO₃)₂ were kept at a high temperature of 1000 C, whereas dioxygen, calcium oxide, and carbonate ions were eliminated from fishbone using the thermal calcination method [27]. Two distinct procedures have been used for isolating nanostructured HAp: calcination at high temperatures in conjunction with the milling process. To prevent grinding, researchers created the alkaline hydrolysis approach for isolating nanostructured HAp from bovine bone [28,29].

HAp has previously been extracted from numerous species of fish bone using the thermal calcination method, including Pintado (Pseudoplatys tomacorruscans), Jau (Paulice alutkeni), Cachara (P. fasciatum), Japanese sea bream fish [30–32]. However, the nanostructured HAp was obtained using a milling method [28,29]. The alkaline method used to produce the nanosized HAp from bone of fish is a novel method for creating huge volumes of nanostructured HAp without milling.

Inspired from study [23] our study isolated HAp from discarded Red big eye (Priacanthus macracanthus) bone using thermal calcination and alkaline hydrolysis. Thus, the alkali hydrolysis method is a novel technique for isolating nanostructured hydroxyapatite from fish bone, whereas the calcination using high temperature is a traditional method for extracting high-crystalline pure HAp [33]. Alkaline hydrolysis and thermal calcination-produced HAp were also contrasted. The alkaline hydrolysis method involved treating 2 g of pulverized Priacanthus macracanthus bone with 2 M sodium hydroxide at 250°C for 5 h. The solid-liquid ratio was maintained at 1:30. The process was repeated to ensure thorough removal of organic components, followed by filtration and drying at 100 °C. The conditions were optimized based on previous studies that demonstrated efficient extraction of high-purity HAp with minimal organic residue.

Alkaline hydrolysis produces HAp that is purer and has larger crystals than thermal calcination. In order to lessen the impact of pollution on environment and make use of trash that has been treated for biological purposes, this work recovered that red giant eye bone, a novel marine source of nanostructured HAp [34].

Fig. 1 shows natural bone, while Figs. 2 and 3 show HAps made using the described processes. Fig. 1 depicts typical SEM images of natural fish bone with inorganic minerals and collagen organic moieties. HAp microcrystals longer than a few micrometres are seen in bone. Alkaline hydrolysis maintains crystal size, while heat calcination agglomerates them. Fig. 2 displays nanoparticles from alkaline hydrolysis of the HAp. Fig. 3 illustrates heat calcination-induced microstructure formation in derived HAp. Alkaline hydrolysis of bovine bone types I collagen produces nanoparticles. However, heat calcination causes nanoparticle aggregation, giving HAp a microstructure. High process temperatures may cause this. Based on the results, alkaline hydrolysis HAps do not contain nanoparticles. Despite having larger particles than alkaline hydrolysis, thermal calcination eliminated the organic portion. Without altering crystal size, alkaline hydrolysis surpasses thermal calcination. We found that raw bone formed microstructure HAp when thermally calcined and nanostructured particles when alkaline hydrolysed [34].

Fourier transform infrared spectroscopy (FT-IR) serves as a valuable tool for probing the structural characteristics of phosphate compounds, shedding light on the origins of their vibrational modes. Fig. 4(a–c) showcases the FTIR spectra obtained from untreated bone, bone subjected to alkaline hydrolysis for hydroxyapatite extraction, and bone exposed to high-temperature heating [35]. Analysis of these spectra reveals significant differences in spectral features between untreated bone and hydroxyapatite derived through the suggested process.

Upon treatment of red big eye bone, FT-IR spectra exhibited distinct bands corresponding to ions such as PO₄³⁻, -OH, and HCO₃⁻. These findings align with referenced spectra reported in literature [11,12], with characteristic bands observed at frequencies of 1455, 1417, 1099, 1044, 963, 875, 632, 603, and 566 cm⁻¹. Notably, Fig. 4(a–c) elucidates specific vibrational modes, including -OH bending at 632 cm⁻¹ and phosphate group stretching in the range of 500–1100 cm⁻¹, consistent with pure hydroxyapatite obtained via the suggested procedures [36].

- Phosphate (PO₄³⁻) stretching modes at 1044 cm⁻¹, 963 cm⁻¹, and 566 cm⁻¹.
- Hydroxyl (OH⁻) bending at 632 cm⁻¹. These values align with the characteristic absorption bands for HAp reported in the literature, indicating successful extraction and identification of HAp.

The observed spectral features provide compelling evidence that the organic constituents of raw bone have been effectively removed through the applied treatment processes [37]. This conclusion is supported by the absence of characteristic bands associated with organic matter in the

FT-IR spectra of treated bone samples. These results underscore the efficacy of the suggested methods in isolating hydroxyapatite from bone matrices, paving the way for further exploration of its biomedical applications.

Overall, FT-IR spectroscopy emerges as a powerful analytical technique for elucidating structural transformations and compositional changes in biomaterials, providing invaluable insights into the underlying chemistry of bone-derived hydroxyapatite. The observed spectral signatures offer crucial evidence of successful organic removal and the generation of pure hydroxyapatite, highlighting the potential of these methods for producing biocompatible materials with tailored properties for biomedical engineering endeavors.

Analysis of surface area, crucial for understanding crystal size, often relies on X-ray diffraction (XRD) techniques. Schwertmann and Latham (1986) established that peak broadening in XRD spectra serves as a reliable parameter for determining mean crystallite dimensions. The Debye-Scherrer equation, frequently employed for this purpose, relates the mean crystallite size (d) to X-ray wavelength (λ), the angle of reflection (θ), and the full width at half maximum (β) of diffraction peaks. The broadening of peaks in XRD patterns can thus provide valuable insights into the crystallinity and grain size of materials.

Fig. 5(a)–(c) illustrate XRD reflections obtained from bone samples subjected to alkaline treatment, thermal calcination, and untreated red big eye bone. The broad peaks observed in raw bone spectra indicate low crystallinity, likely due to the presence of an organic matrix that

R. Gnanasekaran et al.                                                     Sustainable Chemistry for the Environment 7 (2024) 100142

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Fig. 4. (a–c) representing the frequency stretching of Hydroxyapatite from natural fish bone, A-Hap, and T-Hap.

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Fig. 5. (a–c) show XRD reflections of raw red big eye bone, treated bone, and thermal-calcinated bone.

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undergoes physiological calcification. In contrast, treated bone samples, as depicted in Figs. 5(b) and 6, exhibit sharper peaks, indicative of reduced organic content and higher crystallinity post-treatment. Notably, the XRD pattern of alkaline hydrolyzed bone (Fig. 3(b)) displays sharper peaks compared to thermally calcinated bone (Fig. 5(c)), underscoring the superior crystallinity achieved through alkaline hydrolysis. The XRD analysis provided further confirmation of HAp. The prominent peaks at 2θ values of 25.9°, 32.2°, 39.8°, and 46.7° correspond to the crystalline planes (002), (211), (310), and (222) of HAp, respectively. These peaks are in agreement with the standard HAp pattern (JCPDS file no. 09-0432), affirming the high crystallinity and purity of the extracted HAp.

Comparative analysis of XRD patterns reveals that untreated raw bone exhibits the lowest degree of crystallinity. The peak widths in XRD spectra indicate that the crystallite size obtained from raw bone is relatively small. Alkaline calcinated bone displays the narrowest peak widths, followed by thermally calcinated and untreated raw bone, suggesting a trend towards increased crystallinity post-treatment. The highest peak intensity in alkaline calcinated bone occurs at 31.94 degrees (hkl value: 211), whereas for raw bone, it is at 32.4 degrees (hkl: 112). These observations align with previous studies [38–40], which attribute variations in XRD patterns to changes in crystal size.

Further analysis reveals specific parameters characterizing the crystalline structure of hydroxyapatite produced through different methods. Alkaline hydrolyzed HAp demonstrates planar spacing, intensities, and angles akin to conventional HAp parameters, indicating its structural similarity. In contrast, thermally generated HAp exhibits variations in these parameters, suggesting differences in crystal structure. Notably, alkaline hydrolysis emerges as the preferred method for HAp synthesis, given its lower overall relative error compared to HAp produced via thermal calcination.

Observations from XRD patterns of Fe-Al-based mesoporous metal oxides (BMO) further highlight the utility of XRD in characterizing crystalline materials. The presence of strong Cu Kα peaks in BMO XRD patterns supports the crystallinity of BMO. Changes in BMO structure upon heat treatment are discernible through XRD analysis at different temperatures. While line widths in XRD patterns may resemble those of nanoparticles, the Scherrer equation estimates a crystallite size of 10 nm for mesoporous metal oxides. This estimation aligns with FESEM images depicting spherical mesoporous metal oxide particles with a consistent size of approximately 100 nm. FESEM analysis revealed that the HAp particles derived from Priacanthus macracanthus bone exhibit a spherical morphology with an average particle size ranging from 50 to 80 nm. The particle size distribution was relatively narrow, indicating uniformity in particle dimensions.

Comparatively, HAp derived from other biological sources, such as bovine bone, typically shows particle sizes in the range of 100–200 nm, while synthetic HAp often has particle sizes below 50 nm. The nanostructured HAp from Priacanthus macracanthus bone thus offers a unique balance of size and uniformity, which could be advantageous for specific biomedical applications where both properties are critical.

High-resolution transmission electron microscopy (HRTEM) images provide additional evidence of mesoporous metal oxides, revealing their atomic crystallinity and mesoscopic order.

In conclusion, X-ray diffraction emerges as a powerful technique for characterizing the crystalline properties of materials, providing valuable insights into grain size, crystallinity, and structural changes induced by different synthesis methods and treatments. These insights are crucial for understanding material behavior and optimizing synthesis processes for various applications in materials science and biomedical engineering.

Numerous cell experiments have been conducted to evaluate the potential toxicity of nanoparticles, as highlighted in literature [28]. Fig. 6(a)–(b) depict the outcomes of experiments conducted on MG-63 human osteosarcoma cells, assessing both cytotoxicity and proliferation in response to A-HAp and T-HAp crystals across varying dosages and durations. Cytotoxicity and cell proliferation assays were conducted using human osteoblast-like MG-63 cells. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 μg/ml penicillin-streptomycin. The cells were seeded at a density of 2 × 10³ cells per well in 96-well plates and incubated for 24 h at 37 °C in a 5% CO₂ atmosphere.

For the cytotoxicity test, HAp samples were prepared at concentrations of 12.5 and 25 μg/ml. The cells were exposed to these samples for 24 h, and cytotoxicity was assessed using the MTT assay. The results indicated no significant cytotoxic effects, demonstrating the biocompatibility of the HAp samples.

Remarkably, the results indicate that HAp exhibits non-cytotoxic behavior towards osteosarcoma MG-63 cells, with increasing doses correlating with enhanced cell viability. Furthermore, it was observed that MG-63 cells exhibited accelerated growth on treated HAp particles compared to cells cultured on control plates. This suggests a stimulatory effect of HAp on cell proliferation, indicating its potential for promoting tissue regeneration. The current analysis underscores the relatively benign nature of nano and micro-sized HAp particles on the MG-63 cell line. Comparative analysis between A-HAp and T-HAp revealed a gradual increase in cell viability, with A-HAp exhibiting significantly higher cell viability than T-HAp, as depicted in Fig. 6(a & b) and Fig. 7, respectively. This observation underscores the importance of synthesis methods in influencing the biological response of cells to HAp crystals.

Interestingly, the study [41] uncovered that prolonged exposure to elevated concentrations of hydroxyapatite, both in macro and nanoparticle forms, could exert deleterious effects on osteosarcoma MG-63 cell proliferation. While cells cultured on control plates displayed robust growth rates, those exposed to HAp particles exhibited restricted and attenuated levels of cell expansion. This suggests a dose-dependent relationship between HAp concentration and cell proliferation, with

excessive concentrations potentially inhibiting cell growth. Notably, the rate of cell proliferation on HAp-treated plates was significantly lower compared to control plates, indicating a modulatory effect of HAp on cell proliferation dynamics.

These findings underscore the intricate interplay between HAp characteristics, such as particle size and concentration, and their impact on cellular responses. Further elucidating these relationships is essential for harnessing the full therapeutic potential of HAp in biomedical applications. While the stimulatory effect of HAp on cell proliferation holds promise for tissue engineering and regenerative medicine, careful consideration must be given to dosage and exposure duration to mitigate potential adverse effects. Additionally, continued research is needed to unravel the underlying mechanisms governing the cellular response to HAp and optimize its utilization for clinical applications.

Fish consumption is a global phenomenon, with recent years witnessing a significant increase in production. However, a large portion of fish waste is currently either discarded or dumped into land and water bodies, leading to adverse environmental consequences. Recovering fish waste oil presents an opportunity to produce biofuels, potentially mitigating congestion issues associated with other biofuel sources due to their cost-effectiveness. Nonetheless, the fatty acid composition of fish waste varies among species and seasons, affecting the quality of biofuels produced. While biofuels derived from fish discards may not meet global consumer demands alone, they could complement other biofuel sources. The ideal solution lies in implementing fish farming systems, yet this approach remains largely untapped due to limited knowledge in the field. Integrating fish farming could balance fish production with waste generation and create employment opportunities within the farming community. Further research at commercial scales is crucial for advancing this system effectively.

promising prospects of utilizing natural nanoHAp derived from Priacanthus bone for biomedical applications. By leveraging innovative synthesis methods and rigorous characterization techniques, we aim to contribute to the development of advanced biomaterials that offer both clinical efficacy and environmental sustainability. This study successfully demonstrates the extraction and characterization of biocompatible HAp from Priacanthus macracanthus bone. The observed properties suggest potential applications in biomedical fields, including tissue engineering. However, further research is necessary to experimentally validate these potential applications.

In order to extract pure natural nano HAp from Priacanthus bone, a combination of thermal calcination and alkali hydrolysis methods were employed. This approach not only allows for the isolation of nanoHAp from fresh Priacanthus bone but also presents an eco-friendly solution by repurposing processed bone waste, thereby reducing environmental pollution. Through extensive characterization using techniques such as Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), and scanning electron microscopy (SEM), we confirmed that the isolated HAp exhibited no traces of collagen or other organic constituents, ensuring its purity and suitability for biomedical applications. Comparative analyses revealed that alkaline calcination yielded HAp crystals with enhanced crystallinity compared to thermal hydrolysis, underscoring the superiority of the former method in terms of crystalline structure refinement. This improvement in crystallinity is particularly advantageous for biomedical applications, as it enhances the material’s mechanical strength and biological activity. Furthermore, the alkaline hydrolysis technique offers the added benefit of producing carbonated HAp, a form of HAp highly favored in several biomedical applications due to its enhanced bioactivity and biodegradability. The versatility of the proposed alkaline hydrolysis approach lies in its ability to generate HAp with tailored physicochemical properties, making it suitable for a diverse range of biomedical applications. Whether as a bone substitute material, a coating for medical implants, or a component in drug delivery systems, the synthesized HAp holds promise in various fields of regenerative medicine and tissue engineering. In light of the detailed characterization of biowaste-derived HAp and its comparative assessment with conventional synthesized HAp, our findings highlight its utility, suggesting its suitability for broad biomedical and environmental applications. In-vitro assessments using selected cell lines and comprehensive physicochemical analyses are warranted to fully elucidate the material’s biological responses and ensure its safe clinical application. Overall, these results demonstrate the

Future research will focus on conducting comprehensive in vivo studies to assess the long-term biocompatibility and potential cytotoxic effects of hydroxyapatite (HAp) derived from Priacanthus macracanthus bones. Animal models, such as rats, will be employed to validate the suitability of this biowaste-derived HAp for biomedical applications. These studies will provide critical insights into the material’s interaction with biological tissues over extended periods, including any inflammatory responses or toxic effects that may arise from prolonged exposure. The outcomes of these experiments will be crucial for confirming the safety and effectiveness of HAp for clinical use, especially in applications such as bone grafts and implants. Additionally, future research will explore the functionalization of HAp with various biomolecules or therapeutic agents. This could involve coating the HAp particles with proteins, peptides, or growth factors that promote cell attachment and differentiation, thereby enhancing its applicability in bone regeneration and tissue engineering. Functionalization could also extend to drug delivery systems, where HAp particles are used as carriers for controlled release of drugs, potentially improving the treatment of diseases such as osteoporosis or bone infections. Investigating these functional applications will help in tailoring the material for specific medical applications, making it a versatile tool in the field of regenerative medicine. Furthermore, the feasibility of using biowaste-derived HAp in environmental applications will be explored through targeted experimental studies. These studies will focus on the material’s potential for water purification and pollutant adsorption, given its high surface area and chemical reactivity. Experiments will aim to determine the efficiency of HAp in removing contaminants like heavy metals, organic pollutants, and pathogenic microbes from water sources. This line of research not only provides a sustainable approach to recycling waste materials but also addresses significant environmental challenges by offering a cost-effective and eco-friendly solution for pollution mitigation. Comparative studies with commercially available HAp will also be a key focus. Detailed analyses will be conducted to assess the performance, cost-effectiveness, and environmental impact of biowaste-derived HAp in comparison to its commercially synthesized counterparts. Parameters such as mechanical strength, bioactivity, and longevity of the material will be evaluated. Additionally, economic assessments will consider the cost of raw materials, production processes, and potential savings from waste reduction. Environmental impact analyses will account for factors such as energy consumption, chemical usage, and carbon footprint. Overall, these studies

R. Gnanasekaran: Supervision, Project administration. Yuvaraj Dinakarkumar: Writing – review & editing, Validation, Supervision, Conceptualization. C.M. Mathan Muthu: Investigation, Data curation, Conceptualization. R. Ashwin: Writing – original draft, Methodology Investigation, Formal analysis. K. Kaarthikeyan: Writing – original draft, Investigation, Formal analysis. V. Vasanth Kumar: Writing –

original draft, Investigation, Formal analysis. R. Jothi Ramalingam: Project administration, Funding acquisition. Hamad Al-Lohedan: Funding acquisition. Koteswara Reddy: Project administration.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: There is no conflict of interest between the authors of this manuscript.

Data will be made available on request.

The authors are extending their appreciation to Deanship of Scientific research, King Saud University for financial support through vice Deanship of Research Chairs. Research chair of surfactant.

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