[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]
Calcitonin gene-related peptide (CGRP) is synthesized and secreted by trigeminal ganglion neurons, and is a key neuropeptide involved in pain and immune regulation. This study investigates the expression of CGRP in the trigeminal ganglion (TG) and its regulatory role in the polarization of macrophages in rats with temporomandibular arthritis. A rat model of temporomandibular arthritis was established using CFA. Pain behavior was then observed. Temporomandibular joint (TMJ) and the TG were collected, and immunohistochemistry, immunofluorescence (IF) staining, and RT-qPCR were used to examine the expression of CGRP and macrophage-related factors. To investigate the impact of CGRP on macrophage polarization, both CGRP and its antagonist, CGRP 8-37, were separately administered directly within the TG. Statistical analysis revealed that within 24 h of inducing temporomandibular arthritis using CFA, there was a significant surge in CD86 positive macrophages within the ganglion. These macrophages peaked on the 7th day before beginning their decline. In this context, it’s noteworthy that administering CGRP to the trigeminal ganglion can prompt these macrophages to adopt the M2 phenotype. Intriguingly, this study demonstrates that injecting the CGRP receptor antagonist (CGRP 8-37) to the ganglion counteracts this shift towards the M2 phenotype. Supporting these in vivo observations, we found that in vitro, CGRP indeed fosters the M2-type polarization of macrophages. CGRP can facilitate the conversion of macrophages into the M2 phenotype. The phenotypic alterations of macrophages within the TG could be instrumental in initiating and further driving the progression of TMJ disorders.

Keywords: Calcitonin gene-related peptide · Macrophages · Neuroinflammation · Temporomandibular joint osteoarthritis · Trigeminal ganglion

Approximately 10–15% of adults suffer from temporomandibular arthritis and masticatory muscle dysfunction and pain (Gonçalves et al. 2011). Chronic pain, the primary complaint of temporomandibular disorders (TMD) patients, not only diminishes their quality of life but also imposes significant health issues and a considerable societal burden. Despite its prevalence, the precise cause of TMD pain remains elusive. Recently, neuroimmune factors, which are potential targets for the prevention and treatment of inflammation, have garnered attention in temporomandibular arthritis research (Maruyama et al. 2017).

The TMJ receives its sensory innervation primarily from the auriculotemporal and masseter nerves. Both of these nerves are branches of the mandibular division of the trigeminal nerve (Kucukguven et al. 2022). Structurally, the TMJ is

Junli Tao and Xiaohui Wang have equally contributed as co-first authors.

* Jie Xu
xujie@hospital.cqmu.edu.cn

Junli Tao
2021110506@stu.cqmu.edu.cn

Xiaohui Wang
2021110508@stu.cqmu.edu.cn

1 College of Stomatology, Chongqing Medical University, Chongqing, China

2 Chongqing Key Laboratory for Oral Diseases and Biomedical Sciences, Chongqing, China

3 Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing, China

Received: 23 October 2023 / Accepted: 19 January 2024
© The Author(s) 2024

composed of the mandibular fossa, mandibular condyle, and the articular disc, all of which are enveloped by the articular capsule. This capsule comprises an outer fibrous layer and an inner synovial membrane. Within the synovial membrane, two distinct cell types can be identified: fibroblast-like stromal cells and lining cells (Nozawa-Inoue et al. 2003). Moreover, immune cells, such as macrophages, neutrophils, and mast cells, are also present in this membrane (Hayashi et al. 2002; Nozawa-Inoue et al. 2003). It’s noteworthy that primary headaches, including migraines and TMD, often involve the trigeminal nervous system in their pathogenesis. Calcitonin gene-related peptide (CGRP), a neuropeptide, has been implicated in the etiology of migraines. Furthermore, its role in the pathogenesis of TMD and the onset of cranial hypersensitivity makes it a promising target for TMD treatments (Cady et al. 2011; Romero-Reyes et al. 2015; Shu et al. 2020).

CGRP, synthesized and secreted by trigeminal ganglion neurons, is a pivotal neuropeptide in pain and immune regulation. Its release from peripheral or central terminals and neuronal cell bodies serves to activate other neurons and satellite glial cells through autocrine or paracrine mechanisms. Notably, during the chronic pain phase of OA, an upsurge in trigeminal ganglion’s CGRP is observed (Duffadar et al. 2009; Miller et al. 2017). This neuropeptide, predominantly generated by neurons, is released from neural endings to exercise its modulatory effects. Upon release, it binds to CGRP receptors, thereby modulating the immune functionalities of macrophages (Ma et al. 2010; Mikami et al. 2013). Further investigations highlight a potential significant association between CGRP-induced macrophage modulation and the pro-inflammatory cytokine TNF-α (Li et al. 2010). Such research underscores the extensive immunoregulatory role of both neurogenic and macrophage-derived CGRP, laying a solid foundation for examining their impacts on the immune response in temporomandibular arthritis.

Recent studies have demonstrated that immune cells, which infiltrate injured nerves, play multifaceted roles in pathological neurons (Stratton and Shah 2016). Macrophages, for instance, are pivotal in regulating inflammation resolution. These immune cells promote tissue homeostasis by transitioning their phenotype from the pro-inflammatory M1 state to the anti-inflammatory M2 state (Alvarez et al. 2016). Despite being a form of neurogenic inflammation, the impact of macrophage phenotypic shifts in the TG on peripheral nerve repair and hyperalgesia during the progression of temporomandibular joint arthritis remains unexplored. To address this gap, we established a rat model of temporomandibular arthritis. This study delves into the polarization of macrophages in the trigeminal nervous system both morphologically and molecularly during arthritis progression. Additionally, we probed the role of macrophage infiltration in the rat’s trigeminal ganglion during temporomandibular arthritis and examined the interplay between CGRP and macrophage polarization. Our findings lay the groundwork for further research into the therapeutic potential of macrophage-targeted treatments for temporomandibular arthritis and offer new insights for neuropathic pain management.

In our study, we acquired 48 seven-week-old male Sprague–Dawley (SD) rats, each weighing between 220 and 270 g, from the Experimental Animal Centre of Chong Qing Medical University. These rats were maintained in a specific pathogen-free (SPF) laboratory environment. Our experimental procedures were thoroughly reviewed and approved by the Ethics Committee for Animal Research at the School and Hospital of Stomatology, Chong Qing Medical University, China, with the given approval number: Ethics Review 2022 (119).

After a one-week isolation period, we randomly divided the rats into 8 groups, with each group consisting of 6 SD rats. The first group received saline injections in the temporomandibular joint (TMJ). Groups two to five had CFA injected into the TMJ, with subsequent observations at 1, 3, 7, and 14 days, respectively. The sixth group underwent a CFA injection in the TMJ and a simultaneous saline injection in the trigeminal ganglion. The seventh group received CFA in the TMJ and CGRP in the ganglion. The eighth group was administered CFA in the TMJ and CGRP 8-37 in the ganglion. For the administration of drugs via ganglion injection, after establishing an arthritis model in SD rats through CFA injection into the temporomandibular joint, drugs were administered into the trigeminal ganglion of the rats. Anesthesia was administered using 1.5% isoflurane in oxygen, sourced from Abbott Laboratories, North Chicago, IL, USA. Following the established protocol (Kameoka et al. 2010), a 27-gauge 0.5-inch needle was precisely inserted at the anterosuperior portion of the zygomatic arch root of the rat. We then administered a bilateral slow injection of 50 µL of either saline or CFA (product number F5881 from Sigma-Aldrich, USA) into the upper compartment of the TMJ.

In the CFA-injected group, rats were categorized into three subgroups for trigeminal ganglion injection: CGRP, CGRP 8-37, and normal saline, with 6 rats in each subgroup. We

prepared the solutions of rat CGRP (10⁻⁵ M, MCE) and CGRP 8-37 (a CGRP antagonist, 10⁻⁵ M, MCE), both at a concentration of 10⁻⁵ M, in normal saline (Afroz et al. 2019). These were aliquoted and stored at −20 °C in accordance with the manufacturer's guidelines. For TG intra-ganglionic (IG) drug delivery, we followed the previously published protocols (Neubert et al. 2005). A 26-gauge Hamilton syringe with a 10 μL capacity was used for IG administration. The entry point for the needle was the infra-orbital foramen, positioned 1 mm medial to the zygomatic process of the maxilla. We inserted the needle approximately 22 mm, angling it toward the midline by roughly 10 degrees and 15 degrees downward from the plane of the parietal bone (Villa et al. 2010). This procedure allowed the needle to pass through the infraorbital canal and reach the ipsilateral trigeminal ganglion located in Meckel's cave. To verify the accuracy of our injections, methylene violet was injected to visualize the trigeminal ganglion (Fig. 1E).

temporomandibular joint on both sides. Von Frey filaments were specifically employed to assess the TMJ mechanical sensitivity, with measurements conducted at 1, 3, 7, and 14 days post-injection. For mechanical sensitivity testing, Von Frey filaments were applied in ascending order, starting with a 2 g filament for control animals and a 0.16 g filament for inflamed animals. Each filament was applied five times at intervals of a few seconds, and the response threshold was determined by the minimal force that induced three or more head withdrawal reactions during the five applications. We maintained a consistent 2-min interval between subsequent filament applications. All testing procedures were conducted in a tranquil setting to preclude the influence of diurnal variations.

The THP-1 human monocytic leukemia cell line, authenticated by short tandem repeat spectrometry, was acquired from Procell Life (Wuhan, China). It was cultured in RPMI 1640 medium (Procell Life, Wuhan) supplemented with 10% heat-inactivated fetal calf serum (FCS), and the cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. After cultivating, the THP-1 cells underwent centrifugation at 800 RPM/min for 5 min. Post centrifugation, the supernatant was removed, and the cell density was adjusted to 1 × 10^6/mL using fresh culture medium. Subsequently, Phorbol ester (PMA) was introduced to the cell suspension until it reached a concentration of 100 ng/ml. Following gentle mixing, the cell suspension was allocated into 6-well plates, with each well containing 2 ml. These plates were then incubated under controlled light exposure. After a 48-h treatment with PMA, a notable change was observed in the cells: they transitioned from being in suspension to displaying protruding pseudopods as adherent cells. This transformation was confirmed under a microscope. Eventually, the THP-1 cells differentiated into M0 macrophages, which were then cleansed twice with sterile PBS. To understand the impact of CGRP on the inflammatory response of these macrophages, cells were pre-treated with CGRP (10^−6 M, MCE) for 30 min. Thereafter, they were either cultured in a medium containing 50 ng/mL LPS (MCE) + 20 ng/mL IFN-γ (MCE) for 24 h or in another medium with 20 ng/mL IL-4 (MCE) + 20 ng/mL IL-13 (MCE), specifically for a 72-h period targeting M0 macrophages.

Rats were euthanized using an overdose of isoflurane for a humane endpoint. We meticulously extracted the temporomandibular joint tissues, including the mandibular condyle disc, retro-discal area, fossa, and the trigeminal ganglion, from each rat across all groups. These tissues were then fixed in 4% paraformaldehyde for a 24-h period. For histopathological evaluation, the TMJ specimens were subjected to demineralization in a 19% solution of EDTA, with the solution being refreshed every three days. Meanwhile, TG tissues were prepared for dehydration by immersion in 30% sucrose solution overnight.

Total RNA was isolated from cells utilizing the RNAiso Plus reagent (TaKaRa), which was then reverse-transcribed into cDNA with TaKaRa’s reverse transcription kit. The quantitative real-time PCR (qRT-PCR) was performed using SYBR Green (TaKaRa) as the DNA-binding dye, on the 7500 Fast Real-Time PCR System (Applied Biosystems). For normalization purposes, the expression of β-actin mRNA was used as a reference against the expression of the target mRNA. For each cell sample, three replicate wells were set up to eliminate technical errors in the experiment. The specific sequences for the PCR primers utilized in this investigation were shown below: CD86 F_5ʹ-GTTTCATTCCCTGATGTTACGAG-3ʹ R_5ʹ-GAGAAAGGTGAAGATAAAAGCCG-3ʹ, CD206 F_5ʹ-GCTGAAAGGTGACCCTACTATGT-3ʹ R_5ʹ-GCTCAGGTTTTGGTGTTTGTC-3ʹ.

Demineralized TMJs from two groups (CFA-treated, n = 6 joints; Saline-treated, n = 6 joints) were embedded in paraffin and cut into 5 μm thick mid-sagittal sections. These sections were then dewaxed, rehydrated, and triple-washed with PBS to prepare for antigen retrieval. For this process, the slides were immersed in sodium citrate (pH 6.0) and heated to 95 °C for 20 min. Following antigen retrieval, the sections were washed in PBS for 10 min and blocked with 10% bovine serum albumin for one hour at room temperature. Subsequently, these tissues were exposed to primary antibodies: CD86 (Huabio, ER1906, 1:400) and CD206 (CST, 24595, 1:500) and incubated overnight at 4 °C. Upon completion, after three PBS washes, the sections were introduced to a horseradish enzyme-labeled streptavidin solution (Abcam; dilution, 1:500) for an hour at room temperature. The final step involved incubation in diaminobenzidine (DAB, Sigma-Aldrich) for coloration and a counterstain using hematoxylin. In this experiment, the immunohistochemistry assay was performed in triplicate on joint tissue from each identical biological sample, with the results being averaged across these technical replicates.

Statistical analyses were conducted utilizing GraphPad Prism version 9.5 and SPSS version 25. All results are expressed as mean ± standard deviation (SD). We employed one-way analysis of variance (ANOVA) followed by Dunnett's t-test to compare means, assuming normality and homogeneity of variances across samples. For all identical experiments, the number of technical replicates (NTR = 3) is three to exclude technical errors. Levels of statistical significance were denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

TG tissues were prepared by embedding them in an optimal cutting temperature compound (OCT) (Sakura, USA) and then sectioning them serially at a thickness of 10 μm. First, the specimens underwent a 10-min wash using phosphate-buffered saline (PBS). This was followed by antigen retrieval, which entailed heating the specimens in citrate using a microwave oven for 25 min. To prevent nonspecific binding, we treated the tissues with 5% bovine serum albumin (BSA) for 1 h at 37 ℃. Subsequently, the tissues were incubated with primary antibodies: CGRP (CST, ab81887, 1:1000), CD206 (CST, 24595, 1:500), and CD86 (ABclonal, A1199, 1:100) overnight at 4 ℃. As a next step, the tissues were treated with goat anti-rabbit Alexa Fluor 488 (CST, 4412s, 1:1000) secondary antibodies for 1 h at 37 ℃. For cell nuclei visualization, DAPI (Beyotime, C1006, 1:100) was added, with a 5-min incubation period. Images were captured using a fluorescence microscope (Carl Zeiss, Baden-Wurttemberg, Germany). Within the TG, enumerations of both CGRP-positive neurons and total neurons, which were labeled with DAPI, were conducted across five fields of each section. Two independent evaluators performed these counts, using a 20× objective lens for CGRP-labeled immunofluorescence, and a 40× objective lens for CD86 and CD206 labeled immunofluorescence. The data was assessed based on the count of positive cells and the intensity of immunofluorescence. In the technical replicates for both immunofluorescence and immunohistochemistry experiments, this study employed a consistent procedure, where the results for each biological sample were derived from the average of three identical assays. Image J software was employed for all immunofluorescence data analyses.

Immunofluorescence indicates that neurons labeled with CGRP are mainly found at the junction of the trigeminal ganglion and the mandibular branch. Concurrently, this study delineates the temporal changes in the size (μm²) of these neurons in the trigeminal ganglion following CFA-induced temporomandibular joint arthritis in rats (Fig. 2). A marked increase in the mean fluorescence intensity of CGRP compared to the control group was observed on days 1, 3, 7, and 14 post-induction (p = 0.0372, p = 0.0044, p = 0.001, and p < 0.001, respectively), with the most pronounced changes on days 7 and 14 (Fig. 2G). Notably, CGRP-labeled trigeminal neurons in cases of temporomandibular arthritis were predominantly within the 200 to 400 μm² range (Fig. 2B–F). These findings underscore the crucial involvement of CGRP-labeled neurons in the development of temporomandibular arthritis pain and their impact on both the peripheral and central nervous systems.

To assess the changes in M1 and M2 macrophage populations in the trigeminal ganglion after CFA-induced temporomandibular joint arthritis, we examined the expression of CD206 and CD86 in the TG using immunofluorescence in frozen rat sections (Fig. 3A). We identified both CD206-positive M2 and CD86-positive M1 macrophages in the TGs of both naive and CFA-treated rats. Quantitative analysis showed an increase in CD86-positive M1 macrophages post-CFA treatment, peaking at day 7 (Fig. 3B).

We established the pain model of temporomandibular arthritis in rats by CFA injection and conducted behavioral monitoring. Initially, within 7 days post-injection, we documented a time-dependent increase in pain via HWT measurements (Fig. 1B). A significant reduction in HWT was observed immediately following the injection, with the lowest HWT recorded on the 7th day (Fig. 1C). In the later phase, beyond 7 days, HWT began to recover but was still lower than the saline-injected control rats. The HWT was notably reduced on days 1, 3, 7, and 14 post-injection when compared to controls (p = 0.2747, p = 0.0102, p = 0.0017, and p = 0.3946, respectively). Treatment with CGRP 8-37 in the ganglion resulted in a significant increase in HWT relative to saline injections (Fig. 1D), particularly on days 1, 3, and 7 (p = 0.0017, p = 0.049, p = 0.0005, respectively). On the other hand, CGRP injections without the antagonist led to decreased HWT at these same time points compared to the saline group (p = 0.0024, p = 0.3595, p = 0.0436, respectively). Overall, the CFA injection induced a time-dependent pattern of pain, peaking on day 7. The application of CGRP 8-37 effectively mitigated this pain.

These macrophages increased progressively on days 1, 3, and 7 compared to the control group, with the peak at day 7 and a subsequent decrease by day 14 (p = 0.7376, p = 0.1193, p < 0.0001, and p < 0.0001, respectively). In contrast, the number of CD206-positive M2 macrophages declined within 24 h after CFA treatment, reaching the lowest level on day 3, and began to recover, returning to baseline levels by day 7 (Fig. 3C). This trend was significant on day 3 (p = 0.0218) but not on other days when compared to the control group. The M1/M2 macrophage ratio in the TG rose until the end of the first week and then decreased by the end of the second week (Fig. 3D), with the ratio changes being statistically significant on day 3 and day 7 (p = 0.0008, p < 0.001, respectively). Notably, CD206-positive M2 macrophages were observed surrounding TG neurons, displaying distinct morphology and size compared to the neurons. These findings highlight a possible link between CGRP-expressing neurons and macrophage polarization, which may play a role in the modulation of neuroinflammation.

To explore the influence of CGRP on macrophage polarization in the TG, we administered CGRP and its antagonist, CGRP 8-37, directly into the TG. The application of CGRP promoted a shift in macrophages towards the M2 phenotype compared to the control. Conversely, the administration of CGRP 8-37 impeded this M2 polarization (Fig. 4B, C). Relative to the control, the M1/M2 ratio notably increased following the injection of CGRP 8-37, with significant statistical differences (Fig. 4D). After the TG was injected with CGRP 8-37 and CGRP, CD86-positive macrophages were significantly increased and decreased, respectively (p < 0.001 and p = 0.1939, respectively), while CD206-positive macrophages exhibited an inverse pattern (p = 0.6133 and p = 0.0403, respectively). The ratio of CD86-labeled M1 to CD206-labeled M2 macrophages correspondingly increased and decreased (p = 0.002 and p = 0.0037, respectively). Moreover, in vitro studies corroborated that CGRP

induces a transition towards the M2 phenotype and deters the transition towards the M1 phenotype under inflammatory conditions (Fig. 4E). Specifically, CGRP treatment inhibited the differentiation of M0 macrophages into M1 macrophages and facilitated their differentiation into M2 macrophages (p = 0.0280 and p = 0.0226, respectively), confirming the modulatory role of CGRP in macrophage polarization.

To elucidate the M1/M2 macrophage dynamics in temporomandibular joint tissues affected by CFA-induced inflammation, immunohistochemistry was used to assess CD86, CD206, and CGRP expressions in rat TMJ (Fig. 5A). Both control and CFA-treated rats displayed CD86-positive M1 and CD206-positive M2 macrophages in the TMJ tissues. Notably, CD86-positive M1 macrophages significantly increased one day after CFA treatment, peaking at day 7 (Fig. 5B). These macrophages in the TMJ's surrounding soft tissues surged initially and then diminished from day 14 onwards (p = 0.6672, p = 0.1276, p < 0.0001, p = 0.0033, respectively). Likewise, CD206-positive M2 macrophages increased, reaching a maximum at two weeks post-inflammation (Fig. 5C). Their numbers rose progressively on days 1, 3, 7, and 14 (p = 0.0999, p = 0.0008, p = 0.0003, p < 0.0001, respectively). CGRP expression, primarily around blood vessels, significantly spiked one day post-CFA and then declined (Fig. 5D), with the highest expression on day 1, decreasing on days 3, 7, and 14 (p < 0.0001, p = 0.0001, p = 0.0381, p = 0.9095, respectively). These observations demonstrate the critical role of the M1–M2 macrophage shift in the inflammation and progression of TMJ arthritis.

To assess the impact of CGRP and CGRP 8-37 on macrophage polarization in the temporomandibular joint vicinity, we administered these compounds into the trigeminal ganglion of rats with CFA-induced TMJ arthritis. Using immunohistochemistry, we detected CD86, CD206, and CGRP expressions, as shown in Fig. 6A. CGRP 8-37 injections significantly increased CD86 levels in the TMJ adjacent tissues, with no notable change in CD206 levels and a reduction in CGRP compared to the control (Fig. 6B–D). In contrast, exogenous CGRP injections decreased CD86 levels while increasing CD206 and CGRP expressions significantly. Specifically, the number of CD86-positive M1

This study pioneered an exploration into the temporal dynamics and spatial localization of CGRP expression within the trigeminal ganglion and the regulation of the M1/M2 macrophage balance throughout the progression of TMJ in rats. We observed that as the model induction time increased, CGRP expression in both the trigeminal ganglion and the temporomandibular joint progressively increased, correlating with heightened pain behavior. Triggered by peripheral inflammation and nerve injury, there is an upregulated release of CGRP from neurons in the trigeminal node.

macrophages rose with CGRP 8-37 and fell with CGRP treatment (p < 0.0001, p = 0.045, respectively), while CD206-positive M2 macrophages showed the opposite trend (p = 0.7932, p = 0.0272, respectively). CGRP levels followed a similar pattern, decreasing with CGRP 8-37 and increasing with CGRP injection (p = 0.0467, p < 0.0001, respectively). These results underscore the regulatory effects of CGRP on macrophage polarization, which may contribute to the pathophysiology of TMJ arthritis.

an upregulated release of CGRP from neurons in the trigeminal node. When exogenous CGRP is administered directly into the trigeminal ganglion in vivo, there's notable promotion in the macrophages' transformation into M2 within the trigeminal ganglion, accompanied by an augmented CGRP expression in the temporomandibular joint tissues. Contrastingly, an injection of the CGRP receptor-specific inhibitor, CGRP 8-37, into the trigeminal ganglion relieved TMJ pain and concurrently lowered the CGRP expression levels in peripheral TMJ tissues.

CGRP-positive neurons are notably prevalent within the TMJ complex, including areas like the joint capsule, synovial membrane, temporomandibular joint posterior disc tissue, masticatory muscles, tendons, and fascia (Azuma and Sato 2017; Lindquist et al. 2021; Suttle et al. 2023). Echoing our findings, CGRP was predominantly expressed around the blood vessels in the temporomandibular joint tissues of rats. Known for its extensive presence in both central and peripheral nervous systems and in pain pathways of unmyelinated A δ and C sensory nerve fibers (Chen and Wu 2020; Iyengar et al. 2019), CGRP is chiefly discharged from peripheral nerve fibers, such as those found in the trigeminal ganglion.

Originating from the cell bodies of sensory neurons, it is then conveyed to their peripheral endpoints (Ulrich-Lai et al. 2001).

CGRP is prominently expressed in the trigeminal nervous system, with its levels notably rising in the trigeminal ganglion during the chronic pain phase of OA (Appelgren et al. 1995; Jiang et al. 2022; Miller et al. 2017). This molecule, while not directly instigating the disease’s acute inflammatory process, is pivotal in modulating inflammation. Supporting its significance, research indicates that the specific CGRP receptor antagonist MK-8825 can substantially mitigate facial pain and curtail the widespread release of pro-inflammatory cytokines (Romero-Reyes et al. 2015). These findings align with our experimental study results. When CFA is administered into the temporomandibular joint, there’s not only an upregulation of CGRP expression in the sensory system of trigeminal nerve injury but also an augmentation in the peripheral distribution of CGRP within the temporomandibular joint—a phenomenon closely linked to pain behavior. In the TG context, utilizing the CGRP antagonist CGRP 8-37 has shown pain-relieving effects. Yet, the introduction of exogenous CGRP in the TG leads to diminished peripheral pain thresholds, accompanied by enhanced CGRP expression in the vicinity of the temporomandibular joint tissues.

Macrophages located within sensory ganglia are pivotal in regulating inflammation, facilitating nerve tissue repair, and modulating neuropathic pain post-peripheral nerve injury. Existing research has highlighted the swift proliferation of ganglionic tissue-resident macrophages after such injuries. These macrophages infiltrate the spaces between satellite glial cells and neurons, directly engaging with the latter. Characteristically behaving as the M2 type and with minimal neuronal cell death, it is inferred that this neuron-macrophage interaction is instrumental for tissue repair (Iwai et al. 2021). In this study, our analysis indicated a rise in CD86-labeled M1 macrophages within the trigeminal ganglion during acute inflammation following the intra-TMJ injection of CFA. Concurrently, there was an initial decline followed by an upsurge in CD206-labeled M2 macrophages.

This dynamic played a pivotal role in preserving the inflammatory homeostasis within the trigeminal ganglion. Intriguingly, the elevation in CGRP expression within the trigeminal ganglion correlated with an increase in M2-labeled macrophages. When exogenous CGRP was injected into the TG, the count of CD206-labeled M2 macrophages rose, while CD86-labeled M1 macrophages decreased. Furthermore, the introduction of exogenous CGRP into the trigeminal ganglion enhanced CGRP expression in the surrounding TMJ tissues. This was accompanied by a rise in CD206-labeled macrophage expression in adjacent TMJ tissues and a reduction in CD86-labeled macrophages. However, the administration of CGRP 8-37 diminished both CGRP expression and TMJ pain, with a concurrent relative reduction in CD206-labeled macrophages within TMJ tissues.

In this study involving CFA-induced TMJ, both M1 and M2 macrophages in the trigeminal ganglion predominantly encircle CGRP-labeled neurons. This arrangement suggests a modulatory role of CGRP in macrophage activation and polarization within the ganglion. Macrophages have the capability to navigate via diverse signaling routes, adapting to microenvironmental changes. Numerous research studies have characterized the subset of macrophages responsive to tissue injury as "activated macrophages." While their appropriate activation is crucial for upholding physiological harmony in the body, any aberrant activation could induce tissue impairment and immune dysfunctions, leading to various ailments. It is documented that macrophages are instrumental in the onset, progression, and development of nerve injuries and peripheral sensitization, a trait attributed to the profound functional plasticity of these cells (T'Jonck et al. 2018; Zhang et al. 2016).

CGRP has been identified to have anti-inflammatory and immunoregulatory roles in inflammatory responses. Serving as key modulators for tissue stability and repair, macrophages can switch their phenotype from pro-inflammatory to either anti-inflammatory or pro-healing (Körner et al. 2019). Consistent with this, research has demonstrated that CGRP can specifically influence this phenotypic transition, promoting IL-4-induced M2 macrophages while inhibiting

In rats with CFA-induced temporomandibular arthritis, we observed a significant recruitment and polarization of macrophages within the trigeminal system. Delving into the findings, the study primarily reveals two major conclusions:

1. Within the trigeminal ganglion, the presence of CGRP facilitates the conversion of macrophages to the M2 phenotype, effectively mitigating the inflammatory processes.
2. Macrophages are integral to the neuroinflammatory response. Significantly, their phenotypic changes within the TG are pivotal in initiating and further driving the progression of temporomandibular arthritis.

...macrophages in the TG undergo significant phenotypic transformations, which are pivotal in the initiation and progression of temporomandibular arthritis (Fig. 7).

Author Contributions  The study’s design and execution were overseen by JT. XW and JX were responsible for the collection and analysis of data. The initial draft of the manuscript was composed by JT. Subsequently, JX and XW provided critical revisions to the draft. Finally, all authors have read and consented to the published version of the manuscript. The contributions of JT and XW to this research are equally important.

Data Availability  The datasets utilized and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflict of interest  The authors declare no conflicts of interest in relation to this study.

Ethical Approval  The experiments reported in this manuscript adhered to all relevant international, national, and institutional guidelines for the care and use of animals. The study was conducted in compliance with the current laws of the country where the experiments were performed, under the approval number: Ethics Review 2022 (119).

Consent to Participate  All experiments in this manuscript were performed following the ethical standards of the Animal Research Ethics Committee at the College of Stomatology, Chongqing Medical University, China.

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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[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]
Aerogel possesses distinctive features rendering it widely applicable in thermal insulation. Nevertheless, supercritical fluid drying (SCFD) method and freeze-drying employed for aerogels necessitates the complex process, resulting in heightened energy consumption and more risk. To achieve the cost-effective preparation of silica aerogel, we introduce an in-situ acid catalysis strategy for rapidly crafting high-performance SiO2 aerogel materials under ambient conditions. The resultant SiO2 aerogels exhibit remarkable porosity (95%) and a substantial specific surface area (759 m²/g). Furthermore, SiO2 aerogel composites display low thermal conductivity (0.015 W·m⁻¹ K⁻¹), coupled with commendable mechanical property. More importantly, we reveal the mechanism of the in-situ acid catalysis (ISAC) strategy. The entire production cycle of SiO2 aerogel composites and powder is only 6 and 4 h respectively, which greatly reduces the preparation time while ensuring excellent performances. This study introduces a novel approach for the industrial, low-cost, and rapid preparation of SiO2 aerogel materials through ambient pressure drying.

Received: 28 May 2024 / Accepted: 11 August 2024 / Published online: 16 August 2024
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

Zun Zhao¹ · Yuelei Pan¹ · Mingyuan Yan¹ · Yueyue Xiao¹ · Hui Yang¹ · Xudong Cheng¹

Aerogels have a highly porous microstructure with an air volume of 85–99.8% [1–3], with a nacre chain network composed of loosely stacked and bonded particles or fibers [4, 5]. Due to these structural characteristics, silica aerogels exhibit low density (typically 30–180 mg cm–3), low refractive index (1.0–1.05), high surface area (500–1200 m2 g–1), and high mesopore volume (1–5 cm3 g–1) [6–8]. One of the major characteristics of silica aerogels is their very low thermal conductivity, typically of the order of 0.01 W m–1 K–1 at ambient temperature, pressure, and relative humidity. These values are significantly lower than the conductivity of air under the same conditions, e.g., 0.025 W m–1 K–1. Thus, silica aerogels are among the best-known thermal insulating materials [9].

In the process of preparation of SiO2 aerogels, the drying is a key step in the transition from wet gel to dry gel. There are three kinds of commonly used drying methods: supercritical fluid drying (SCFD), freeze-drying and ambient pressure drying (APD). SCFD, which is the earliest proposed and most widely used drying method, involves placing the wet gel and solvent in a high-pressure container [10, 11]. The pressure and temperature are adjusted to exceed the corresponding critical point of the solvent, rendering it in a supercritical state. The solvent is then maintained in this state for a period of time before slowly returning it to normal temperature and pressure conditions. During the drying process, the aerogel does not lose its structure due to the absence of a distinct gas-liquid phase differentiation caused by surface tension [12]. Currently SCFD used in industrial production are EtOH supercritical drying and CO2 supercritical drying. In EtOH supercritical drying process, the temperature will exceed 260 °C and the pressure will reach 6.3 MPa. The supercritical point of CO2 is 31 °C and 7.29 MPa. Therefore, the SCFD is considerably costly and risky.

Different from SCFD, freeze-drying is a method which converting liquid-gas interface to solid-gas interface under low temperature and low-pressure conditions. This method utilizes the sublimation of solvent in wet gel to avoid the effect of the capillary stresses [13]. The structural control and performance enhancement of aerogels can be realized by freeze-drying. This method is considered as a green and inexpensive drying method [14]. However, the freeze-drying method is too complex to be applied in large-scale industrial production at present.

The fundamental principle of APD involves initially treating the gel to mitigate capillary force. Common treatment methods include aging to reinforce the gel structure, surface modification using silane coupling agents, and substituting high surface tension solvents in the gel pores with low surface tension alternatives. This approach effectively removes the solvent from the nanopores under normal pressure, while preventing the collapse of the nanopores [15, 16]. The traditional APD method to preparing SiO2 aerogel composites includes: sol–gel synthesis, ion exchange, ageing, solvent replacement, hydrophobic modification and drying. It usually takes 1–7 days to prepare aerogel composites by traditional methods because of the process of solvent replacement and ion exchange. Simultaneously, they possess a relatively high thermal conductivity (0.021–0.049 W m–1 K–1) [17–22].

Herein, for the first time, we present an innovative in-situ acid catalysis (ISAC) strategy for achieving the rapid ambient drying preparation of aerogels. Its production equipment is simple. The process is relatively safe because all of the reaction takes place at ambient pressure. Compared with traditional ambient drying, the process of solvent replacement and ion exchange are avoided in this work. The entire production cycle of SiO2 aerogel composites (120*30 cm2) and powder is only 6 and 4 h respectively. The thermal conductivity of the in-situ acid catalysis aerogel (ISACA) composites is 0.015 W m–1 K–1 at room temperature. This is a great improvement compared to previous methods. Additionally, ISAC strategy is less costly and more environmentally friendly. Using water-glass as the silicon source, an inorganic compound, substantially reduces the cost of industrial production while minimizing the consumption of organic solvents (Comparison of different fabrication methods of aerogel composites in Supplementary Table S1).

Glass-fiber felts were purchased locally from Taobao. Water glass (wt:35%, Na2O:SiO2 = 1:3.3) used was purchased from Jiashan Yourui Refractory Co., LTD. Anhydrous alcohol (EtOH, 99%), hydrochloric acid (HCl, AR, 36.0–38.0%) and hexamethyldisiloxane (HMDSO) were obtained from Shanghai Aladdin Biochemical Technology Co., ltd., all analytical pure. The water used in the preparation process is deionized water.

Initially, 24–82 ml of deionized water, 60 ml of anhydrous ethanol and 58–116 ml of HCl (11.9 mol/L) were added to the beaker and mixed well. Besides, 100 ml of water glass diluent (water glass: deionized water = 1:1, 0.88 mol/L) was added under vigorous stirring. The gel formed in 3–130 min. And then put the gel in 65 °C water bath pot aging for 1 h. Subsequently, 600 ml of hydrophobic modifier was added to

the beaker, which was then heated and stirred in a water bath at 65 °C for 2 h. Finally, the modified powder was placed in a drying oven at 120 °C for 1 h to obtain ISACA powder.

The procedure of fabrication of ISACA composites is schematically presented in Fig. 1a. Firstly, the glass fiber felt was immersed in the prepared sol, which was configured according to the dilution of water glass (0.88 mol/L): hydrochloric acid (HCl, 11.9 mol/L): ethanol: water = 2.5:2.5:1.5:1. The gelation usually occurred in 3 min. After aging at 75 °C for 1 h, the wet gel composite is immersed in hydrophobization agent for hydrophobic modification at 75 °C for 4 h. Finally, under ambient pressure drying at 200 °C for 1 h, ISACA composites can be obtained.

The pore size distribution was measured using a fully automated specific surface area and porosity analyzer (TriStar II 3020M). The microstructure of the samples was characterized by scanning electron microscopy (SEM, SU8220). XPS (ESCALAB 250Xi) were conducted to analyze the elemental composition. XRD (TTR-III) analysis was used to analyze the phases. The densities of aerogels were assessed based on the standard of ISO 845:2006. The Brunauer–Emmett–Teller (BET) method obtained the specific surface area. The pore size distribution was determined using the Barret-Joyner-Halenda (BJH) method and the desorption branch of the isotherms. A Fourier transform infrared (FTIR) spectra was obtained on KBr pellets with a Nicolet iN10 infrared spectrometer (Supplementary Fig. S2).

Mechanical properties were studied by using the universal testing machine (Instron E3000K8953) and dynamic thermomechanical analyzer (DMA, TA Q800). The thermal conductivity of sample was measured employing the Hot Disk technique (TPS2500S). Thermogravimetry-differential scanning calorimetry (TG-DSC) (SDT Q600) was performed to analyze the pyrolysis process. The thermal insulation property was characterized by the infrared thermal imager (FLIR A655sc).

The ISAC strategy can be utilized for the preparation of a larger-scale ISACA sample (120 × 30 cm²), as illustrated in Fig. 1b, showcasing the method's scalability. Besides, the ISACA composites exhibit high machinability and can be customized into various shapes to meet the complex geometric requirements of practical applications (Fig. 1c). Also, we drop stained water onto the composite, and the water forms droplets on the surface. This phenomenon indicates the extraordinary hydrophobic properties of the composite (Fig. 1d, Supplementary Fig. S1). After being burned by the outer flame of the alcohol lamp, the structure remains intact, which proves that it has excellent high temperature resistance (Fig. 1e).

The ISAC strategy significantly reduces raw materials and processing time. The entire production cycle takes

only 6 h, including gel, aging, hydrophobic modification and APD. The solvent exchange step has been removed, which is important to the traditional preparation process. The acidic catalyst used in the gelation and the modification has been added to the sol in advance. The hydrogen ion concentration in the sol can be controlled by adjusting the addition amount of hydrochloric acid. The addition amount of each component and the gel time under different hydrogen ion concentrations are shown in Table 1. The progressive acidification process of sodium monosilicate ions in a strong acid solution is shown in Fig. 2a. In a higher acid concentration, the process of sol gelling is shown in Fig. 2b.

The pore size distribution (PSD) of silica aerogel powder S1–S5 is displayed in Fig. 3a. The produced aerogels' PSD had a distinct unimodal distribution. A more homogeneous PSD and a smaller pore size were the outcomes of raising the [H+]. A wide PSD with a peak value of about 15 nm was seen when the [H+] = 2.0 mol/L (S1). This indicates that the PSD is inhomogeneous. There was a narrow peak about 8 nm as the [H+] increased to 4.0 mol/L (S5). This indicates that when [H+] = 4 mol/L, the pore size in the sample is uniformly distributed around 8 nm. As shown in Fig. 3b, as the concentration of hydrogen ions in the sol continues to increase, the density of the powder continues to decrease and the specific surface area continues to increase. The porosity of the samples is shown in Supplementary Table S1. Figure 3c shows the XRD patterns of the ISACA. The broad diffraction peaks were observed at 2θ = 22°. A low crystallinity degree is indicated by the characteristic peaks of the SiO2 aerogels with alkyl groups. Figure 3d illustrated SiO2 aerogels' formation in the ISAC by X-ray photoelectron spectroscopy (XPS) results. When [H+] = 4.0 mol/L, the aerogel has the best performance. When the [H+] exceeds 4.0 mol/L, stable gel cannot be formed, which is not suitable for this study. Therefore, the maximum [H+] of this work is 4.0 mol/L.

The microstructure of the aerogels (Fig. 4) can also reflect the condition mentioned above. It can be seen that when the [H+] = 2.5 mol/L, there are fewer pores between aerogels, and no clear skeleton structure can be observed. When the [H+] increases, it can be intuitively observed that the pores in the aerogel structure continue to increase, the aerogel skeleton becomes more and more obvious, and even a "fibrous" skeleton structure appears. When the [H+] in the sol reaches 4.0 mol/L, the aerogel structure is the most stable.

All samples: Water glass 100 ml, EtOH 60 ml

The conventional steps for preparation of aerogel composites are: gel, aging, displacement, modification, and drying. However, the ISAC strategy has the advantage of eliminating the displacement step, which is extremely time

Fig. 2 a The progressive acidification process of sodium monosilicate ions in a strong acid solution. b The sol–gel process in higher acid concentrations

The surface of silica hydrogels undergoes a transformation to nonpolarity following the grafting of alkyl or aryl groups throughout the surface modification process. This alteration effectively prevents the condensation reaction between the Si–OH groups within the pores of the silica hydrogels [24]. In this study, silica hydrogels underwent modification through the substitution of –OH groups with –Si(CH₃)₃. The hydrolytically stable –Si(CH₃)₃ groups present in HMDSO are capable of replacing Si–OH groups

on the surface of silicon oxide (Fig. 5b). The inset of Supplementary Fig. S1 displays a photograph of water droplets on the surface of SiO₂ aerogel composites. The aerogels’ superhydrophobic properties further demonstrate the successful hydrophobization of the silica hydrogels via the ISAC method.

In practical production applications, SiO₂ aerogel composites must be capable of withstanding long-term and high-frequency mechanical shocks. To investigate the elasticity and fatigue resistance of ISACA composites, experiments

were conducted using a universal testing machine. A sequence of tests involving 1000 loading–unloading cycles was performed to assess the fatigue resistance of the ISACA composites at a maximum strain (ε) of 40%, with a loading rate of 60 mm/min. The ISACA composites demonstrated the ability to endure up to 40% elastic deformation (ε) (Fig. 6).

Compression tests along the layer-stacking direction at various ε values of 15, 30, and 40% showed that ISACA composites possess considerable mechanical robustness. The compression stress was recorded at 100 kPa for 30% strain, increasing to 200 kPa at a compression strain of 40% (Fig. 7a). ISACA composites exhibited outstanding cyclic durability in loading–unloading fatigue test at 40% strain (Fig. 7b), with only a 3% deformation observed after 1000 cycles. Young’s modulus was measured following 1000 compression cycles (Fig. 7c). After undergoing 1000 cycles, ISACA composites maintained a low energy loss coefficient of nearly 0.04 from the 10th cycle, preserving over 90% of maximum stress. The Young’s modulus of the composite exhibited a slight increase before stabilizing. The low

energy loss coefficient signifies minimal energy dissipation throughout the loading–unloading cycles, while the silica aerogel’s three-dimensional structure contributes to the composite’s high compression resistance. Dynamic mechanical analysis (DMA) test was conducted to evaluate the mechanical robustness and fatigue resistance of ISACA composites under 1% oscillatory strain across 100,000 cycles, further investigating their fatigue resistance. Remarkably, the outcomes of these tests on ISACA composites were unexpected (Fig. 7d). The storage and loss moduli of the ISACA composites were comparable to those of the previously reported super-elastic aerogels [25–27], indicating outstanding fatigue resistance. Meanwhile, dynamic compression test, conducted at an oscillatory strain (ε) of 1%, revealed a nearly constant storage modulus, loss modulus, and damping ratio across a frequency range of 0.1–30 rad/s, further highlighting the robust mechanical properties of ISACA composites (Fig. 7e). The maximum bending modulus of glass fiber (GF) registers at 7.25 kPa. When integrated with the ISAC strategy and SiO₂ aerogels, the composite material exhibits a maximum bending modulus of 202.85 kPa, representing an approximate 28-fold increase. This enhancement has been validated through three-point bending and uniaxial compression tests (Fig. 7f). The ISACA composites exhibit significantly enhanced impact resistance and mechanical robustness compared to similar elastomeric materials documented in previous reports (Fig. 8) [28–33].

An infrared thermal imager was utilized to capture the temperature distribution and its changes over time in order to more precisely describe the ISACA composites’ thermal

insulation capabilities. First, a 300 °C hot plate simulated a moderately high-temperature operating environment. Different types of samples (GF, ASW, and ISACA composites) were placed on the hot plate (Fig. 9a). The infrared thermal imager continuously recorded the upper surface temperature of the samples (Fig. 9c). As illustrated in Fig. 9b, upon heating, the sample surface temperature increased gradually before stabilizing. After 30 min of heating, the upper surface temperature of GF reached 100.7 °C, while that of ASW was 90.4 °C. In comparison, the upper surface temperature of the ISACA composite was 61.1 °C. These results indicate that ISACA composites adequately fulfill the thermal insulation requirements for these temperature scenarios.

In addition, we built ourselves a device to test the thermal insulation properties of the material. The device is heated by a Muffle furnace, which simulates the actual application of the sample in everyday life. Infrared thermograph, thermocouple and module are used to monitor the surface temperature change of the test sample, and computer is used to record and process the data (Fig. 10a). Measurements of the heat insulation and fire resistance of GF, ASW, and ISACA composite (1 cm thickness) are shown in an optical picture (Fig. 10c). Using an infrared camera and thermocouple temperature collector, the sample’s qualitative thermal insulation on the self-built platform is captured, displaying a gradient temperature distribution on the back of ISACA composites (Fig. 10c). At first, the back-side surface’s temperature increased gradually. The samples’ backside surface temperature became smooth and steady after 30 min of exposure to the heat radiation from the Muffle Furnace, reaching a maximum temperature of roughly 101.3 °C (GF), 98.4 °C (ASW), and 88.6 °C (ISACA composite) (Fig. 10b). The temperature difference

was maintained at 300 °C in comparison to a high heating source of 400 °C, demonstrating the enormous potential of ISACA composite in high temperature applications [34].

As seen in Fig. 11a, thermogravimetric analysis (TGA) was used to examine the ISACA composites’ tolerance to high temperatures. Upon reaching approximately 423 °C, a reduction in mass was observed, suggestive of a small number of organic molecules and physically adsorbed water evaporating, contributing to 5.99% of the total mass of ISACA composite. A further mass loss, constituting 3.79% of the overall mass, was noted. This additional decrease in mass is associated with the process of hydroxyl group condensation, leading to dehydration and the formation of Si-O-Si bonds [35], thereby enhancing the SiO₂ network, as demonstrated by the reaction:

≡Si–OH + HO–Si≡ → ≡Si–O–Si≡ + H₂O

Additionally, at various temperatures, we examined the ISACA composites’ thermal conductivity variation law (Fig. 11b). The findings demonstrated that thermal conductivity progressively rose as temperature rose, mostly as a result of an increase in thermal radiation at high temperatures.

Heat conduction through fibers and aerogel, heat transfer through air, and thermal radiation are all included in the heat transfer mode for ISACA composites. (Fig. 12a) [36]. Heat transmitted through (1) aerogel Qₐₑ, (2) aerogel and fibers Qₐₑ+f, (3) air Qₐᵢᵣ, and (4) radiation heat through

The heat radiation can be compared to the radiation between two virtual planes of incoming and outgoing with temperatures \(T_1\) and \(T_2\), since it is assumed that there is a one-dimensional steady-state heat transfer. The radiant heat flux can be stated as follows in the radiant heat transfer energy balance equation:

When \(l_r = L_0\), the thermal resistance to radiation is provided.

Consequently, the ISACA composites’ room temperature thermal conductivity formula (Eq. (4)).

former pathways \(Q_r\) make up the total heat flux. The parallel thermal resistance of the appropriate part equals the total equivalent thermal resistance of the ISACA composites (Fig. 12b). The formula for total thermal resistance (Eq. (1)) can be obtained using the Fourier equation \(Q = \Delta T / R = \lambda_t S_t / l_t \Delta T\).

where \(\nu\) and \(x\) represent the relative volume fractions of air and fiber in ISACA composites. The air section’s thermal conductivity is represented by \(\lambda_{air}\), while SiO\(_2\)’s thermal conductivity is represented by \(\lambda_{ae}\). The thermal radiation synthesis constant is given by \(F = 4G\varepsilon\sigma\), where \(G\) is the radiation shape factor and has a range of 0 to 1. The average temperature of the unit cube is \(T_m\), and it may be found by

Supplementary information The online version contains supplementary material available at https://doi.org/10.1007/s10971-024-06518-2.

Acknowledgements This work was financially supported by Collaborative Innovation Program of Universities in Anhui Province (GXXT-2022-018), National Natural Science Foundation of China (No. 52374238, 52104226 and U2233202), Youth Innovation Promotion Association (No. CX2320007001), Key Laboratory of Fire Protection Technology for Industry and Public Building, Ministry of Emergency Management (No. 2022KLIB02).

In summary, we report a new strategy for the ultra-rapid synthesis of silica aerogels featuring exceptional thermal insulation, high porosity, and extensive specific surface area, achieved through in situ acid catalysis. The synthesized ISACA composites exhibit remarkable elasticity, boasting a bending resistance up to 30 times greater than previously observed. Furthermore, these composites demonstrate outstanding thermal insulation, characterized by an ultra-low thermal conductivity of merely 0.01499 W·m\(^{-1}\) K\(^{-1}\) at ambient temperature. Crucially, the mechanism behind gel modification through the in-situ acid catalysis method is clarified by contrasting it with gel modifications in HMDSO. Because the acid catalyst is included in the sol prior to gelation, it is homogeneously dispersed through the mesopores of the gel, resulting in much reduced hydrophobization time. Additionally, we are able to modify the pore structure of silica aerogels by varying the concentration of hydrogen ions during gelation. We contend that this study offers novel insights into the cost-effective and expedited synthesis of silica aerogels.

Author contributions Zun Zhao conducted the experiment and wrote the main manuscript text. Yuelei Pan and Xudong Cheng devised the project, the main conceptual ideas and proof outline. Mingyuan Yan guided the writing of the entire manuscript text. Yueyue Xiao prepared figure 12 and performed the analytic calculations. Hui Yang and Xudong Cheng were involved in planning and supervised the work. All authors reviewed the manuscript.

\[
\frac{1}{R_t} = \frac{1}{R_{ae}} + \frac{1}{R_{ae+f}} + \frac{1}{R_{air}} + \frac{1}{R_r} \tag{1}
\]

\[
Q_r = \lambda_r S_r / l_r \Delta T = S_r G \varepsilon \sigma (T_1^4 - T_2^4) = 4 S_r G \varepsilon \sigma T_m^3 \Delta T \tag{2}
\]

\[
\lambda_r = 4 L_0 G \varepsilon \sigma T_m^3 = F L_0 T_m^3 \tag{3}
\]

\[
\lambda_r = (1 - \nu^{1/2})\lambda_{ae} + x(\lambda_{air} - \lambda_{ae}) + F \alpha / \nu^{1/2} T_m^3 \tag{4}
\]

\[
T_m^3 = (T_1^3 + T_1^2 T_2 + T_1 T_2^2 + T_2^3) / 4
\]

Conflict of interest The authors declare no competing interests.

Cellulose Nanofibers. ACS Nano 12(1):140–147. https://doi.org/10.1021/acsnano.7b04246

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