Triamcinolone Modulates Chondrocyte Biomechanics and Calcium-Dependent Mechanosensitivity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is an in vitro study which examines the dose-dependent effects of triamcinolone ac-15 etonide (TA) on chondrocyte biomechanics and mechanosensitivity using primary human chondrocytes derived from articular cartilage samples obtained from patients undergoing total knee arthroplasty. Their main conclusion is that TA increases chondrocyte stiffness through cytoskeletal remodeling and enhances Ca²⁺-dependent mechanotransduction, suggesting a dual biomechanical response of osteoarthritic chondrocytes to corticosteroid exposure.

Although the AFM data are convinced, the conclusions remain insufficiently supported at the biological and mechanistic level. The dose-dependent effects of TA on the mechanical properties of chondrocytes and their mechanosensing capabilities are described but the downstream mechanosensing signaling pathway activated by mechanical stimuli is not investigated at all . Consequently, the results remain limited and do not fully elucidate the biological mechanisms underlying the observed effects.

I would suggest also to:

• Improve figure 2: it is difficult to appreciate changes in actin cytoskeleton. Higher-resolution imaging or confocal microscopy would significantly improve the clarity of cytoskeletal alterations.
• Gene expression analysis: The results are not statistically significant but show a trend with increasing Sox9 expression alongside decreasing COL2 expression upon stress induction. Since Sox9 is a known positive regulator of COL2 transcription, this apparent contradiction requires further explanation. How many biological replicates were analysed?

Author Response

Comment 1: [Reviewer: it is difficult to appreciate changes in actin cytoskeleton. Higher-resolution imaging or confocal microscopy would significantly improve the clarity of cytoskeletal alterations.]

Figure 1 has been fully renewed to clarify the cellular changes between the different cocentrations of TA in higher resolutions.

Comment 2: [The results are not statistically significant but show a trend with increasing Sox9 expression alongside decreasing COL2 expression upon stress induction. Since Sox9 is a known positive regulator of COL2 transcription, this apparent contradiction requires further explanation. How many biological replicates were analysed?]

As the reviewer mentioned this critical point in regards of SOX9 to COL2A1 balance, we re-run the statistics on PCR data and analyzed the delta CT values, with statistically more robust results in our opinion. Here we obtained positive results on MMP1 reduction by TA, so that the result and discussion parts have been re-written.

Focusing on the reviewers question of SOX9, see lines 350-362: SOX9 and COL2A1 remained their contradictory trends, which did not reach significant level. as the reviewer described, SOX9 level can activate type II collagen expression. The observed contradictory phenomen can be explained on the one hand that, when SOX9 is overexpressed at high level, it can also induce an inhibition of COL2A1 (Magdalini et al 2003). But more likely is that reduction of COL2A1 and SOX9 is known to be suppressed in OA chondrocytes (Lee et al 2010 and Zhong et al 2016), which fit the reduced level of COL2A1 in our study, as the study cohorts consist of 9 patients with higher levels of OA (Kellgren-Lawrence grade III and IV). Additionally, the exposure to TA did not lead to the expected reduction of SOX9 expression in the OA progress, but it is a fact that TA can enhance the genetic expression of SOX9, which is consistent with previous report (Tempfer et al 2009), which probably mildered the OA effect of SOX9 reduction. 

Details to references please see reference list in updated manuscript

Reviewer 2 Report

Comments and Suggestions for Authors

Glucocorticoids (GCs) are steroid hormones commonly used for their anti-inflammatory and immunosuppressive properties, including in the management of osteoarthritis (OA). Their effects on articular chondrocytes are complex and can influence disease progression. GCs suppress the production of pro-inflammatory cytokines and enzymes like matrix metalloproteinases (MMPs) that degrade cartilage matrix. This reduces inflammation-associated cartilage breakdown, thereby potentially slowing cartilage destruction. However, chronic exposure can induce chondrocyte apoptosis or hypertrophy, which may accelerate cartilage degeneration. GCs can suppress the synthesis of extracellular matrix components like collagen type II and aggrecan, impairing cartilage repair and regeneration. GCs may induce phenotypic changes in chondrocytes, including promoting hypertrophic differentiation, which is associated with cartilage calcification and degeneration in OA. So, while glucocorticoids can reduce inflammation and cartilage degradation in osteoarthritis, their direct effects on chondrocytes may also impair cartilage repair mechanisms and promote hypertrophy or apoptosis if used long-term. Therefore, their therapeutic use requires careful consideration of timing, dosage, and duration to balance anti-inflammatory benefits against potential detrimental effects on cartilage health. Therefore, the study of Dr. Liang et al. is very important.

Comments:

1. Abstract: The authors claim that intra-articular GCs may provoke “oxidative stress and chondrotoxicity” but the paper does not explore these issues. The authors should adjust their research for the correct rationale.
2. Abstract: The authors should indicate the number of patients studied and whether these were healthy or OA patients.
3. Introduction, Line 57: The authors should add the characteristics and compare the efficacy of GCs used for OA, which they mention. They should explain, why TA was selected.
4. Lines 86 and 220-222: If clinical TA dosing is from 10 mg to 80 mg while 0.2 mM is equal to 1.13 mg TA, why concentration range of 2 µM to 2mM was used in the study? This should be explained.
5. Line 111, 119: The authors should clearly indicate the total number of the examined patients and present their clinical characteristics. They also should indicate the number of patients involved in each experiment.
6. Lines 126-132: the figures should be placed next to their description.
7. Line 133: The thesis “Since cellular stiffness influences Ca2+ dynamics” is not clear. The authors should clarify it and present corresponding references.
8. Lines 138, 154: Enhanced Ca2+ influx and turnover may result in mitochondria disruption, cellular dysfunction, contributing to conditions such as insulin resistance and other metabolic diseases. It also can trigger apoptosis, water loss due to activation of potassium channels. Why the authors considered the concentration of 0.2 mM as optimal? It could be a borderline concentration, however, harmless concentration is lower and the effects of the harmless concentration should have been explored thoroughly in the study. This should be addressed.
9. Line 133: Could the authors explain if there is any association of cellular stiffness and a clinical stiffness observed in OA patients?
10. Line 190: The critical roles of ion channels should be described in the Introduction. Here the authors should present a reference.
11. Figure 5, line 306: According to ddCt method the control value should be equal to 1. Therefore, the corrected results may not support the authors statement. The calculations should be corrected. As the number of patients was less than 30, the non-parametric statistics should be used while the graphs should be constructed using box and violin approach.
12. Lines 261,263,265: The abbreviation for PCM should be disclosed.
13. Line 325, 337: The number of patients used in these experiments should be indicated.
14. Line 420-441: The detailed protocol of PCR examination should be presented.
15. Lines 442-448: As the number of patients was less than 30, the non-parametric statistics should be used while the graphs should be constructed using box and violin approach.
16. The conclusion should be rewritten in accordance with corrections applied.

 

Author Response

Comment 1: [Abstract: The authors claim that intra-articular GCs may provoke “oxidative stress and chondrotoxicity” but the paper does not explore these issues. The authors should adjust their research for the correct rationale.]

We appreciate the reviewers comment, the rationale have been corrected and specified in line 13 - 18.

Comment 2: [Abstract: The authors should indicate the number of patients studied and whether these were healthy or OA patients.]

Line 20: number of patients (n=23) was added. The samples between the experimental steps have to be collected from different patients. As the samples only source from OA patients, yield of each patient was quite low, so that we needed to ensure enough probe for each experimental step.

Comment 3: [Introduction, Line 57: The authors should add the characteristics and compare the efficacy of GCs used for OA, which they mention. They should explain, why TA was selected.]

the sentence in line 60 – 63 might be misleading, so that we have corrected to a clearer explanation, that TA appears to the most effective glucocorticoid to reach clinical relief.

Comment 4: [Lines 86 and 220-222: If clinical TA dosing is from 10 mg to 80 mg while 0.2 mM is equal to 1.13 mg TA, why concentration range of 2 µM to 2mM was used in the study? This should be explained.]

Section 2. determination of optimal TA concentrations for effective experimental outcomes has been re-written. In summary, at 2 mM, a clear crystalization can be observed macroscopically (see attached file, Figure 1), so that no higher dose can be dissolved without supplementation of clinically used auxillary substances such as benzyl-alcohol. That’s why we started with the highest range of 2mM downwards and at 0.2mM, no crystallization can be found.

Comment 5: [Line 111, 119: The authors should clearly indicate the total number of the examined patients and present their clinical characteristics. They also should indicate the number of patients involved in each experiment.]

Number of patients were provided in the section materials and methods, now also added in abstract. N= number of patients, n = cells per patient. and Clinical characteristics for the OA grade were added in Line 413-415.

Comment 6: [Lines 126-132: the figures should be placed next to their description.]

Figure description are placed in the next line after each figure in each figure. 

Comment 7: [Line 133: The thesis “Since cellular stiffness influences Ca2+ dynamics” is not clear. The authors should clarify it and present corresponding references.]

Line 147: we replaced the word “influence” by “induce” to specify the reported outcomes, 3 references are added. 

Comment 8: [Lines 138, 154: Enhanced Ca2+ influx and turnover may result in mitochondria disruption, cellular dysfunction, contributing to conditions such as insulin resistance and other metabolic diseases. It also can trigger apoptosis, water loss due to activation of potassium channels. Why the authors considered the concentration of 0.2 mM as optimal? It could be a borderline concentration, however, harmless concentration is lower and the effects of the harmless concentration should have been explored thoroughly in the study. This should be addressed.]

We also appreciated the reviewers comment on this issue, which is now adressed in lines 339 – 352: The common recommended clinical dosage is at 20-80mg, but with shown safety in use, so we can assume that not all levels of enhanced Ca2+influx result in total apoptosis of chondrocytes and cartilage degradation. Anish et al 2009 (DOI: 10.1002/jor.20809) showed that there is almost a linear increase in percentual cell death of chondrocytes in a calcium medium with a concentration from 0 to 20mM. As the intracellular calcium level cannot reach 0, a certain percentual cell death needs to be determined, at which “not too many” chondrocytes are lost, but the chondroprotective effect can still be used, when applicating TA. In our study, we observed enhanced calcium influx, but not irreversibly on the gene level, as PIEZO1, 2 and TRPV4 were not significantly influenced. In order of that, we prefer that at this dose, the chondrocytes excite changes in F-actin reorganization, but still have recovery potential. Additional the chondroprotective effect like modulation of MMP1 expression like in our renewed results under this concentration may be used at this concentration. Lower concentration did not show changes in F-actin reorganization and in expression of cytokines, so we assumed that this was the most effective dosage. Also, Porter et al 2024 (DOI: 10.1002/jor.25913) report minimal negative effect on viability, proliferation and anabolic activity, but significant inhibition of catabolic activitiy, when exposed to 1nM to 0.2mM, supporting our choice of 0.2mM TA as probably most effective dosage with least damage (line 260-263). 

Comment 9: [Line 133: Could the authors explain if there is any association of cellular stiffness and a clinical stiffness observed in OA patients?]

There are many studies implicating the change of elastic (young’s) modulus in OA chondrocytes, but it has not reached its agreement, whether hardening or softening, whereas the reviewer mentioned “clinical” stiffness, which we interpret as cartilage or matrix stiffness, can be assumed as a highly sensitive marker for OA, see lines 272-279. A global systemic or joint stiffness can be modified by application of glucocorticoids in arthritis patients, but there are barely any study investigating global systemic or joint stiffness in relation to chondrocyte stiffness in OA patients, which is also not part of our study. But as an example for this issue, Ahmed et al. 2012 linked the progression of joint stiffness in experimental OA mice to proteasome activity by inhibition with MG132 (DOI: 1016/j.pain.2011.08.001).

Comment 10: [Line 190: The critical roles of ion channels should be described in the Introduction. Here the authors should present a reference.]

Lines 74 – 79 have been added in the introduction to summarize roles of PIEZO1, 2 und TRPV4.

Comment 11: [Figure 5, line 306: According to ddCt method the control value should be equal to 1. Therefore, the corrected results may not support the authors statement. The calculations should be corrected. As the number of patients was less than 30, the non-parametric statistics should be used while the graphs should be constructed using box and violin approach.]

Here we want to thank the reviewer for the valuable opinion. In correction as the reviewer suggested, we re-run the statistics on the delta CT data. Due to a clear distribution in QQ-Plot (see attached file Figure 2) and confirmed by normality tests, we applied one way ANOVA and found significant MMP1 modulation, which fits the preliminary results from our following unpublished study. Therefore, we have rewritten the passages: Results, Discussion and Conclusion and changed figure 5. But to verify the data results, we also applied Friedmanns test and Wilcons test and obtained the same results like in ANOVA, which speaks for the reliability of data as shown in the revised manuscript. 

Comment 12: [Lines 261,263,265: The abbreviation for PCM should be disclosed.]

The word “pericellular matrix” is now introduced in line 280.

Comment 13: [Line 325, 337: The number of patients used in these experiments should be indicated.]

as commented in Comment 2, we added number of patients in the different sections. 

Comment 14 [Line 420-441: The detailed protocol of PCR examination should be presented.]

Detailed protocol was followed analogous to previous reports of our group, references see line 511

Comment 15: [Lines 442-448: As the number of patients was less than 30, the non-parametric statistics should be used while the graphs should be constructed using box and violin approach.]

see our answer in Comment 11. 

Comment 16: [The conclusion should be rewritten in accordance with corrections applied.]

See Line 542 - 549: Conclusion has been adjusted. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I reviewed the manuscript again, and for me the revision done by the authors is okay.