The Susceptibility of Tenocytes from Different Ages of Donors Towards Dexamethasone and Ascorbic Acid can be Screened in a Microfluidic Platform

Round 1

Reviewer 1 Report

I think the paper is not appropriate for this journal. The title is misleading and too long. The paper does not have any microfluidic components. They merely using a device that might have a microfluidic part inside. The conclusion is drawn from only 2 samples. It is not clear how the statistical analysis is done on a single sample and what is used as control. I think the presentation should be improved and the paper transfers to a biomechanic journal.   

 

Author Response

Reviewer 1

 

The title is misleading and too long.

 

Reply: Thanks for the comment. We tried to confirm tenocytes from different ages of patients can proliferate inside the microfluidic system and respond to different concentrations of dexamethasone and ascorbic acid accordingly.

 

The paper does not have any microfluidic components. They merely using a device that might have a microfluidic part inside.

 

Reply: Thanks for the comment. Tenocytes from different ages of patients were cultured in a “microfluidic device” (xCELLigence system (Roche/ACEA Biosciences, San Diego, CA)). The advantage of using a microfluidic system here is because freshly cultured tenocytes are not manifold available in a sufficient amount in most tenocytes research.[1] The microscale dimensions of microfluidic xCELLigence system provide a real-time, label-free and non-invasive method to see cell proliferation under different circumstances.[2]

 

The conclusion is drawn from only 2 samples. It is not clear how the statistical analysis is done on a single sample and what is used as control.

 

Reply: Thanks for the comment. We only include two samples in this study is because tenocytes tend to have phenotypic drift between individuals and passages. Close cell apposition appears to be lost early, from passage 0 to passage 1, with ‘‘rounding’’ becoming prominent at passage 4.[3] Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if tenocytes passaged multiple times are used.[4] Therefore, we only recruited a single patient in each age group rather than mixed samples from different patients of the same age, to decrease the confounding factors from multiple patients.

 

References

[1]       Almarza AJ, Augustine SM, Woo SL. Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts. Ann Biomed Eng 2008;36(12):1927-33.

[2]       Kho D, MacDonald C, Johnson R, Unsworth CP, O'Carroll SJ, du Mez E, et al. Application of xCELLigence RTCA Biosensor Technology for Revealing the Profile and Window of Drug Responsiveness in Real Time. Biosensors (Basel) 2015;5(2):199-222.

[3]       Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM. Phenotypic drift in human tenocyte culture. Tissue Eng 2006;12(7):1843-9.

[4]       Mazzocca AD, Chowaniec D, McCarthy MB, Beitzel K, Cote MP, McKinnon W, et al. In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers. Knee Surg Sports Traumatol Arthrosc 2012;20(9):1666-72.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

The author screens the best dexamethasone and ascorbic acid combination treatment for tenocytes from young and old donors via a high throughput microfluidic platform xCELLigence system. Individual effect of dexamethasone and ascorbic acid on tenocytes from young and old donors is investigated at first. Then three combinations of dexamethasone and ascorbic acid are used to treat the tenocytes. The author finds that tenocytes from old donor have best proliferation when they are treated by 0.5 mg/mL dexamethasone and 250 μg/mL ascorbic acid, while tenocytes from young donor are more resistant to the cytotoxicity of 2.5 mg/mL dexamethasone when they are treated along with 500 μg/mL ascorbic acid. These results are significantly beneficial for personalized treatment of tenocytes used for anterior cruciate ligament reconstruction and thus greatly contribute to medical applications of microfluidics.

 

Overall, this manuscript is very well written and logically organized. I highly recommend this manuscript for publication. Here I still want to ask the author two questions:

1. Does the author consider using tenocytes from male donors aged 30 years old?

2. The two samples are only from two individual patients. Are the results affected by other factors? And why does the author not use more samples from different patients of the same age?

Author Response

Reviewer 2

 

1.Does the author consider using tenocytes from male donors aged 30 years old?

Reply: Thanks for the comment. Yes, we have considered using tenocytes from male donors aged 30 years old. However, the main objective of this study is to confirm tenocytes from different ages of patients can proliferate inside the microfluidic system and respond to different concentrations of dexamethasone and ascorbic acid accordingly. If the results are positive, we can confirm the clinical application of the microfluidic system as a powerful screening tool for drug cytotoxicity because dexamethasone is believed to increase apoptosis and senescence of tenocytes,[1] while ascorbic acid can protect hamstring-derived tenocytes from oxidative stress.[2] Our study demonstrated the hypothesis and hence, the optimal combination dose of ascorbic acid and dexamethasone could be determined before clinical application. The reason why we used 20 and 41-year-old patients in this study is that CD 34+ cells are more prevalent in adolescent ACL remnants and related tissues and decrease with age. [3] CD34+ cells sorted from ACL-ruptured tissue exhibit a high potential for proliferation and multilineage differentiation.[4] They also have a greater effect on the maturation of bone-tendon healing in a rat ACL reconstruction model compared with cells collected from older patients.[5] In our institute, most ACL reconstructions are done between 20 to 40 years old. Therefore, we select patients with relatively extreme ages we commonly dealt with in this study and try to figure out if age is a determining factor regarding the tenocytes susceptibility towards dexamethasone and ascorbic acid.

 

2.The two samples are only from two individual patients. Are the results affected by other factors? And why does the author not use more samples from different patients of the same age?

 

Reply: Thanks for the comment. We only include two individual patients in this study is because tenocytes tend to have phenotypic drift between individuals and passages. Close cell apposition appears to be lost early, from passage 0 to passage 1, with ‘‘rounding’’ becoming prominent at passage 4.[6] Therefore, only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models, and one has to be aware of the phenotypic changes if tenocytes passaged multiple times are used.[7] Therefore, we only recruited a single patient in each age group rather than mixed samples from different patients of the same age, to decrease the confounding factors from multiple patients. The other advantage of using a microfluidic system as a screening platform is because freshly cultured tenocytes are not manifold available in a sufficient amount in most tenocytes research.[8] The microscale dimensions of microfluidic xCELLigence system provide a real-time, label-free and non-invasive method to see cell proliferation under different circumstances.[9]

 

References

[1]       Jo CH, Lee SY, Yoon KS, Shin S. Effects of Platelet-Rich Plasma With Concomitant Use of a Corticosteroid on Tenocytes From Degenerative Rotator Cuff Tears in Interleukin 1beta-Induced Tendinopathic Conditions. Am J Sports Med 2017;45(5):1141-50.

[2]       Poulsen RC, Carr AJ, Hulley PA. Protection against glucocorticoid-induced damage in human tenocytes by modulation of ERK, Akt, and forkhead signaling. Endocrinology 2011;152(2):503-14.

[3]       Uefuji A, Matsumoto T, Matsushita T, Ueha T, Zhang S, Kurosaka M, et al. Age-Related Differences in Anterior Cruciate Ligament Remnant Vascular-Derived Cells. Am J Sports Med 2014;42(6):1478-86.

[4]       Matsumoto T, Ingham SM, Mifune Y, Osawa A, Logar A, Usas A, et al. Isolation and characterization of human anterior cruciate ligament-derived vascular stem cells. Stem Cells Dev 2012;21(6):859-72.

[5]       Nakano N, Matsumoto T, Takayama K, Matsushita T, Araki D, Uefuji A, et al. Age-dependent healing potential of anterior cruciate ligament remnant-derived cells. Am J Sports Med 2015;43(3):700-8.

[6]       Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM. Phenotypic drift in human tenocyte culture. Tissue Eng 2006;12(7):1843-9.

[7]       Mazzocca AD, Chowaniec D, McCarthy MB, Beitzel K, Cote MP, McKinnon W, et al. In vitro changes in human tenocyte cultures obtained from proximal biceps tendon: multiple passages result in changes in routine cell markers. Knee Surg Sports Traumatol Arthrosc 2012;20(9):1666-72.

[8]       Almarza AJ, Augustine SM, Woo SL. Changes in gene expression of matrix constituents with respect to passage of ligament and tendon fibroblasts. Ann Biomed Eng 2008;36(12):1927-33.

[9]       Kho D, MacDonald C, Johnson R, Unsworth CP, O'Carroll SJ, du Mez E, et al. Application of xCELLigence RTCA Biosensor Technology for Revealing the Profile and Window of Drug Responsiveness in Real Time. Biosensors (Basel) 2015;5(2):199-222.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors here determined the effects of ascorbic acid and dexamethasone, alone, and in combination, on tenocytes isolated from young and aged donors. All experiments was done on a commercial microfluidic platform, xCelligence, that allows continual monitoring of cell adhesion properties and results were expressed as Cell Index measurements.

The introduction and methods were well presented, giving ample background and clarity in approach respectively. The results does narrow down the range of dose of both ascorbic acid and dexamethasone.

The journal should expect a custom-'micromachine' as compared to the use of a commercially avail product, that the authors have also used in a series of similarly conducted papers.

Chiu et al. (2019), Real-Time Monitoring of Ascorbic Acid-Mediated Reduction of Cytotoxic Effects of Analgesics and NSAIDs on Tenocytes Proliferation, Int'l Dose Response Society. Drugs: Bupivacaine, ascorbic acid and ketorolac tromethamine. Chiu et al. (2017), Comparison between xCELLigence biosensor technology and conventional cell culture system for real-time monitoring human tenocytes proliferation and drugs cytotoxicity screening, J Ortho Surg Res. Drugs: Ketorolac tromethamine, bupivacaine, methylprednisolone, and betamethasone.

In 2. the biology was more in-depth with gene expression studies, a direction that authors should continue along to provide more impact to the current submission. Other assays (protein secretion profile, surface markers specific to tenocytes) should even make the study more significant. In addition, the use of tenocytes from aged (n=1) and young (n=1) patients might not be representative of populations due to statistical insignificance. 

These points should be addressed. It will show that the use of such commercial micromachines/microdevices are capable of generating biological readouts as well, to compliment just cell adhesion. 

Author Response

Other assays (protein secretion profile, surface markers specific to tenocytes) should even make the study more significant. In addition, the use of tenocytes from aged (n=1) and young (n=1) patients might not be representative of populations due to statistical insignificance. These points should be addressed. It will show that the use of such commercial micromachines/microdevices are capable of generating biological readouts as well, to compliment just cell adhesion. 

 

Reply: Thanks for your kindly comment. This manuscript focused on the clinical application of a well-established microfluidic system (xCELLigence). As some published articles confirmed, tenocytes from patients with different ages and gender revealed different cell growth capacities.^1-3 Also, human tenocytes change the expression level of surface markers and their cytokine release in response to complex stimulation mixtures originating from activated human immune cells as well as the single proinflammatory cytokines.⁴ To control the variables, we used tenocytes only from 2 patients with same gender and disease but different ages, to see the proliferative capacity and their real-time responses to different stimuli inside microfluidic system. As you mentioned, our group has confirmed gene expression is identical in both conventional and xCELLigence system.⁵ Therefore, we did not perform “protein secretion profile and surface markers specific to tenocytes” in this study because there was not enough material to perform all of the tests on all samples. Most of the specimen was used for ACL reconstruction rather than performing the study. On the other hand, only the first 3 passages of tenocytes were used in this study (edited in the revised manuscript in the section of “Isolation of human tenocytes”) because it is known that tenocytes quickly lose their specific phenotype in vitro⁶ and only cells within the first 3 passages should be used as a precaution for in vitro monolayer cell models.⁷ The study also confirms the advantages of using a microfluidic system for drug screening because it provides high throughput capacity and high content data from a relatively limited cell source.

 

References

Klatte-Schulz F, Pauly S, Scheibel M, et al. Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors. PLoS One 2013;8:e67209. Klatte-Schulz F, Pauly S, Scheibel M, et al. Influence of age on the cell biological characteristics and the stimulation potential of male human tenocyte-like cells. European cells & materials 2012;24:74-89. Pauly S, Stahnke K, Klatte-Schulz F, Wildemann B, Scheibel M, Greiner S. Do patient age and sex influence tendon cell biology and clinical/radiographic outcomes after rotator cuff repair? Am J Sports Med 2015;43:549-56. Stolk M, Klatte-Schulz F, Schmock A, Minkwitz S, Wildemann B, Seifert M. New insights into tenocyte-immune cell interplay in an in vitro model of inflammation. Sci Rep 2017;7:9801. Chiu CH, Lei KF, Yeh WL, et al. Comparison between xCELLigence biosensor technology and conventional cell culture system for real-time monitoring human tenocytes proliferation and drugs cytotoxicity screening. J Orthop Surg Res 2017;12:149. Stoll C, John T, Endres M, et al. Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering. J Orthop Res 2010;28:1170-7. Mazzocca AD, McCarthy MB, Chowaniec D, et al. Bone marrow-derived mesenchymal stem cells obtained during arthroscopic rotator cuff repair surgery show potential for tendon cell differentiation after treatment with insulin. Arthroscopy 2011;27:1459-71.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Noting is changed in the paper, even the title (still too long a misleading). I do not see any reason to change the review I provided as a result.

No new data is presented in the author's response. My point about using a commercial microfluidic system is also confirmed in the response. The paper does not add anything to “microfluidic technology”, just use a commercially available system that happened to have microfluidics part. therefore, it might be more appreciate for a biomechanic journal.

 

My point about to patients was more about the statistical conclusions in the results, not the nature of the problem.

Author Response

The title is still too long and misleading.

 

Reply: Thanks for your kindly comment. Do you consider it more appropriate if the title is changed into “The susceptibility of tenocytes from different ages of donors towards dexamethasone and ascorbic acid can be screened in a microfluidic platform.”?

 

My point about to patients was more about the statistical conclusions in the results.

 

Reply: As some published articles confirmed, tenocytes from patients with different ages and gender revealed different cell growth capacities.^1-3 Also, human tenocytes change the expression level of surface markers and their cytokine release in response to complex stimulation mixtures originating from activated human immune cells as well as the single proinflammatory cytokines.⁴ To control the variables, we used tenocytes only from 2 patients with same gender and disease but different ages, to see the proliferative capacity and their real-time responses to different stimuli inside microfluidic system. Each experiment was performed six times. To compare CI among different culture conditions, analysis of variance (ANOVA) followed by Bonferroni post hoc test was used. A p-value of <0.05 was considered significant.

 

 

References

Klatte-Schulz F, Pauly S, Scheibel M, et al. Characteristics and stimulation potential with BMP-2 and BMP-7 of tenocyte-like cells isolated from the rotator cuff of female donors. PLoS One 2013;8:e67209. Klatte-Schulz F, Pauly S, Scheibel M, et al. Influence of age on the cell biological characteristics and the stimulation potential of male human tenocyte-like cells. European cells & materials 2012;24:74-89. Pauly S, Stahnke K, Klatte-Schulz F, Wildemann B, Scheibel M, Greiner S. Do patient age and sex influence tendon cell biology and clinical/radiographic outcomes after rotator cuff repair? Am J Sports Med 2015;43:549-56. Stolk M, Klatte-Schulz F, Schmock A, Minkwitz S, Wildemann B, Seifert M. New insights into tenocyte-immune cell interplay in an in vitro model of inflammation. Sci Rep 2017;7:9801.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The authors addressed my concerns regarding the number of sample size. However, secretion and genetic changes should still be assessed given that these are different drugs used from prior studies. Suggest referencing studies that indicate that genetic or protein markers do not differ if not in the position to carry out extra experiments. no further comment.

Author Response

Thanks for the comment. Poulsen et al¹ assessed cell survival, collagen production, and the induction of signaling pathways in primary human tenocytes treated with dexamethasone and the antioxidant effects when they were cotreated with ascorbic acid. Dexamethasone treatment resulted in reduced viable cell number, cell proliferation, and collagen production. Dexamethasone induced reactive oxygen species generation in tenocytes and strongly up-regulated the stress-response transcription factors FOXO1 and FOXO3A. Phosphorylation of ERK and protein kinase B/Akt, which regulates cell proliferation and also inhibits forkhead activity, was decreased after dexamethasone treatment. Chemical inhibition of ERK or Akt activity significantly reduced the tenocyte cell number. Ameliorating the dexamethasone-induced reduction in ERK or Akt activity by cotreatment with ascorbic acid protected against the reduction in cell number. Ascorbic acid cotreatment therapy also silenced FOXO1 and prevented the dexamethasone-induced reduction in collagen 1⍺1 expression. Therefore, the ascorbic acid injection may be a clinically useable co-therapy to reduce side effects associated with dexamethasone therapy.¹ As the results were well- established by Poulsen et al¹, which is referenced in the manuscript (Reference No. 21), we did not study further genetic marker because we focused on the clinical application of the high throughput microfluidic system in this study.

 

References

Poulsen RC, Carr AJ, Hulley PA. Protection against glucocorticoid-induced damage in human tenocytes by modulation of ERK, Akt, and forkhead signaling. Endocrinology 2011;152:503-14.

Author Response File: Author Response.pdf