Bioinspired Nanofiber Scaffold for Differentiating Bone Marrow-Derived Neural Stem Cells to Oligodendrocyte-Like Cells: Design, Fabrication, and Characterization
Authors: Shohreh Mashayekhan
Fatemeh Rasti Boroojeni
Mohamadhasan Ansarizadeh
Hojjat-Allah Abbaszadeh
Maryam-Sadat Khoramgah
Vafa Rahimi Movaghar
Journal: International Journal of Nanomedicine (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: central nervous system; controlled triiodothyronine release; nanofibers scaffold; oligodendrocyte cells; polyaniline graphene.
Affiliations: Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran., Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linköping University, Linköping, Sweden., Hearing Disorders Research Center, Loghman Hakim Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran., Laser Application in Medical Sciences Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran., Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland., Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Abstract:  Background: Researchers are trying to study the mechanism of neural stem cells (NSCs) differentiation to oligodendrocyte-like cells (OLCs) as well as to enhance the selective differentiation of NSCs to oligodendrocytes. However, the limitation in nerve tissue accessibility to isolate the NSCs as well as their differentiation toward oligodendrocytes is still challenging. Purpose: In the present study, a hybrid polycaprolactone (PCL)-gelatin nanofiber scaffold mimicking the native extracellular matrix and axon morphology to direct the differentiation of bone marrow-derived NSCs to OLCs was introduced. Materials and methods: In order to achieve a sustained release of T3, this factor was encapsulated within chitosan nanoparticles and chitosan-loaded T3 was incorporated within PCL nanofibers. Polyaniline graphene (PAG) nanocomposite was incorporated within gelatin nanofibers to endow the scaffold with conductive properties, which resemble the conductive behavior of axons. Biodegradation, water contact angle measurements, and scanning electron microscopy (SEM) observations as well as conductivity tests were used to evaluate the properties of the prepared scaffold. The concentration of PAG and T3-loaded chitosan NPs in nanofibers were optimized by examining the proliferation of cultured bone marrow-derived mesenchymal stem cells (BMSCs) on the scaffolds. The differentiation of BMSCs-derived NSCs cultured on the fabricated scaffolds into OLCs was analyzed by evaluating the expression of oligodendrocyte markers using immunofluorescence (ICC), RT-PCR and flowcytometric assays. Results: Incorporating 2% PAG proved to have superior cell support and proliferation while guaranteeing electrical conductivity of 10.8 × 10-5 S/cm. Moreover, the scaffold containing 2% of T3-loaded chitosan NPs was considered to be the most biocompatible samples. Result of ICC, RT-PCR and flow cytometry showed high expression of O4, Olig2, platelet-derived growth factor receptor-alpha (PDGFR-α), O1, myelin/oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) high expressed but low expression of glial fibrillary acidic protein (GFAP). Conclusion: Considering surface topography, biocompatibility, electrical conductivity and gene expression, the hybrid PCL/gelatin scaffold with the controlled release of T3 may be considered as a promising candidate to be used as an in vitro model to study patient-derived oligodendrocytes by isolating patient's BMSCs in pathological conditions such as diseases or injuries. Moreover, the resulted oligodendrocytes can be used as a desirable source for transplanting in patients. 

Retracted: No

PubPeer comments:
 This article is getting severe criticism on Twitter. This thread summarises one issue and links to threads on other, more classic, problems. https: twitter.com sTeamTraen status 1295098216271286272 , 
 Supplementary Table 3 shows the population proportions that were used to calculate the weightings. It includes data from the age categories 10-14, 15-19, and 20-24 (5 years each). But the authors divided people into age groups 13-17 (5 years), 18-21 (4 years), and 22-24 (3 years). What assumptions were made in converting from the US Census data to the actual percentage weights used, to account for (a) different category age bounds, (b) different category sizes in 2 cases, and (c) the lack of information about the precise population in the 13-24 age range, given than there were no participants aged 10-12? ![file] , 
 Continuing with Supplementary Table 3, the numbers of males and females sum to 253,915,197. It seems unlikely that over 73 million Americans would fail to report their sex in the census. This number does, however, correspond exactly to the total number of people aged 18 or over (see second images, from the Census page linked in the Supplementary Table). ![file] ![file] In the Census, the percentage of males was 48.7. In Supplementary Table 3 this has become 49.2. It is not clear what explains this difference (which may, however, be in the right direction, given that the sex ratio of 1 males per female in the adult population is mostly due to more females at the top of the age distribution). As a minimum, however, it suggests that the numbers in Supplementary Table 3 are of limited value in explaining the weighting process. Additionally, if the weights assigned were 49.2 and 50.8 for males and females, this leaves zero weighting for people whose sex was recorded as  Other . Yet, apparently, these people had non-zero numbers on all of the outcome variables apart from  Never-users of e-cigarettes Positive COVID-19 test . It is not clear to me how a group that has been given a weighting of zero can have cases. , 
 The article u201cAssociation Between Youth Smoking, Electronic Cigarette Use, and Coronavirus Disease 2019u201d, by S M Gaiha et al (Journal of Adolescent Health (2020) 1-5, https: doi.org 10.1016 j.jadohealth.2020.07.002) concludes that vaping and dual cigarette usage u201c*are significant underlying risk factors for COVID-19*u201d. This claim rests on weak and speculative arguments, as the survey utilized by the authors did not inquire about hospitalization nor progression to severe health conditions (a shortcoming they recognize). If vaping was a serious risk factor for COVID-19, a large presence of vapers among hospitalized COVID-19 patients would have been detected by now. While lack of data at the outset of the pandemic (March April) could justify speculation on the plausibility of vaping as COVID-19 risk factor arguing that a presence of vapers could have remained below the radar, this argument is hard to sustain after six months within the pandemic given the wealth of information on patients comorbidities in hundreds of published studies undertaken in in many countries. u00a0u00a0 The authors examined the incidence of positive negative self-reported results (via a survey that is not publicly accessible) in PCR tests. The latter are merely indicative (without 100% certainty) of SARS-COV-2 contagion, not of disease and thus do not provide information on actual health risks of those infected. At best, the study by Gaiha et al might be revealing that young people who vape and or smoke are more likely to assume risks associated with COVID-19 infection and thus are also more likely to undertake testing (an important covariate because of their higher propensity to assume risks). However, the same can be said of young people who have never vaped or smoked but assume other risky behaviors. The authors surveyed and controlled for socioeconomic factors, ethnicity and compliance with confinement precautions, but not for other risk behaviors besides smoking or vaping (such as alcohol drinking, substance usage, unprotected sex). The lack of this important comparative context further weakens the conclusions of this study. , 
 On behalf of six co-authors, I posted this critique on Qeios: https: www.qeios.com read A58MQC , 
 Drs Farsalinos and Niaura have also posted a critique on Qeios: https: www.qeios.com read TCEJ7G , 
 # Devastating post-publication peer reviews but the paper, PR and politics remain untouched ## Problems with the paper There are now two substantive and highly critical post-publication peer reviews of the Gaiha, Cheng and Halpern-Felsher paper on COVID-19 risks and adolescent vaping **[1]**: Firstly, Gitchell et al. describe six problems with the paper: **[2]**: 1. Implausible Testing Counts 2. No difference in COVID-19 positivity 3. Implausible statistics 4. Unrepresentative sample 5. Lack of biological plausibility 6. Causal Inference with Cross-Sectional Data Secondly, Farsalinos and Niaura summarise their concerns as follows: **[3]** In a recent study, Gaiha et al. examined the association between e-cigarette use and COVID-19 in an online cross-sectional study of people aged 13-24 years conducted from May 6 to May 14, 2020. We have noticed serious issues in population weighting, response bias and biological implausibility. The suggested conclusions and interpretation of the study findings cannot be considered reliable. These issues raise the question of retracting the study. In addition to the GRIM problems and other inconsistencies described above by Nicholas Brown, these two reviews set out serious issues with the paper and the claims made for it by the authors and the institution, Stanford University. There is enough *obviously* wrong with this paper to conclude its findings are unreliable and that it should be retracted. So far there has been no response from the authors to any of the concerns raised. Yet the COVID-19 pandemic is a fast-moving and politically-charged crisis in which authors should publish responsibly and correct or retract responsively before misinformation and misunderstanding cause further harm. They need to act without further delay. ## Problems with the public relations offensive Flaws in the paper itself are only one part of the problem that needs to be addressed by the authors. The other is the public relations offensive mounted on the back of the paper once it was published in the Journal of Adolescent Health on August 11. The authors had no hesitation or qualification in asserting an extravagant causal claim - that e-cigarette use raises the risk of COVID-19 by 5-7 times for young vapers in their press release **[4]**. Among young people who were tested for the virus that causes COVID-19, the research found that those who vaped were five to seven times more likely to be infected than those who did not use e-cigarettes. u201cTeens and young adults need to know that if you use e-cigarettes, you are likely at immediate risk of COVID-19 because you are damaging your lungs,u201d said the studyu2019s senior author, Bonnie Halpern-Felsher, PhD, professor of pediatrics. Similar statements, which go far beyond the reach of the study were published for the pediatric health community, in a summary that asserts a causal claim in its title: *Stanford-led study finds vaping significantly increases risk of COVID-19 in teens* **[5]**: u201cYoung people may believe their age protects them from contracting the virus or that they will not experience symptoms of COVID-19, but the data show this isnu2019t true among those who vape,u201d said the studyu2019s lead author, postdoctoral scholar Shivani Mathur Gaiha, PhD. u201cThis study tells us pretty clearly that youth who are using vapes or are dual-using [vapes and cigarettes] are at elevated risk, and itu2019s not just a small increase in risk, itu2019s a big one,u201d Gaiha said. Unsurprisingly, such an incendiary claim generated considerable media coverage, even though there is no basis for it in fact or evidence. The very best that the authors could claim is that vapers have elements of lifestyle or situation that makes it more likely that they will receive a COVID-19 test. They certainly cannot claim any causal relationship between vaping and COVID-19. ## Problems with the politics Finally, there was a political dimension. On the same day as the article was published, August 11, 2020, Illinois Congressman Raja Krishnamoorthi, Chairman of the Subcommittee on Economic and Consumer Policy, found time to write a press release **[6]** and letter to the Commissioner of the Food and Drug Administration, Dr. Stephen Hahn **[7]** demanding FDA  *clear the market of e-cigarettes* in response to the study. Dr. Bonnie Halpern-Felsher of Stanford University released a seminal study in the Journal of Adolescent Health today showing in a national sample that adolescent vapers aged 13 to 24 are five times more likely than non-vapers to be diagnosed with COVID-19. Those individuals who have vaped and smoked combustible cigarettes in the last 30 days (dual users) are nearly seven times more likely than non-users to be diagnosed with COVID-19, and almost five times more likely to experience symptoms. E-cigarette users are also posing an outsized burden on our coronavirus testing resources. The Stanford University-led study found that past 30-day dual users are nine times more likely to have been tested for COVID-19 than non-users. And those using e-cigarettes alone in the past 30 days are 2.6 times more likely to be tested. If we reduce the number of vapers in America, we will reduce the unnecessary stress we are putting on our testing system. People should not have to wait weeks for COVID-19 test resultsu2014removing the risk posed by vaping will help. Again a causal claim is at the heart of this call. It is assuming that removing vaping products would reduce testing burdens and COVID-19 cases. There is no evidence in the paper to support that claim. If vaping is a marker for other risk behaviours or circumstances (poverty, employment, housing etc) that make COVID tests more likely then these risk would persist after e-cigarettes were  cleared from the market . Representative Krishnamoorthi s proposal is grossly irresponsible for a wide range of additional reasons - perhaps causing millions of vapers to revert to smoking, promoting black markets and criminality, and encouraging consumer workarounds that put them at greater risk. Representative Krishnamoorthi s letter to FDA and press release appeared on the same day, August 11, as the publication of the paper and PR offensive. While I recognise that this claim is made by a politician and not directly the authors, the timing means it is likely that the authors and politician were in contact. Even if there was no contact, responsible authors would have written immediately to Representative Krishnamoorthi to explain the limitations of their study, suggest he withdraw the letter and to prevent their work being used wilfully or accidentally to mislead Congress or to make a baseless and damaging proposal to the FDA. ## References **[1]** Gaiha SM, Cheng J, Halpern-Felsher B. Association Between Youth Smoking, Electronic Cigarette Use, and Coronavirus Disease 2019. J Adolesc Heal. 2020 Aug;0(0). [[link] 30399-2 fulltext)] **[2]** Gitchell JG, Kleykamp B, Niaura R, Shiffman S, Cummings KM, Sweanor D, et al. Bad Data and Bad Conclusions Will Lead to Bad Policy u2013 Implausible Claims that Vaping Increases COVID-19 Risk for Youth and Young Adults. Qeios. 2020 Sep 8; [[link] ] **[3]** Farsalinos K, Niaura R. E-cigarette use and COVID-19 in youth and young adults: serious questions about data reliability and call for retraction. Qeios. 2020 Sep 15; [[link] ] **[4]** Vaping linked to COVID-19 risk in teens and young adults, Stanford University Press Release, August 11, 2020 [[link] ] **[5]** Kate DeTrempe, Stanford-led study finds vaping significantly increases risk of COVID-19 in teens, Stanford Children s Health, August 11, 2020 [[link] ] **[6]** US Congressman Raja Krishnamoorthi, _Chairman Krishnamoorthi Renews Demand For FDA To Clear The Market Of E-Cigarettes Following Study Finding Young Vapers Are 5 Times More Likely To Catch Coronavirus Than Peers_, August 11, 2020. [[link] ] **[7]** US Congressman Raja Krishnamoorthi, Letter to Food and Drug Administration Commissioner Stephen Hahn, August 11, 2020. [[link] ] ,