Can mesenchymal stem cell and retinoic acid therapies more effectively reduce lung damage due to bronchopulmonary dysplasia?

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SENEM ALKAN ÖZDEMİR, Eda Açıkgöz, Aslı Çelik, Osman Yılmaz, Meral Sarper, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6387279/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Aim: The purpose of the present study was to assess the efficacy of retinoic acid (RA) treatment combined with mesenchymal stem cells (MSCs) compared to MSCs alone in lung injury due to bronchopulmonary dysplasia. Methods: 29 Wistar baby rats on the postnatal third day were used. The experiment started on the postnatal 3rd day after the birth of the cubs. It was continued until the postnatal 14th day. Hyperoxia system was created with the help of Plexiglas chambers. Bone marrow-derived stem cells (1x10 6 ) were administered intraperitoneally. Retinoic acid (500 mcg/kg) was administered intraperitoneally every day starting from the third postnatal day and continued until the 13th postnatal day. Morphometric analyses and histological analyses were performed to provide more detailed assessments of lung structure changes. To assess fibrosis, Masson's trichrome staining was used to show collagen deposition in lung sections. Immunohistochemistry and immunofluorescence staining were performed to assess alpha-smooth muscle actin (α-SMA) expression levels. Results: The enlarged alveolar distribution decreased in the group receiving MSCs, whereas the MSCs+RA group had regular alveolar structures similar to the healthy controls. morphometric analyzes were performed by measuring the mean linear intercept (MLI), alveolar tissue distribution, alveolar size and vessel wall thickness. MLI, alveolar size and vessel wall thickness were significantly higher in the hyperoxic group compared to the control group, whereas alveolar tissue distribution was decreased. In the treatment groups, α-SMA expression was found to be at lower levels compared to hyperoxic group. the results revealed that lung fibrosis-associated α-SMA expression was reduced by MSCs and MSCs+RA treatments. Conclusion: The combined treatment significantly decreased fibrosis and inflammation in the BPD model, and the best reduction was achieved with the combination of the two therapies. bronchopulmonary dysplasia lung injury preterm retinoic acid stem cell Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by requiring mechanical ventilation and oxygen therapy. It is characterized by restricted lung growth, alveolar and blood vessel development and impaired pulmonary function 1 . Despite the innovations in neonatology, bronchopulmonary dysplasia (BPD) remains a significant concern. BPD is a disease with a high risk of mortality and morbidity 2 . The lung triggers a pro-inflammatory response that promotes the release of free radicals in response to elevated oxygen levels and mechanical ventilation. Disruption of alveolar architecture results in fewer alveoli, simpler structures and less surface area for gas exchange. Alveolar septal fibrosis also develops in conjunction with modest thickening of the smooth muscles of the airways 3 . Vitamin A and its active metabolite, retinoic acid (RA), play an important role in the growth, proliferation and differentiation of most cells 4 . Retinoids are effective in the branching and development of the lung. Particularly, it plays a role in the differentiation of epithelial cells in the respiratory tract 3 , 4 . Administration of vitamin A to very low birth weight infants reduces the frequency of BPD and may provide a protective/repairing effect especially for immature lungs 5 . Research involving the administration of mesenchymal stem cells (MSCs) to animal models of lung damage caused by hyperoxia has yielded encouraging results. Neonatal lung injury has been lessened by MSCs given intravenously, intraperitoneally, or intratracheally, as evidenced by reduced lung inflammation 6 , avoidance of vascular damage and alveolar development arrest, and suppression of lung fibrosis 6 – 7 . It was hypothesized that the combination of two useful applications could be more effective and the study was designed around this concept. This study is the first in the literature to use retinoic acid and MSCs therapy together in a neonatal hyperoxia model. Materials and Methods Experimental procedure 29 Wistar baby rats on the postnatal third day, which were obtained from Animal Experimental Research Unit of Dokuz Eylul University Faculty of Medicine, were used. In order to form our working groups; Since the average number of offspring of Wistar albino pregnant rats was 6–8, four pregnant mothers were followed and n = 29 baby rats were housed with their mothers during the study. After the study, the mothers were returned to the laboratory. The subjects were housed and cared for during the study by this unit. This present study was approved by the Dokuz Eylul University Animal Experiments Local Ethics Committee in Izmir (Approval number: 2021/011). There were 29 Wistar-Albino rats in total amount of male and female between each group. Study groups consist of the rats who were on the 3th postnatal day and weighting between 3 and 9 g. Due to the presence of Wistar Albino strain in Dokuz Eylul University Faculty of Medicine Experimental Animals Laboratory and compatibility with the literature, this type was preferred as a subject. All animals were kept at controlled temperature (21 \(\:^\circ\:\) C-23ºC) and 12 h light 12 h dark condition. Considering that their mothers were breastfeeding, the baby rats used in our study were made using cotton as much as possible in order to prevent cannibalism. Figure 1 shows the experimental site and set-up. The experiment started on the postnatal 3rd day after the birth of the cubs. It was continued until the postnatal 14th day. Hyperoxia system was created with the help of Plexiglas chambers. In this way, oxygen concentration was ensured to be ≥ 90. Oxygen monitoring was checked twice a day. Humidity was set to 80% and CO 2 accumulation was prevented 8 . Cubs were checked regularly every day and survival was noted. Application of Mesenchymal Stem Cells and Retinoic Acid MSCs were isolated in bone marrow based on the presence of cell-specific markers CD105, CD90, and CD73, absence of surface markers CD45, CD34, CD14, CD11b, CD79a, CD19, or HLA-DR, plastic adherence, and in vitro differentiation potential. MSCs have limited immunogenicity due to low expression of major histocompatibility antigens, allowing for allogeneic treatment without immunosuppression. These cells may be grown in vitro in an undifferentiated form, allowing for clinical application and cryopreservation. They may impact the immunological response to injury, pathogen defense, and tissue healing. All GMP tests were performed at Health Science University Gulhane Faculty of Medicine Department of Stem Cell Laboratory. Species-matched bone marrow-derived mesenchymal stem cells (1x10 6 ) were administered intraperitoneally. Prior to application, the quantity and viability of mesenchymal stem cells were assessed, and then they were used. Retinoic acid (500 mcg/kg) was administered intraperitoneally every day starting from the third postnatal day and continued until the 13th postnatal day. The study groups were mentioned at the below. The flowchart of the study is shown in Fig. 2 . The study groups were formed as follows; Group 1 , Naive Control (NC) Group n = 5, The group to be monitored in room air, no intervention group. Group 2 , Hyperoxic Control (HC) Group n = 8, BPD model developed and intraperitoneal saline (0.3 ml) Group 3 , Mesenchymal Stem Cell (MSCs) Group, n = 8, BPD model developed ve intraperitoneal MSCs + intraperitoneal saline (0.3 ml) Group 4 , Retionic Acid (RA) Group, n = 8, BPD model developed and given intraperitoneal MSCs + intraperitoneal RA (500 mcg/kg, 0.3 ml) Animals were sacrificed on postnatal day 21 with 120 mg/kg ketamine and 12 mg/kg xylazine (high dose anesthesia). Histopathological evaluation The lungs were resected via thoracotomy and fixed overnight in 10% buffered formalin under a constant inflation pressure of 20 cm H 2 O. Afterwards, the lungs were transferred into freshly prepared 10% buffered formalin and stored for 24 hours. Lung samples were subjected to routine histological tissue processing and embedded in paraffin, as in the previous study 8 . Paraffin-embedded tissues were cut into 5 µm sections with a microtome (Leica) and mounted on poly-l-lysine coated slides. Sections were stained with hematoxylin-eosin (H&E) and imaged using an Olympus BX53 microscope (Olympus) with an Olympus DP74 camera attachment. Morphometric analysis of lung structure Morphometric analyses based on previous studies were performed to provide more detailed assessments of lung structure changes 9 , 10 , 11 . Mean linear intercept measurement was performed at 90 random fields for each animal. Alveolar tissue distribution was measured by modifying Moreira et al 10 technique. Measurements were performed with Image J by obtaining 30 images from each lung (outside the major airways and vascular system). To assess vessel wall tihickness, five different areas for each animal were measured by adjusting the areas containing vessels in the cross-section. Masson’s Trichrome (MT) staining To assess fibrosis, Masson's trichrome staining was used to show collagen deposition in lung sections. Masson's trichrome staining was performed using a kit (04-010802 kit, Bio-Optica) according to the manufacturer's instructions. Immunohistochemistry and immunofluorescence staining Histologically, BPD is characterized by pulmonary vascular remodeling. In this context, immunohistochemistry and immunofluorescence staining were performed to assess alpha-smooth muscle actin (α-SMA) expression levels. The lung sections were stained as previously described with some modifications 9 , 10 , 11 , 12 . Briefly, for inhibition of endogenous peroxidase, 5-µm-thick sections were immersed in 3% H 2 O 2 in methanol for 10 minutes at room temperature and then washed with PBS. To antigen retrieval, samples were heated in citrate buffer (10 mM, pH 6.0) at 90 º C for 10 minutes. After blocking, lung sections were incubated with α-SMA (ab 7817, Abcam) primary antibody at 4°C overnight. For immunohistochemistry staining, secondary antibody (Thermo Scientific, Ultravision Large Volume Detection System Anti-Polyvalent, HRP, TP-125-BN) and streptavidin peroxidase (Thermo Scientific, TS-125-HR) were applied to the samples for 10 minutes, respectively, and washing was carried out with PBS after each step. Subsequently, the sections were subjected to 3,3′-Diaminobenzidine tetrahydrochloride (DAB) (ab64238, Abcam) as a chromogen and finally counterstained with Mayer's hematoxylin. In immunofluorescence staining, samples were incubated with FITC-conjugated secondary antibody for 1 hour at room temperature and covered with mounting medium containing 4´,6-diamidino-2-phenylindole (DAPI, sc-24941, Santa Cruz). Finally, stained lung sections were visualized using an Olympus BX53 microscope with an Olympus DP74 camera attachment. In immunofluorescence staining, α-SMA intensity was measured using Image J (Fiji, version 1.2). Statistical analysis The values obtained in the experiment were analyzed using the SPSS 25.0 program for Windows. The conformity of univariate data to normal distribution will be evaluated by Shapiro-Wilk test and variance analysis will be evaluated by Levene's test. To compare more than two groups according to quantitative data, parametric data will be analyzed with One-Way Anova and nonparametric data will be analyzed with Mann-Whitney U tests. Variables will be analyzed at 95% confidence interval and will be considered significant if p < 0.05. Results The study involved 29 rats (Fig. 2 ). Although there was no difference in average birth weight between the groups, animals under hyperoxia demonstrated a considerable drop in body weight. (p < 0.05, 20.1 ± 1.5 g). Of the rats included in the study, 55% were male and 45% were female. Mesenchymal stem cell and retinoic acid treatments alleviate morphological changes in the lung due to bronchopulmonary dysplasia To evaluate the effects of hyperoxia and being subjected treatments (MSCs and MSCs + RA) protocols on lung tissue architecture, H&E staining was performed and the images were examined under a light microscope. As shown in Fig. 3 , lung alveoli characterised by regular small alveoli were distinguished in the control group, whereas in hypoxia (HC) the lung structure was disrupted and the alveoli were markedly enlarged with a heterogeneous distribution (Fig. 3 A). It was observed that the enlarged alveolar distribution decreased in the group receiving MSCs, whereas the MSCs + RA group had regular alveolar structures similar to the healthy control group (Fig. 3 A). Depending on the histopathological findings, morphometric analyzes were performed by measuring the mean linear intercept (MLI), alveolar tissue distribution, alveolar size and vessel wall thickness. MLI, alveolar size and vessel wall thickness were significantly higher in the hyperoxic group compared to the control group, whereas alveolar tissue distribution was decreased (p < 0.0001) (Fig. 3 B). Compared with the HC group, there was a significant decrease in MLI, alveolar size and vessel wall thickness parameters in the MSCs and MSCs + RA groups (p < 0.0001) (Fig. 3 B). In the analysis of MSCs and MSCs + RA groups, hypoxia-induced lung injury was significantly improved in the MSCs + RA group, similar to the control group. This suggested that combined treatment may be more effective. MT staining was used to assess collagen accumulation in lung tissue. As seen in Fig. 3 C, more significant collagen accumulation was observed in the HC group compared to the control group. There was a significant decrease in collagen accumulation in MSCs and MSCs + RA groups compared to the HC group. Reduced collagen levels were observed to be quite significant in the MSCs + RA group. These results showed that MSCs + RA treatment significantly prevented lung fibrosis due to collagen deposition. High α-SMA expression in the lungs of rats exposed to hyperoxia was suppressed by mesenchymal stem cell and retinoic acid treatments To assess lung fibrosis, immunohistochemical staining was used to assess α-SMA expression levels, a key biomarker of BPD. Dark brown areas representing SMA expression were observed to be more intense in the HC group. Treatment groups exhibited reduced α-SMA expression compared to HC (Fig. 4 A). To both confirm the IHC images and quantify α-SMA expression, IF staining was performed and α-SMA intensity was measured using Image J. Consistent with IHC results, α-SMA intensity was significantly higher than NC. In the treatment groups, α-SMA expression was found to be at lower levels compared to HC. A statistically significant difference in α-SMA expression was found between all groups. Consistent with IHC results, α-SMA intensity was significantly higher than NC (p < 0.0001) (Fig. 4 B and 4 C). In the treatment groups, α-SMA expression was found to be at lower levels compared to HC (Fig. 4 B). A statistically significant difference in α-SMA expression was found between all groups (Fig. 4 C). Taken together, the results revealed that lung fibrosis-associated α-SMA expression was reduced by MSCs and MSCs + RA treatments. Discussion The present treatment for BPD is confined to moderately active medications including caffeine, vitamin A, and dexamethasone, which have been linked to serious long-term neurodevelopmental side effects 13 . Improved treatment choices for preterm babies with BPD can improve lung health and prevent problems, leading to a better quality of life. The limits of medication for BPD have led to a quest for alternative treatment approaches. Stem-cell therapy is a promising possibility for treating several medical disorders. In this study, we aimed to compare the effect of the combined use of mesenchymal stem cells as a promising treatment for the prevention and control of BPD with the accepted treatment of vitamin A with intact controls. In a study on neonatal rats, transdifferentiation of type II alveolar epithelial cells (AT2) into type I alveolar epithelial cells (AT1) was found to increase under hyperoxic treatment 14 . In the developing lung, alveolar septation and angiogenesis are regulated by lung-resident mesenchymal stem/stromal cells. Alveolar septation and angiogenesis are regulated by mesenchymal stem cells in the developing lung 13 – 14 . MSCs have limited immunogenicity due to low expression of major histocompatibility antigens, allowing for allogeneic treatment without immunosuppression. They may be grown in vitro while remaining undifferentiated, allowing for sufficient quantities for clinical usage as well as cryopreservation before to use. They can impact the immunological response to injury, pathogen defense, and tissue healing. MSCs are a promising therapeutic tool in regenerative medicine for their effects on regeneration, repair, immune response, angiogenesis, and tissue protection 15 . Multiple studies have demonstrated that MSCs or MSC-conditioned media can improve hyperoxia-induced BPD in mouse models utilizing various methods of delivery 16 – 18 . MSC treatment in animal models reversed BPD, avoided halted alveolar development, and restored lung alveolarization and vascularization 19 – 21 . MSC therapy improved lung architecture, reduced fibrosis, and increased survival rates in BPD mice 22 . It also reduced the expression of profibrotic factors such as angiotensin II, angiotensin II type 1 receptor, and angiotensin-converting enzyme 23 . MSCs have been shown to modulate the immune system, reduce inflammation, and lower levels of inflammatory mediators 24 . MSCs positive effects are believed to be due to their focus on angiogenesis, immunomodulation, wound healing, and cell survival 25 . Our results provide a new idea and reveal that species-matched systemic MSCs treatment plays a therapeutic role in neonatal rats with BPD, and its use in combination with retinoic acid is more effective, thus clearly demonstrating the additive effect of MSCs. We established a classical BPD newborn rats model, and confirmed that transplantation of MSCs can improve lung structure in rats with BPD. As known, vitamin A plays an important role in pulmonary development. In the connective tissue matrix surrounding the distal airspaces, BPD has demonstrated an abnormal quantity and distribution of α-SMA expression levels, which results in diminished septation and fewer alveoli. One actin isoform that is crucial to fibrogenesis is alpha-SMA. Blood arteries, myofibroblasts, and smooth muscle cells all contain alpha-SMA 26 . The activation of fibroblasts to myofibroblasts is linked to alpha-SMA. The capacity to contract sets myofibroblasts apart from fibroblasts. Transforming growth factor-beta controls the myofibroblast phenotype that produces extracellular matrix compound and expresses α-SMA 27 . According to Rao et al 28 , myofibroblasts' contractile qualities are linked to α-SMA expression and are involved in inflammation, wound healing, fibrosis, and carcinogenesis. α-SMA is also expressed by cancer cells that develop into mesenchymal cells 29 . MSCs have been shown to increase lifespan and exercise tolerance, decrease alveolar loss and inflammation, diminish fibrotic alterations, lower α-smooth muscle actin expression, and avoid pulmonary hypertension 30 . In addition, MSCs attenuated the increased collagen activation. The activation of lung fibrosis due to collagen deposition is the major pathogenic contributor to BPD through its function in the modulaton of collagen deposition 31 . Our findings suggest that fibrosis inhibition in response to MSCs transplantation played an important role in alleviating at least some of the adverse pulmonary effect of hyperoxia. Currently, the precise molecular mechanisms of these action remain to further investigation. The results imply that MSCs may shield human newborns' other organs from O 2 -induced harm in addition to the lung. In infants who subsequently develop BPD, an excessive inflammation occurs especially in the first few days of life. Low vitamin A levels in extremely low birth weight infants are associated with an increased risk of BPD 32 . Vitamin A and its active metabolite retinoic acid play an important role in the growth, proliferation and differentiation of most cells. It is effective in the branching and development of the lung and reduces fibrosis. However, immunomodulatory/anti-inflammatory effect has not been demonstrated. Although 5000 IU vitamin A supplementation administered intramuscularly three times a week for four weeks decreased the rate of BPD, the effect rate was moderate 33 . Indeed, in the largest evaluation, the researchers found a statistically significant reduction in the rate of death or BPD, but the results were as follows: 55% in the RA group versus 62% in the placebo group 34 . Compared with the control group, there was a small benefit of vitamin A in reducing the risk of death or oxygen requirements at one month of age 35 . Indeed, no benefit on long-term respiratory or neurodevelopmental prognosis was observed. This demonstrated the need for combined therapies. In this study, we investigated the effect of mesenchymal stem cell therapy, which has anti-inflammatory effects, and retinoic acid treatment, which is the only currently accepted method of retinoic acid application. It is the only study in which the two were used together and it was clearly shown that the best results can be achieved with the combined application of the them. Few studies have compared MSCs from various sources for their therapeutic effectiveness against BPD. Placenta, adipose tissue and bone- marrow may be a more convenient supply of tissue. Standardized parameters for cultivating MSCs from these sources, including culture media composition and oxygen content in growth chambers, are not addressed. It is also unclear how different culture conditions affect the therapeutic efficacy of MSCs. Third, for cell-based treatments to be effective, there must be a consistent supply of vast amounts of cells. It is vital to standardize culture methodologies that produce cells with consistent effectiveness. Another problem in MSCs-based therapy is understanding whether the therapeutic potential of MSCs stays consistent when cells are passaged to increase cell quantity. Although MSCs are known to multiply and grow throughout passage, it is unclear if their therapeutic effectiveness changes with recurrent passages. This must be addressed in order to standardize the maximum number of MSCs that may be passed through culture without losing therapeutic effectiveness 36 . Our study's most potent feature is that mesenchymal stem cell viability and quantity were assessed right before delivery. MSCs treatment in animals resulted in normal alveolar numbers and significantly decreased lung neutrophil and macrophage buildup 37 . Morphometric measurements of the lung were performed in our study. It was observed that the enlarged alveolar distribution was decreased in the group that received MSCs, and regular alveolar structures were observed in the MSC + RA group, similar to the healthy control group. Bone marrow-derived mesenchymal stem/stromal cells (MSCs) have been used in many research studies due to their easy accessibility. Although several studies using bone marrow-derived MSCs in a hyperoxia-induced environment showed improvement in alveolar structure in a neonatal lung injury model 16 – 18 it has not been found that concomitant use with retinoic acid may have a similar effect on healthy lung. To determine the optimal timing, Chang et al 38 compared early and late stem cell administration. Early administration was shown to be better than late administration in reducing inflammation in the tissue. In our study, rats in the treatment group received early MSCs with hyperoxic injury and one group received combined treatment (MSCs + RA). Early systemic administration of MSCs was found to significantly reduce inflammation. Combined administration of MSCs and RA was shown to be the most effective way in this study. The implications of this work for neurodevelopmental status will be presented in Part-2. While stem cells may be useful therapeutic techniques for the treatment of BPD, various practical obstacles to using stem cell-based therapy in clinical practice must be addressed. Stem cells require standardized procedures for isolation and storage. They must be assessed for their consistent response, which may differ depending on the tissue from which they are separated. This is critical for their future use as potential biotherapeutics in human clinical studies. Conclusion This is the first study to evaluate the combined effect of mesenchymal stem cells and retinoic acid versus mesenchymal stem cells alone on hyperoxic lung injury. We have showed that the combined treatment significantly decreased fibrosis and inflammation in the BPD model, and the best reduction was achieved with the combination of the two therapies. The results of this study suggest that the combined use of RA-MSCs instead of retinoic acid alone may be instructive for clinical use. Declarations Ethics approval and consent to participate This present study was approved by the Dokuz Eylul University Animal Experiments Local Ethics Committee in Izmir (Approval number: 2021/011). Consent for publication Written informed consent was obtained from the patients to publish this paper. Competing interests All authors have no conflict of interest. Funding This research received no external funding. Author contributions Conceptualization, S.AÖ.; data curation, S.AÖ. A.Ç., O.Y. Stem cell isolation and tests: M.S. and G.G. formal analysis, S.AO and E.A.; methodology, S.AO.E.A., and M.Y.; supervision, S.C., and T.GY.; writing-original draft, E.A. and S.AO.; writing-review & editing, S.AO.,E.Ö, and G.Ö. All authors have read and agreed to the published version of the manuscript. Data availability The datasets generated and analyzed for the study are available from the corresponding author upon reasonable request. References Gilfillan M, Bhandari A, Bhandari V. 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Evidence for the Management of Bronchopulmonary Dysplasia in Very Preterm Infants. Child (Basel). 2021;8(4):298. Omar SA, Hafez AA, Ibrahim S, Pillai N, Abdulmageed M, et al. Stem-Cell Therapy for Bronchopulmonary Dysplasia (BPD) in Newborns. Cells. 2022;11(8):1275. Aslam M, Baveja R, Liang OD, Fernandez-Gonzalez A, Lee C, Mitsialis SA, Kourembanas S. Bone Marrow Stromal Cells Attenuate Lung Injury in a Murine Model of Neonatal Chronic Lung Disease. Am J Respir Crit Care Med. 2009;180:1122–30. Chang YS, Choi SJ, Ahn SY, Sung DK, Sung SI, Yoo HS, Oh WI, Park WS. Timing of umbilical cord blood derived mesenchymal stem cells transplantation determines therapeutic efficacy in the neonatal hyperoxic lung injury. PLoS ONE. 2013;8(1):e52419. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 12 Apr, 2026 Reviewers agreed at journal 26 Jun, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 23 Apr, 2025 First submitted to journal 15 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6387279","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452219785,"identity":"88dbe908-08ab-49a0-8e3f-0168c7f9b977","order_by":0,"name":"SENEM ALKAN ÖZDEMİR","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDCCA1CajwdIfABiNnZitbABtTDOADGYSdHCDLKJgZAWvtvHHz4uqNgmz8Zz+Nljm1/b5PmYGRg/fMzBrUXyXI6x8Ywztw3beNvMjXP7gAxmBmbJmdtwazE4w8Mmzdt2m7GNn8FMOrcHyGAGeocXrxb25795/922b+Nn/yZt2QNkENbCYMbM23A7sY23x0ya4QeQQUiL5BkeY2meY7eT23jOlEn2NgAZzIzNeP3Cd4b94Weemtu2/Tzp2yR+/LltO7+9+eCHj3i0oALGNjDZQKx6EPhDiuJRMApGwSgYKQAAT89MzDdHJwMAAAAASUVORK5CYII=","orcid":"","institution":"Dr. Behcet Uz Cocuk Hastanesi: TC Saglik Bakanligi SBU Dr Behcet Uz Cocuk Hastaliklari Ve Cerrahisi Egitim Ve Arastirma Hastanesi","correspondingAuthor":true,"prefix":"","firstName":"SENEM","middleName":"ALKAN","lastName":"ÖZDEMİR","suffix":""},{"id":452219786,"identity":"cc048726-d9c8-48d9-ba50-e868e83df700","order_by":1,"name":"Eda Açıkgöz","email":"","orcid":"","institution":"Yüzüncü Y?l Üniversitesi: Van Yuzuncu Yil Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Eda","middleName":"","lastName":"Açıkgöz","suffix":""},{"id":452219787,"identity":"1679bf01-81ce-4db2-96cd-6966273a6ca8","order_by":2,"name":"Aslı Çelik","email":"","orcid":"","institution":"Dokuz Eylul University Faculty of Medicine: Dokuz Eylul Universitesi Tip Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"Aslı","middleName":"","lastName":"Çelik","suffix":""},{"id":452219788,"identity":"ec2dc765-3683-42ce-8325-da9c78982129","order_by":3,"name":"Osman Yılmaz","email":"","orcid":"","institution":"Dokuz Eylul University Faculty of Medicine: Dokuz Eylul Universitesi Tip Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"Osman","middleName":"","lastName":"Yılmaz","suffix":""},{"id":452219789,"identity":"cf4092ac-8a78-415c-b814-512717e5fe5c","order_by":4,"name":"Meral Sarper","email":"","orcid":"","institution":"Gülhane Training and Research Hospital: Ankara Gulhane Egitim ve Arastirma Hastanesi","correspondingAuthor":false,"prefix":"","firstName":"Meral","middleName":"","lastName":"Sarper","suffix":""},{"id":452219790,"identity":"6f771382-3ddc-4ebb-bfd8-3196d1adb11d","order_by":5,"name":"Gurur Garip","email":"","orcid":"","institution":"Izmir Tinaztepe University: Izmir Tinaztepe Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Gurur","middleName":"","lastName":"Garip","suffix":""},{"id":452219791,"identity":"7923547d-692d-46d1-8045-b27610250faa","order_by":6,"name":"Esra Özer","email":"","orcid":"","institution":"İzmir Tınaztepe Üniversitesi Tıp Fakültesi: Izmir Tinaztepe Universitesi Tip Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"Esra","middleName":"","lastName":"Özer","suffix":""},{"id":452219792,"identity":"9414e52c-1078-432c-8031-b869cbe9d2a3","order_by":7,"name":"Gülperi Öktem","email":"","orcid":"","institution":"Ege University Faculty of Medicine: Ege Universitesi Tip Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"Gülperi","middleName":"","lastName":"Öktem","suffix":""}],"badges":[],"createdAt":"2025-04-06 14:24:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6387279/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6387279/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82267196,"identity":"61167649-0636-4656-b138-e62b873b0000","added_by":"auto","created_at":"2025-05-08 13:30:41","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":376386,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental site and set-up.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6387279/v1/1a378ba0b855bf9e63bbc094.jpeg"},{"id":82267194,"identity":"14499143-f702-4591-86a7-1ffa58eb1794","added_by":"auto","created_at":"2025-05-08 13:30:40","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126947,"visible":true,"origin":"","legend":"\u003cp\u003eThe flowchart of the study\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6387279/v1/64aed888ff82b64ecad464cc.jpeg"},{"id":82267204,"identity":"c47a1a8d-d552-46cc-a94f-e638c02a71f9","added_by":"auto","created_at":"2025-05-08 13:30:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3106424,"visible":true,"origin":"","legend":"\u003cp\u003eAdministration of MSCs and retinoic acid alleviates hyperoxia-induced lung injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Representative lung tissue of each group with H-E staining (Scale bar: 200 µm and 100 µm). \u003cstrong\u003eB)\u003c/strong\u003e Morphometric analyzes of lung tissue including mean linear intercept (MLI), alveolar tissue distribution, alveolar size and vessel wall thickness (****p\u0026lt;0.0001). \u003cstrong\u003eC)\u003c/strong\u003eMasson Trichrome staining of lung tissue. Blue areas represent collagen content in fibrotic areas (Scale bar: 200 µm and 100 µm).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6387279/v1/03982c931484b9798213785a.jpeg"},{"id":82267203,"identity":"e04e94a2-a150-47be-a0e8-a355253607f2","added_by":"auto","created_at":"2025-05-08 13:30:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2294592,"visible":true,"origin":"","legend":"\u003cp\u003eα-SMA expression in lung tissue. \u003cstrong\u003eA)\u003c/strong\u003e Immunohistochemistry staining of α-SMA. DAB-stained brown areas represent the α-SMA expression level (Scale bar: 100 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB)\u003c/strong\u003e Fluorescence intensity of α-SMA ((Scale bar: 100 µm). \u003cstrong\u003eC)\u003c/strong\u003e Bar graph of mean fluorescence intensity of α-SMA (****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6387279/v1/d04a462e9a41386c575e5230.png"},{"id":82269878,"identity":"80287633-ac89-4781-902b-3e8dd52f867a","added_by":"auto","created_at":"2025-05-08 13:54:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6194152,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6387279/v1/3f645741-4763-4abd-a9d3-b60079b9e7b4.pdf"}],"financialInterests":"","formattedTitle":"Can mesenchymal stem cell and retinoic acid therapies more effectively reduce lung damage due to bronchopulmonary dysplasia?","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by requiring mechanical ventilation and oxygen therapy. It is characterized by restricted lung growth, alveolar and blood vessel development and impaired pulmonary function \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Despite the innovations in neonatology, bronchopulmonary dysplasia (BPD) remains a significant concern. BPD is a disease with a high risk of mortality and morbidity \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The lung triggers a pro-inflammatory response that promotes the release of free radicals in response to elevated oxygen levels and mechanical ventilation. Disruption of alveolar architecture results in fewer alveoli, simpler structures and less surface area for gas exchange. Alveolar septal fibrosis also develops in conjunction with modest thickening of the smooth muscles of the airways \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVitamin A and its active metabolite, retinoic acid (RA), play an important role in the growth, proliferation and differentiation of most cells \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Retinoids are effective in the branching and development of the lung. Particularly, it plays a role in the differentiation of epithelial cells in the respiratory tract \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Administration of vitamin A to very low birth weight infants reduces the frequency of BPD and may provide a protective/repairing effect especially for immature lungs \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResearch involving the administration of mesenchymal stem cells (MSCs) to animal models of lung damage caused by hyperoxia has yielded encouraging results. Neonatal lung injury has been lessened by MSCs given intravenously, intraperitoneally, or intratracheally, as evidenced by reduced lung inflammation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, avoidance of vascular damage and alveolar development arrest, and suppression of lung fibrosis \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt was hypothesized that the combination of two useful applications could be more effective and the study was designed around this concept.\u003c/p\u003e \u003cp\u003eThis study is the first in the literature to use retinoic acid and MSCs therapy together in a neonatal hyperoxia model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental procedure\u003c/h2\u003e \u003cp\u003e29 Wistar baby rats on the postnatal third day, which were obtained from Animal Experimental Research Unit of Dokuz Eylul University Faculty of Medicine, were used. In order to form our working groups; Since the average number of offspring of Wistar albino pregnant rats was 6\u0026ndash;8, four pregnant mothers were followed and n\u0026thinsp;=\u0026thinsp;29 baby rats were housed with their mothers during the study. After the study, the mothers were returned to the laboratory. The subjects were housed and cared for during the study by this unit. This present study was approved by the Dokuz Eylul University Animal Experiments Local Ethics Committee in Izmir (Approval number: 2021/011). There were 29 Wistar-Albino rats in total amount of male and female between each group. Study groups consist of the rats who were on the 3th postnatal day and weighting between 3 and 9 g. Due to the presence of Wistar Albino strain in Dokuz Eylul University Faculty of Medicine Experimental Animals Laboratory and compatibility with the literature, this type was preferred as a subject. All animals were kept at controlled temperature (21\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:^\\circ\\:\\)\u003c/span\u003e\u003c/span\u003eC-23\u0026ordm;C) and 12 h light 12 h dark condition. Considering that their mothers were breastfeeding, the baby rats used in our study were made using cotton as much as possible in order to prevent cannibalism. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the experimental site and set-up. The experiment started on the postnatal 3rd day after the birth of the cubs. It was continued until the postnatal 14th day. Hyperoxia system was created with the help of Plexiglas chambers. In this way, oxygen concentration was ensured to be \u0026ge;\u0026thinsp;90. Oxygen monitoring was checked twice a day. Humidity was set to 80% and CO\u003csub\u003e2\u003c/sub\u003e accumulation was prevented \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Cubs were checked regularly every day and survival was noted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eApplication of Mesenchymal Stem Cells and Retinoic Acid\u003c/h3\u003e\n\u003cp\u003eMSCs were isolated in bone marrow based on the presence of cell-specific markers CD105, CD90, and CD73, absence of surface markers CD45, CD34, CD14, CD11b, CD79a, CD19, or HLA-DR, plastic adherence, and in vitro differentiation potential. MSCs have limited immunogenicity due to low expression of major histocompatibility antigens, allowing for allogeneic treatment without immunosuppression. These cells may be grown in vitro in an undifferentiated form, allowing for clinical application and cryopreservation. They may impact the immunological response to injury, pathogen defense, and tissue healing. All GMP tests were performed at Health Science University Gulhane Faculty of Medicine Department of Stem Cell Laboratory. Species-matched bone marrow-derived mesenchymal stem cells (1x10\u003csup\u003e6\u003c/sup\u003e) were administered intraperitoneally. Prior to application, the quantity and viability of mesenchymal stem cells were assessed, and then they were used.\u003c/p\u003e \u003cp\u003eRetinoic acid (500 mcg/kg) was administered intraperitoneally every day starting from the third postnatal day and continued until the 13th postnatal day. The study groups were mentioned at the below. The flowchart of the study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe study groups were formed as follows;\u003c/p\u003e \u003cp\u003e \u003cem\u003eGroup 1\u003c/em\u003e, Naive Control (NC) Group n\u0026thinsp;=\u0026thinsp;5, The group to be monitored in room air, no intervention group.\u003c/p\u003e \u003cp\u003e \u003cem\u003eGroup 2\u003c/em\u003e, Hyperoxic Control (HC) Group n\u0026thinsp;=\u0026thinsp;8, BPD model developed and intraperitoneal saline (0.3 ml)\u003c/p\u003e \u003cp\u003e \u003cem\u003eGroup 3\u003c/em\u003e, Mesenchymal Stem Cell (MSCs) Group, n\u0026thinsp;=\u0026thinsp;8, BPD model developed ve intraperitoneal MSCs\u0026thinsp;+\u0026thinsp;intraperitoneal saline (0.3 ml)\u003c/p\u003e \u003cp\u003e \u003cem\u003eGroup 4\u003c/em\u003e, Retionic Acid (RA) Group, n\u0026thinsp;=\u0026thinsp;8, BPD model developed and given intraperitoneal MSCs\u0026thinsp;+\u0026thinsp;intraperitoneal RA (500 mcg/kg, 0.3 ml)\u003c/p\u003e \u003cp\u003eAnimals were sacrificed on postnatal day 21 with 120 mg/kg ketamine and 12 mg/kg xylazine (high dose anesthesia).\u003c/p\u003e\n\u003ch3\u003eHistopathological evaluation\u003c/h3\u003e\n\u003cp\u003eThe lungs were resected via thoracotomy and fixed overnight in 10% buffered formalin under a constant inflation pressure of 20 cm H\u003csub\u003e2\u003c/sub\u003eO. Afterwards, the lungs were transferred into freshly prepared 10% buffered formalin and stored for 24 hours. Lung samples were subjected to routine histological tissue processing and embedded in paraffin, as in the previous study \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Paraffin-embedded tissues were cut into 5 \u0026micro;m sections with a microtome (Leica) and mounted on poly-l-lysine coated slides. Sections were stained with hematoxylin-eosin (H\u0026amp;E) and imaged using an Olympus BX53 microscope (Olympus) with an Olympus DP74 camera attachment.\u003c/p\u003e\n\u003ch3\u003eMorphometric analysis of lung structure\u003c/h3\u003e\n\u003cp\u003eMorphometric analyses based on previous studies were performed to provide more detailed assessments of lung structure changes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Mean linear intercept measurement was performed at 90 random fields for each animal. Alveolar tissue distribution was measured by modifying Moreira et al \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e technique. Measurements were performed with Image J by obtaining 30 images from each lung (outside the major airways and vascular system). To assess vessel wall tihickness, five different areas for each animal were measured by adjusting the areas containing vessels in the cross-section.\u003c/p\u003e\n\u003ch3\u003eMasson’s Trichrome (MT) staining\u003c/h3\u003e\n\u003cp\u003eTo assess fibrosis, Masson's trichrome staining was used to show collagen deposition in lung sections. Masson's trichrome staining was performed using a kit (04-010802 kit, Bio-Optica) according to the manufacturer's instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eHistologically, BPD is characterized by pulmonary vascular remodeling. In this context, immunohistochemistry and immunofluorescence staining were performed to assess alpha-smooth muscle actin (α-SMA) expression levels. The lung sections were stained as previously described with some modifications \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Briefly, for inhibition of endogenous peroxidase, 5-\u0026micro;m-thick sections were immersed in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in methanol for 10 minutes at room temperature and then washed with PBS. To antigen retrieval, samples were heated in citrate buffer (10 mM, pH 6.0) at 90 \u003csup\u003e\u0026ordm;\u003c/sup\u003eC for 10 minutes. After blocking, lung sections were incubated with α-SMA (ab 7817, Abcam) primary antibody at 4\u0026deg;C overnight. For immunohistochemistry staining, secondary antibody (Thermo Scientific, Ultravision Large Volume Detection System Anti-Polyvalent, HRP, TP-125-BN) and streptavidin peroxidase (Thermo Scientific, TS-125-HR) were applied to the samples for 10 minutes, respectively, and washing was carried out with PBS after each step. Subsequently, the sections were subjected to 3,3\u0026prime;-Diaminobenzidine tetrahydrochloride (DAB) (ab64238, Abcam) as a chromogen and finally counterstained with Mayer's hematoxylin. In immunofluorescence staining, samples were incubated with FITC-conjugated secondary antibody for 1 hour at room temperature and covered with mounting medium containing 4\u0026acute;,6-diamidino-2-phenylindole (DAPI, sc-24941, Santa Cruz). Finally, stained lung sections were visualized using an Olympus BX53 microscope with an Olympus DP74 camera attachment. In immunofluorescence staining, α-SMA intensity was measured using Image J (Fiji, version 1.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe values obtained in the experiment were analyzed using the SPSS 25.0 program for Windows. The conformity of univariate data to normal distribution will be evaluated by Shapiro-Wilk test and variance analysis will be evaluated by Levene's test. To compare more than two groups according to quantitative data, parametric data will be analyzed with One-Way Anova and nonparametric data will be analyzed with Mann-Whitney U tests. Variables will be analyzed at 95% confidence interval and will be considered significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe study involved 29 rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although there was no difference in average birth weight between the groups, animals under hyperoxia demonstrated a considerable drop in body weight.\u003c/p\u003e \u003cp\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 20.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 g). Of the rats included in the study, 55% were male and 45% were female.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMesenchymal stem cell and retinoic acid treatments alleviate morphological changes in the lung due to bronchopulmonary dysplasia\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effects of hyperoxia and being subjected treatments (MSCs and MSCs\u0026thinsp;+\u0026thinsp;RA) protocols on lung tissue architecture, H\u0026amp;E staining was performed and the images were examined under a light microscope. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, lung alveoli characterised by regular small alveoli were distinguished in the control group, whereas in hypoxia (HC) the lung structure was disrupted and the alveoli were markedly enlarged with a heterogeneous distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). It was observed that the enlarged alveolar distribution decreased in the group receiving MSCs, whereas the MSCs\u0026thinsp;+\u0026thinsp;RA group had regular alveolar structures similar to the healthy control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDepending on the histopathological findings, morphometric analyzes were performed by measuring the mean linear intercept (MLI), alveolar tissue distribution, alveolar size and vessel wall thickness. MLI, alveolar size and vessel wall thickness were significantly higher in the hyperoxic group compared to the control group, whereas alveolar tissue distribution was decreased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Compared with the HC group, there was a significant decrease in MLI, alveolar size and vessel wall thickness parameters in the MSCs and MSCs\u0026thinsp;+\u0026thinsp;RA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the analysis of MSCs and MSCs\u0026thinsp;+\u0026thinsp;RA groups, hypoxia-induced lung injury was significantly improved in the MSCs\u0026thinsp;+\u0026thinsp;RA group, similar to the control group. This suggested that combined treatment may be more effective. MT staining was used to assess collagen accumulation in lung tissue. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, more significant collagen accumulation was observed in the HC group compared to the control group. There was a significant decrease in collagen accumulation in MSCs and MSCs\u0026thinsp;+\u0026thinsp;RA groups compared to the HC group. Reduced collagen levels were observed to be quite significant in the MSCs\u0026thinsp;+\u0026thinsp;RA group. These results showed that MSCs\u0026thinsp;+\u0026thinsp;RA treatment significantly prevented lung fibrosis due to collagen deposition.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHigh α-SMA expression in the lungs of rats exposed to hyperoxia was suppressed by mesenchymal stem cell and retinoic acid treatments\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo assess lung fibrosis, immunohistochemical staining was used to assess α-SMA expression levels, a key biomarker of BPD. Dark brown areas representing SMA expression were observed to be more intense in the HC group. Treatment groups exhibited reduced α-SMA expression compared to HC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo both confirm the IHC images and quantify α-SMA expression, IF staining was performed and α-SMA intensity was measured using Image J. Consistent with IHC results, α-SMA intensity was significantly higher than NC. In the treatment groups, α-SMA expression was found to be at lower levels compared to HC. A statistically significant difference in α-SMA expression was found between all groups. Consistent with IHC results, α-SMA intensity was significantly higher than NC (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the treatment groups, α-SMA expression was found to be at lower levels compared to HC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A statistically significant difference in α-SMA expression was found between all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Taken together, the results revealed that lung fibrosis-associated α-SMA expression was reduced by MSCs and MSCs\u0026thinsp;+\u0026thinsp;RA treatments.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present treatment for BPD is confined to moderately active medications including caffeine, vitamin A, and dexamethasone, which have been linked to serious long-term neurodevelopmental side effects \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Improved treatment choices for preterm babies with BPD can improve lung health and prevent problems, leading to a better quality of life. The limits of medication for BPD have led to a quest for alternative treatment approaches. Stem-cell therapy is a promising possibility for treating several medical disorders. In this study, we aimed to compare the effect of the combined use of mesenchymal stem cells as a promising treatment for the prevention and control of BPD with the accepted treatment of vitamin A with intact controls.\u003c/p\u003e \u003cp\u003eIn a study on neonatal rats, transdifferentiation of type II alveolar epithelial cells (AT2) into type I alveolar epithelial cells (AT1) was found to increase under hyperoxic treatment \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In the developing lung, alveolar septation and angiogenesis are regulated by lung-resident mesenchymal stem/stromal cells. Alveolar septation and angiogenesis are regulated by mesenchymal stem cells in the developing lung \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. MSCs have limited immunogenicity due to low expression of major histocompatibility antigens, allowing for allogeneic treatment without immunosuppression. They may be grown in vitro while remaining undifferentiated, allowing for sufficient quantities for clinical usage as well as cryopreservation before to use. They can impact the immunological response to injury, pathogen defense, and tissue healing. MSCs are a promising therapeutic tool in regenerative medicine for their effects on regeneration, repair, immune response, angiogenesis, and tissue protection \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Multiple studies have demonstrated that MSCs or MSC-conditioned media can improve hyperoxia-induced BPD in mouse models utilizing various methods of delivery \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. MSC treatment in animal models reversed BPD, avoided halted alveolar development, and restored lung alveolarization and vascularization \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. MSC therapy improved lung architecture, reduced fibrosis, and increased survival rates in BPD mice \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. It also reduced the expression of profibrotic factors such as angiotensin II, angiotensin II type 1 receptor, and angiotensin-converting enzyme \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. MSCs have been shown to modulate the immune system, reduce inflammation, and lower levels of inflammatory mediators \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. MSCs positive effects are believed to be due to their focus on angiogenesis, immunomodulation, wound healing, and cell survival \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Our results provide a new idea and reveal that species-matched systemic MSCs treatment plays a therapeutic role in neonatal rats with BPD, and its use in combination with retinoic acid is more effective, thus clearly demonstrating the additive effect of MSCs. We established a classical BPD newborn rats model, and confirmed that transplantation of MSCs can improve lung structure in rats with BPD. As known, vitamin A plays an important role in pulmonary development. In the connective tissue matrix surrounding the distal airspaces, BPD has demonstrated an abnormal quantity and distribution of α-SMA expression levels, which results in diminished septation and fewer alveoli. One actin isoform that is crucial to fibrogenesis is alpha-SMA. Blood arteries, myofibroblasts, and smooth muscle cells all contain alpha-SMA \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The activation of fibroblasts to myofibroblasts is linked to alpha-SMA. The capacity to contract sets myofibroblasts apart from fibroblasts. Transforming growth factor-beta controls the myofibroblast phenotype that produces extracellular matrix compound and expresses α-SMA \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. According to Rao et al \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, myofibroblasts' contractile qualities are linked to α-SMA expression and are involved in inflammation, wound healing, fibrosis, and carcinogenesis. α-SMA is also expressed by cancer cells that develop into mesenchymal cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMSCs have been shown to increase lifespan and exercise tolerance, decrease alveolar loss and inflammation, diminish fibrotic alterations, lower α-smooth muscle actin expression, and avoid pulmonary hypertension \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In addition, MSCs attenuated the increased collagen activation. The activation of lung fibrosis due to collagen deposition is the major pathogenic contributor to BPD through its function in the modulaton of collagen deposition \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our findings suggest that fibrosis inhibition in response to MSCs transplantation played an important role in alleviating at least some of the adverse pulmonary effect of hyperoxia. Currently, the precise molecular mechanisms of these action remain to further investigation. The results imply that MSCs may shield human newborns' other organs from O\u003csub\u003e2\u003c/sub\u003e-induced harm in addition to the lung.\u003c/p\u003e \u003cp\u003eIn infants who subsequently develop BPD, an excessive inflammation occurs especially in the first few days of life. Low vitamin A levels in extremely low birth weight infants are associated with an increased risk of BPD \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Vitamin A and its active metabolite retinoic acid play an important role in the growth, proliferation and differentiation of most cells. It is effective in the branching and development of the lung and reduces fibrosis. However, immunomodulatory/anti-inflammatory effect has not been demonstrated. Although 5000 IU vitamin A supplementation administered intramuscularly three times a week for four weeks decreased the rate of BPD, the effect rate was moderate \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Indeed, in the largest evaluation, the researchers found a statistically significant reduction in the rate of death or BPD, but the results were as follows: 55% in the RA group versus 62% in the placebo group \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Compared with the control group, there was a small benefit of vitamin A in reducing the risk of death or oxygen requirements at one month of age \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Indeed, no benefit on long-term respiratory or neurodevelopmental prognosis was observed. This demonstrated the need for combined therapies. In this study, we investigated the effect of mesenchymal stem cell therapy, which has anti-inflammatory effects, and retinoic acid treatment, which is the only currently accepted method of retinoic acid application. It is the only study in which the two were used together and it was clearly shown that the best results can be achieved with the combined application of the them.\u003c/p\u003e \u003cp\u003eFew studies have compared MSCs from various sources for their therapeutic effectiveness against BPD. Placenta, adipose tissue and bone- marrow may be a more convenient supply of tissue. Standardized parameters for cultivating MSCs from these sources, including culture media composition and oxygen content in growth chambers, are not addressed. It is also unclear how different culture conditions affect the therapeutic efficacy of MSCs. Third, for cell-based treatments to be effective, there must be a consistent supply of vast amounts of cells. It is vital to standardize culture methodologies that produce cells with consistent effectiveness. Another problem in MSCs-based therapy is understanding whether the therapeutic potential of MSCs stays consistent when cells are passaged to increase cell quantity. Although MSCs are known to multiply and grow throughout passage, it is unclear if their therapeutic effectiveness changes with recurrent passages. This must be addressed in order to standardize the maximum number of MSCs that may be passed through culture without losing therapeutic effectiveness \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our study's most potent feature is that mesenchymal stem cell viability and quantity were assessed right before delivery.\u003c/p\u003e \u003cp\u003eMSCs treatment in animals resulted in normal alveolar numbers and significantly decreased lung neutrophil and macrophage buildup \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Morphometric measurements of the lung were performed in our study. It was observed that the enlarged alveolar distribution was decreased in the group that received MSCs, and regular alveolar structures were observed in the MSC\u0026thinsp;+\u0026thinsp;RA group, similar to the healthy control group. Bone marrow-derived mesenchymal stem/stromal cells (MSCs) have been used in many research studies due to their easy accessibility. Although several studies using bone marrow-derived MSCs in a hyperoxia-induced environment showed improvement in alveolar structure in a neonatal lung injury model \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e it has not been found that concomitant use with retinoic acid may have a similar effect on healthy lung.\u003c/p\u003e \u003cp\u003eTo determine the optimal timing, Chang et al \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e compared early and late stem cell administration. Early administration was shown to be better than late administration in reducing inflammation in the tissue. In our study, rats in the treatment group received early MSCs with hyperoxic injury and one group received combined treatment (MSCs\u0026thinsp;+\u0026thinsp;RA). Early systemic administration of MSCs was found to significantly reduce inflammation. Combined administration of MSCs and RA was shown to be the most effective way in this study. The implications of this work for neurodevelopmental status will be presented in Part-2.\u003c/p\u003e \u003cp\u003eWhile stem cells may be useful therapeutic techniques for the treatment of BPD, various practical obstacles to using stem cell-based therapy in clinical practice must be addressed. Stem cells require standardized procedures for isolation and storage. They must be assessed for their consistent response, which may differ depending on the tissue from which they are separated. This is critical for their future use as potential biotherapeutics in human clinical studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first study to evaluate the combined effect of mesenchymal stem cells and retinoic acid versus mesenchymal stem cells alone on hyperoxic lung injury. We have showed that the combined treatment significantly decreased fibrosis and inflammation in the BPD model, and the best reduction was achieved with the combination of the two therapies. The results of this study suggest that the combined use of RA-MSCs instead of retinoic acid alone may be instructive for clinical use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThis present study was approved by the Dokuz Eylul University Animal Experiments Local Ethics Committee in Izmir (Approval number: 2021/011).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eWritten informed consent was obtained from the patients to publish this paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eAll authors have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eConceptualization, S.A\u0026Ouml;.; data curation, S.A\u0026Ouml;. A.\u0026Ccedil;., O.Y. Stem cell isolation and tests: M.S. and G.G. formal analysis, S.AO and E.A.; methodology, S.AO.E.A., and M.Y.; supervision, S.C., and T.GY.; writing-original draft, E.A. and S.AO.; writing-review \u0026amp; editing, S.AO.,E.\u0026Ouml;, and G.\u0026Ouml;. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated and analyzed for the study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGilfillan M, Bhandari A, Bhandari V. Diagnosis and management of bronchopulmonary dysplasia BMJ. 2021;375:n1974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonadies L, Moschino L, Valerio E, Giordano G, Manzoni P, Baraldi E. Early Biomarkers of Bronchopulmonary Dysplasia: A Quick Look to the State of the Art. Am J Perinatol. 2022;39(S 01):S26\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiusto K, Wanczyk H, Jensen T, Finck C. Hyperoxia-induced bronchopulmonary dysplasia: better models for better therapies. Dis Model Mech. 2021;23(2):14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Wang S, Chen M, Lv Y, Chen X, Yang C. The value of hematocrit for predicting bronchopulmonary dysplasia in very low birth weight preterm infants. Med (Baltim). 2023;102(39):e35056.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris C, Greenough A. The prevention and management strategies for neonatal chronic lung disease. Expert Rev Respir Med. 2023;17(2):143\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenny M, Courchia B, Shrager S, Sharma M, Chen P, et al. Comparative Effects of Bone Marrow-derived Versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia. Stem Cells Transl Med. 2022;11(2):189\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePurcell E, Shah J, Powell C, Nguyen T, Zhou L, et al. Umbilical cord blood-derived therapy for preterm lung injury: a systematic review and meta-analysis. Stem Cells Transl Med. 2024;15(7):606\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira A, Winter C, Joy J, Winter L, Jones M, Noronha M, Porter M, Quim K, Corral A, Alayli Y, et al. Intranasal Delivery of Human Umbilical Cord Wharton\u0026rsquo;s Jelly Mesenchymal Stromal Cells Restores Lung Alveolarization and Vascularization in Experimental Bronchopulmonary Dysplasia. Stem Cells Transl Med. 2020;9:221\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlkan Ozdemir S, Ozdemir N, Aksan O, Kınalı B, Bilici G\u0026uuml;ler G, Erbil G, Ozer E, Ozer E. Effect of humic acid on oxidative stress and neuroprotection in hypoxic-ischemic brain injury: part 1. 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Curr Opin Crit Care. 2015;21:20\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurley GF, Hayes M, Ansari B, Shaw G, Ryan A, Barry F, O\u0026rsquo;Brien T, O\u0026rsquo;Toole D, Laffey JG. Mesenchymal Stem Cells Enhance Recovery and Repair Following Ventilator-Induced Lung Injury in the Rat. Thorax. 2012;67:496\u0026ndash;501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAslam M, Baveja R, Liang OD, Fernandez-Gonzalez A, Lee C, Mitsialis SA, Kourembanas S. Bone Marrow Stromal Cells Attenuate Lung Injury in a Murine Model of Neonatal Chronic Lung Disease. Am J Respir Crit Care Med. 2009;180:1122\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Wang H, Shi Y, Peng W, Zhang S, Zhang W, Xu J, Mei Y, Feng Z. Role of Bone Marrow-Derived Mesenchymal Stem Cells in the Prevention of Hyperoxia-Induced Lung Injury in Newborn Mice. Cell Biol Int. 2012;36:589\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Haaften T, Byrne R, Bonnet S, Rochefort GY, Akabutu J, Bouchentouf M, Rey-Parra GJ, Galipeau J, Haromy A, Eaton F, et al. Airway Delivery of Mesenchymal Stem Cells Prevents Arrested Alveolar Growth in Neonatal Lung Injury in Rats. Am J Respir Crit Care Med. 2009;180:1131\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoreira A, Winter C, Joy J, Winter L, Jones M, Noronha M, Porter M, Quim K, Corral A, Alayli Y, et al. Intranasal Delivery of Human Umbilical Cord Wharton\u0026rsquo;s Jelly Mesenchymal Stromal Cells Restores Lung Alveolarization and Vascularization in Experimental Bronchopulmonary Dysplasia. Stem Cells Transl Med. 2020;9:221\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeluzzo AM, Autieri MV Challenging the Paradigm: Anti-Inflammatory Interleukins and Angiogenesis., Cells, Abiramalatha T, Ramaswamy VV, Bandyopadhyay T, Somanath SH, Shaik NB, Pullattayil AK, Weiner GM. Interventions to prevent Bronchopulmonary Dysplasia in preterm infants: An umbrella review of systematic reviews and meta-analyses JAMA Pediatr. 2022;176(5):502\u0026ndash;516.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Wang H, Shi Y, Peng W, Zhang S, Zhang W, Xu J, Mei Y, Feng Z. Role of Bone Marrow-Derived Mesenchymal Stem Cells in the Prevention of Hyperoxia-Induced Lung Injury in Newborn Mice. Cell Biol Int. 2012;36:589\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen CM, Chou HC. 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Biophys Acta Mol Basis Dis. 2017;1863(1):298\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun KH, Chang Y, Reed N, Sheppard D. α-Smooth muscle actin is an inconsistent marker of fibroblasts responsible for force-dependent TGFβ activation or collagen production across multiple models of organ fibrosis. Am J Physiol Lung Cell Mol Physiol. 2016;1(9):L824\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalathi KBR, Rajan NNS. S.T.,(2014).Evaluation of Myofibroblasts By Expression of Alpha Smooth Muscle Actin: A Marker in Fibrosis, Dysplasia and Carcinoma,J Clin of Diagn Res. 8(4), ZC14\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnggorowati N, Ratna Kurniasari C, Damayanti K, Cahyanti T, Widodo I, Ghozali A, Romi MM, Sari DC, Arfian N. Histochemical and Immunohistochemical Study of α-SMA, Collagen, and PCNA in Epithelial Ovarian Neoplasm. 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PLoS ONE. 2013;8(1):e52419.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"italian-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"itjp","sideBox":"Learn more about [Italian Journal of Pediatrics](http://ijponline.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ITJP/default.aspx","title":"Italian Journal of Pediatrics","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"bronchopulmonary dysplasia, lung injury, preterm, retinoic acid, stem cell","lastPublishedDoi":"10.21203/rs.3.rs-6387279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6387279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eAim:\u003c/strong\u003e The purpose of the present study was to assess the efficacy of retinoic acid (RA) treatment combined with mesenchymal stem cells (MSCs) compared to MSCs alone in lung injury due to bronchopulmonary dysplasia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 29 Wistar baby rats on the postnatal third day were used. The experiment started on the postnatal 3rd day after the birth of the cubs. It was continued until the postnatal 14th day. Hyperoxia system was created with the help of Plexiglas chambers. Bone marrow-derived stem cells (1x10\u003csup\u003e6\u003c/sup\u003e) were administered intraperitoneally. Retinoic acid (500 mcg/kg) was administered intraperitoneally every day starting from the third postnatal day and continued until the 13th postnatal day. Morphometric analyses and histological analyses were performed to provide more detailed assessments of lung structure changes. To assess fibrosis, Masson's trichrome staining was used to show collagen deposition in lung sections. Immunohistochemistry and immunofluorescence staining were performed to assess alpha-smooth muscle actin (α-SMA) expression levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe enlarged alveolar distribution decreased in the group receiving MSCs, whereas the MSCs+RA group had regular alveolar structures similar to the healthy controls. morphometric analyzes were performed by measuring the mean linear intercept (MLI), alveolar tissue distribution, alveolar size and vessel wall thickness. MLI, alveolar size and vessel wall thickness were significantly higher in the hyperoxic group compared to the control group, whereas alveolar tissue distribution was decreased. In the treatment groups, α-SMA expression was found to be at lower levels compared to hyperoxic group. the results revealed that lung fibrosis-associated α-SMA expression was reduced by MSCs and MSCs+RA treatments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e The combined treatment significantly decreased fibrosis and inflammation in the BPD model, and the best reduction was achieved with the combination of the two therapies.\u003c/p\u003e","manuscriptTitle":"Can mesenchymal stem cell and retinoic acid therapies more effectively reduce lung damage due to bronchopulmonary dysplasia?","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 13:30:36","doi":"10.21203/rs.3.rs-6387279/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2026-04-13T03:57:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-26T07:18:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-05T17:04:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-23T12:50:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Italian Journal of Pediatrics","date":"2025-04-16T03:03:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"italian-journal-of-pediatrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"itjp","sideBox":"Learn more about [Italian Journal of Pediatrics](http://ijponline.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ITJP/default.aspx","title":"Italian Journal of Pediatrics","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5c9462ef-2487-49e6-9d88-c3abff87ee0e","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-30T11:05:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 13:30:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6387279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6387279","identity":"rs-6387279","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}