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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 46  |  Issue : 3  |  Page : 172-182

The possible therapeutic role of mesenchymal stem cells in amiodarone-induced lung injury in adult male albino rats


Department of Anatomy and Embryology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission20-Jan-2018
Date of Acceptance14-Mar-2018
Date of Web Publication28-Feb-2019

Correspondence Address:
Maram M.M Ghabrial
Department of Anatomy and Embryology, Faculty of Medicine, Tanta University, Tanta
Egypt
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DOI: 10.4103/tmj.tmj_4_18

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  Abstract 


Background and aim Lung injury and even pulmonary fibrosis represent a known adverse effect of amiodarone. No effective treatment was reported for pulmonary fibrosis except lung transplantation. Stem cell therapy holds a great promise for the repair of injured tissues. Mesenchymal stem cells (MSCs) have the potential to present a new trend of treatment. This work aimed to study the effect of bone marrow-MSCs on amiodarone-induced lung injury in albino rats by histological methods.
Materials and methods Forty-two adult male albino rats were used. Seven rats were used as a source of bone marrow-MSCs. Thirty-five rats were divided into the following groups: negative control group included seven rats that received no treatment; vehicle control group included seven rats that received polysorbate 80; amiodarone group included 14 rats that received daily oral amiodarone for 6 weeks and were subdivided into two equal subgroups as subgroup 3a (amiodarone administrated) and subgroup 3b (kept for 4 weeks after amiodarone stoppage); and stem cell-treated group included seven rats that received stem cells after amiodarone stoppage. Lung specimens were examined histologically.
Results Amiodarone group showed disrupted lung architecture, collapsed alveoli, and significant increase in the thickness of interalveolar septa. Bronchioles showed thickened smooth muscle layer and obliterated lumens. Highly significant increase in area percent of collagen fibers was observed. In stem cell group, there was improvement of these histological changes.
Conclusion MSCs can improve the deleterious effects associated with amiodarone-induced lung injury.

Keywords: amiodarone, lung injury, mesenchymal stem cells


How to cite this article:
Ghabrial MM, Salem MF, El Ela AM, El Deeb SA. The possible therapeutic role of mesenchymal stem cells in amiodarone-induced lung injury in adult male albino rats. Tanta Med J 2018;46:172-82

How to cite this URL:
Ghabrial MM, Salem MF, El Ela AM, El Deeb SA. The possible therapeutic role of mesenchymal stem cells in amiodarone-induced lung injury in adult male albino rats. Tanta Med J [serial online] 2018 [cited 2019 Oct 16];46:172-82. Available from: http://www.tdj.eg.net/text.asp?2018/46/3/172/253202




  Introduction Top


Amiodarone is an effective antiarrythmic drug. However, it causes a variety of pulmonary adverse effects such as chronic interstitial pneumonia, diffuse alveolar damage, and even life-threating pulmonary fibrosis through different mechanisms including direct toxicity to lung tissue and increased oxidative stress [1]. No effective treatment was reported for pulmonary fibrosis except lung transplantation. So, new trends of treatment are in mass need [2].

Stem cell therapy holds a great promise for the repair of injured tissues. Stem cells are unspecialized cells having the ability of self-renewal and giving rise to different specialized cell types [3]. Adult stem cells can be isolated from many sources such as bone marrow, adipose tissue, and umbilical cord blood [4].

Bone marrow-mesenchymal stem cells (BM-MSCs) have the ability to differentiate into various types of cells as hepatocytes, chondrocytes, osteoblasts, and neurons [5]. Moreover, they can migrate through the blood to reach the injured tissues and exert therapeutic effects. Consequently, mesenchymal stem cells (MSCs) become an attractive choice for the possible development of clinical applications [6],[7].

Therefore, this study was performed to evaluate the possible therapeutic role of BM-MSCs on the lung injury induced by amiodarone administration in adult male albino rats.


  Materials and methods Top


Animals

Forty-two adult male albino rats weighing about 200 g each were used in this study. They were obtained from the animal houses, Faculties of Medicine, Cairo and Tanta Universities. The animals were kept in clean properly ventilated cages and received a balanced diet and free water supply. Care of animals and all steps of the experiment were carried out according to the rules and regulations laid down by the ethical committees of animal’s experimentation of Cairo and Tanta Universities.

Drug

Amiodarone (Cardio-Mep, Mepaco-Medifood at Heliopolis) was used as 200 mg tablets that were manufactured by Arab Company of pharmaceuticals and medicinal plants (Mepaco-Medifood). Each tablet was grinded, and the required dose for each rat was weighed and dissolved in 1 ml polysorbate 80, which is the solvent for amiodarone (El-Gomhoria Company, Tanta Al Gharbia, Egypt).

The experimental design

Seven rats from the animal house, Faculty of Medicine, Cairo University, were used as bone marrow donors. The other 35 rats obtained from the animal house, Faculty of Medicine, Tanta University, were divided into the following groups:
  1. Group 1 (negative control group): it consisted of seven rats that received no treatment.
  2. Group 2 (vehicle control group): it consisted of seven rats that received 1 ml polysorbate 80 (orally through oro-gastric tube) daily for 6 consecutive days per week for 6 weeks [8].
  3. Group 3 (amiodarone-treated group): it consisted of 14 rats, where each rat received amiodarone (30 mg/kg body weight) dissolved in 1 ml polysorbate 80 (orally through oro-gastric tube) daily for 6 consecutive days per week for 6 weeks [9]. These rates were divided into two equal subgroups:
    1. Subgroup 3a: The seven rats were killed the next day after stopping of amiodarone administration.
    2. Subgroup 3b: The seven rats were kept without medication for another 4 weeks after amiodarone stoppage and then killed.
  4. Group 4 (BM-MSCs treated group): it consisted of seven rats. Each rat received amiodarone (30 mg/kg body weight) dissolved in 1-ml polysorbate 80 (orally through oro-gastric tube) daily for 6 consecutive days per week for 6 weeks. Then, the next day after stopping of amiodarone, each rat was injected intravenously with a single dose of BM-MSCs (3×106) in the tail vein. Then they were left for 4 weeks without any medication and then were killed [10].


Bone marrow-mesenchymal stem cells isolation, culture, and labeling

Bone marrow isolation

Seven rats were used as bone marrow donors. They were anesthetized by inhalation of light ether and then were killed. Rat’s skin of the hind limb was sterilized with betadine before incision of the skin. The femurs were carefully dissected and cleaned from the surrounding tissues, and then they were immersed in 70% alcohol for 1–2 min. The bones were delivered to the laminar flow to extract the bone marrow. After removal of epiphyses of both ends, the bone marrow was flushed from the medullary cavities using gauge needle with 2 ml of complete media. The marrow plugs were expelled in sterilized tissue culture tube of 15 ml [11].

Mesenchymal stem cell culture

These steps were done at Cell Culture Unit of Egyptian Society for Progenitor Cell Research in Cairo.
  1. The marrow plugs were centrifuged at 1200 rpm for 10 min and the supernatant was removed. Overall, 3 ml of complete media was added to the pellet and gentle rapping was applied till pellet dissolved and full suspension was formed [11].
  2. Cells from this suspension, were resuspended in 20 ml of the prewarmed complete media. Cell suspension was added to a tissue culture flask 25. The flask containing the bone marrow cells was incubated in a humidified incubator at 37°C in 5% CO2 and 95% air. At the first day of the culture, the cultured cells were examined using the inverted microscope to show the presence of rounded floating cells. Periodic examination by inverted microscope was done to follow-up the growth of the cells and to exclude the presence of any infection in the culture [12].
  3. After 6 days, the nonadherent cells were removed by aspiration using a sterile pipette. The adherent cells were then washed twice with PBS. Finally 10 ml of fresh complete media was added to the flask. MSCs became obvious and began to acquire a spindle shape. The media were removed every 3 days and replaced with 10 ml of prewarmed another complete media. MSCs in culture were characterized by their adhesiveness to tissue culture floor and fusiform shape [13].
  4. After 2 weeks, the MSCs confluence became 90%. The cells were detached using 2 ml of 0.25% trypsin-EDTA (EDTA). The flask was incubated at 37°C and monitored periodically for cell detachment under the inverted microscope. Cell detachment was evidenced by changing from spindle to round shape. This process took 5–7 min. Moreover, 5 ml of prewarmed complete media was added to the flask to neutralize the trypsin-EDTA [14].
  5. After detachment, the cells were transferred to a 15-ml conical tube and centrifuged at 1200 rpm for 5 min. The supernatant was removed to leave the pellet. The cell pellet was resuspended in a small amount of prewarmed complete media. Then the cells were checked for viability using 0.4% trypan blue and transferred using a pipette into the chambers of clean hemocytometer slide. The viable cells appear transparent and glistening (unstained), whereas the nonviable cells appear blue (stained) [15]. For counting the viable cells in the hemocytometer, all cells in the 1-mm center square and 4-mm corner squares were counted. The cell viability could be determined from this equation: cell viability (%)=total viable cells (unstained)/total cells (stained and unstained)×100 [16].


Labeling of mesenchymal stem cells

BM-MSCs were labeled by incubation with a complex of ferumoxide and poly-l-lysine hydrobromide 375 ng/ml [17].

For light microscopic study

Animals were killed according to their scheduled time under light ether anesthesia. Lung specimens were prepared and subjected to the following stains:
  1. Hematoxylin and eosin stain for detection of the general histological structure [18].
  2. Mallory’s trichrome stain for detection of collagen fibers [19].
  3. Prussian blue stain for localization of the iron-labeled stem cells [20].


Morphometric study

Using Leica Qwin 500 (Leica quin a National research center Dokki, Cairo, Egypt) image analyzer computer system, counting of type II pneumocytes was done in hematoxylin and eosin-stained sections. The area percentage of collagen fibers was measured in Mallory’s trichrome-stained sections. These measurements were performed in 10 different fields at magnification of 400/slide in the control and experimental groups [21].

Statistical analysis

Statistical analysis was conducted by analysis of variance, using the mean and standard deviation by using SPSS program. P values less than 0.05 were considered statistically significant, and highly significant differences were achieved if P values were less than 0.001 [22].


  Results Top


Assessment of morphological changes of the bone marrow-mesenchymal stem cells

Examination of the primary culture of BM-MSCs at the sixth day was done by using inverted microscope, which revealed most of the BM-MSCs were arranged in chains or colonies whereas other cells appeared elongated with spindle-shaped appearance. These cells became crowded and very close to each other covering the floor of the flask (confluence 90%) at the 14th day of culturing ([Figure 1]).
Figure 1 A photomicrograph of bone marrow-mesenchymal stem cells (BM-MSCs) culture (a) at the sixth day showing crowded BM-MSCs with different arrangement. Most of them are arranged in chains (curved arrows) or colonies (double head arrows) whereas other cells appear elongated with spindle-shaped appearance (single head arrow). (b) A photomicrograph at the 14th day showing very crowded cells close to each other (inverted microscope ×100).

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Histological and histochemical results

Hematoxylin and eosin stain

Groups 1 and 2 (negative and vehicle control groups): Examination of lung sections of adult control albino rats (groups 1 and 2) showed the histological features of normal lung in the form of patent alveoli, alveolar sacs, and thin interalveolar septa. Alveoli were lined mainly by type I pneumocytes, few type II pneumocytes, and some macrophages. Bronchioles with patent lumen surrounded by regular smooth muscle layer and lined by ciliated simple columnar epithelial cells with oval and vesicular basal nuclei were observed ([Figure 2]).
Figure 2 A photomicrograph of a lung section of an adult control albino rat showing (a) many patent alveoli (a), alveolar sacs (as) and thin interalveolar septa (I) (H&E, ×200); (b) alveoli lined mainly by type I pneumocytes (p1), few type II pneumocytes (p2), and some macrophages (M) (H&E, ×1000); (c) rounded bronchiole (Br) with patent lumen surrounded by regular smooth muscle layer (sm) (H&E, ×400); and (d) ciliated simple columnar epithelial cells (arrows) with oval basal nuclei (N) lining a bronchiole (H&E, ×1000).

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Group 3 (amiodarone-treated group): Examination of lung sections of subgroup 3a revealed disrupted lung architecture and variable-sized alveoli either collapsed or dilated. Dilated alveolar sacs, markedly thickened interalveolar septa with massive inflammatory cellular infiltration, and interstitial hemorrhage were found. Higher magnifications showed nearly absent type I pneumocytes in the alveolar lining epithelium with abundant type II pneumocytes and many macrophages ([Figure 3]a and b).
Figure 3 A photomicrograph of a lung section of an adult albino rat (subgroup 3a) showing the following: (a) variable sized alveoli either collapsed (a) or dilated (a1), alveolar sacs (as), interstitial hemorrhage (arrows), and massive inflammatory cellular infiltration (curved arrows) (H&E, ×200); (b) nearly absent type I pneumocytes in the alveolar lining epithelium with abundant type II pneumocytes (p2) and many large macrophages (M) (H&E, ×1000); (c) two irregular bronchioles (Br) with obliterated lumens by cellular debris (d) and thickened surrounding smooth muscle layer (sm). Peribronchial cellular infiltration (straight arrows) and area of hemorrhage (curved arrow) are also seen (H&E, ×400); and (d) a bronchiole lined by disrupted epithelium detached from the basement membrane (red arrows) with sloughing of some epithelial cells into the lumen as debris (d), destruction of the apical cilia (black arrows), and dark nuclei at different levels (N) (H&E, ×1000).

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Bronchioles exhibited thick surrounding smooth muscle layer and obliterated lumens. They were lined by disrupted epithelium detached from the basement membrane with destruction of the apical cilia and dark nuclei. Peribronchial cellular infiltration and areas of hemorrhage were found ([Figure 3]c and d).

Examination of the lung sections of subgroup 3b showed collapsed alveoli and dilated others with expanded alveolar sacs and thick interalveolar septa with massive inflammatory cellular infiltrations. Alveoli were lined by few type I pneumocytes with predominance of type II pneumocytes and many macrophages. Bronchioles with obliterated lumens and irregular thickened smooth muscle layer with peribronchial cellular infiltration were seen. They were lined by disrupted epithelium with destructed cilia and dark pyknotic nuclei with irregular arrangement ([Figure 4]).
Figure 4 A photomicrograph of a lung section of an adult albino rat (subgroup 3b) showing (a) collapsed alveoli (a), dilated others (a1), expanded alveolar sacs (as) and thick interalveolar septa (I) with inflammatory cellular infiltration (arrows) (H&E, ×200); (b) alveoli lined by few type I pneumocytes (p1) and predominance of type II pneumocytes (p2). Many large macrophages (M) are seen (H&E, ×1000). (c) Bronchiole (Br) with obliteration of its lumen by cellular debris (d) and irregular surrounding smooth muscle layer (sm). Peribronchial cellular infiltration (arrows) is seen (H&E, ×400). (d) A part of bronchiole lined by disrupted epithelium showing area of cytoplasmic vacuolation (v) with destructed cilia (arrows), dark pyknotic nuclei with irregular arrangement (N), and thickening of the surrounding smooth muscle layer (sm) (H&E, ×1000).

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Group 4 (BM-MSCs treated group): After 4 weeks of injection of BM-MSCs, lung sections showed partial regaining of the normal lung structure, patent alveoli, alveolar sacs, and thin interalveolar septa but with mildly congested blood capillaries. The alveolar lining epithelium regained its normal structure with type I pneumocytes and some type II pneumocytes with few macrophages ([Figure 5]a and b).
Figure 5 A photomicrograph of a lung section of stem cell-treated adult albino rat (group 4) showing (a) patent alveoli (a), alveolar sacs (as), and thin interalveolar septa (I) with mildly congested pulmonary capillaries (c) (H&E, ×200). (b) Reappearance of the flat type I pneumocytes (p1), some type II pneumocytes (p2), and few macrophages (M) (H&E, ×1000). (c) Bronchiole (BR) with patent lumen, mildly thickened surrounding smooth muscle layer (sm), and less peribronchial cellular infiltrations (arrow) (H&E, ×400). (d) A part of bronchiole lined by ciliated columnar epithelial cells (arrows) with basal nuclei (N) but few cells show basal vacuolation (v) of their cytoplasm (H&E, ×1000).

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Bronchioles with patent lumen and mildly thickened surrounding smooth muscle layer with less peribronchilal cellular infiltrations were seen. They were lined by ciliated columnar epithelium with basal nuclei, but few cells showed cytoplasmic vacuolation ([Figure 5]c and d).

Mallory’s trichrome stain

Minimal collagen fibers were found in the interalveolar septa, around bronchioles, and blood vessels of lung sections of control rats. However, subgroups 3a and 3b showed extensive collagen fibers deposition. After stem cells injection, moderate collagen fibers deposition was observed ([Figure 6]).
Figure 6 A photomicrograph of a Mallory’s trichrome-stained lung section of (a) an adult control albino rat showing minimal collagen fibers (arrows) in the interalveolar septa (I), around bronchioles (Br) and blood vessel (bv). (b, c) Adult albino rat (subgroup 3a and 3b respectively) showing extensive collagen fibers (arrows) in interalveolar septa (I), around bronchiole (Br), and blood vessel (bv). (d) Stem cell-treated adult albino rat (group 4) showing moderate collagen fibers (arrows) in the interalveolar septa (I), around bronchiole (Br) and blood vessel (bv) (Mallory’s trichrome, ×200).

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Prussian blue stain

Only the iron-labeled stem cells in group 4 showed Prussian blue-positive stain in the interalveolar septa, near the blood vessels, and in the adventitia of some bronchioles ([Figure 7]).
Figure 7 A photomicrograph of a lung section of stem cell-treated adult albino rat group 4) showing (a) Prussian blue-positive iron-labeled stem cells (curved arrows) in the interalveolar septum (I) and near the adventitia of a bronchiole (Br) (Prussian blue ×200) and (b) Prussian blue-positive iron-labeled stem cells (curved arrows) near a blood vessel (bv) (Prussian blue ×200).

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Statistical results

Mean number of type II pneumocytes

The mean number of type II pneumocytes showed significant increase in subgroups 3a and 3b in comparison with control groups. In contrary, the stem cell-treated group (group 4) showed no significant difference with control groups but with a significant decrease in comparison with both amiodarone subgroups ([Table 1] and [Figure 8]).
Table 1 Mean number of type II pneumocytes in different groups

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Figure 8 Histogram showing mean number of type II pneumocytes in different groups.

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Mean surface area percentage of collagen fibers

The mean surface area percentage of collagen fibers showed a highly significant increase in subgroups (3a and 3b) in comparison with control groups. In contrary, the stem cell-treated group (group 4) showed a highly significant decrease in comparison with amiodarone subgroup with no significant difference between it and control groups ([Table 2] and [Figure 9]).
Table 2 Mean surface area percentage of collagen fibers in different groups

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Figure 9 Histogram showing mean surface area percentage of collagen fibers in different groups.

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  Discussion Top


Amiodarone-induced pulmonary toxicity is a distinctive form of drug-induced lung injury. Diffuse alveolar damage, interstitial pneumonia, and even pulmonary fibrosis are recognized patterns of amiodarone lung injury [23].

The new era of stem cell therapy reveals promising benefits in experimental models of lung disease caused by drug administration. BM-MSCs are multipotent stem cells with unique properties that make them an attractive candidate for stem cell-based therapy. They are useful for regeneration and regulation of immune and inflammatory responses in injured tissues [24],[25].

In this work, amiodarone administration in subgroup 3a showed collapsed alveoli and thickened interalveolar septa. Alveolar collapse could be explained by the imbalance between production and degradation of surfactant. Excessive production of surfactant seemed to exceed the ability of alveolar macrophages to degrade it [26],[27]. Large areas of collapsed alveoli could be attributed to the combined endothelial and epithelial injury that led to basement membrane denudation and collapse of alveoli [28].

Type II pneumocytes might constitute the reserve epithelial cells of alveoli. This could explain the significant increase in mean number of type II pneumocytes as a compensatory mechanism to regenerate the injured epithelium [29].

In this work, obliteration of the lumen of bronchioles, detachment of the bronchiolar lining epithelium, destructed cilia, and dark pyknotic nuclei were in accordance with Barker et al. [30] who referred obliteration of lumen of most bronchioles to the direct toxic effect of amiodarone on mucosal lining the bronchioles and injury of bronchiolar epithelium as well as inflammation of subepithelial structures.

The highly significant increase in the mean surface area percent of collagen fibers was concomitant with that reported by previous researches that observed increased collagen deposition in amiodarone-induced lung injury on rat models. It could be attributed to the immune and inflammatory mechanisms that lead to fibroblasts stimulation and collagen deposition [31],[32].

The oxidative stress contributes in the fibrogenesis by inducing genetic overexpression of fibrogenic cytokines such as connective tissue growth factor and platelet-derived growth factor. These factors stimulate the differentiation of fibroblasts from epithelial and endothelial cells with increased synthesis and deposition of collagen [33].

Papiris et al. [34] suggested that amiodarone was found to upregulate angiotensinogen messenger RNA, and angiotensin II was shown to promote fibrosis through stimulation of transforming growth factor-β1.

The present study revealed persistent lung damage after amiodarone stoppage (subgroup 3b). This was owing to slow elimination of amiodarone because of having long half-life. In addition, amiodarone and its principal metabolite (DEA) have a tendency to accumulate in the lung [35].

After BM-MSCs injection, there was partial improvement with regain of the normal lung architecture. Thin interalveolar septa with few inflammatory cellular infiltrations were observed with a highly significant decrease in the mean surface area percentage of collagen fibers in comparison with amiodarone-treated group. These results could be explained by the ability of MSCs to secrete many cytokines and growth factors that have antiapoptotic activity and play an essential part in tissue regeneration and reducing inflammation [36].

Moreover, MSCs can inhibit the activation of T-lymphocytes and natural killer cells, reduce the secretion of inflammatory cytokines such as tumor necrosis factor α and interferon γ, and increase the release of anti-inflammatory cytokines such as interleukin 10 and interleukin 4, resulting in decreasing of inflammation [37],[38].

In the present study, MSCs therapy showed restoration of the normal epithelium lining the alveoli, which is confirmed by statistic data. This finding coincides with Ortiz et al. [39] who delivered BM-MSCs to mice after bleomycin administration and found that the donor cells homed to the injured lung and adopted epithelial phenotypes including pneumocytes of alveolar epithelial cells.

In this work, the highly significant decrease in the mean surface area percent of collagen fibers in comparison with both subgroups 3a and 3b indicates the improvement of pulmonary fibrosis after MSCs therapy. The role of MSCs in improvement of pulmonary fibrosis could be explained by their ability to secrete a wide array of cytokines that contribute to fibrosis reduction. Meanwhile, MSCs could directly dissolve fibrosis as it was found that the MSCs were able to produce the matrix metalloproteinase. This enzyme is capable of degrading the extracellular matrix which alleviates fibrosis directly [40],[41].

Follow-up of MSCs in Prussian blue-stained sections revealed Prussian blue-positive cells found in the interalveolar septa and near the blood vessels. This result was previously recorded by Sueblinvong and Weiss [42] who postulated that following systemic administration of MSCs in lung injury, the administered MSCs were localized or retained inside lung tissue and resulted in engraftment of the cells such as epithelial, vascular endothelial, or interstitial cells. Prussian blue-positive cells were also found in adventitia of bronchioles. This is in line with Chistiakov [43] who reported that to restore airway function, epithelium has to be rapidly repaired along with regeneration of its structure and integrity. He added that stem cell populations are capable of differentiation, leading to restoration of a completely differentiated airway epithelium.


  Conclusion Top


From this study, it could be concluded that amiodarone has adverse effects on lung tissue. However, BM-MSCs can improve the deleterious effects associated with amiodarone-induced lung injury.

Recommendations
  1. Further investigations for ensuring stem cells safety for human therapy application are recommended.
  2. Other stem cell sources should be tried to detect the best source of these cells.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

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