Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 5 (may 2021) : 530-535

In vitro Cytotoxicity Analysis of Hybrid Graphene Oxide (hGO) Nano Structures in Caprine Wharton’s Jelly Derived Mesenchymal Stem Cells (WJ-MSCs)

S.A. Dhenge1,*, N.E. Gade1, O.P. Mishra1, Abinash Kumar1, V.N. Khandait2
1Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Anjora, Durg-491 001, Chhattisgarh, India.
2College of Veterinary and Animal Sciences, Udgir, Latur-413 517, Maharashtra, India.
Cite article:- Dhenge S.A., Gade N.E., Mishra O.P., Kumar Abinash, Khandait V.N. (2020). In vitro Cytotoxicity Analysis of Hybrid Graphene Oxide (hGO) Nano Structures in Caprine Wharton’s Jelly Derived Mesenchymal Stem Cells (WJ-MSCs) . Indian Journal of Animal Research. 55(5): 530-535. doi: 10.18805/ijar.B-3992.

ABSTRACT

Background: Nanotechnology is used in stem cell culture as well as in vivo delivery and tracking of stem cells. Graphene oxide (GO) is a carbon based nanomaterial and it has large surface area as well as good biocompatibility and heteroatoms doped GO exploit its properties. Hybrid GO (hGO) nano structures biocompatibility is depends on its size, dose and exposure time as well as in vitro cell models and hence, need thorough cytotoxicity studies in different species in vitro cell models. 

Methods: Caprine Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) were isolated, characterized and dose dependent (100, 50, 25, 10 and 0µg /ml) in vitro cytotoxicity of  three different hGO nano structures (phosphorus doped graphene oxide titanium oxide tubes, rods and sheets) were  analysed in caprine WJ-MSCs by studying cell cytotoxicity assays.  

Result: All three hGO nano structures were damaged cell morphology at 100 and 50 µg /ml doses, however, morphologically more good cells were observed in hGO tubes treated group than hGO rods and hGO sheets at 25 and 10 µg/ml doses as compared to control. Cell viability percentage was significantly (P<0.01) decreased at dose 100 µg/ml and it was significantly (P<0.01) increased at 25 µg/ml dose as compared to 50, 10 and 0 µg/ml doses. But, hGO tubes significantly (P<0.01) increased cell viability % as compared to hGO rods and hGO sheets. Cell population doubling time (PDT) was not altered significantly by all hGO nano structures, but 100 and 50 µg/ml doses significantly (P<0.01)increased cell PDT as compared to 25, 10 and 0 µg/ml doses. All hGO nano structures were non significantly altered growth curve, however, all hGO nano structures at 25 µg /ml dose altered (inclined) shape of growth curve, while 100 and 50 µg /ml doses significantly declined growth curve shape as compared to 10 and 0 µg /ml doses. Cell proliferation % was significantly (P<0.01) increased at 25 and 10 µg/ml doses, while, it was significantly (P<0.01) decreased at 100 µg /ml dose as compared to 50 and 0 µg /ml. However, there was no significance difference was observed in cell proliferation % in groups treated by different hGO nanostructures. In last, it was concluded as, hGO nano structures cytotoxicity was dose dependent and hGO nano tubes were least cytotoxic in caprine WJ-MSCs. 

INTRODUCTION

In recent, nanotechnology is applied in biomedical sciences including stem cell research (Deb et al., 2012) and stem cells are progenitor cells that differentiate into diverse cell lineages. Mesenchymal stem cells (MSCs) are adult stem cells and isolated from different sources like bone marrow, adipose tissue and fetal adnexa in different species such as caprine (Elkhenany et al., 2015), bovine (Cardoso et al., 2012), ovine (Eswari et al., 2016). MSCs are differentiate into osteocytes, chondrocytes, cardiac myocytes and pancreatic cells and were used to treat different regenerative diseases (Sangeetha et al., 2017) because, there is no risk of teratoma formation after in vivo MSCs administration and their immunomodulatory properties make them comparatively more suitable in regenerative therapy (Bhat et al., 2018). Caprine Wharton’s jelly derived MSCs (WJ-MSCs) are reliable stem cell source and differentiated into osteocytes, adipocytes and chondrocytes (Somal et al., 2016). MSCs are surface adherent cells and continuous stimulation is required to their rapid in vitro expansion and differentiation. Therefore, nanomaterials are used to stem cell expansion, differentiation and transplantation (Guo et al., 2017) as well as in vivo stem cells imaging (Bhat et al., 2018). In vitro cytotoxicity of different nanomaterials was studied in stem cells, however, nanomaterials cytotoxicity depends on size, composition and exposure time as well as cell models (Sohaebuddin et al., 2010). Graphene oxide (GO) is carbon based nanomaterial and was applied stem cell research (Elkhenany et al., 2015) and exhibited dose dependent cytotoxicity in 264.7 RAW macrophage (Figarol et al., 2015) and caprine WJ-MSCs (Dar et al., 2015; Gade et al., 2015). Titanium (Ti) based nano substrates were also used in stem cell research and it did not induce significant cytotoxicity in human bone marrow derived MSCs (hBM-MSCs) (Qu et al., 2016). Ti, GO coated Ti (Ti/GO) constructs were used as vehicles for delivery of bone morphogenic protein-2 (BMP-2) and substance P (SP) in osteoblasts and hBM-MSCs respectively, (La et al.,2014). However, modification of GO with organic or inorganic nanomaterials exploit their properties (Zhuang et al., 2018), biocompatibility (Nishida et al., 2014) as well as stem cells expansion and differentiation (Ravindran et al., 2016). Graphene coated Ti substrates were enhanced adhesion of human adipose derived stem cells (hASCs) and hBM-MSCs and promoted their in vitro as well as in vivo osteogenic differentiation (Ming et al., 2018). However, in vitro cytotoxicity reports on hybrid GO (hGO) (phosphorus doped graphene oxide titanium oxide) (P-GO-TiO2) nano structures are scanty and their details experimental in vitro cytotoxicity analysis is prerequisite. Therefore, the present study was carried out with the objective to analyse dose dependent in vitro cytotoxicity of hGO nano structures (P-GO-TiO2 tubes, P-GO-TiO2 rods and P-GO-TiO2  sheets) in caprine WJ-MSCs.

MATERIALS AND METHODS

Isolation of caprine WJ-MSCs
 
The present study was carried out in the year 2018 at Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Anjora, Dist. Durg (CG) with the approval of Institution Animal Ethics Committee (No. VPB/PhD-1/2017). Caprine gravid uteri (nearly 45 day age) were collected from local abattoir and washed with sterile normal saline solution. Caprine WJ-MSCs were isolated as per the method described by Babaei et al., (2008) and maintained in CO2 incubator at 37°C temperature and 5% CO2 environment and observed periodically. Dulbecco’s modified eagles’ medium (DMEM) fortified with 15% fetal bovine serum was replaced every after 4th day and third passaged cells were characterized and were used to study in vitro cytotoxicity assays.
 
Characterization of caprine WJ-MSCs
 
Caprine WJ-MSCs were stained with Alzarin red staining as per method reported by Baghaban et al., (2008) to detect the mineralized matrix in cells. Clonogenic property of caprine WJ-MSCs was evaluated by colony forming unit (CFU) assay as per the method described by Dar et al., (2015). The in vitro cytotoxicity of hGO nano structures (P-GO-TiO2 tubes, P-GO-TiO2 rods and P-GO-TiO2 sheets) was analysed at doses 100, 50, 25, 10 and 0µg /ml media by studying following cell cytotoxicity assays.
 
Cell morphology
 
Confluent caprine WJ-MCSs were treated with different hGO nanostructures as per designed doses and morphological changes were assessed after 72 hrs.
 
Cell viability
 
Caprine WJ-MCSs were treated with hGO nano structures as per given doses and cell viability was assessed after 72 hrs with trypan blue dye exclusion technique described by Bregoli et al., (2009).
 
Cell Growth Kinetic (GK)
 
Caprine WJ-MSCs were seeded and treated with different hGO nanostructures as per given doses and cells were harvested and counted with hemocytometer every after 48 hrs during 14 consecutive days and growth curves were plotted.
 
Cell population doubling time (PDT)
 
Caprine WJ-MSCs were cultured with hGO nano structures cells were harvested and counted using a hemocytometer daily during three consecutive days and cell PDT was calculated using equation described by Gade et al., (2015).
 
Cell proliferation
 
Caprine WJ-MSCs proliferation rate with the exposure of hGO nanostructures was evaluated by using Tetrazolium dye 3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay kit as per the manufacturer instructions. Caprine WJ-MSCs were cultured in a 96 well cell culture plate and treated with different doses of hGO nano structures for 24 hrs. Cell monolayer was treated with MTT reagent and incubated for 4 hrs in CO2 incubator and observed periodically. The dark coloured MTT formazan crystals were dissolved with solubilization solution and absorbance was read thrice at 620 nm on ELISA plate reader and cell proliferation % was determined.
 
Statistical analysis
 
Data was analysed with Full Factorial design (SPSS statistic 25 software) and P<0.01 values were considered as statistically significant.

RESULTS AND DISCUSSION

Caprine WJ-MSCs were exhibited heterogeneous morphology including flattened, polygonal and fusiform or spindle shape and mostly fibroblastoid morphology and reached confluent stage on day 14 (Fig 1) and this is in accordance with previous findings in caprine (Somal et al., 2016). In caprine WJ-MSCs mineralized matrix was formed and it was positively stained with red colour (Fig 2A) which showed their capability to differentiate into osteocytes and these findings are in accordance with earlier reports in caprine (Somal et al., 2016). CFU assay is a gold standard test used to characterize MSCs and caprine WJ-MSCs formed colonies (Fig 2B) and clonogenic property of caprine WJ-MSCs in present study is like earlier findings reported by Dar et al., (2015).
 

Fig 1: Caprine WJ-MSCs on day 14.


 

Fig 2: Characterization of caprine WJ-MSCs [A] Alzarin red staining and [B] CFU assay.


 
Cell morphology
 
Comparatively, hGO tubes were least damaged cell morphology and more good cells were observed than hGO rods and hGO sheets treated groups at 25 and 10 µg/ml doses (Fig 3). However, all hGO nano structures significantly disturbed cell morphology at doses 100 and 50 µg/ml as compared to 25, 10 and 0 µg/ml doses (Fig 3). Each hGO nano structures at 25 and 10 µg / ml doses stimulated cell growth and this is confirmed with the earlier reports of Figarol et al., (2015) who observed as no significant impact of pristine and acid functionalized carbon nano tubes (CNTs) and graphene on RAW 264.7 macrophages at doses 15, 30, 60 and 120 µg/ml but, cytotoxicity was dose dependent. However, GOs were internalized in HeLa cells and damaged it while, Au-GO hybrid nanostructures were significantly enhanced cell survival and proliferation rate (Liu et al., 2012) in support to present findings. But, hBM-MSCs morphology did not differ significantly which cultured on Ti based SiO2 substrates synthesized under oxygen, nitrogen and argon atmosphere (Qu et al., 2016) similar to this study.
 

Fig 3: Caprine WJ-MSCs morphology exposed with different hGO nano structures (A-100 µg/ml, B- 50 µg/ml, C- 25 µg/ml, D- 10 µg/ml, E- 0 µg/ml).


 
Cell viability
 
Cell viability % was significantly (P<0.01) increased by hGO tubes as compared to hGO rods and hGO sheets after 72 hrs (Table 1) and significantly (P<0.01) higher cell viability % was observed at 25 and 10 µg / ml doses and it was significantly (P<0.01) decreased at 100 and 50 µg / ml doses as compared to control (Table 2). In this study, significant increase in cell viability% with treatment of hGO tubes might be due to their shape as tubes can easily penetrate through cell membrane and increase cell metabolism because nanoparticles have an intrinsic property which promote stem cells proliferation (Ravindran et al., 2016). TiO2, SiO2 and multi walled carbon nano tubes (MWCNTs) were significantly (P<0.01) reduced 3T3 fibroblast, 264.7 macrophage and human bronchiolar epithelial cells viability at doses 100 and 1000 µg/ml but, 10 µg/ml dose did not reduce cell viability (Sohaebuddin et al., 2010) similar to present study. Also, as like present study graphene quantum dots (GQDs) (Dar et al., 2015) and GO-Iron oxide nanocomposites (GO-Fe2O3 NCs) (Gade et al., 2015) at 50 and 100 µg/ml doses were significantly (P<0.01) decreased caprine WJ-MSCs viability %. But, GO substrates did not evoke any significant cytotoxicity in murine MSCs (C3H10T1/2) (Kim et al., 2018) and in support to present study findings, Mirza et al., (2019) observed that hybrid GO (GO-poly methyl methacrylate) substrates significantly increased hBM-MSCs viability.
 

Table 1: In vitro effect of hGO nano structures on caprine WJ-MSCs viability, PDT and proliferation (Mean ± S.E.).


 

Table 2: In vitro effect different doses of hGO nano structures on caprine WJ-MSCs viability, PDT and proliferation (Mean±S.E.).


 
Cell growth kinetics
 
Cell growth curves with the treatment of different hGO nano structures during 0-14 days were not altered significantly and followed normal cell growth pattern (Fig 4). However, 100 and 50 µg/ml doses of hGO nano structures were significantly (P<0.01) changed shape of growth curves (declined growth) as compared to 25, 10 and 0 µg/ml doses (normal pattern) and 25 µg/ml dose showed slight increased cell growth rate during 0-14 days (Fig 5). In support to present findings, similarly, BMP2 with Ti-GO construct increased alkaline phosphatase activity and Runx-2 expression profile of human osteoblasts did not change significantly as compared with control (La et al., 2014). However, MWCNTs were cytotoxic and genotoxic in human umbilical vein endothelial cells (Guo et al., 2011), single walled carbon nano tubes (SWCNTs) increased apoptosis in rat pheochromocytoma cells (PCl2) (Wang et al., 2011). But, CNTs, graphene and carbon black were showed dose dependent cytotoxicity in 264.7 cells (Figarol et al., 2015) like the present study.
 

Fig 4: Effect of different hGO nano structures on caprine WJ-MSCs growth curve.


 

Fig 5: Effect of different doses of hGO nano structures on caprine WJ-MSCs growth curve.


 
Cell PDT
 
hGO nano structures were significantly (P<0.01) increased cell PDT at 100 and 50 µg/ml doses as compared to 25, 10 and 0 μg/ml (Table 2). But, cell PDT difference was non significant within different hGO nano structures groups (Table 1). In agreement to these findings, there are earlier similar reports by Dar et al., (2015) and Gade et al., (2015) who observed GQDs and GO-Fe2O3 NCs, respectively, increased caprine WJ-MSCs PDT at 50 and 100 µg/ml doses as compared to 10 and 0 ìg/ml doses. However, GO stimulate stem cell proliferation which was similarly justified by Elkhenany et al., (2015) in caprine BM-MSCs that, GO reduced cell PDT with >95% viability as compared to control. Similarly, grapheme substrates increased human neural stem cells (hNSCs) proliferation and hNSCs differentiated into neurite cells (Park et al., 2011). GO doped Gelatin-Hydroxyapatite (G-HA) scaffolds (0.5 wt.% GO in GO- Gelatin HA) stimulate human adipose tissue derived MSCs (hAD-MSCs) differentiation into osteocytes than pure Gelatin HA scaffolds (Nair et al., 2015) and 1.0 µg/ml GO in GO-Collagen (GO-COL) scaffolds significantly stimulated MC3T3-E1 cells proliferation rate (Nishida et al., 2014) like in the present study.
 
Cell proliferation
 
In the present study, cell proliferation % did not significantly differ within different hGO nano structure groups (Table 1) but, it significantly (P<0.01) decreased at 100 µg/ml dose and significantly (P<0.01) increased cell proliferation % was observed at 25 µg/ml and 10 μg/ml doses as compared to 50 µg/ml and 0 µg/ml doses (Table 2). In this study, each hGO nano structures inhibited cell proliferation % at 100 µg/ml dose and maximum tolerable dose was 50 µg/ml. However, 25 and 10 µg/ml doses accelerated cell growth. SWCNTs and MWCNTs were significantly reduced lung epithelial (A549) cell viability and MWCNTs more cytotoxic but, Ti nanotubes did not induce significant cytotoxicity in A549 cells (Wadhwa et al., 2011) in support to present findings. Oxidized MWCNTs-F, functionalized MWCNTs-NH (ethylene diamine) and pristine MWCNTs were significantly (P<0.01) decreased murine macrophage (RAW-264.7) cell viability and proliferation at 80 µg/ml dose (Szczypta et al., 2012) like present study. Similarly, TiO2 nano belts were significantly (P<0.01) decreased small air way and intestinal epithelial cells proliferation rate at 100 µg/ml (Tilton et al., 2014). In accordance with the findings of the present study, earlier Rodriguez et al., (2017) also reported as nanocrystalline glass like carbon (NGLC) microflakes were decreased substantia nigra dopaminergic (SN4741) cells viability at dose 50 µg/ml but, NGLC film stimulate SN4741 cells proliferation and differentiation into neuronal cells.

CONCLUSION

In the present study, it was concluded as, cytotoxicity of hGO nano structures was dose dependent and all hGO nano structures were biocompatible in caprine WJ-MSCs at 25 and 10 µg/ml doses. However, comparatively, hGO nano tubes were least cytotoxic to caprine WJ-MSCs and therefore, hGO tubes can be used in stem cell research.

ACKNOWLEDGEMENT

All authors thankful to the Dean, College of Veterinary Sciences and Animal Husbandry, Anjora, Dist. Durg (CG) -for permitting to carry this study as well as ICAR for providing financial support.

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