Stem cell growth and proliferation on RGD bio-conjugated cotton fibers

Abstract

BACKGROUND:

Merging stem cells with biomimetic materials represent an attractive approach to tissue engineering. The development of an alternative scaffold with the ability to mimic the extracellular matrix, and the 3D gradient preventing any alteration in cell metabolism or in their gene expression patterns, would have many medical applications.

OBJECTIVE:

In this study, we introduced the use of RGD (Arg-Gly-Asp) bio-conjugated cotton to promote the growth and proliferation of mesenchymal stem cells (MSCs).

METHODS:

We measured the expression of stem cell markers and adhesion markers with Q-PCR and analyzed the transcriptomic. The results obtained showed that the MSCs, when cultured with bio-conjugated cotton fibers, form aggregates around the fibers while proliferating. The seeded MSCs with cotton fibers proliferated in a similar fashion to the cells seeded on the monolayer (population doubling level 1.88 and 2.19 respectively).

RESULTS:

The whole genome sequencing of cells adhering to these cotton fibers and cells adhering to the cell culture dish showed differently expressed genes and pathways in both populations. However, the expression of the stem cell markers (Oct4, cKit, CD105) and cell adhesion markers (CD29, HSPG2 and CD138), when examined with quantitative RT-PCR, was maintained in both cell populations.

CONCLUSION:

These results clearly show the ability of the cotton fibers to promote MSCs growth and proliferation in a 3D structure mimicking the in vivo environment without losing their stem cell phenotype.

Keywords

Stem cell markers adhesion markers genome sequencing cotton 3D scaffold mesenchymal stem cells (MSCs)

1. Introduction

In the last two decades, several natural polymers such as proteins and polysaccharides have been extensively investigated as natural hydrogels for stem cell growth and differentiation due to their biocompatibility and cell-mediated scaffold degradation characteristics [1]. Therefore, developing materials with the ability to mimic the complex arrangement of fibers and fibrils of the extracellular matrix will pave the way for a wide range of novel applications such as artificial organs, implant tissue and wound and medical textile materials [2]. In the last two decades, several biopolymers, including PLLA, PLGA, PLLA-PLGA copolymers, and other natural biomaterials including collagen gels, alginate, agarose, silk, and chitosan, have been investigated as an alternative scaffolding for stem cells growth and differentiation [1]. These endeavours have considerably improved our understanding of the material-cell interactions process. Unfortunately, cells isolated directly from higher organisms frequently have their metabolism or their gene expression patterns altered when cultured with these types of scaffolds [2]. Protein-based biomaterials have been for a long time an ideal candidate for tissue engineering approaches due to their structural properties, in particular, their ability to promote stem cell growth and differentiation. Michelini et al. have reported the growth and differentiation of monkey embryonic stem cells into both endothelial and neural phenotypes when cultured inside the three-dimensional culture system made of collagen. Collagen can be isolated from an array of tissues, usually of animal origin, such as tendon, bone or skin. Im et al. have also reported the use of fibrin, a non-globular protein involved in blood clotting, as a scaffold material for stem cell growth and differentiation. Where Mesenchymal stem cells (MSCs) are seeded inside fibrin clots and differentiated into bone cells [3]. Furthermore, exotic materials such as silk have also been uninvestigated as a culturing system for ligament regeneration [4] and for the culture of human Schwann cells, for the purposes of nerve cells regeneration [5]. Also, Kaplan and co-workers have demonstrated that human MSCs combined with silk scaffolds can be implemented in bone regeneration. During this study, they established that the flow conditions around, and the properties of the scaffold have a big impact on the rate of calcium deposition, which is an important factor for bone tissue engineering [6]. In addition, hybrid scaffolds made of freeze-dried porous silk-fibroin and chitosan have promote human umbilical cord blood MSCs to attach, proliferate and differentiate into chondrocytes cells [7,8]. In conjunction with protein-based scaffold, natural polysaccharides such as agarose, alginate and chitosan also showed a capability to imitate to some extent, the extracellular matrix [9]. For example, hyaluronan, also known as hyaluronic acid (high molecular weight polysaccharide), is one of the major components of the extracellular matrix which contributes significantly to cell migration and proliferation. Martínez-Ramos et al. reported the use of Hyaluronan as a culturing system to grow U-87 cancer stem cells (CSCs) and exploited it as a model for anti-cancer drug screening [10]. Furthermore, Johnstone and his team have used hyaluronan as a scaffold to grow and proliferate MSCs as an approach to restoring cartilage both in vitro and in vivo [11]. In addition, Wang et al. have recently reported the use of a combination of a couple treated cotton fibers in agarose gel to grow and enrich cancer stem cells (PC12) [12]. The modification of these cotton fibers was performed using harsh NH₃ plasma to create enough positive charge to allow better adhesion of the cells onto the cotton fibers [12]. Recently, biodegradable synthetic materials such as polyester, polybutylene succinate, have demonstrated their ability to promote MSCs cells to grow and differentiate into osteocytes cells [13]. Similarly, a titanium-based scaffold with controlled pore size was also developed, to promote the growth of the MSCs [14]. A hybrid scaffold between strontium hydroxyapatite and nanocrystals was also investigated for the growth of the MSCs [15]. In recent years a new type of hybrid materials that combines synthetic materials such as PLGA and natural ones such as silk has been investigated as a candidate for the transplantation of osteoblasts, where these microfibers sustained their viability and promoted the formation of bones in vivo. Recently, carbonized cotton fibers coated with polydopamine have been successfully used to grow and differentiate rat PC12 cells into neurons, using electrical current as external stimulus [16]. Besides these achieved objectives, a significant constraint of natural-based scaffolds, is most of them are animal-derived biomaterials which can cause a major compatibility issue. Furthermore, these natural-based scaffolds often contain the residue of growth factors and undefined proteins that may cause downstream problems such as immune responses upon transplantation; animal pathogen contamination; batch to batch variation; and complications during developmental studies arising from undefined factors within the cultural media. Therefore, developing hybrid material without some of the above limitations, which can promote stem cell proliferation without differentiating them, will represent a milestone of immense proportions. In this study, we investigated the use of modified natural cotton fibers for MSC growth and proliferation, since cotton fibers can be easily chemically modified.

Our cotton fibers modification was performed with RGD using a simple chemical procedure that generates fibers with enhanced capability toward stem cells attachment via their integrin receptor. Natural cotton fibers have attractive properties such as a slow rate of degradation, the ability to produce a variety of structures, desirable mechanical properties and low cytotoxicity [17]. The RGD peptides have been extensively used to promote stem cell adhesion and spreading. These peptides are recognized by surface receptors on stem cells; they bind to the VLA-5 integrin receptor found on stem cells [18]. MSCs are heterogeneous non-immunogenic stem cell populations that can be found in various tissues, like bone marrow, adipose tissue and cord blood [19]. They are multipotential stem cells, mainly obtained from adult tissue, that are capable of self-regeneration, and that are able to differentiate into multiple cell types. As such, they represent a promising tool for regenerative medicine [20]. In this study we evaluated the capacity of the mesenchymal stem cells to adhere to RGD modified cotton fibers and proliferate without losing their stem cell phenotype. We have achieved this by measuring the proliferation of the cells and also quantified the stem cell markers and cell adhesion markers. We furthermore performed the whole genome sequencing for cell population cultured and adhered to cotton fibers and cells cultured without cotton fibers.

2. Materials and methods

2.1. Materials

Mesenchymal stem cells (MSCs) from human bone marrow (Stem Cell Technologies, Cambridge, MA, USA) were cultured in fetal bovine serum qualified for mesenchymal stem cell growth, DMEM with low glucose containing glutamine and Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Carlsbad, CA, USA). The same batch number for serum was used for all experiments. RNA was extracted using RNeasy extraction Kit (Qiagen, Hilden, Germany). Primers were designed using the Invitrogen Custom DNA Oligos and primers were ordered (Eurofins MWG/Operon, Louisville, USA). The cDNA was synthesized with high capacity cDNA Reverse transcription kit and Fast SYBR green Master Mix (Applied Biosystems, Carlsbad, CA, USA) and Q-PCR was carried out (Fast Real-time PCR System 7900 HT, Applied Biosystems, Waltham, MA, USA). Whole genome sequencing was run with Hiseq2000 Sequencer (Illumina, San Diego, CA, USA).

2.2. Statistical analysis

The experiments were performed in triplicate and the data were reported as the mean $\pm$ standard deviation (SD). Statistical analysis was performed using TTEST comparing two means. $^*=p < 0.05$ , $^{**}=p < 0.01$ , $^{***}=p < 0.001$ were considered statistically significant.

2.3. Methods

2.3.1. Cotton bio-conjugation with RGD

Dry cotton (10 grams) was dissolved in 40 mL DMF. Potassium carbonate (2.76 g, 20 mmol) was then added to the solution and the reaction mixture was allowed to stir for 15 min at room temperature. 7-Chloroheptanal (2.97 g, 20 mmol) was then added dropwise to the solution and the reaction mixture was allowed to stir for 24 hours at room temperature. After completion, the reaction mixture was filtered out using Buchner funnel followed by the addition of 40 mL of DMF. The mixture was washed with Ethanol (3 × 40 mL) to remove the extra Potassium carbonate and the unreacted 7-Chloroheptanal. The dry cotton was then dissolved again in 50 ml of ethanol and 1% acetic acid. RGD (Arg-Gly-Asp) (300 mg, 0.8 mmol) dissolved in 10 ml of water was added slowly to the mixture and left to magnetically stir for 15 min at 60 °C. Then the resulting suspension was stirred at room temperature overnight. The mixture was filtered and washed 10 times with distilled water to remove unbound RGD and all traces of the remaining reagents. The functionalized cotton was then dried for three days within a vacuum in a desiccator.

2.3.2. Cell cultivation

MSCs from bone marrow were revived and cultured in DMEM supplemented with 10% serum and 1% Penicilli-Streptomycin (Complete medium). Cells were passaged when 80% confluent. They were then detached with 0.25% w/v trypsin-EDTA in phosphate-buffered saline (PBS; pH 7.4) for 5 min in an incubator. The trypsin was deactivated with a complete medium. Detached cells were transferred to a 15 ml tube and centrifuged at 1500 rpm for 5 min. The supernatant was discarded and the pellet resuspended in 1 ml of complete medium. Cell number and viability were measured with trypan blue for cell viability prior to seeding.

Cells were seeded in three wells of a 6-well plate at 0.5 × 10⁶ with cotton and left for 24 and 48 hours in culture. The cells were detached from the cotton by washing the fibers with a medium. For control experiments, cells were seeded in a monolayer without cotton in three wells of a 6-well plate at 0.5 × 10⁶. Cells were detached with trypsin and counted with trypan blue. Experiments were repeated at least three times.

2.3.3. Q-RT-PCR for stem cell and cell adhesion markers

MSCs were seeded at a density of 0.25 × 10E⁶ per well on six well plates, with or without cotton (three wells each). The same concentration of cells was kept as a pellet at −80 °C to be used as a control. Cotton was washed three times to remove the cells attached to it. The cells seeded without cotton were detached with trypsin. Cells were centrifuged and the pellet resuspended in buffer RLT. Complementary DNA from each well was synthesized from 500 ng of RNA using High Capacity cDNA Reverse transcription. Ten nanograms of cDNA were amplified with SyberGreen and loaded in a Fast Real-Time PCR system 7900HT LightCycler system (Roche). All samples were run in triplicate in 10 μl reactions. The standard PCR conditions were 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min, and 72 °C for 30 s. Primer validation was first carried out. Stem cell markers for MSC (Oct4, cKit and CD105) and cell adhesion markers (HSPG2, CD29 and Syndecan) were tested (Table 1 shows the sequence of the primers used). Expression levels of these six target genes were calculated by the comparative Ct method (2–^ΔΔ Ct formula), after being normalized to the Ct value of the Beta-actin housekeeping gene. The levels of beta actin were similar in all samples (data not shown).

Table 1
Sequence of primers used for qPCR analysis

Primer name Primer sequence

CD138 Forward CCGCTGCCACGTTGGA

CD138 Reverse TGAAGGCTGAGTCCCAGCAT

CD105 Forward TCTGCACATGGGAACAATGG

CD105 Reverse CCCAGGTTCAAGCGATTCTC

HSPG2 Forward TCTCAATGCCCCAAGAAGTC

HSPG2 Reverse TCCAGCTGATGTCAGGAGTG

Oct4 Forward CGACCATCTGCCGCTTTG

Oct4 Reverse GCCGCAGCTTACACATGTTCT

CD29 Forward CAACACCAGCTAAGCTCAGGAA

CD29 Reverse CTAAATGGGCTGGTGCAGTTC

cKit Forward TTTTCTTTGGGAGCTGTTCTCTTT

cKit Reverse AGAACTTAGAATCGACCGGCATT

Beta actin Forward GATGAGATTGGCATGGCTTT

Beta actin Reverse CACCTTCACCGGTCCAGTTT

Primer name	Primer sequence
CD138	Forward	CCGCTGCCACGTTGGA
CD138	Reverse	TGAAGGCTGAGTCCCAGCAT
CD105	Forward	TCTGCACATGGGAACAATGG
CD105	Reverse	CCCAGGTTCAAGCGATTCTC
HSPG2	Forward	TCTCAATGCCCCAAGAAGTC
HSPG2	Reverse	TCCAGCTGATGTCAGGAGTG
Oct4	Forward	CGACCATCTGCCGCTTTG
Oct4	Reverse	GCCGCAGCTTACACATGTTCT
CD29	Forward	CAACACCAGCTAAGCTCAGGAA
CD29	Reverse	CTAAATGGGCTGGTGCAGTTC
cKit	Forward	TTTTCTTTGGGAGCTGTTCTCTTT
cKit	Reverse	AGAACTTAGAATCGACCGGCATT
Beta actin	Forward	GATGAGATTGGCATGGCTTT
Beta actin	Reverse	CACCTTCACCGGTCCAGTTT

2.3.4. Preparation of cDNA library and sequencing

The RNA from cells forming aggregates around cotton fibers and cells grown on monolayer were isolated using the RNAesy. One microgram of RNA was isolated for the analysis of the whole transcriptomic with Illumina using a Hiseq2000 Sequencer. The RNA concentration was measured with the Cubit, and the purity measured with the ratio A260/A280 was >1.8.

The preparation of the cDNA library was performed according to the manufacturer protocol. In brief, single-strand cDNA synthesis was accomplished using 1 microgram of RNA. Double-stranded cDNA was synthesized by primer extension using Ex Taq polymerase. Double stranded cDNA was fragmented to 300 bp to 500 bp sizes by sonication to generate libraries for sequencing. Fragments were purified with Ampure beads (Agencourt, USA). Sequencing libraries were prepared from sheared cDNA using TruSeq Paired-End Cluster Kit v2.0 (Illumina, USA) and 200 cycle TruSeq SBS HS v2 kit (Illumina, USA) generating 100 bp reads. An Electropherogram was run to check the size of the library and quality.

2.3.5. Analysis of differently expressed genes and their expression profiles

Adaptors sequences reads with unknown sequences ‘N’ and low-quality sequences were cleaned using a trimmomatic tool [21] (http://www.usadellab.org/cms/?page=trimmomatic). The clean reads were mapped to reference sequences Human Reference Genome GRCh37 Version using a CLCGenomic workbench 6.5 (http://www.clcbio.com/products/clc-genomics-workbench/) with default parameters. The number of annotated clean reads for each gene was calculated and normalized to reads per kilobase per million (RPKM). Gene and transcript-wise expressions for each population were calculated with custom Perl scripts, from the expression tables output from CLC Genomics workbench. The list of genes differentially expressed in each cell line was compared using Venny, an online, interactive tool for comparing lists with Venn diagrams [22]. The comparison was done based on genes that are expressed in both cells adhering to cotton, and cells adhering as a monolayer. If the genes appeared in both lists, they are classified as common genes. If they appear in either list they are classified as unique to the subgroup. The genes that were differently expressed were taken for Gene Ontoloy (GO), and pathway analysis.

2.3.6. Gene Ontology and pathway analysis

The analyses were done to obtain Transcriptomic RNA-Seq data sets and Individual Gene expression profiles by comparing expression profiles between both populations, and by pathway analysis. The Gene Ontology (GO) and pathway analysis were done using the DAVID Functional Annotation tool (http://david.abcc.ncifcrf.gov/) [23,24].

3. Results and discussion

In this work, the cotton fibers bio-conjugation was fabricated using classic organic synthesis protocols as described in the methods section.Twenty-four hours after seeding, cells formed aggregates around the bio-conjugated cotton fibers. At 48 hours, cells formed hanging grapes like spheroids around the cotton fibers as shown in (Fig. 1A and 1B), as compared to the cells seeded in a monolayer without cotton fibers (Fig. 1C). Cells that adhered to the surface had the typical fibroblast-like phenotype allowing cell-surface connection and cell-cell connection in 2D structure. However, the cells cultured on the bio-conjugated cotton stayed in suspension in spheroidal-like shape. This condition allowed cell-cell connection and cell-fibers connection in 3D structure, mimicking the in vivo environment. Cells formed also aggregates around the cotton fiber when seeded with lower concentration: 0.25 × 10⁶. Cells forming aggregates around cotton fibers proliferated and showed high viability in similar fashion to the non-cotton cells.

Fig. 1.

Cells were seeded in six well plates at 0.5 × 10E⁶ and left for 48 hours. (A) and (B) cells seeded in a medium containing bio-conjugated cotton formed aggregates around the fibers. (C) Cells seeded without cotton attached to the culture surface and proliferated.

The number of cells forming aggregates almost doubled (0.95 × 10E⁶) compared to the seeding density (0.5 × 10E⁶) (Fig. 2). The viability of the cells at 48 hours forming aggregates with cotton was 91% similar to the viability of the cells grown without cotton and in monolayer (90%). Cells proliferated in both conditions with or without cotton demonstrated a population doubling level of 1.88 and 2.19 for cells grown in cotton and in monolayer respectively. Most of the cells seeded with cotton were recovered only by washing with a medium instead of trypsinization, which limits the denaturation of surface protein of the cells. No cells remained on the cotton fibers when checked under the microscope (data not shown).

Fig. 2.

Cells in suspension at 48 hours seeded with or without cotton compared to cells seeded at T = 0 hour (control). Cells seeded with cotton fibers proliferated compared to seeding density (control). Cells in suspension when seeded with no cotton were significantly low. Data presented are the mean value of three independent experiments ± S.D. Cells seeded at T = 0 were compared to cells in suspension with and without cotton. ***=p < 0.001.

Quantitative RT-PCR was run to check the effect of cotton fibers on the expression of stem cell markers and cell adhesion markers. Cells seeded in a monolayer were also characterized for these markers. Mesenchymal stem cells are reported to be positive for Oct4, CD105 and cKit [25]. Cell adhesion markers Beta integrin 1 (CD29), Heparan Sulfate Proteoglycan (HSPG2) and Syndecan 1 (CD138) are involved in cell adhesion. These markers mediate the attachment of the cells to the extracellular matrix [26]. Proteoglycans are the major constituents of the extracellular matrix (ECM) involved in cell proliferation, differentiation and gene expression [27]. Heparan sulfate chains interact with growth factors and morphogens (FGF, Wnts), their receptors (FGFRs) and ECM structural molecules (Fibronectin) [27]. Heparan Sulfate is a cofactor in cell adhesion, motility, proliferation, differentiation and tissue morphogenesis. Cell marker CD29 was already reported to be expressed in human MSCs [25]. CD138 was shown to promote the proliferation of undifferentiated adipocyte progenitor and inhibits their adipogenic differentiation [28].

Stem and cell adhesion markers were tested with quantitative RT-PCR at 24 and 48 hours (Fig. 3). The expression of all stem cell markers (CD117, CD105, Oct4) and cell adhesion markers (HSPG2, CD138, CD29) was maintained in the cells seeded with cotton. The increase in CD117 and the decrease in CD138 and CD29 were not significant in cells seeded with cotton. The expression of CD117, Oct4, CD138 and CD29 were significantly reduced in cells seeded in a monolayer and without cotton. At 48 hours, the expression of CD117, Oct4 was normalized in cells seeded without cotton compared to the control. As described in the material and method section, the control cells were cells that had not been in culture and have been frozen at the same concentration.

Fig. 3.

Quantitative RT-PCR gene analysis of stem cell markers (Oct4, cKit, CD105) and cell adhesion markers (HSPG2, CD138 and CD29). Cells were left 24 and 48 hours in culture with cotton (+cotton) or without cotton (-cotton) and compared to the control. Control was similar for all conditions. The relative expression of the genes is expressed as the 2–dd-Ct formula. The bars represent the standard deviation. **=p < 0.01.

The whole genome sequencing was run with Illumina Hiseq 2000 sequencing for cells growing as aggregates on cotton and cells seeded in monolayer and left 48 hours in culture. The results showed that the two populations had genes commonly expressed and genes uniquely expressed within each population (Fig. 4). Cells adhering to cotton had 511 differently expressed genes (DEG) compared to the cells grown on monolayer, which had 632 DEG. Cells from both populations had 14611 commonly expressed genes. The analysis was done without RPKMP filtration so the expression could be as low as 0.001 to tens of thousands. Reasons for the difference in gene expression between the two populations are not clear but it could be some genes are affected by the cotton fibers, or that the cells were being affected by the surface culture. Cells from both conditions expressed the stem cell markers (CD105, CD117 and Oct4) and cell adhesion markers (CD29, CD138 and HSPG2) as examined with q-RT-PCR.

Fig. 4.

Venn diagram representing differently expressed genes (DEG) in the cell population seeded with cotton and adhering to the fiber (511 genes), and in the cell population seeded without cotton and adhering in a monolayer (632 genes). Both cell populations had 14611 commonly expressed genes.

Table 2 presents some of the genes that are differently expressed and could affect cell proliferation, adhesion and migration. Other genes not presented in the table, like CD53, were only expressed in the cell population seeded without cotton. This gene, a member of the tetraspanins family, is involved in cell adhesion and motility, cell activation and proliferation, development, growth, differentiation, and cancer [29]. Another gene, FGF17 was expressed in the cell population seeded with cotton and growing in aggregates. FGF17 is involved in embryonic development and cell growth, morphogenesis, tissue repair, and tumor growth and invasion [30]. Sox2 was only expressed in the cell population seeded with cotton. This gene is a marker of the stem and progenitor cells in diverse adult tissues [31]. The ITGAL was also expressed in the cells seeded with cotton and has a role in cell adhesion. Those differences in adhesion markers like SELL, ITGAD, CTNNA3, CLDN6, CEACAM6, F11R, and migration markers like NCAN in the cells seeded with cotton (Table 3), could influence the adhesion of the cells to cotton fibers, and migration in vivo to the target tissue. It was previously shown that increasing the expression of the cell adhesion markers will improve the homing and engraftment capacities of the cells when are transplanted to target organs [32,33]. Hence, the expression of the adhesion and migration markers in the cell population adhering to cotton fiber will be of great interest for cell transplant in regenerative medicine.

Table 2

Presentation of some genes differently expressed in the cells seeded with cotton and genes expressed in cells seeded without cotton

Genes expressed in cells seeded without cotton	Function	Genes expressed in cells seeded with cotton	Function
ITIH5	Extracellular matrix stabilization	SELL	Cell surface adhesion, belongs to a family of adhesion/homing receptors
Sox10	Regulation of embryonic development and in the determination of the cell fate	PRKCB	Apoptosis induction, endothelial cell proliferation
FGF22	Embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion	ITGAD	Activation and adhesion functions of leukocytes
EPCAM	Epithelial cell adhesion	CTNNA3	Cell-cell adhesion
TNF	Cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation	CLDN6	Cell-to-cell adhesion in epithelial or endothelial cell sheets
CXCR3	Integrin activation, cytoskeletal changes and chemotactic migration	NCAN	Cell adhesion and migration
HS6ST2	Cell growth, differentiation, adhesion, and migration	CEACAM6	Cell adhesion
MMP28	Breakdown of extracellular matrix	F11R	Regulator of tight junction assembly, cell-to-cell adhesion

Cells seeded with cotton and cells without cotton had four differently expressed pathways and one was expressed in common (Table 3). The impact of these pathways might be tested by knocking down key genes involved to examine their effect on MSCs phenotype and function.

Table 3

Pathway analysis in the cell population seeded with cotton and the cell population seeded without cotton

Pathways expressed in cells seeded with cotton	Pathways in cells seeded without cotton
Cytokine-cytokine receptor interaction	Leucocyte transendothelial migration
Neuroactive ligand-receptor interaction	Neuroactive ligand-receptor interaction
Systemic lupus erythematous	Cell adhesion molecules (CAMs)
Retinol metabolism	Tight junction
Primary immunodeficiency	Calcium signalling pathway

Many scaffold systems have been developed to allow the growth of stem cells in 3D environment. Organic polymeric materials, such as polylactic acid (PLA), polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) are most widely used, but all of them have serious disadvantages. They show low hydrophobicity, low cell adhesion ability and low mechanical strength [34]. Their acid degradation causes aseptic inflammation.

Regeneration of tissue needs a considerable number of stem cells to transplant to replace the defective tissue or organ. MSCs can be expanded more than 20 passages with more than 50 population doubling [27], but this is not enough to overcome the demand in regenerative medicine. Developing a scaffold allowing stem cells to proliferate and at the same time keep their stem cell markers is of great interest for regenerative medicine. Our aim was to use RGD modified cotton fibers that could allow the stem cells to grow, form 3D structure and keep their stem cell phenotype.

Cotton is formed mainly by cellulose (90%), a major component of plant cell walls, and can be degraded by cellulase [35]. Previous reports showed that there is an increased interest in the use of cellulose-based materials as scaffold to grow stem cells and progenitor cells. Cellulose based- scaffolds have already been used for mammalian cell culture [36,37]. It was shown that bacterial cellulose could be used as a scaffold to grow human adipose derived stem cells (HASC) [34]. The HASC were successfully differentiated under the right conditions to form osteogenic cells within the bacterial cellulose. It also allowed the cells to be transplanted into injured animals and repair the damage caused to animal bone [34]. Nanofibrillar cellulose hydrogel also promoted the functional 3D spheroid formation of human liver cells [38]. Lou et al. showed that the nanofibrillar cellulose, which is isolated from the walls of wood and plants, may be used as hydrogel. The nanofibrillar cellulose hydrogel can form a flexible 3D environment for the proliferation and differentiation of human pluripotent stem cells [38].

Hepatic progenitor cells were previously grown on wood-derived nanofibrillar cellulose [39]. It induced the formation of 3D multicellular spheroids and supported the mRNA expression of hepatocyte markers (albumin and CYP3A4) and metabolic activity of CYP3A4 activity in the HepaRG cell culture. In other words, the main CYP3A4 enzyme activity was not compromised when grown as spheroids on cellulose-based scaffold.

Bacterial cellulose membranes were used for the transdermal administration of diclofenac. It showed good transdermal permeation [40]. Bacterial cellulose is biodegradable as opposed to the plant cellulose [34]. Apple-derived cellulose scaffolds were generated after decellularization and used to grow cells. The mammalian cell line Huh7 proliferated on 3D apple-derived cellulose scaffolds in vitro for 12 weeks [35].

We demonstrated that cotton fibers sustained MSCs growth and viability comparable to the cells seeded in monolayer. Our data also showed that cotton did not decrease the expression of the stem cell markers and the cell adhesion markers as shown in Fig. 3. White cotton fibers are made of 94 to 96% of cellulose, a naturally occurring sugar [41]. They represent a promising tool to grow the MSCs in a scaffold without compromising their stem cell and adhesion markers. One of the major limitations resulting from transplantation with MSCs is their low homing and engraftment capacities as they are entrapped in the vasculature of the lungs after intravenous infusion [32,33]. Maintaining the expression of the cell adhesion markers like SELL, NCAN, CEACAM6 will improve the engraftment capacities of the cells to the target organ. This will need to be further tested in in vivo studies.

This study showed that MSCs seeded with cotton keep proliferating under normal cell culture conditions while keeping their stem cell markers. Cotton is a cheap naturally occurring fiber and a promising material to allow stem cells to proliferate and grow in 3D structure. Having such a scaffold will allow us to grow the cells to the required cell number for transplantation and for in vitro assays. The 3D structure mimics the in vivo environment, where all cells and organs are organized in 3D. Apart from the cells growing in an in vivo like environment, they will not be subject to the effect of the trypsin, which is known to denature the cell surface proteins and the extracellular matrix.

The main drawback of using scaffolds in vivo is the generation of an inflammatory response once they degrade [42]. The cells growing on the cotton fibers could be isolated from the fibers with cellulase, which degrades the cellulose – the main component of the cotton fiber – leaving the cells in spheroids hence in 3D structure. In our study, the cells grown on cotton fibers in spheroids can overcome the inflammatory response in vivo observed with other materials as stated earlier. This latter can only be confirmed when performing in vivo studies. A cotton-based scaffold could be developed in future studies to allow stem cells to grow in spheroids without losing their phenotype. The scaffold could be used as a mold for different organs or tissues – that is, to grow stem cells for regenerative medicine.

4. Conclusion

As demonstrated in this study, RGD bio-conjugated cotton fibers offer a platform for stem cell growth and proliferation and enable the cells to maintain their stem cell and adhesion markers and keep their spherical shape, because it mimics a tissue-like environment in vitro.

The 3D structure has many advantages like the cell-cell interaction, cell-matrix interaction, and its ability to keep the cells in a spherical shape, this being a more natural shape for the cells compared to the cells adhering on a surface. Cotton also has superior properties compared to synthetic polymers, especially in terms of its biocompatibility properties. Cotton can be used as a mold to shape organs or grow tissue to be transplanted. Another advantage for the use of bio-conjugated cotton fibers as a scaffold, is the elimination of the trypsinization process - this, as stated earlier, avoids any degradation of the surface protein of the cells that might be caused during the dissociation of the cells from the culture dish surface. Cotton is widely available and cheap compared to silk fibers, which makes it cost-effective to use. Future experiments should be performed over a longer period of time to assess the viability of the cells with the scaffold. These experiments should also include differentiation of the MSCs to the three lineages: chondrocytes, adipocytes and osteocytes. We believe that our cotton fibers can be a promising 3D scaffold model for regenerative medicine, and for drug screening and development, to the benefit of patients.

Footnotes

Acknowledgements

The authors would like to thank Mr. Christopher Smith for his English language editing.

Conflict of interest

The authors have no conflicts of interest to report.

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