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. 2019 Dec 4;14(1):73–77. doi: 10.1049/iet-nbt.2019.0278

Efficient co‐cultivation of human fibroblast cells (HFCs) and adipose‐derived stem cells (ADSs) on gelatin/PLCL nanofiber

Marziyeh Ranjbar‐Mohammadi 1, Elham Mousavi 2, Mohammad Mostakhdem Hashemi 3, Mahdi Abbasian 3, Jahanbakhsh Asadi 3, Ehsan Esmaili 4, Mehrafarin Fesharaki 5, Pouyan Asadi 6, Zahra Arab‐Bafrani 3,7,8,✉
PMCID: PMC8676645  PMID: 31935681

Abstract

In this study, we investigated whether the nanofibers produced by natural‐synthetic polymers can probably promote the proliferation of co‐cultured adipose‐derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L‐lactide co‐ɛ‐caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real‐time polymerase chain reaction were applied to assess the proliferation of co‐cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor‐basic and transforming growth factor‐β1 were also measured in culture medium of co‐cultured ADSs/HDCs using enzyme‐linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co‐culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non‐coated plate. Also, synthesis of collagen and cytokines secretion of co‐cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non‐coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co‐cultured ADSs/HFCs in wound healing process.

Inspec keywords: biomedical materials, enzymes, adhesion, fluorescence, polymer fibres, tissue engineering, wounds, nanofibres, cellular biophysics, molecular biophysics, gelatin, biochemistry, nanomedicine, field emission scanning electron microscopy, nanofabrication

Other keywords: cell morphology, cell proliferation, efficient cocultivation, HFCs, ADSs, gelatin‐PLCL nanofiber, natural‐synthetic polymers, cocultured adipose‐derived stem cells‐human fibroblast cells, FESEM, fluorescent microscopy, MTS assay, quantitative real‐time polymerase chain reaction, collagen type I synthesis, collagen type III synthesis, cytokines, transforming growth factor‐β1, fibroblast growth factor‐basic growth factor‐β1, culture medium, enzyme‐linked immunosorbent assay assay, structural properties, toxicity analysis, cellular adhesion, physiological characteristics in vivo, wound healing

1 Introduction

Wound healing, as a very complicated process, has been a major challenge in modern medicine. Previous studies have exhibited that treatment of wound site with co‐cultured stem cells and fibroblasts could increase the rate of wound healing through synthesising collagen and promoting the proliferation and migration of fibroblasts [1, 2]. Meanwhile, adipose‐derived stem cells (ADSs) are a valuable source of stem cells that can be easily isolated. Many researchers have been interested to study skin regeneration mechanisms using ADSs as these cells participate directly or indirectly in the process of wound healing [3]. The positive effects of ADSs treatment have been reported on chronic wound healing, such as diabetic and radiogenic wounds [4, 5, 6]. Hence, the efficient transplantation of these cells to the injury site and development of optimal co‐culture system may be an impressive strategy for wound healing [2, 7].

Co‐culture systems can reliably provide complex interactions occurring among different cell types that are affected by topography, components and physical properties of extracellular matrix (ECM) [8, 9, 10]. Recently, application of ECM‐based techniques for co‐culture system has become interested as a new research area [11]. In recent years, there has been significant development leading to fabrication of scaffold in tissue engineering. Among them, electrospun nanofibrous scaffolds have been introduced as an effectual scaffold that can perfectly simulate the structure and topography of native ECM and provide the specific condition for controlling and enhancing tissue regeneration. The interconnected and porous structure of the nano‐fibers can facilitate the transfer of nutrients and the removal of waste materials leading to speed up tissue regeneration. Synthetic polymeric nanofibers are known as promising scaffolds due to desirable stability and enough mechanical properties in aqueous media [12, 13, 14]. Moreover, alongside the physical and mechanical properties of ECM, the other main factors that can influence multiple cellular processes including cell adhesion, differentiation and migration are ECM components. Synthetic polymers are usually hydrophobic and have few sites for adhesion of cells, hence they are unable to properly simulate cell–cell and cell–ECM interactions due to the lack of ECM components [15, 16].

Although, native polymers can provide biological cues to cells and surrounding tissues, low mechanical strength and quick degradation rate limit their utilisation in culture medium (CM).

Recently, adding the native proteins like collagen to synthetic‐based nanofiber composition presented new platform to assess better imitation of in vivo characteristics. [17, 18, 19, 20]. Currently a hydrolysed form of collagen, Gelatin (Gel), as an inexpensive, non‐immunogenic, and biodegradable natural polymer is being widely considered in the production of blended nanofibers [21]. Several studies have shown that blending of Gel with synthetic polymers such as poly (l‐lactic acid) (PLLA), poly (ɛ ‐caprolactone) and poly (L‐lactide‐co‐ɛ ‐caprolactone) (PLCL), could produce nanofibers with high biocompatibility and mechanical properties [22, 23, 24]. In the present study, for the first time in literature to our knowledge, the role of electrospun Gel/PLCL nanofibers on the proliferation of co‐cultured (ADSs/human fibroblast cells (ADSs/HFCs)) and collagen synthesis have been investigated.

2 Material and methods

2.1 Fabrication of Gel/PLCL nanofibers

PLCL (70:30) with molecular weight of 150 kDa, gelatin (Merck, Germany) and 1,1,1,3,3,3‐hexafluor‐2‐propanol (HFP) (Sigma–Aldrich) were used. First, gelatin and PLCL were dissolved in HFP at the ratio of 50:50 (wt%), to reach 11% (w/v) solution and stirred for 12 h. For fabrication of electrospun nanofibers from PLCL and gelatin compositions, both solutions were individually injected in 10 mL syringes related with blunted steel needles of 27 G size. The syringe pump was used to run a flow rate of 1 mL/h. Nanofibers were gathered on the flat grounded steel plate at a distance of 14 cm from the needle tip and applied voltage of 15 kV, then they were kept in vacuum desiccator to remove residual solvent into mat structure.

Scanning electron microscopy (SEM) (Seron technology, AIS2300C, Korea) was utilised to study the surface morphology of fabricated electrospun nanofiber at an accelerating voltage of 15 kV. Samples were sputter‐coated with a thin layer of gold. The average nanofiber diameter was calculated by means of 100 random measurements using web plot digitiser software based on SEM images.

To perform the biodegradability test, the fibres were immersed in phosphate buffer saline (PBS) (pH = 7.4) and incubated for a period of 40 days at 37°C. After 40 days, the scaffold was washed and subsequently dried in a vacuum oven for 48 h. The morphology changes were reported by field emission scanning electron microscopy (FESEM).

To calculate weight loss of samples, nanofibers were immersed in PBS and incubated in vitro at 37°C for 1 to 24 days. Weight loss percentages were calculated using equation below where Wi and Wf are specimen weights before and after soaking in PBS.

Weightloss%=Wi−WfWi×100

2.2 HFCs attachment on Gel/PLCL nanofiber

HFCs were isolated from dermal layer of scalp and cultivated in Dulbecco's Modified Eagle's Medium (DMEM) supplied by 10% fetal bovine serum (FBS), (Gibco‐Invitrogen) and 1% penicillin/streptomycin (Sigma‐Aldrich). The cells were stored at humidified atmosphere at 37°C with 5% CO2. The cells' medium was changed every two days. When the cells reached >80% of confluency, they were trypsinised and seeded on 24‐well plates coated with sterilised nanofiber substrate. After six days, to evaluate cell attachment to nanofibers, the cells were washed twice with PBS and then fixed in 2.5% glutaraldehyde for 2 h, rinsed thrice with PBS for 12 h, immersed in osmium tetroxide 0.1% for 30 min and dried in different concentration of acetone, i.e. 20, 30, 50, 70, 90 and 100% for 10 min each. Finally, they were freeze‐dried and sputter‐coated with gold for cell morphology studies by FESEM.

2.3 Co‐cultured ADSs and HFCs on Gel/PLCL nanofiber

ADSs were isolated from scalp adipose tissue of healthy volunteer. Briefly, 1 × 1 mm2 piece of scalp adipose tissue were cultivated in CM (DMEM/F12 and modified Eagleed(d scalp Gibco BRL, Paisley), with 12% FBS (Gibco, UK) as reported on our previous study [25]. After three passages, cell population was enriched with ADSs. For co‐culturing, 2 × 104 ADSs and HFCs (co‐cultured ADSs/HFCs) were mixed and seeded on six‐well Gel/PLCL nanofiber and non‐coated plates.

2.4 Fluorescence viability staining

For detecting live cells, seven days co‐cultured ADSs/HFCs were washed three times with PBS then fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature, followed by permeabilisation in 1.0% Triton X‐100 for 15 minutes. The samples were incubated by Calcein AM that stains alive cells in green for 30 min at 37°C and washed with PBS. The green‐stained samples were subjected to fluorescent microscopy with a 490 nm excitation filter and a 520 nm emission filter.

2.5 Proliferation assay

Influence of Gel/PLCL nanofiber on the proliferation of co‐cultured ADSs/HFCs was analysed with MTS‐assay. 2 × 103 cells were seeded on both coated and non‐coated 96‐well plates. Cell proliferation was evaluated three and seven days after culture with the Cell Titer 96 Aqueous One Solution Reagent (Promega, USA). Briefly, RPMI (100 ml) was supplemented with MTS solution 20 µ l/well, incubated for 2 h, and then the optical density was read at 490 nm wavelength by a plate reader (Bio‐Rad, USA). Cell proliferation was calculated based on [26, 27]

Cellproliferation=meancellviabilityintestwellsmeancellviabilityincontrolwells. (1)

2.6 Quantitative real‐time polymerase chain reaction (qRT‐PCR)

Total RNA was extracted from co‐cultured ADSs/HFCs on Gel/PLCL and non‐coated plate using GeneAll RiboEx kit (GeneAll Biotechnology, Korea) according to the manufacturer's protocol [28]. The SuperScript II reverse transcriptase (Invitrogen) was used for the synthesis of complementary DNA (cDNA). The yielded cDNA was then recruited for the qRT‐PCR using SYBR green PCR Core Reagents (Thermo Scientific) with ABI 7300 instrument (Applied Biosystems). The change in relative mRNA expression of collagen type I and collagen type III were assessed using the standard curve method [29]. All samples were normalised to GAPDH gene expression as the internal control.

2.7 Enzyme‐linked immunosorbent assay (ELISA) of CM

After 72 hours, culture media of co‐cultured ADSs/HFCs on Gel/PLCL and non‐coated plate were collected, centrifuged and filtered. The concentrations of two cytokines including fibroblast growth factor‐basic (FGF‐basic) and transforming growth factor (TGF)‐β 1 were measured using Abcam ELISA kits (lot numbers: ab99979 and ab100647, respectively) according to manufacturer's instructions.

In brief, the series of standard solutions were prepared in 2 ml sterilise polystyrene microtubes exactly prior to use. Then, 100 µl of the standard solutions and samples were added to the microplate wells. They were covered and incubated at room temperature for 2.5 h with gentle shaking. After 4× washing, 100 µl of biotin‐antibody was added to each well and incubated at room temperature for 1 h after shaking. 4× washing was performed and the prepared HRP‐streptavidin solution was added in 100 µl volume to each well and incubated for 45 min with gentle shaking. In this step the final 4× washing was performed and 100 µl of TMB one‐step substrate was added to each well and incubated at room temperature in darkness for 30 min with shaking. Finally, 50 µl of stop solution was added to each well and the absorbance was read at 450 nm using ELISA reader (statfax 4300, chromate, USA).

2.8 Statistical analysis

Differences between two groups were analysed with the t ‐test. Differences <0.05 (P ‐value<0.05) were considered statistically significant. All experiments were performed in triplicate and repeated at least three times.

3 Results

3.1 Gel/PLCL nanofiber morphology

Electrospun nanofibers formulation is one of the most important factors that can affect the interaction between the scaffold and cell surface receptors. Existence of natural polymers such as gelatin in scaffold structure improves the biological characteristic of the mat. When natural and synthetic polymers are blended for fabrication of nanofibers, biocompatibility and stability of scaffolds in biological media might increase. Fig. 1 exhibits the SEM images of PLCL and Gel/PLCL 50:50, along with their diameter distribution.

Fig. 1.

Fig. 1

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SEM images and fibre diameter of

(a) PLCL, (b) Gel/PLCL

The images show three‐dimensional interconnected networks of nanofibers without any cracks or bead in their structure (Fig. 1). Fiber diameter was estimated to be 382 ± 21 nm and 316 ± 22 nm for PLCL and Gel/PLCL 50:50, respectively.

3.2 Biodegradation of Gel/PLCL and PLCL nanofibers

The SEM micrographs related to degradation behaviour of electrospun nanofibrous mats are shown in Fig. 2. As shown in Fig. 2, combining PLCL with gelatin not only increases the degradation rate of the PLCL nanofibers after passing 40 days which made cell infiltration and increased adhesion but also results in better maintenance of the fabricated mat integrity. This can be occurred because of the presence of more hydrophilic groups in the gelatin structure. As a result, the water penetration into nanofibrous structures has been simplified and the higher rate of hydrolysis has been taken place. Fig. 3 depicts weight loss profiles of PLCL and Gel/PLCL nanofibers in determined times (days 1, 5, 14, 21, and 24). After passing 24 days, PLCL and Gel/PLCL nanofibers exhibited weight loss about 6 and 69%, respectively.

Fig. 2.

Fig. 2

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Degradation behaviour of nanofibers after passing 40 days

(a) PLCL nanofibers, (b) Gel/PLCL nanofiber

Fig. 3.

Fig. 3

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Weight loss of PLCL and Gel/PLCL

3.3 High growth rate of HFCs on Gel/PLCL

The morphologies of HFCs on PLCL and Gel/PLCL nanofibers were investigated by FESEM analysis after three days (Fig. 4). HFCs covered throughout the surface of nanofibers are showing an extended spindle‐like morphology on Gel/PLCL scaffolds, however, the morphology of cells on PLCL nanofibers is circular and their number is limited. The higher amount of cell growth on Gel/PLCL nanofibers is related to the excellent biological characteristics of gelatin which leads to more hydrophilicity along with higher protein adsorption of the nanofibrous scaffold.

Fig. 4.

Fig. 4

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Cell culture behaviour of fabricated nanofibers

(a) PLCL, (b) Gel/PLCL nanofibers

3.4 Morphology changes of co‐cultured ADSs/HFCs on Gel/PLCL nanofiber

Regarding higher growth and adhesion of HFCs on Gel/PLCL, co‐culture was performed on Gel/PLCL nanofibers. The effects of Gel/PLCL nanofiber on co‐cultured ADSs/HFCs morphology were evaluated during different intervals. As shown in Figs. 5 a and 5 b, the cell morphology was different between coated and non‐coated plates. In non‐coated plates, the cells were dispersed over the surface while in plates coated with Gel/PLCL nanofiber, the cells aggregated and condensed, especially after seventh day of culture. Fig. 5 c shows the alive cells on Gel/PLCL nanofiber. The round morphology specified the early stage of differentiated cells and polygonal or cobblestone morphology indicated differentiated cells.

Fig. 5.

Fig. 5

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Co‐cultured ADSs/HFCs on

(a) Non‐coated plate, (b) Plate coated with Gel/PLCL nanofiber at different days, (c) Fluorescence microscopy image of them on Gel/PLCL nanofiber

3.5 Proliferation enhancement of co‐cultured ADSs/HFCs on Gel/PLCL nanofiber

As shown in Fig. 6, cell proliferation was significantly higher on plates coated with Gel/PLCL nanofiber than non‐coted plates (P ‐value < 0.05), also the difference was increased from day 3 to day 7 which means that proliferation rate was higher on Gel/PLCL nanofiber.

Fig. 6.

Fig. 6

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Proliferation rate of co‐cultured ADSs/HFCs on Gel/PLCL nanofiber and non‐coated plate

3.6 Increased collagen production and cytokines secretion on Gel/PLCL nanofiber

The qRT‐PCR analysis showed collagen type I and III syntheses were significantly upregulated on Gel/PLCL nanofiber (P ‐value < 0.05, Fig. 7).

Fig. 7.

Fig. 7

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Relative expression of collagen of co‐cultured ADSs/HFCs on Gel/PLCL and non‐coated plate

In addition, the concentration of FGF‐basic and TGF‐β 1 in CM of co‐cultured ADSs/HFCs on Gel/PLCL nanofiber was significantly higher than the non‐coated plate (P ‐value < 0.05, Fig. 8).

Fig. 8.

Fig. 8

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Secretion of cytokines in CM of co‐cultured ADSs/HFCs on Gel/PLCL and non‐coated plate

4 Discussion

Nowadays, ADSs transplantation has been considered as a pioneering treatment method for chronic and immedicable wounds. ADSs would improve wound healing rate through the enhancement of fibroblast proliferation and stimulation of collagen synthesis. However, the poor retention of ADSs in the wound bed limit their wound healing potential. The chemical and mechanical cues of the ECM are important factors that influence the maintenance and behaviour of ADSs. Protein‐based nanofibers have been proposed for stem cell culture due to the perfect simulation of the ECM topography and providing numerous adhesion sites [24]. In recent years, gelatin as a hydrolysed form of collagen is widely used in these nanofibers. In our previous work, FTIR analysis of Gel/PLCL nanofiber revealed the presence of gelatin with PLCL in nanofibers composition. However, mechanical strength analysis showed higher strength for Gel/PLCL composition compared with PLCL scaffold. It showed that Gel/PLCL is resistant against any stress compared with PLCL scaffold [30]. In agreement with previous studies [25, 31, 32], cultivation of ADSs on Gel‐PLCL nanofibers increased cell proliferation. It was suggested that interaction of gelatin ligands, specifically RGD (Arg‐Gly‐Asp) as a key recognition motif for cell attachment, with cellular integrins is responsible for activation of signalling pathways that can terminate cell differentiation and proliferation [33, 34]. Moreover, nanoscale features of nanofibers would increase the accessibility of these ligands to interact with cellular integrins.

In addition, intercellular interaction with adjacent cells influences stem cells behaviour [35]. Sivamani et al. [35] revealed that mesenchymal stem cells (MSCs) can differentiate into early myofibroblasts and neural lineages, when they were cultured without any physical contact with keratinocytes, while MSCs will differentiate towards epithelial lineage in contact co‐culture. Our results showed that the contact co‐culture of ADSs/HFCs on the Gel/PLCL nanofiber synergistically increased cellular adhesion and proliferation compared to the non‐coated plate. Also, in parallel with the previous study [36], the appearance of rounded epithelia and fibroblast‐like cell morphology could be exhibited as a hallmark of the ADSs differentiation to epidermal cells, due to the improvement of cell–cell and cell‐ECM interaction on Gel/PLCL nanofiber.

The other mechanism of ADSs in wound curing is suggested by the secretion of factors including cytokines and growth factors that motivate collagen synthesis pathways in fibroblast cells [2, 37].

In parallel with the previous study [38], our results showed that the nanofiber can upregulate collagen expression and motivate the secretion of ADSs – cytokines in comparison with non‐coated plate. Besides, the role of growth factors and cytokines, interaction of gelatin RGD motifs with fibroblast integrin can also induce activity of periodontal ligament fibroblasts and promote collagen production in fibroblast.

In this work, the effects of Gel/PLCL nanofiber on the proliferation of co‐cultured ADSs/HFCs, synthesis of collagen and secretion of growth factors, as the main factors in wound healing, were studied. Our results suggest that Gel/PLCL nanofiber can be considered as an appropriate scaffold for the imitation of physiological characteristics occurred in vivo and the enhancement of the efficacy of co‐cultured ADSs/HFCs in wound healing.

5 Acknowledgments

The authors are highly thankful to all technicians who provided support during the course of research. The part of this work was supported by Metabolic Disorders Research Center, Golestan University of Medical Science (Grant no: 110 988)

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