Fabrication and investigation of nanofibrous matrices as esophageal tissue scaffolds using human non-keratinized, stratified, squamous epithelial cells

Abstract

Clinical conditions of the esophagus are conventionally treated by autologous grafts and are generally associated with complications such as leakage, infection and stenosis necessitating an alternative synthetic graft with superior outcomes. Therefore, in the present study, we have fabricated polymeric random and aligned nanofibers of PHBV, PHBV-gelatin, PCL and PCL-gelatin using electrospinning. The cell–matrix interaction, an essential parameter to obtain proper tissue regeneration, has been investigated using non-keratinized, stratified, squamous epithelial cells. Immunostaining of the filamentous actin morphology of epithelial cells on the nanofibers revealed the elongated and spread morphology demonstrating that the nanofibers provide proper contact guidance and right milieu for the cell growth and proliferation. The aligned nanofibrous scaffolds exhibited oriented cell growth along the fiber axis, which was not observed in the random nanofibers. PCL-gelatin nanofibers exhibited maximum cell proliferation when compared to other types of scaffolds investigated. There was no significant difference observed in the gene expression and cell proliferation between the random and aligned nanofibers (p > 0.05). However, gelatin incorporated PCL scaffold showed significantly higher cell proliferation and function gene expression levels than the PCL nanofibers (p < 0.05). Thus the study concludes that the nanofibrous scaffolds may have potential for the regeneration of mucosal layer of the esophagus tissue.

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Introduction

The esophagus has a complex anatomy with four different layers which as the inner mucosal layer, sub-mucosa, middle muscularis externa layer and adventitia as the outer-most layer.¹ The mucosal layer of the esophagus consists of non-keratinized, stratified, squamous epithelial cells, which protect the esophagus from mechanical abrasion caused by the food bolus.² Clinical disorders of the esophagus (congenital diseases/trauma or malignancy) are difficult to repair due to the poor regenerative ability of tissue.^3–5

Tissue engineering strategy utilizing biodegradable synthetic grafts could be promising alternative to the existing conventional grafts (autologous or allografts or xenografts). Various types of porous scaffolds developed using natural polymers (collagen, chitosan, etc.,) and synthetic polymers (PCL, PLLC, PLLA, PLGA, etc.,) through several techniques (solvent casting, salt leaching, gas foaming and electrospinning) have been used as scaffolds for esophageal tissue engineering.^1–3,6,7 Usually, a basement membrane protein such as collagen or fibronectin was coated on the surface of synthetic polymeric scaffolds to enhance the bio-functionality of the scaffolds.^1,6 Due to the high cost and poor processibility of collagen or fibronectin, in the present study, we have employed gelatin, a hydrolyzed product of collagen that retains the cell recognition motif (RGD motif) of collagen.^8–10

PHBV is a biodegradable and biocompatible polymer investigated for tissue engineering applications due to its oxygen permeability, moderate degradation and ductility.¹¹ PCL is a food and drug administration (FDA) approved slow degrading polymer, which is also biodegradable and biocompatible with various cell types including esophageal epithelial cells.¹² Both PHBV and PCL are hydrophobic polymers and do not possess cell recognition motif and therefore requires blending with another constituent that possesses bio-recognition motifs.

Basement membrane of the esophagus is made of nanofibers that provides the structural and mechanical stability to the growing epithelial cells. Most of the studies on esophageal regeneration have focused on the use of decellularized matrices from various tissues.^13–15 Though these matrices possess adequate strength and structural properties, they are still associated with clinical constraints such as infection, inflammation and enhanced risk of disease transmission. In addition, special attention is required for the decellularization process since enzymes or detergents used for the process may alter the architecture of the scaffold.¹⁶ Nanofibers possess excellent characteristics such as higher surface area-to-volume ratio that facilitates better cell adhesion and proliferation, mimics the native extracellular matrix, and can be tailored to match the properties of the native tissue (mechanical or degradation or porosity).¹⁷ Moreover, nanofibrous scaffolds have been effectively used for various tissue engineering applications.^18–25 In the context of esophageal tissue engineering, only few reports are available on the use of nanofibrous scaffolds to support the adhesion and proliferation of epithelial cells that have also been shown to be effective in in vivo animal models.²⁶ Poly(lactide-co-caprolactone) copolymer was electrospun to form a nanofibrous scaffold that was further modified with fibronectin through surface grafting.⁶ In vitro studies employing porcine esophageal epithelial cells revealed that the scaffold supported the adhesion, growth, proliferation of the cells with retention of the epithelial functional markers. The same group had also reported that electrospun porous poly(lactide) nanofibers promoted esophageal epithelial cell adhesion and proliferation due to their ability to absorb protein and drive cell adhesion through formation of focal contacts on the nanoporous surface.²⁷ Recently, a three layered scaffold comprising of electrospun nanofibrous layers of poly(caprolactone) sandwiching a layer of silk fibroin has been fabricated and their efficacy for regeneration of esophagus was investigated in rat models with circumferential defects created on the esophagus.²⁸ The scaffolds were able to promote healing of the defect in a two-week period and the scaffold remained fused with the regenerated tissue. However, long-term evaluation of this scaffold is required to assess its translational potential. Our group has also been actively involved in development of polymeric nanofibrous scaffolds to support the adhesion and proliferation of epithelial and smooth muscle cells.^29–31 From the scan of literature, it is evident that more number of novel nanofiber-based scaffolds needs to be identified to obtain better outcomes for esophageal tissue regeneration. In the present study, we have focused on the fabrication of random and aligned nanofibers of PHBV, PHBV-gelatin, PCL and PCL-gelatin and investigated their in vitro potential using non-keratinized stratified squamous epithelial cells. In a recent report, Li et al., demonstrated the significance of aligned topography in scaffolds, which guides oriented cell growth and influences cytoskeletal skeletal protein reorganization, extracellular matrix protein remodeling, alteration in the gene expression and membrane protein relocation.³² Therefore, in the current study we have compared the influence of random and aligned nanofibers of the selected polymers on the cellular behavior.

Experimental

Materials

PHBV with a molecular weight (M_w) of 450 kDa (Goodfellow Cambridge Ltd, UK), gelatin with a M_w of 50 kDa (Sigma Aldrich, USA), and PCL with a M_w of 300 kDa (Lakeshore Biomaterials, USA) were used for scaffold fabrication. The solvents chloroform (CHCl₃), dimethyl formamide (DMF) (Merck, India) and hexafluoroisopropanol (HFIP) (Sigma Aldrich, USA) of analytical grade were used in the study. The nanofibers were fabricated using the Nano Fiber Electrospinning Unit, HO-NFES-040, Holmarc, India. Human non-keratinized, stratified, squamous epithelial cells were a kind gift from Sankara Nethralaya Eye Hospital, India. Cell culture media, antibody and other chemicals used in the in vitro study were procured from Gibco, USA, Invitrogen, USA, Promega, USA and Molecular probe, USA.

Methods

Preparation of random and aligned nanofibers. Random and aligned nanofibers were fabricated using electrospinning. Briefly, PHBV with a concentration of 19% w/v was dissolved in a 9 : 1 mixture of DCM and DMF. For fabricating a physical blend of PHBV and gelatin, PHBV-gelatin (7 : 3) blend with a concentration of 12% w/v was dissolved in HFIP. The nanofibrous scaffold of PCL was fabricated using PCL at a concentration of 7% dissolved in a 7 : 3 mixture of CHCl₃ : DMF and PCL-gelatin (7 : 3) blend with a concentration of 5% dissolved in HFIP was used to prepare PCL-gelatin blend nanofibers. The flow rate of the polymer solution from the syringe was regulated using syringe pump (Holmarc, India). The positive terminal of a high voltage power supply was connected at the tip of the needle to overcome the surface tension leading to the formation of an electrically charged jet that moved towards the collector (static plate for random nanofibers or rotating dynamic mandrel for aligned nanofibers) due to the potential difference between the needle tip and the collector. The evaporation of the solvent during its travel to the collector results in the formation solid nanofibers on the collector. The electrospinning parameters were optimized to obtain defect-free random and aligned nanofibrous scaffolds made of PHBV, PHBV-gelatin, PCL and PCL-gelatin. The morphology of random and aligned nanofibers was observed through field emission scanning electron microscopy (FE-SEM, JSM 6701F, JEOL, Japan). The average mean space between the fibers of nanofibrous scaffolds was analyzed using the scanning electron micrographs.

Investigation of cell–scaffold interactions.
Epithelial cell culture. Non-keratinized, stratified, squamous epithelial cells were cultured in Dulbecco's modified Eagle medium/Ham's F12 (DMEM/F12, Gibco, USA) with 1% epithelial growth factor supplement (Cat # S-009-5, Gibco, USA), 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin–streptomycin (P/S, Gibco, USA) in humidified atmosphere with 5% CO₂ at 37 °C. The culture medium was changed every alternate day until confluence followed by detachment of cells with 0.05% trypsin–EDTA (Gibco, USA). Cells were mixed with trypan blue, loaded in hemocytometer and counted using phase contrast microscope (CKX41, Olympus, Japan). Nanofibrous scaffolds with 12 mm diameter were sterilized with UV (ultraviolet radiation) and placed in a 48-well cell culture plate followed by sterilization in 100% ethanol.^12,33 The scaffolds were washed thrice with D-PBS (Dulbecco's and phosphate-buffered saline, Gibco, USA). The sterilized scaffolds were pre-conditioned with culture media overnight and then seeded with 50 000 cells for the assessment of morphology, viability and gene expression while 25 000 cells were seeded for proliferation studies.
Cell attachment, morphology, and viability. The cell attachment on the nanofibrous matrices was examined using a field emission scanning electron microscope (FE-SEM). In brief, cell seeded scaffolds were fixed with 4% glutaraldehyde at 4 °C overnight followed by washing with phosphate buffered saline (PBS) and drying at room temperature. Dried samples were sputter-coated with gold and the cell morphology was observed through FE-SEM. For evaluation of the morphology of actin filaments, the cell-seeded scaffolds were removed and washed with PBS followed by incubation with rhodamine-phalloidin (Invitrogen, USA) for one hour and counter-stained with Hoechst (Molecular probe, USA) for 15 minutes.¹¹ For viability assessment, cellular scaffolds were removed and incubated with calcein AM and ethidium bromide (Molecular probe, USA) for thirty minutes. The stained samples were observed using laser scanning confocal microscopy (LSCM, FV1000, Olympus, Japan). Further, the cell infiltration into the nanofibrous scaffolds was assessed by Z-sectioning obtained using confocal images. The cell viability was quantitatively measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, CellTiter® 96 AQueous one solution, Promega, USA) assay. Epithelial cell seeded scaffolds was removed at pre-determined time points (3, 7 and 10 days) and incubated with MTS and fresh culture medium for 2 hours in the dark at 37 °C and the absorbance was measured at 490 nm using multimode reader (Infinite 200M, Tecan, USA).⁹
Real time RT-PCR. The expression of functional genes by the cells cultured on the random and aligned nanofibers was investigated using real time RT-PCR. Total RNA was extracted from the cell-seeded nanofibrous matrices at various time intervals (3, 7 and 10 days) according to the acid-phenol guanidinium method using Trizol reagent (Invitrogen, USA) and RNeasy isolation kit (Qiagen, USA). Total RNA was quantified by using UV-Visible spectrophotometer (NanoDrop 2000, Thermo scientific, USA) followed by synthesis of complementary DNA (cDNA) through Qiagen reverse transcriptase kit as per the manufacturer's instructions. cDNA samples were evaluated for genes of interest such collagen type IV, laminin, cytokeratin 4 and 15 and the housekeeping gene, GAPDH, in independent reactions using SYBR-Green maters mix (Qiagen, USA) in thermo cycler (Eppendorf AG22331, Germany) according to the manufacturer's instructions. Table 1 lists the primers used in this study. Relative expression levels for each gene of interest were calculated by normalizing the target gene transcript level (Ct) to the respective GAPDH level and TCPS control using the delta–delta CT method.³⁴

Table 1 The forward and reverse primers used in this study

Gene name	Gene sequences
Cytokeratin 4	F: 5′-AGCTAGATACCTTGGGCAATG-3′
	R: 5′-CACAAAGTCATTCTCGGCTG-3′
Cytokeratin 15	F: 5′-TGCGACTACAGCCAATACTTC-3′
	R: 5′-GGCCAGCTCATTCTCATACTTG-3′
Collagen IV	F: 5′-TGTGGATCGGCTACTCTTTTG-3′
	R: 5′-TAGTAATTGCAGGTCCCACG-3′
Laminin	F: 5′-AATGGAGTGAGACAGGAACAAG-3′
	R: 5′-TGACCATACGATGCCTTGATG-3′
GAPADH	F: 5′-ACATCGCTCAGACACCATG-3′
	R: 5′-TGTAGTTGAGGTCAATGAAGGG-3′

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Statistics. Two-factor analysis of variance (ANOVA) was performed for cell proliferation and gene expression studies. The statistical significance was evaluated at p < 0.05 using Tukey test.

Results

Morphology of random and aligned nanofibers

In the present study, electrospinning was used to fabricate defect-free random and aligned nanofibrous scaffolds of PHBV, PHBV-gelatin, PCL and PCL-gelatin. The structural morphology of electrospun random and aligned nanofibers was analyzed by scanning electron microscopy (SEM). SEM images revealed that electrospun scaffolds were beadless with nanoscale fibrous morphology with different orientation (random or aligned) based on the electrospinning parameters used (Fig. 1). PHBV (19%, w/v) dissolved in DCM : DMF (9 : 1 ratio) was successfully electrospun to produce defect-free random nanofibers at 8 kV and at a tip-to-target distance of 12 cm (Fig. 1A1 & A2). Aligned PHBV fibers were formed at 17 kV applied voltage and at a tip-to-target distance of 12 cm (Fig. 1B1 & B2). For the preparation of nanofibrous scaffolds of PHBV and PCL blended with gelatin, a different solvent system was employed. A common solvent is required to dissolve both polymers when preparing the physical blend to avoid phase separation during the electrospinning process. Phase separation is sometimes not desirable, as it will result in defects in the fibers and also causes heterogeneity. Earlier reports have shown that the physical blend of PHBV and gelatin can be fabricated using trifluoroethanol solvent for biomedical applications.³⁵ In the present study, we have chosen HFIP as solvent for preparing PHBV-gelatin blend nanofibers because it is a polar solvent that can form extensive hydrogen bonds with amides. Defect-free PHBV-gelatin blend random fibers were obtained at 12% (w/v) polymer blend concentration, 5 kV applied potential, and at tip-to-target distance of 12 cm (Fig. 1C1 & C2). Aligned fibers of PHBV-gelatin were achieved at 10 kV applied potential with 12 cm tip-to-target distance (Fig. 1D1 & D2). Similarly random and aligned PCL and PCL-gelatin nanofibers were fabricated according to the conditions reported by our group.^9,12 Fig. 1E–H shows the defect-free random and aligned PCL and PCL-gelatin nanofibers. The fiber dimensions of random fibers of PHBV ranged between 350 nm and 850 nm while their aligned counter parts ranged from 150 nm and 600 nm. PHBV-gelatin random fibers of dimensions 250 nm and 650 nm and aligned PHBV-gelatin fibers between 150 nm and 800 nm were obtained. In the case of PCL, the dimensions of random fibers varied from 200–500 nm and aligned fibers exhibited a range between 100–200 nm. PCL-gelatin blend fibers were found to possess dimensions from 150–350 nm in the random orientation and 50–300 nm when aligned axially. The fibrous proteins in the esophageal basement membrane have been reported to exist between the dimensions of 28–165 nm.²⁶ Among fabricated scaffolds, the fiber diameters of all systems except pristine PHBV random fibers were to have similar dimensions and hence may provide proper contact guidance to the growing epithelial cells.

Fig. 1 Scanning electron micrographs show the morphology at low and high magnifications of [A1 & A2] random PHBV nanofibers; [B1 & B2] aligned PHBV nanofibers; [C1 & C2] random PHBV-gelatin nanofibers; [D1 & D2] aligned PHBV-gelatin nanofibers; [E1 & E2] random PCL nanofibers; [F1 & F2] aligned PCL nanofibers; [G1 & G2] random PCL-gelatin nanofibers and [H1 & H2] aligned PCL-gelatin nanofibers.

Investigation of cell–scaffold interactions

The efficacy of the nanofibrous scaffolds to support cell growth and expansion were explored using human non-keratinized, stratified, squamous epithelial cells. The four types of nanofibrous matrices were investigated for cell attachment, morphology, proliferation, viability and functional gene expression. Fig. 2 shows the scanning electron micrographs of human non-keratinized, stratified, squamous epithelial cells on the random and aligned nanofibrous matrices at various culture intervals. The micrographs reveal that the epithelial cells adhered on all the nanofibrous scaffolds under investigation by 2 hours of culture. The cells displayed a typical spherical morphology after 2 hours, which then transformed to an elongated and well spread morphology completely covering the scaffold surface after 1 and 3 days, demonstrating an intense proliferation activity of the cells. Epithelial cell viability on these matrices was assessed qualitatively as well as quantitatively using live-dead assay and MTS assay respectively. Fig. 3 shows the viability of non-keratinized, stratified, squamous epithelial cells on the random and aligned nanofibrous scaffolds. It is observed that cells cultured on blend nanofibrous scaffolds show a well-extended morphology when compared to those cultured on the pristine nanofibers. The cell extension was more distinct on aligned nanofibers than the random nanofibers. Interestingly, cells cultured on the aligned nanofibrous systems exhibit oriented growth parallel to the fiber axis suggesting that the aligned geometry provides topographical cues to the cells to extend along the fiber axis. The morphology of cells on the random and aligned nanofibers was assessed by immunostaining the cells with cytoskeletal marker protein filamentous actin (Fig. 4 & 5). The results indicate that both random and aligned nanofibrous scaffolds of PHBV, PHBV-gelatin, PCL and PCL-gelatin support the adhesion of non-keratinized, stratified, squamous epithelial cells and also provide the right milieu for their well-extended growth. The cell elongation along the fiber axis is once again clearly discernible on the aligned scaffolds through the cytoskeletal staining. However, the cells cultured on the random nanofibers, though with an extended morphology, did not exhibit the oriented cell growth.

Fig. 2 Scanning electron micrographs of human non-keratinized, stratified, squamous epithelial cells cultured on random and aligned nanofibrous scaffolds of PHBV, PHBV-gelatin, PCL and PCL-gelatin at various time intervals.

Fig. 3 CLSM images showing the viability of human non-keratinized, stratified, squamous epithelial cells cultured on PHBV, PHBV-gelatin, PCL and PCL-gelatin random and aligned nanofibrous scaffolds after 3 days of culture.

Fig. 4 CLSM images of human non-keratinized, stratified, squamous epithelial cells cultured on random and aligned nanofibrous scaffolds of PHBV, and PHBV-gelatin after 3 days of culture. Cell nuclei (blue) and actin filaments (red) were stained by Hoechst and rhodamine-phalloidin respectively on [A1 & A2] random PHBV nanofibers (low magnification); [A3 & A4] random PHBV nanofibers (high magnification); [B1 & B2] aligned PHBV nanofibers (low magnification); [B3 & B4] aligned PHBV nanofibers (high magnification); [C1 & C2] random PHBV-gelatin nanofibers (low magnification); [C3 & C4] random PHBV-gelatin nanofibers (high magnification); [D1 & D2] aligned PHBV-gelatin nanofibers (low magnification); [D3 & D4] aligned PHBV-gelatin nanofibers (high magnification).

Fig. 5 CLSM images of human non-keratinized, stratified, squamous epithelial cells cultured on random and aligned nanofibrous scaffolds of PCL, and PCL-gelatin after 3 days of culture. Cell nuclei (blue) and actin filaments (red) were stained by Hoechst and rhodamine-phalloidin respectively on [A1 & A2] random PCL nanofibers (low magnification); [A3 & A4] random PCL nanofibers (high magnification); [B1 & B2] aligned PCL nanofibers (low magnification); [B3 & B4] aligned PCL nanofibers (high magnification); [C1 & C2] random PCL-gelatin nanofibers (low magnification); [C3 & C4] random PCL-gelatin nanofibers (high magnification); [D1 & D2] aligned PCL-gelatin nanofibers (low magnification); [D3 & D4] aligned PCL-gelatin nanofibers (high magnification).

The quantitative measurement of cell viability on the nanofibrous scaffolds was carried out using MTS assay (Fig. 6). It was observed that there was no significant difference between the cell viability on random and aligned nanofibers in both PHBV and PHBV-gelatin nanofibrous scaffolds at all-time points. PHBV nanofibers exhibited higher cell proliferation than the PHBV-gelatin nanofibers after 7 and 10 days of culture (p < 0.05). This may be due to the poor stability of the blend PHBV-gelatin nanofibers in the aqueous environment that tends to degrade faster within 10 days. However, TCPS exhibited maximum proliferation than the nanofibrous scaffolds (p < 0.05). Most of the literature reports have employed gelatin composite nanofibers that were cross-linked before the in vitro study to improve their stability in aqueous environment.^36,37 However, the cross-linking may increase the strength, decrease the flexibility and also reduce the porosity of the nanofibrous scaffolds.³⁸ The nanofiber cross-linking also leads to the fiber fusion, which compromises the topographical stimulus and the residual cross-linking agent in the systems, if present, may cause undesirable cytotoxicity.³⁹ Hence, cross-linking was not attempted in the present study. It was observed that at initial time point (3 days), the cell viability on the PCL and PCL-gelatin nanofibrous matrices are comparable (p > 0.05) regardless of the orientation. However, after 7 days of culture PCL aligned nanofibers exhibited higher cell viability than their random counterparts (p < 0.05) but the difference disappeared after 10 days of culture (p > 0.05). The cell viability on the PCL nanofibers and TCPS control was comparable after 7 and 10 days of culture (p > 0.05). Another interesting observation was that the PCL-gelatin nanofibers exhibited superior cell viability than the PCL nanofibers after 7 and 10 days of culture, which may be due to the presence of RGD motif in gelatin. However, there was no significant difference observed in the cell viability between the random and aligned PCL-gelatin nanofibers after 7 and 10 days of culture (p > 0.05).

Fig. 6 MTS result showing the viability of human non-keratinized, stratified, squamous epithelial cells cultured on random and aligned nanofibrous scaffolds (p < 0.05).

The functional genes such as collagen type IV, laminin, CK 4 and CK 15 expressed by non-keratinized, stratified, squamous epithelial cells on the nanofibrous matrices were quantified through real time RT-PCR (Fig. 7A1–A4 & B1–B4). Hence, we have assessed the collagen type IV and laminin in the present study. Fig. 7 illustrates the gene expression profile of human non-keratinized, stratified, squamous epithelial cells on the nanofibrous systems. Collagen type IV expression on the PHBV and PHBV-gelatin was not upregulated in all the time points under investigation. However, the expression was stable at all the time points. It was also observed that PHBV aligned nanofibers exhibited higher collagen type IV expression than the PHBV-gelatin aligned nanofibers after 1 day of culture but was not statistically significant at 3 and 7 days of culture.

Fig. 7 Gene expression levels of cytokeratin 4, cytokeratin 15, collagen IV and laminin on random and aligned nanofibrous scaffolds of PHBV, PHBV-gelatin, PCL, and PCL-gelatin at various time intervals (p < 0.05).

The fiber orientation (random and aligned nanofibers) of the scaffolds has profound effect on the cellular behaviors such as adhesion, proliferation, and matrix gene expression. The extracellular matrix gene (collagen) expression is influenced by scaffold properties such topography, mechanical strength, pore size, porosity and chemical nature. We have measured the average space between the fibers of the nanofibrous scaffolds to understand the influence on the collagen gene expression and the results are shown in Table 2. The result demonstrates that PHBV random nanofibers exhibit significantly higher mean space distance between the fibers than the PHBV aligned nanofibers. A similar trend was observed in PHBV-gelatin as well as PCL nanofibers. However, the mean space distance between the fibers of PCL-gelatin random and aligned nanofibers has not shown significant difference. Among the different type of scaffolds investigated, PHBV random nanofibers exhibited maximum mean space fiber distance followed by PHBV-gelatin random, PHBV aligned, PCL random, PHBV-gelatin aligned, PCL aligned, PCL-gelatin random and PCL-gelatin aligned nanofibers. Though, we have observed significant difference in the mean space distance between the fibers of PHBV random and aligned nanofibers but those effects was not observed in the expression collagen type IV gene. Hence, it is evident that there are other factors that influence the collagen expression levels in the cells.

Table 2 The average mean space between the fibers and average fiber dimensions of the nanofibrous scaffolds. (*p < 0.05 significant difference between the random and aligned nanofibers; #p < 0.05 significant difference between the materials)

Nanofibrous matrices	Average fiber diameters (nm)	Average mean space between the fibers (μm)
PHBV random nanofibers	583 ± 90*#	1.28 ± 0.65*#
PHBV aligned nanofibers	330 ± 85#	0.53 ± 0.23
PHBV-gelatin random nanofibers	432 ± 60*	1.08 ± 0.54*
PHBV-gelatin aligned nanofibers	402 ± 124	0.44 ± 0.20
PCL random nanofibers	324 ± 50*#	0.48 ± 0.24*#
PCL aligned nanofibers	166 ± 39	0.30 ± 0.18
PCL-gelatin random nanofibers	242 ± 30*	0.30 ± 0.16
PCL-gelatin aligned nanofibers	155 ± 55	0.27 ± 0.14

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The upregulation of collagen type IV was observed in the PCL nanofibers unlike the PCL-gelatin nanofibers after 1 day of culture followed by down regulation after 3 and 7 days of culture. There was no significant difference observed in the expression of collagen between the random and aligned nanofibers in both the cases. However, PCL aligned nanofibers exhibited significantly higher collagen type IV expression when compared with the PCL-gelatin aligned nanofibers after 24 hours (p < 0.05). The collagen expression was stable on the both random and aligned PHBV, PHBV-gelatin, PCL and PCL-gelatin nanofibers demonstrating that the scaffolds mimics the natural basement membrane and thereby the cells grow normally on these nanofibrous matrices. Laminin was upregulated in all the scaffolds at all the time points investigated. It was also observed that the expression of laminin was not significantly different between the scaffolds at all the time points. The laminin expression levels gradually increased and attained peak levels after 7 days of culture.

It was found in the present study that the expression levels of CK 4 and CK 15 were upregulated in both PHBV and PHBV-gelatin nanofibrous scaffolds after 1 and 3 days of culture followed by down regulation at 7 days of culture. PCL aligned nanofibers exhibited maximum CK 4 expression, which was significantly higher than the PCL-gelatin aligned nanofibers (p < 0.05) after 3 days of culture. However, PCL random, PCL-gelatin random and aligned nanofibers exhibited decreased CK 4 expression levels after 1, 3, and 7 days of culture. Similarly, CK 15 expression was up regulated on the random and aligned nanofibers of PCL and PCL-gelatin after 1 day of culture followed by down regulation at 3 and 7 days of culture. The CK 15 expression was not significant by different between the scaffolds (p > 0.05) at all the time points.

Discussion

Electrospinning is an effective and simple method for fabricating polymeric nanofibers with different orientations. The structural morphology, fiber dimensions and orientations have been controlled by optimizing the various solution and process parameters such as polymer concentration, tip–target distance, needle size, flow rate, applied potential and speed of the rotating mandrel during the electrospinning process. Achievement of defect-free nanofibers with uniform dimensions is a crucial aspect in tissue engineering as it serves to mimic the topography of the native extracellular matrix and hence serves as a biologically recognizable bioscaffold.⁴⁰ Earlier studies have shown that electrospinning of PHBV can be carried out using different solvents like chloroform, hexafluoroisopropanol (HFIP), trifluoroethanol (TFE), and chloroform with dimethylformamide.^41–45 Many studies have reported formation of PHBV fibers with dimensions between 1000 and 4000 nm when chloroform was used as the solvent, 300 and 600 nm using HFIP as solvent and 50–500 nm using TFE.⁴⁴ Thus, it is evident that choice of the solvent plays a major role in controlling the fiber dimensions as well as to form defect-free fibers. In the present study, the pristine polymers were electrospun using DCM : DMF (9 : 1) and the blend systems were electrospun using HFIP.

Though all scaffolds supported the adhesion of the epithelial cells, the extended morphology of the cells cultured on the blend scaffolds compared with pristine PHBV and PCL scaffolds may be attributed to the presence of the integrin binding RGD motif in gelatin that facilitates superior cell adhesion, spreading and proliferation. The formation of well-extend cell morphology on the nanofibrous scaffolds suggests their ability to mimic the basement membrane of the esophageal tissue. Our group had previously demonstrated that human dermal fibroblasts cultured on PHBV films formed through solvent casting exhibited rounded morphology unlike the native extended morphology when they were cultured on the PHBV nanofibers, which confirms the significance of scaffold topography on the cell elongation.¹¹ Similar observations were reported by the Meng et al., on PHBV-gelatin scaffolds seeded with NIH 3T3 cells.³⁵

PHBV-gelatin scaffolds degraded rapidly in the aqueous environment and hence the proliferation of cells was distinctly reduced on this scaffold when compared with the other systems. The quantification of cells at different time points on the PCL-gelatin scaffolds with random and aligned orientation revealed no significant differences after 7 and 10 days of culture. Similar trend was observed by Ghasemi-Mobarakeh et al., for nerve stem cells cultured on PCL scaffolds.⁴⁶ Similarly, in another study no significance difference in the MC3T3-E1 cell proliferation between the random and aligned nanofibers of PLGA and PLGA-gelatin observed.⁴⁷ In the present study, the lack of difference in the viability of cells cultured on random and aligned nanofibrous scaffolds of PCL-gelatin at the later time points suggests that while cell orientation and elongation are strongly influenced by the fiber orientation and topography, cell viability is strongly influenced by the cell–scaffold interaction. The RGD motif in gelatin enables close interaction of the cells with the scaffold through binding of integrin receptors leading to their clustering on the cell membrane thereby activating a biochemical signaling cascade through mechanotransduction. In the pristine scaffolds the strong hydrophobic interactions between the cells and scaffold influenced the cell viability rather than their orientation.

Basement membranes are made of solid nanofibers that support the epithelial cells.⁴⁸ Collagen type IV and laminin are the main structural components that provides the structural and mechanical support to the growing cells/tissues in the native environment.⁴⁹ The collagen expression was found to be stable in all scaffolds. Zhu et al., reported that porcine epithelial cells cultured on the fibronectin (Fn) modified poly(L-lactide-co-caprolactone) (PLLC) nanofibers showed higher collagen type IV synthesis than the TCPS control and PLLC nanofibers. The authors claim that Fn-PLLC nanofibers support the growth and proliferation of functional epithelial cells.⁶ A similar conclusion can be arrived in the present study, which also suggests that epithelial cells are functional when they were cultured on these nanofibrous scaffolds. Laminin is a non-collagenous heterodimeric glycoprotein that plays a vital role in cell-fate process such as cell adhesion, motility, differentiation and proliferation along with collagen type IV, which stabilizes the basement membranes in biological tissues.⁵⁰ The expression of laminin by the epithelial cells confirms that the functionality and proliferative behavior of the cells are retained on these nanofibrous matrices.

Cytokeratins are intermediate filament proteins and their expression levels are tissue-specific, which are divided into type I (acidic) and type II (basic or neutral).⁵¹ Epithelial cells express keratin from type I and type II that aid in the formation of intermediate filaments, which provide structural integrity to the epithelial cells, maintain the mechanical properties of cells, and also play a vital role in tissue differentiation.⁵² Cytokeratin 4 (CK 4, type II) and cytokeratin 15 (CK 15, type I) are key proteins expressed in the stratified epithelium.^53,54 CK 15 is expressed in the basal layer of terminally differentiated cells while the CK 4 is expressed in the suprabasal layer of early differentiated cells.⁵⁵ Both CK 4 and 15 are expressed by the non-keratinized, stratified, squamous epithelial cells. Previous reports suggests that CK 4 (58 000 Da), CK 6 (56 000 Da), and CK 13 (52 000 Da) were expressed in the normal human esophageal epithelium.⁵⁶ The epithelial cells (non-keratinized, stratified, squamous epithelial cells) found in the upper digestive track also express the CK 4.⁵⁷ The CK 4 expression was decreased in the diseased conditions especially dysplasia and esophageal carcinoma when compared to normal epithelial cells.⁵⁸ The expression levels of CK 4 and CK 15 genes by the epithelial cells demonstrate that the structural integrity of the cells was maintained when cultured on these nanofibrous matrices. The aligned topography seemed to be a dominant factor in driving cell orientation rather than the expressions of cytokeratins and laminin while collagen IV levels remained stable in all nanofibrous scaffolds suggesting that the nanofibrous morphology promotes collagen deposition.

Conclusions

PHBV, PHBV-gelatin, PCL and PCL-gelatin random and aligned nanofibers were fabricated through electrospinning. The fiber orientation was optimized and the results exhibited good degree of alignment. PHBV and PCL-gelatin aligned nanofibers exhibited superior epithelial proliferation than the other scaffolds. Interestingly, the aligned nanofibers were found to guide the cells to extend along the fiber orientation, highlighting the significance of the aligned geometry in the scaffolds. The expression of epithelial cell-specific functional genes confirms the functionality of the cells when cultured on these nanofibrous scaffolds. The results of study suggest that PHBV and PCL-gelatin aligned nanofibers could be promising scaffolds for esophageal tissue regeneration. Among the four types of scaffolds investigated, PCL-gelatin aligned fibers exhibited maximum cell viability while the cell-specific gene expressions levels on this scaffold were comparable to the other scaffolds.

Acknowledgements

Indian Council of Medical Research, India (no. 35/21/2010-BMS), Nano Mission (SR/S5/NM-07/2006 & SR/NM/PG-16/2007), Department of Science & Technology, India, and FIST, Department of Science & Technology, India (SR/FST/LSI-327/2007 & SR/FST/LSI-058/2010) are acknowledged for their financial support. SASTRA University is also acknowledged for infrastructure support. First author would like to thank the Council of Scientific and Industrial Research for the Senior Research Fellowship (09/1095/(0003)/2013/EMR-I). The authors also acknowledge Dr S. Anuradha, Centre for Nanotechnology & Advanced Biomaterials, for the help with the pore analysis.

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