2.1

Preparation of CH solution

A solution of Low molecular weight (LMw) (50–190 kDa) (Sigma–Aldrich, UK) CH was prepared at 4.5 wt% in a combination of 3 wt% acetic acid (AA) with dimethyl sulfoxide (DMSO) (ratio 10:1). Ultrahigh molecular weight polyethylene oxide (PEO) (5,000,000 Mw) (Alfa Aesar. UK) was added to get a final CH:PEO ratio of 95:5. To ensure maximum dissolution, CH was firstly mixed in distilled water (DiH ₂ O) and heated to 60 °C for 30 min followed by dropwise addition of AA and allowing a further 15 min of mixing. PEO was then added to the solution and after another 30 min of mixing, DMSO (2 ml) was finally added and left stirring for 24 h at room temperature. After 24 h, the solution was centrifuged at 45000 rpm for 5 min to remove undissolved impurities. Each prepared electrospinning solution was utilized within 24 h of preparation.

2.2

Electrospinning

PEO-doped CH solutions were loaded into two 1 ml syringes each with a needle gauge of 4.699 mm and placed onto an automated syringe pump (World Precision Instruments, Sarasota, Florida) and dispensed at a constant rate of 1 ml/h. Voltage at the needle was set between 17.8 kV and 22 kV. Fibers were collected on a sheet of aluminum foil wrapped around a rotating steel drum. The distance between the needle tip and the collector drum (6 cm diameter) was 15 cm. The collector was set to rotate at 160 rpm for random fibers and 2500 rpm for aligned fibers. After completion of spinning, fibers were carefully removed from the collector and placed under vacuum for 24 h at room temperature to remove any remaining solvent. They were then stored in a sealed plastic bag at room temperature (25 °C). For swelling ratio, degradation and cell culture studies, fibers were neutralized with a solution of 1 M sodium hydroxide (NaOH) and 50% Ethanol (Fisher Scientific UK) (1:1) for 45 min and followed by washing twice with distilled water for 15 min each.

2.3

Scanning electron microscopy

SEM was employed to study the surface morphology of electrospun fibers (spot size: 3.0, voltage range 5–10 kV, Philips X-L 20 microscope). Samples were mounted on aluminum stubs with double-sided carbon adhesive dots (Agar Scientific, UK) and were sputter coated under vacuum with carbon using Speedivac carbon coating unit (Model 12E6/1598). Images obtained from SEM were scaled using Image J software (NIH, USA) and fiber diameter was measured. Frequency histograms of fiber diameter were plotted using Graphpad prism software version 6.0 and directionality histograms were obtained with ImageJ2 (Fiji) (NIH, USA) software using directionality plug-in for generating orientation graphs.

2.4

Fourier transform infrared (FTIR) spectroscopy

FTIR-ATR spectra were obtained using a Thermo Scientific iS50™ FTIR spectrophotometer, which is coupled with an ATR sampling accessory. Spectra were collected in the mid infrared region (4000–650 cm ⁻¹ ) at 4 cm ⁻¹ resolution accumulating 64 number of scans. ATR had a diamond crystal mounting. Spectral data was processed using a Thermo Nicolet OMNIC™ software (Thermo Scientific, Madison, WI, USA).

2.5

Mechanical properties

Electrospun fibrous scaffolds were mechanically tested in tension under dry and wet conditions on a BOSE ELF 3200™ (BOSE ElectroForce System groups, BOSE, Minnesota, USA) using a 22.5 N load strained at a rate of 0.1 mm/s to failure and a gauge length of 6 mm. From the obtained stress–strain curves, the point at which the samples snapped was used to calculate the ultimate tensile strength (UTS) and the strain (%), while the initial linear gradient was taken as the Young’s modulus ( E ). Specimens were cut into rectangles with dimensions 5 mm × 20 mm × T , where T is the thickness of the membranes under dry or wet conditions.

2.6

Swelling and degradation studies

Samples (13 mm Ø) for use in swelling studies were dried and weighed before storing in phosphate buffered saline (PBS) at 37 °C to allow any water uptake to occur. At time intervals of 0, 15, 30, 60, 120 and 140 min, specimens were removed from PBS and any excess water was removed with tissue paper before weighing samples. The swelling ratio was calculated using the formula: Swell Ratio % ( Q ) = ( W _w − W _d )/ W _d × 100. Where dry weight is given as W _d and wet weight is given as W _w .

For degradation studies, dry samples (13 mm Ø) were weighed and noted as W _o. These were then immersed in degradation media containing PBS and 5 mg/mL of hen egg lysozyme (Sigma–Aldrich. UK). Samples were incubated at 37 °C for 4, 7, 14, 21 and 28 days. Media was renewed after every 3 days and at each time interval samples were washed 3 times with distilled H ₂ O and then dried out thoroughly before weighing them again as W _t . Weight loss was calculated by using the formula: Weight loss % = ( W _o – W _t )/ W _o × 100.

Measurements of the supernatant pH were performed using a pH meter (Mettler Toledo S20 SevenEasy™). Ultraviolet–visible (UV–vis) spectrophotometry was performed (LAMBDA 950 UV Vis spectrophotometer PerkinElmer) on the sample supernatant by taking blank background and PBS was used as a reference against the sample while acquiring the spectra. Analysis was performed within the range of 200–500 nm. Samples were analyzed in quartz cuvettes (Thor Labs UK). Data of the UV–vis was processed by Vision pro™ software by Thermo Scientific™. FTIR-ATR spectroscopic analysis was also carried out on each specimen at each time point.

2.7

Cell culture on fibrous mats

Cell culture was conducted using human osteosarcoma cells (MG63) and human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs). MG63’s were expanded in Dulbecco’s modified Eagle’s medium (DMEM) (Biosera, Ringmer, UK) supplemented with 10% Foetal Calf serum (FCS), 2 mM l -glutamine, 100 μg/mL of penicillin and streptomycin. hES-MPs were expanded on gelatin (0.1% w/v in distilled water) coated surfaces and cultured in Alpha Minimum essential medium (α-MEM) (Lonza, Verviers, Belgium), supplemented with 10% FCS, 2 mM l -glutamine and 100 μg/mL penicillin and streptomycin. Cells were grown in a humidified incubator at 37 °C with 5% CO ₂ with fresh media changes performed every 2–3 days. Cells were grown to 90% confluency and then detached via trypsin-EDTA. MG63s were used between passages 60–68 while hES-MPs were used between passages 3–7. To analyze the viability of osteoblastic cells on different fiber orientations, cells were seeded at a density of 250,000 cells per sample using a marine grade stainless steel seeding ring (internal Ø 10 mm). Prior to cell seeding, the fibers were sterilized with ethanol for 1 h, washed twice with PBS for 15 min, and then coated with either culture medium for 1 h prior to seeding MG63’s or gelatine for 1 h prior to seeding hES-MPs. Cell free fibers were included as controls. All other reagents were purchased from Sigma–Aldrich, Life Sciences, UK.

2.8

Alamar Blue ^® assay

In order to quantify cell attachment and viability, fluorescent measurements of Alamar Blue were obtained after 1, 4 and 7 days of culture. For each time point, cell seeded samples were carefully washed with PBS and 0.5 ml of Alamar blue ^® solution (diluted 1:10 with PBS) was added followed by incubation at 37 °C for 4 h. Fluorescence was measured at 570 nm using a fluorescence plate reader (Bio-TEK, NorthStar Scientific Ltd, UK). Based on cell metabolic activity the system incorporates an oxidation–reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of the growth medium resulting from cell growth. Reduction related to growth causes the REDOX indicator to change from oxidized (blue) form to reduced (red) form. After the measurements were taken, samples were washed with PBS, fresh media was added and samples were further cultured in the incubator until the next time point.

2.9

Collagen and calcium staining

For identification of collagen deposition by cells on the CH membranes, Picro-sirius red staining was performed. Media was removed after 14, 21 and 28 days, and samples were washed with PBS and then fixed with 3.7% formaldehyde for 30 min and samples were then washed with PBS and Sirius red solution (Direct red dye 1 mg/ml in saturated picric acid, both Sigma, UK) was added to fully submerge samples and left for 18 h under mild rocking (20 rpm). Excess dye was washed with distilled H ₂ O and samples were destained for quantitative analysis using a known volume of 0.2 M NaOH and Methanol (v/v) (1:1) for 15 min. The extracted solution was read for absorbance at 490 nm in a 96 well plate reader. Total calcium deposition by hES-MPs was quantified at day 14, 21 and 28 after seeding. Samples were fixed (see collagen staining) followed by distilled H ₂ O washes and then application of 1% Alizarin red solution (pH 4.1) (Sigma, UK) at 1 ml per sample for 20 min on a platform shaker. The unbound dye was removed with distilled water washes. For quantification, the stain was extracted using a known volume of 5% (v/v) perchloric acid to each well for 30 min. The extracted solution was read for absorbance at 405 nm. Data shown is after subtraction of the absorbance reading obtained on blank scaffolds.

2.10

Histological cryosectioning

Histological sectioning of electrospun fibers was taken as complete transverse-sections across the centre of electrospun scaffolds. Samples were fixed with 3.7% formaldehyde, washed 2× PBS for 15 min each and then soaked in 1% sucrose solution for 30 min prior to embedding in OCT™ (Tissue-Tek ^® , Sakura, freezing medium) compound media. Samples were cryo-sectioned (Leica CM1860UV Ag protect) at 7 μm slices and mounted on glass slides (Thermo Scientific, Menzel-Glaser, Saarbruckener, Germany). Staining was performed with Haematoxylin (Harris) and Eosin (Sigma–Aldrich, UK). Stained sections were imaged with a light microscope (Leica, Motic) with 4×, 10× and 20× objectives. Images were scaled on Image J (NIH, USA) software.

2.11

Statistical analysis

Unless stated otherwise, all experiments were conducted at least in triplicates. All presented data refers to mean ± standard deviation (SD). In order to check for any statistically significant differences, One-way ANOVA was performed followed by Tukey’s post hoc test. Results with p -values of ≤ 0.05 (*) were considered statistically significant. All data was analyzed using Graphpad Prism 6.07 software. Mechanical properties were analyzed by Two-way ANOVA, which was performed using the computing environment R (R Development Core Team, 2015). R Core Team (2015).

2.1

Preparation of CH solution

A solution of Low molecular weight (LMw) (50–190 kDa) (Sigma–Aldrich, UK) CH was prepared at 4.5 wt% in a combination of 3 wt% acetic acid (AA) with dimethyl sulfoxide (DMSO) (ratio 10:1). Ultrahigh molecular weight polyethylene oxide (PEO) (5,000,000 Mw) (Alfa Aesar. UK) was added to get a final CH:PEO ratio of 95:5. To ensure maximum dissolution, CH was firstly mixed in distilled water (DiH ₂ O) and heated to 60 °C for 30 min followed by dropwise addition of AA and allowing a further 15 min of mixing. PEO was then added to the solution and after another 30 min of mixing, DMSO (2 ml) was finally added and left stirring for 24 h at room temperature. After 24 h, the solution was centrifuged at 45000 rpm for 5 min to remove undissolved impurities. Each prepared electrospinning solution was utilized within 24 h of preparation.

2.2

Electrospinning

PEO-doped CH solutions were loaded into two 1 ml syringes each with a needle gauge of 4.699 mm and placed onto an automated syringe pump (World Precision Instruments, Sarasota, Florida) and dispensed at a constant rate of 1 ml/h. Voltage at the needle was set between 17.8 kV and 22 kV. Fibers were collected on a sheet of aluminum foil wrapped around a rotating steel drum. The distance between the needle tip and the collector drum (6 cm diameter) was 15 cm. The collector was set to rotate at 160 rpm for random fibers and 2500 rpm for aligned fibers. After completion of spinning, fibers were carefully removed from the collector and placed under vacuum for 24 h at room temperature to remove any remaining solvent. They were then stored in a sealed plastic bag at room temperature (25 °C). For swelling ratio, degradation and cell culture studies, fibers were neutralized with a solution of 1 M sodium hydroxide (NaOH) and 50% Ethanol (Fisher Scientific UK) (1:1) for 45 min and followed by washing twice with distilled water for 15 min each.

2.3

Scanning electron microscopy

SEM was employed to study the surface morphology of electrospun fibers (spot size: 3.0, voltage range 5–10 kV, Philips X-L 20 microscope). Samples were mounted on aluminum stubs with double-sided carbon adhesive dots (Agar Scientific, UK) and were sputter coated under vacuum with carbon using Speedivac carbon coating unit (Model 12E6/1598). Images obtained from SEM were scaled using Image J software (NIH, USA) and fiber diameter was measured. Frequency histograms of fiber diameter were plotted using Graphpad prism software version 6.0 and directionality histograms were obtained with ImageJ2 (Fiji) (NIH, USA) software using directionality plug-in for generating orientation graphs.

2.4

Fourier transform infrared (FTIR) spectroscopy

FTIR-ATR spectra were obtained using a Thermo Scientific iS50™ FTIR spectrophotometer, which is coupled with an ATR sampling accessory. Spectra were collected in the mid infrared region (4000–650 cm ⁻¹ ) at 4 cm ⁻¹ resolution accumulating 64 number of scans. ATR had a diamond crystal mounting. Spectral data was processed using a Thermo Nicolet OMNIC™ software (Thermo Scientific, Madison, WI, USA).

2.5

Mechanical properties

Electrospun fibrous scaffolds were mechanically tested in tension under dry and wet conditions on a BOSE ELF 3200™ (BOSE ElectroForce System groups, BOSE, Minnesota, USA) using a 22.5 N load strained at a rate of 0.1 mm/s to failure and a gauge length of 6 mm. From the obtained stress–strain curves, the point at which the samples snapped was used to calculate the ultimate tensile strength (UTS) and the strain (%), while the initial linear gradient was taken as the Young’s modulus ( E ). Specimens were cut into rectangles with dimensions 5 mm × 20 mm × T , where T is the thickness of the membranes under dry or wet conditions.

2.6

Swelling and degradation studies

Samples (13 mm Ø) for use in swelling studies were dried and weighed before storing in phosphate buffered saline (PBS) at 37 °C to allow any water uptake to occur. At time intervals of 0, 15, 30, 60, 120 and 140 min, specimens were removed from PBS and any excess water was removed with tissue paper before weighing samples. The swelling ratio was calculated using the formula: Swell Ratio % ( Q ) = ( W _w − W _d )/ W _d × 100. Where dry weight is given as W _d and wet weight is given as W _w .

For degradation studies, dry samples (13 mm Ø) were weighed and noted as W _o. These were then immersed in degradation media containing PBS and 5 mg/mL of hen egg lysozyme (Sigma–Aldrich. UK). Samples were incubated at 37 °C for 4, 7, 14, 21 and 28 days. Media was renewed after every 3 days and at each time interval samples were washed 3 times with distilled H ₂ O and then dried out thoroughly before weighing them again as W _t . Weight loss was calculated by using the formula: Weight loss % = ( W _o – W _t )/ W _o × 100.

Measurements of the supernatant pH were performed using a pH meter (Mettler Toledo S20 SevenEasy™). Ultraviolet–visible (UV–vis) spectrophotometry was performed (LAMBDA 950 UV Vis spectrophotometer PerkinElmer) on the sample supernatant by taking blank background and PBS was used as a reference against the sample while acquiring the spectra. Analysis was performed within the range of 200–500 nm. Samples were analyzed in quartz cuvettes (Thor Labs UK). Data of the UV–vis was processed by Vision pro™ software by Thermo Scientific™. FTIR-ATR spectroscopic analysis was also carried out on each specimen at each time point.

2.7

Cell culture on fibrous mats

Cell culture was conducted using human osteosarcoma cells (MG63) and human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs). MG63’s were expanded in Dulbecco’s modified Eagle’s medium (DMEM) (Biosera, Ringmer, UK) supplemented with 10% Foetal Calf serum (FCS), 2 mM l -glutamine, 100 μg/mL of penicillin and streptomycin. hES-MPs were expanded on gelatin (0.1% w/v in distilled water) coated surfaces and cultured in Alpha Minimum essential medium (α-MEM) (Lonza, Verviers, Belgium), supplemented with 10% FCS, 2 mM l -glutamine and 100 μg/mL penicillin and streptomycin. Cells were grown in a humidified incubator at 37 °C with 5% CO ₂ with fresh media changes performed every 2–3 days. Cells were grown to 90% confluency and then detached via trypsin-EDTA. MG63s were used between passages 60–68 while hES-MPs were used between passages 3–7. To analyze the viability of osteoblastic cells on different fiber orientations, cells were seeded at a density of 250,000 cells per sample using a marine grade stainless steel seeding ring (internal Ø 10 mm). Prior to cell seeding, the fibers were sterilized with ethanol for 1 h, washed twice with PBS for 15 min, and then coated with either culture medium for 1 h prior to seeding MG63’s or gelatine for 1 h prior to seeding hES-MPs. Cell free fibers were included as controls. All other reagents were purchased from Sigma–Aldrich, Life Sciences, UK.

2.8

Alamar Blue ^® assay

In order to quantify cell attachment and viability, fluorescent measurements of Alamar Blue were obtained after 1, 4 and 7 days of culture. For each time point, cell seeded samples were carefully washed with PBS and 0.5 ml of Alamar blue ^® solution (diluted 1:10 with PBS) was added followed by incubation at 37 °C for 4 h. Fluorescence was measured at 570 nm using a fluorescence plate reader (Bio-TEK, NorthStar Scientific Ltd, UK). Based on cell metabolic activity the system incorporates an oxidation–reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of the growth medium resulting from cell growth. Reduction related to growth causes the REDOX indicator to change from oxidized (blue) form to reduced (red) form. After the measurements were taken, samples were washed with PBS, fresh media was added and samples were further cultured in the incubator until the next time point.

2.9

Collagen and calcium staining

For identification of collagen deposition by cells on the CH membranes, Picro-sirius red staining was performed. Media was removed after 14, 21 and 28 days, and samples were washed with PBS and then fixed with 3.7% formaldehyde for 30 min and samples were then washed with PBS and Sirius red solution (Direct red dye 1 mg/ml in saturated picric acid, both Sigma, UK) was added to fully submerge samples and left for 18 h under mild rocking (20 rpm). Excess dye was washed with distilled H ₂ O and samples were destained for quantitative analysis using a known volume of 0.2 M NaOH and Methanol (v/v) (1:1) for 15 min. The extracted solution was read for absorbance at 490 nm in a 96 well plate reader. Total calcium deposition by hES-MPs was quantified at day 14, 21 and 28 after seeding. Samples were fixed (see collagen staining) followed by distilled H ₂ O washes and then application of 1% Alizarin red solution (pH 4.1) (Sigma, UK) at 1 ml per sample for 20 min on a platform shaker. The unbound dye was removed with distilled water washes. For quantification, the stain was extracted using a known volume of 5% (v/v) perchloric acid to each well for 30 min. The extracted solution was read for absorbance at 405 nm. Data shown is after subtraction of the absorbance reading obtained on blank scaffolds.

2.10

Histological cryosectioning

Histological sectioning of electrospun fibers was taken as complete transverse-sections across the centre of electrospun scaffolds. Samples were fixed with 3.7% formaldehyde, washed 2× PBS for 15 min each and then soaked in 1% sucrose solution for 30 min prior to embedding in OCT™ (Tissue-Tek ^® , Sakura, freezing medium) compound media. Samples were cryo-sectioned (Leica CM1860UV Ag protect) at 7 μm slices and mounted on glass slides (Thermo Scientific, Menzel-Glaser, Saarbruckener, Germany). Staining was performed with Haematoxylin (Harris) and Eosin (Sigma–Aldrich, UK). Stained sections were imaged with a light microscope (Leica, Motic) with 4×, 10× and 20× objectives. Images were scaled on Image J (NIH, USA) software.

2.11

Statistical analysis

Unless stated otherwise, all experiments were conducted at least in triplicates. All presented data refers to mean ± standard deviation (SD). In order to check for any statistically significant differences, One-way ANOVA was performed followed by Tukey’s post hoc test. Results with p -values of ≤ 0.05 (*) were considered statistically significant. All data was analyzed using Graphpad Prism 6.07 software. Mechanical properties were analyzed by Two-way ANOVA, which was performed using the computing environment R (R Development Core Team, 2015). R Core Team (2015).