Electrospun oriented gelatin-hydroxyapatite fibre scaffolds for bone tissue engineering

Ali A. Salifu1, Constantina Lekakou1 and Fatima Labeed2

1Advanced Materials Group, University of Surrey, Guildford, Surrey GU2 7XH, UK

2Centre of Biomedical Engineering, University of Surrey, Guildford, Surrey GU2 7XH, UK

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Abstract

Tissue engineering of human foetal osteoblast (hFOB) cells was investigated on gelatin-

hydroxyapatite (HA), crosslinked, electrospun oriented fibre scaffolds at the different

hydroxyapatite concentrations of 0, 10, 20 and 25 wt% in the dry fibres and different fibre

diameter, pore size and porosity of scaffolds. Rheological tests and proton NMR

spectroscopy were conducted for all solutions used for electrospinning. It was found that 25

wt% HA-gelatin scaffolds electrospun at 20 kV led to the greatest cell attachment, cell

proliferation and extracellular matrix (ECM) production while fibre orientation improved the

mechanical properties, where crosslinked electrospun 25 wt% HA-gelatin fibre scaffolds

yielded a Young’s modulus in the range of 0.5 to 0.9 GPa and a tensile strength in the range

of 4 to 10 MPa in the fibre direction for an applied voltage of 20 to 30 kV, respectively, in the

electrospinning of scaffolds. Biological characterisation of cell seeded scaffolds yielded the

rate of cell growth and ECM (collagen and calcium) production by the cells as a function of

time; it included cell seeding efficiency tests, alamar blue cell proliferation assay, alkaline

phosphate (ALP) assay, collagen assay, calcium colorimetric assay, fluorescence microscopy

for live and dead cells, and scanning electron microscopy (SEM) for cell culture from 1 to 18

days. After 18 days, cells seeded and grown on the 25 wt% HA-gelatin scaffold, electrospun

at 20 kV, reached production of collagen at 370 g/L and calcium production at 0.8 mM.

Keywords: tissue engineering; osteoblasts; gelatin-hydroxyapatite; electrospinning;

scaffolds; mechanical testing; biological characterisation.

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1. Introduction

Tissue engineering of bone has been studied extensively given the issues associated with the

development of biomimetic bone tissue that could be easily integrated in vivo which are: (a)

the relatively large size of osteoblasts compared with other cells which means that relatively

large scaffold pores may be needed for their migration through the scaffold; (b) bone has high

modulus and strength to provide structural support but is also much denser than other tissues

which would make cell migration difficult; (c) oxygen is a critical nutrient for osteoblasts

and, in the absence of angiogenesis, the populated scaffold needs to have sufficient porosity

to provide access to the culture medium or blood in vivo for oxygen supply to the osteoblasts.

Non-union bone defects, which are defects above the critical-size for which the gap in the

bone is too large with limited vascularisation, are the major target for tissue engineered bone

[1].

The bone extracellular matrix (ECM) is a porous nanocomposite of organic matrix and

uniformly embedded hydroxyapatite (HA) nanocrystals [2]. The organic component, which

makes up about 30% of the ECM, consists of collagen fibres (predominantly type I) (approx.

90%) and proteoglycans and glycoproteins (approx. 10%) [3,4,5]. Natural and synthetic

material-based scaffolds made from ceramics [6,7], polymers [8-12], polymer blends [11,12],

and polymer-ceramic composites [9,11,13-15] have been proposed by various researchers for

bone tissue engineering. Biocompatible scaffolds support cell attachment and proliferation

and bone tissue formation without eliciting a significant or lengthy inflammatory and/or

adaptive immune response from the host [16]. Bioactive scaffolds promote cell attachment on

their surfaces whereas bioinert scaffolds may require surface modification to facilitate cell

adhesion [17]. Biodegradable scaffolds gradually transfer the structural support duties to the

newly formed bone tissue while they gradually degrade and are resorbed into the body.

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Collagen-based nanocomposites, which possess these properties are, therefore, desirable.

Collagen contains the necessary amino acid sequences for binding with the integrins of

osteoblasts and this study uses gelatin as a low cost collagen substitute to promote cell

binding to the scaffold. HA is known to promote osteoblast cell growth and osteogenesis in

both natural [13-14] and synthetic [9,11] polymer scaffolds.

Electrospun gelatin-HA scaffolds fabricated via in situ HA precipitation within the gelatin

matrix have been shown to possess superior mechanical properties [13] and cellular activity

[14] versus their conventionally mixed gelatin-HA counterparts. However, there still exists

the need to improve the mechanical properties of these scaffolds so that they can better

support cell growth and tissue regeneration and maintain the mechanical integrity of the bone

defect site. To date, many electrospun gelatin-HA fibre scaffolds have been fabricated for

bone tissue engineering, but typically low mechanical properties have been reported; such as

a Young’s modulus of 326 MPa and a tensile strength of 5 MPa for a 20 wt% HA-gelatin

scaffold with random fibre orientation [13]. Following the hypothesis that fibre orientation

would improve the mechanical performance of the scaffold, a preliminary study by our group

of producing scaffolds with fibre orientation yielded 60 MPa and 3.9 MPa in the fibre

direction for a 30 g/g HA-gelatin scaffold [18] for the Young’s modulus and tensile strength,

respectively; it is obvious that this fibre-oriented scaffold had not reached its full potential in

mechanical properties which was attributed to the fact that the processing conditions of

electrospinning were not optimised in that preliminary study [18].

It is clear that a comprehensive investigation is required beyond previous studies and our own

preliminary study [18] to optimise material composition and the processing conditions of

electrospinning of HA-gelatin nanocomposite fibre scaffolds in order to maximise fibre

orientation of electrospun single scaffold layers and mechanical performance of scaffolds

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while scaffold composition and microstructure is also optimised for cell attachment and

growth as well as ECM production. A research programme has been compiled for this study

including systematic changes in the concentration of produced HA, in situ precipitated in

gelatin in the form of nanoparticles, changes in the applied electrospinning voltage and

increase of the rotation speed of the collector of electrospun fibres compared to our early

study [18] to improve fibre orientation and increase process productivity. A novel aspect of

the present study includes rheological and NMR characterisation of the viscoelastic HA-

gelatin solutions used for electrospinning which offer novel insights for processing and fibre

formation in electrospinning as well as micro- and nano-structure of the HA-gelatin solutions.

A most detailed physical characterisation of the crosslinked electrospun fibre scaffolds in this

paper comprises full microstructural analysis (including distributions of fibre orientation),

dynamic water contact angle goniometry, XRD, FTIR, AFM and tensile mechanical testing.

In this manner, this study explores the effect of the applied voltage and HA concentration on

the scaffold microstructure, chemical composition, hydrophilicity and mechanical properties

aiming at increasing the mechanical performance of the HA-gelatin scaffolds compared to all

previous studies. The research continues with detailed biological characterisation of these

scaffolds in order to relate the effect of scaffold porosity, pore size, specific pore surface area,

fibre diameter and HA concentration on human foetal osteoblast (hFOB) cell proliferation

and ECM production (collagen and mineral production). Another novel aspect of this paper is

the presentation of the kinetics of hFOB cell growth, collagen and minerals production for all

scaffolds of different microstructures and HA concentration, fully related to the

electrospinning conditions.

2. Materials and methods

2.1 Materials

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Porcine gelatin (Type A gelatin) (Sigma-Aldrich, UK) was used throughout this study.

Hydroxyapatite was prepared in situ in gelatin solution from phosphoric acid (H3PO4) crystals

and calcium hydroxide (Ca(OH)2) powder (Sigma-Aldrich). Sodium hydroxide (NaOH)

(Fisher Scientific, UK) was used as a pH regulator while 2,2,2-Trifluoroethanol (TFE)

(Sigma-Aldrich) was used as a solvent to prepare solutions for electrospinning and 25 % w/v

glutaraldehyde grade II (Sigma-Aldrich) was used to crosslink the electrospun scaffolds.

Ca(OH)2 powder and H3PO4 crystals, at a mass ratio of Ca(OH)2 / H3PO4 = 1.26 (aiming to

stoichiometrically obtain Ca10(PO4)6(OH)2), were separately dissolved in 25 ml of deionised

water. The required amount of gelatin powder (to prepare gelatin-HA nanocomposites of

different compositions) was dissolved in the Ca(OH)2 solution at about 37 °C. The H3PO4

solution was then added to the Ca(OH)2-gelatin solution drop by drop with continuous

stirring. The pH of the resulting solution was adjusted to 10 by adding 1M NaOH solution

dropwise while stirring continued, in order to obtain uniform-sized HA crystals formed in

situ in the gelatin solution. The resulting nanocomposite suspensions were placed in a 37 °C

water bath for 24 hours to allow the HA to mature. All samples were transferred into square

Petri dishes and frozen at -20 °C for 48 hours followed by freeze-drying at -40 °C for a

further 48 hours.

2.2 Electrospinning of fibrous scaffolds

Pure gelatin and the gelatin-HA nanocomposites were dissolved in TFE at a concentration of

10% w/v and each solution was loaded into a 5 ml plastic syringe with an 18-gauge stainless

steel blunt needle at its tip. A syringe pump (Cole-Parmer, USA) was used to deliver each

solution at a constant feed rate of 5 ml/h (against 1.5 ml/h in our previous study [18])

followed by the application of high voltage (20, 25 and 30 kV) from a high voltage power

supply (Glassman High Voltage, USA) to the solutions via a crocodile clip attached to the tip

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of the needle (negative polarity). The distance between the tip of the needle and the collector

was maintained at 15 cm. The electrospun fibres were deposited on a wired drum collector

rotating at a speed of 30 rpm (against 2 rpm in our previous study [18]). Electrospinning was

carried out at 25 oC and 40% relative humidity in a sealed fume cupboard for 2 hours. The

resulting fibre mats were crosslinked with saturated glutaraldehyde vapour in a sealed

desiccator for 2 hours [19].

Solutions of 10% w/v solid in TFE were used for electrospinning where the solid was: 100 wt

% gelatin, 10 wt% HA-90 wt% gelatin, 20 wt% HA-80 wt% gelatin and 25 wt% HA-75 wt%

gelatin. In all these cases, gelatin was fully dissolved in TFE yielding a clear solution

whereas the HA nanoparticles were suspended in the gelatin-TFE solution yielding a whitish

liquid. Solutions of 10 %w/v of (40 wt% HA-60 wt% gelatin) in TFE were also tried for

electrospinning but resulted in spray rather than fibre formation Replacing TFE by water, it

was found that the gelatin and HA-gelatin solutions in water gelled at the nozzle and blocked

the nozzle used for electrospinning, hence, TFE remained the solvent of choice.

The viscoelastic properties of the electrospun solutions were measured in dynamic

rheological studies using a Rheometrics RDA II instrument at 25 oC. The tests included

steady shear rate sweep from 0.005 to 5 s-1 to determine the viscosity under shear, ,

elongational flow at different strain rates to determine the elongational viscosity, el, and a

frequency sweep from 1 to 80 Hz to determine the relaxation time, trel (trel = /G’, where G’ is

the elastic shear modulus). In all tests the experimental data were fitted by the following

power-law relations:

μ=μo γ̇ n1 (1)

μel=μel , o ϵ̇ n 2 (2)

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t rel=t o γ̇n 3 (3)

Table 1 presents the values of the parameters of the power-law relations (1)-(3) after fitting

with the experimental rheological data according to which all solutions exhibited shear and

strain thinning behaviour. Elongational viscosity is of main interest for electrospinning and it

can be seen that the presence of HA nanoparticles reduces both the consistency and the

power-law index in comparison with the pure gelatin solutions, possibly by interrupting the

gelatin chain interactions. Pure gelatin solutions have also longer relaxation time compared to

HA-gelatin nanocomposite solutions, especially at low shear rates.

The next stage was to carry out proton nuclear magnetic resonance (NMR) spectroscopy for

all the electrospun solutions (in TFE) further diluted in CDCl3, a solvent commonly used in

NMR. The experiments were carried out at 25°C using a Bruker Avance III 500 NMR

spectrometer. Figure 1 presents the NMR spectra for different compositions of the gelatin-

HA nanocomposites where the gelatin was fully dissolved in TFE. The observed peaks have

been attributed to the following groups: the peak at 3.57 ppm to proline amino acid and to the

H of the –OH group of TFE; the triplet at 3.9 ppm to degraded collagen products in gelatin

[20] and the hydrogen of the –CH2- group of TFE. Introducing HA nanoparticles in the

gelatin-TFE solution reduces dramatically the triplet intensity at 3.9 ppm (more than the

reduction of gelatin concentration from 100 to 90 wt%) as well as the consistency of the

viscous solution in Table 1 which might be associated with the degradation of gelatin during

the in situ formation of HA nanoparticles; it is noted that the proline-associated peak at 3.9

ppm is eliminated in the presence of HA nanoparticles. As the HA content is increased to 20

wt%, a new peak appears at 4.3 ppm which is reduced and shifted to 4.15 ppm at 25 wt%

HA, which we believe is due to additional gelatin fragments generated during the in situ

formation of an increasing content of HA nanoparticles (in the corresponding presence of

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increased quantities of H3PO4 and Ca(OH)2 solutions) or additional secondary structures due

to hydrogen bonding. The rheological effects of these changes in the gelatin structure

competing with the increased content of HA nanoparticles, would cause the irregular pattern

in the change of the elongational viscosity consistency and relaxation time in Table 1 as the

HA content in increased from 10 to 25 wt%.

2.3 Physical characterisation of scaffolds

X-ray diffraction (XRD) analysis was carried out to detect the presence of the apatite phase

and the crystallinity of HA in the scaffolds. A PANalytical X’Pert powder X-ray

diffractometer was employed using Cu Kα radiation (λ = 1.54 Å). The samples were scanned

between 10° and 90° (2ϴ) at a scan rate of 0.036°/s and step size of 0.0016°.

Fourier transform infrared (FTIR) spectroscopy was carried out to detect the presence of

apatite groups. FTIR spectra were obtained from an Agilent Cary 640 FTIR spectrometer for

each sample at a resolution of 4 cm-1 and 32 scans in the wavenumber range of 4000 to 400

cm-1.

Dynamic contact angle goniometry (CAG) was carried out for a sessile de-ionised water drop

spreading onto each type of scaffold, using the Krüss EasyDrop instrument at 25 oC, in order

to assess the hydrophilicity of each scaffold. The results are reported as initial contact angle,

o and final contact angle, t, at the time the drop has spread over the scaffold, tspread.

Microstructural analysis of scaffolds was conducted from scanning electron microscope

(SEM) images of the scaffolds visualised with a Hitachi S3200N SEM at 15 kV. Each

microstructural analysis included 5 different samples of the same type from which 5 different

SEM images were taken per sample. The SEM images (25 fibres and 15 pores per image)

were analysed using Digimizer® software to determine fibre diameters, fibre orientation, pore

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diameters and porosity. The short and long diagonal lengths of each pore were measured and

averaged to obtain the pore diameter. Scaffold porosity, , was determined using the

gravimetric approach where it was taken that the density of the dry gelatin is 1340 kg m -3 and

the density of HA is 3160 kg m-3. From the values of the scaffold porosity and fibre

diameters, the specific pore surface area (SSA) was estimated assuming aligned cylindrical

fibres. Atomic force microscopy (AFM) in tapping mode (Bruker) was also to obtain

topography and phase maps of scaffolds to investigate the dispersion of the HA solid phase in

the gelatin matrix [19].

The tensile properties of the dry crosslinked scaffolds were determined using an Instron

5500R 6025 machine. The scaffolds were cut into rectangular shapes of 13 mm x 3 mm with

the aid of a “dog bone” specimen cutter, in such a way that tests were performed with the

tensile stress in the average fibre direction (original circumferential collector direction) or

with the tensile stress perpendicular to the fibre direction (original axial collector direction).

Each scaffold was vertically mounted and held in place at both ends by two aligned gripping

units attached to a 10 N load cell. The scaffold specimens were tested at a crosshead speed of

1 mm/min until failure. A total of 6 tests were performed on 6 different samples for each type

of scaffold.

2.4 Cell culture

Human foetal osteoblast (hFOB 1.19) cells (ATCC/LGC Standards, UK) were used. The

complete cell culture medium was prepared from GIBCO® Dulbecco’s Modified Eagle

Medium and Ham’s F12 Medium (1:1) supplemented with 10% foetal bovine serum and 0.3

mg/ml geneticin® selective antibiotic. GIBCO® 0.25% trypsin/EDTA and phosphate buffered

saline (PBS, pH 7.2) were also used. These were all obtained from Life Technologies, UK.

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The crosslinked scaffolds were cut into 22 mm discs and soaked in 1M glycine solution

(Fisher Scientific, UK) for 14 hours to remove residual glutaraldehyde. They were then

soaked twice in PBS, sterilised in 70% ethanol for 4 hours and soaked twice again in PBS

under sterile conditions. The scaffolds were subsequently soaked in the culture medium for

14 hours, removed and hFOB cells between passages 5 and 7 were seeded on one side of the

scaffolds in 12-well plates at a seeding density of 4.0 x 105 cells/ml. After a 3-hour cell

attachment period, 5 ml of culture medium was added to the seeded scaffolds, incubated at 37

oC and 5% CO2. The cells were cultured for a period of 18 days with medium renewal every 2

to 3 days.

2.5 Biological characterisation techniques

All investigations were undertaken on seeded scaffolds on days 1, 4, 7, 11, 14 and 18 in

culture.

2.5.1 Cell seeding efficiency

The cell seeding efficiency on scaffolds was evaluated by dividing the number of attached

cells by the total number of cells initially seeded. After the usual 3-hour cell attachment

period in the incubator at 37 oC and 5% CO2, the scaffolds were taken out of the wells and

rinsed in the culture medium so as to measure only truly adherent cells. The spent medium

and the contents of trypsinised wells were transferred into a centrifuge tube and centrifuged at

1000 rpm for 5 minutes. Thereafter, the cell pellets were resuspended in fresh culture medium

and counted with a C-Chip DHC-N01 disposable haemocytometer (NanoEnTek, South

Korea) to determine the number of unattached cells from which the number of attached cells

was determined.

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2.5.2 Alamar blue cell proliferation assay

The alamar blue assay operates on the principle that metabolically active viable cells convert

the non-fluorescent resazurin into the highly fluorescent resorufin. Alamar blue reagent (Life

Technologies, UK) was diluted 1 in 10 with the culture medium and 0.8 ml of the working

solution was added to triplicate seeded scaffolds and unseeded scaffolds (controls) in 12-well

plates. The samples were incubated at 37 oC and 5% CO2 for 2 hours after which 100 µl

aliquots of each sample were transferred to different wells of a Nunclon Delta white opaque

96-well plate (Sigma-Aldrich, UK) and the fluorescence intensities were measured at 540 nm

excitation and 590 nm emission using a FLUOstar Omega microplate reader (BMG Labtech,

Germany). Corrected fluorescence intensities were obtained by subtracting readings from the

controls.

2.5.3 Alkaline phosphatase (ALP) assay

ALP activity of hFOB cells on the scaffolds was measured using QuantiChromTM ALP assay

kit (BioAssay Systems, USA). ALP catalyses the hydrolysis of the colourless p-nitrophenyl

phosphate into a yellow p-nitrophenol product. The scaffolds were washed twice with PBS

and the cells were lysed in 1 ml 0.2% Triton X-100 in deionised water (Sigma-Aldrich, UK)

by vortexing at room temperature for 20 minutes. Samples were prepared in each well of a

96-well plate by adding 50 µl cell lysates and 150 µl of the assay working solution. The

absorbance of the samples was measured at 405 nm using an ELISA microplate reader (Bio-

Tek Instruments, USA). ALP activity was determined by following the manufacturer’s

instructions.

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2.5.4 Collagen assay

The amount of collagen secreted by hFOB cells on the scaffolds during matrix formation was

determined using Sircol™ collagen assay kit (Biocolor, UK); a colorimetric assay based on

the selective binding of Sirius red dye to soluble collagen to form a collagen-dye complex.

The seeded scaffolds were washed twice in PBS and new collagen was extracted by adding

1.0 ml of pepsin at 0.1 mg/ml in 0.5M acetic acid (Sigma-Aldrich, UK) to each sample and

incubating overnight at 4 oC. Into 1.5 ml Eppendorf Protein LoBind microcentrifuge tubes

(Sigma-Aldrich, UK), 100 µl each of collagen standard solutions at concentrations 5, 10 and

15 µg/100 µl, extracted collagen and controls (from unseeded scaffolds) were transferred and

1.0 ml of Sircol™ dye reagent was added. The manufacturer’s instructions were followed and

the absorbance of 200 µl solution measured at 555 nm with the FLUOstar Omega microplate

reader in 96-well plates. Corrected absorbance values were obtained by deducting readings

from the controls. A collagen standard curve was plotted and used to interpolate the amount

of collagen.

2.5.5 Calcium colorimetric assay

The amount of calcium produced by the hFOB cells on the scaffolds during collagen matrix

mineralisation was monitored with Calcium Colorimetric Assay Kit (BioVision Research

Products, USA), which utilises the reaction between O-cresolphthalein and calcium ions that

forms a chromogenic complex with a measurable absorbance at 575 nm. The scaffolds were

washed twice with PBS and calcium was extracted by adding 0.5 ml 5% trichloroacetic acid

(Sigma-Aldrich, UK) to the scaffolds at room temperature for about 30 minutes. Deionised

water (40 µl) was added to replicate wells of a 96-well plate containing 10 µl of each sample

and its control (unseeded scaffold) and 10 µl calcium standard solutions at 0, 0.4, 0.8, 1.2, 1.6

and 2.0 µg calcium per well. The manufacturer’s instructions were followed and the

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absorbance of the resulting samples was measured at 575 nm with the FLUOstar Omega

microplate reader. Corrected absorbance values were obtained by deducting readings from

the controls. A calcium standard curve was plotted and used to interpolate the amount of

calcium on each scaffold.

2.5.6 Fluorescence microscopy

Live and dead cells were visualised on the scaffolds using a Live/Dead Cell Double Staining

Kit (Sigma-Aldrich, UK) consisting of non-fluorescent solutions of acetoxymethyl ester of

calcein (Calcein-AM) and propidium iodide (PI) for simultaneous fluorescent labelling of

live (green) and dead (red) cells. Calcein-AM permeates live cell membranes and gets

hydrolysed into a green-fluorescent calcein product whereas PI only passes through damaged

dead cell membranes and interacts with the DNA double helix to generate red-fluorescent PI-

DNA complexes. The scaffolds were rinsed briefly in PBS and placed on glass slides. Cover

slips were placed over the scaffolds after 200 µl of the working solution were added to each

scaffold. The slides were then incubated at 37 oC and 5% CO2 for 15 minutes after which live

and dead cells on the scaffolds were observed under a Nikon fluorescence microscope

(Eclipse TS100).

2.5.7 Scanning electron microscopy

The seeded scaffolds were rinsed with PBS, fixed in 4% formaldehyde solution (Sigma-

Aldrich, UK) overnight at room temperature, rinsed with distilled water to remove residual

salts from the PBS and dehydrated in an alcohol graded series (1 hour in 70% ethanol, 1 hour

in 90% ethanol and 2 hours in 100% ethanol). They were then sputter-coated with 30 nm of

gold and visualised with a Hitachi S3200N SEM at an accelerating voltage of 15 kV.

2.6 Statistical analysis

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The results are reported as mean ± standard deviation for n = 3 (or different if otherwise

stated). One-way ANOVA was used to analyze differences between scaffold groups. Two-

way ANOVA with post-hoc Bonferroni corrected t tests was used to analyze differences

between scaffold groups at different culture durations. The differences were statistically

significant at p < 0.05.

3. Results

An increase in the HA concentration in the scaffold or the applied electrospinning voltage

during scaffold fabrication resulted in significantly smaller scaffold diameter, smaller pore

size and lower porosity (p < 0.05) but greater specific pore surface area (SSA), as shown in

Table 2, which presents average values of the microstructural parameters. The reduction of

fibre diameter with increasing electrospinning voltage is as expected in typical

electrospinning processes, and unlikely the reverse trend that was observed in the

electrospinning of similar gelatin-TFE solutions by Elsayed et al in our group [21]. This is

attributed to the negative polarity of the feed solution nozzle in the present study creating an

electric field acting on the polar gelatin which extended the chains; in contrast, Elsayed et al’s

[21] study connected the feed solution nozzle to the positive high voltage pole which

generated an attractive force on the polar gelatin leading to larger fibre diameters at

increasing voltage.

Figure 2 shows the fibre orientation distribution with the 0o angle corresponding to the

circumferential direction of the rotating drum collector in electrospinning. In general, fibre

orientation follows a Gaussian distribution with the peak at 0o angle. Scaffolds of 25 wt% HA

display the highest fibre orientation. Hence, fibres were mostly aligned in the circumferential

drum direction during electrospinning (also observed but not measured in [18]).

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Figure 3 presents AFM micrographs of the 25 wt% HA-gelatin scaffold (dry) including both

a topographical and a surface force map, the latter providing information about hard (dark)

versus soft (white) material phases: it illustrates that the hard (dark) HA phase has aggregated

in the central core of the fibres.

The XRD diffractograms shown in Figure 4 indicate the presence of HA characteristic peaks

(indicated by *) in the composite samples, even though some of the peaks were not distinct

due to the masking effect of the gelatin matrix on the low levels of HA particles. The

composites exhibited low crystallinity, as revealed by the weak splitting of the HA peaks

between (211) and (112), which suggested that the HA phase was poorly crystallised [13].

The broadening of the peak at around 2ϴ = 30° also suggested that the HA phase was

nanocrystalline.

The presence of distinct phosphate (PO43-) bands in the FTIR spectra presented in Figure 5

confirmed the presence of HA in the composites, especially distinct in the case of the 20 wt%

HA scaffold. The characteristic bands for the gelatin matrix included peaks at 1631.9, 1531.1,

1334.9 – 1447.0 and 1237.2 cm-1 representing amide I (C=O stretching), amide II (N-H in-

plane bending and C-N stretching), carboxyl and amide III (C-N and N-H in-plane stretching)

groups, respectively. HA characteristic peaks included peaks at 1025.4 – 1079.7 cm-1 and

525.3 – 598.8 cm-1 representing phosphate groups and a weak carbonate (CO3) peak at 873.1

cm-1.

The CAG measurements of the water contact angle presented in Table 2 demonstrate that all

scaffolds are hydrophilic. The introduction of 10 wt% HA to the gelatin brings an increase in

the water contact angle for the 20 kV electrospun scaffolds; however, when the HA is

increased to 25 wt%, the water contact angle is reduced substantially rendering the 25 wt%

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HA-gelatin scaffolds highly hydrophilic for subsequent good wetting with the water-based

culture medium.

Figure 6 displays the stress-strain curves derived from the tensile testing of scaffolds parallel

and perpendicular to the main fibre direction. As expected, the Young’s modulus and tensile

strength are significantly higher (about double) in the fibre direction compared to the

perpendicular direction (p < 0.05). The scaffold with 25 wt% HA had the highest Young’s

modulus and tensile strength, which were significantly enhanced as the applied

electrospinning voltage was raised from 20 kV to 30 kV (p < 0.05). The values of the scaffold

mechanical properties (fibre direction) are tabulated in Table 2.

The next phase of results comes from the biological characterisation of the scaffolds seeded

with hFOB cells. The alamar blue assay is a non-destructive assay resulting in measurable

fluorescence intensity generated by the cells, providing an indirect way of determining the

number of cells and viable cell proliferation rates. Cell fluorescence on the scaffolds was

monitored for 18 days after cell seeding. Figure 7(a), (b) and (c) present the fluorescence

intensity results for the different seeded scaffolds as a function of cell culture time. Generally,

there was a significant increase in cell proliferation with incubation time up to 18 days (p <

0.05). In particular, cell growth increased sharply from day 1 to day 4 followed by a generally

steady growth until day 18. It can be seen that the optimum scaffolds for hFOB cell response

are those electrospun at 20 kV. Hence, cell seeding efficiency (last row of Table 2) was

measured for those scaffolds.

The results of the amount of newly synthesised collagen on the scaffolds determined using

the Sircol™ collagen assay are presented in Figures 7(d), (e), (f). In general, the amount

collagen steadily increased with incubation time (p < 0.05) as a result of cellular synthesis

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and secretion of collagen with the amount of de novo collagen being about six-fold higher at

day 18 than day 1 values.

The expression of ALP activity by the hFOB cells is indicative of their bone forming ability.

The results of the ALP activity of cells are presented in Figures 7(g), (h), (i). Generally, fairly

constant low levels of ALP activity were recorded for the first 7 days of cell culture (p >

0.05) probably due to the initial colonisation of the scaffold by the cells. Afterwards, there

was a gradual rise in ALP activity between days 7 and 18 (p < 0.05) averaging at about 4 x

10-5 (IU/L)/FU of ALP activity with respect to cell fluorescence during cell proliferation,

ECM synthesis and mineralisation.

The amount of newly produced calcium on the scaffolds was used to determine the extent of

mineralisation of the collagen matrix on the scaffolds; the results are presented in Figures 7

(j), (k), (l). Generally, the amount of calcium produced during collagen matrix mineralisation

was low and not significantly different between scaffolds during the first 7 days (p > 0.05),

corresponding to constant low levels of ALP activity by the cells in Figures 7(g), (h), (i),

which implies that there was no significant mineralisation taking place during this period.

Between days 7 and 18, there were gradual increments in the calcium amounts (p < 0.05), as

a result of increasing matrix mineralisation, also consistent with increased ALP activity by

the hFOB cells. Figure 8 demonstrates the presence of mineral deposits after 18 days of cell

culture on the 25 wt% HA-gelatin scaffold (20 kV).

Fluorescence microscopy and SEM were undertaken on the hFOB seeded 25 wt% HA-gelatin

scaffolds electrospun at 20 kV, and 20 kV pure gelatin counterpart for comparison, since they

outperformed all the other scaffolds based on the biological characterisation results. Green

(live cells) and red (dead cells) fluorescence images are displayed in Figure 9. Table 3

presents the values of cell viability for the two scaffolds as a function of cell culture time

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using the green and red fluorescence images. At day 1, there was a similarity in cell

morphology on the pure gelatin and the 25 wt% HA-gelatin scaffolds. The live cells were

mostly singular and round-shaped, as shown in Figures 9(a) and (f). From the day 1 images,

the average diameter of the hFOB cells was determined to be 18 ± 3 µm (n = 40 cells) using

the Digimizer® software. Cell morphologies were different on the scaffolds as the culture

time increased. On days 4 (Figures 9(b) and (g)), 7 (Figures 9(c) and (h)) and 11 (Figures

9(d) and (i)), there was more clumping together of live cells (denser cellular areas) on the 25

wt% HA-gelatin scaffolds than on the pure gelatin ones, probably as a result of more rapid

cell division taking place in the former. The population of dead cells on both scaffolds also

gradually increased although the overall cell viability was very good (increased) for both

scaffolds, as shown in Table 3, and the differences were not statistically significant (p >

0.05). By day 14 of cell culture (Figures 9(e) and (j)), both the larger cell clumps on the 25 wt

% HA scaffolds and the smaller cell clumps on the pure gelatin scaffolds were fairly

uniformly distributed throughout the scaffolds. However, by day 18, Table 3 shows that cell

viability started decreasing on both scaffolds probably due to overcrowding and reduced

access to nutrients.

Figure 10 displays SEM micrographs illustrating the attachment and proliferation of hFOB

cells on the pure gelatin and 25 wt% HA scaffolds and the subsequent production of mineral

deposits. On day 1 (Figures 10(a) and (e)), the hFOB cells were attached to portions of the

scaffolds with plenty of fibres visible and the cells appeared to align along the direction of

fibres; also illustrating the much smaller fibre diameters and pore size of the 25 wt% HA

scaffold compared to the pure gelatin scaffold. Due to cell proliferation, the cells spread

across the scaffolds and covered more areas of the scaffold surfaces at days 4, 7 and 11 of

cell culture, with more coverage on the 25 wt% HA scaffold than on its pure gelatin

counterpart by virtue of the greater cell proliferation rates on the former. By day 14, there

19

was near total surface coverage on the 25 wt% HA scaffold (Figure 10(h)), and a few

mineralised nodules were observed whereas some fibres were still visible on the pure gelatin

scaffolds (Figure 10(d)).

4. Discussion

Gelatin-hydroxyapatite (HA) fibre scaffolds were fabricated by electrospinning around a

rotating, axially wired drum collector with the fibres generally oriented in the circumferential

drum direction. The scaffolds were subsequently crosslinked in glutaraldehyde vapour for 2

hours. About 75% of the fibres were aligned within ±15o for the 25 wt% HA-gelatin fibre

scaffold which is a novel feature of HA-gelatin scaffolds compared to past studies of such

random fibre orientation scaffolds used for bone tissue engineering [13, 14]. This

electrospinning study has, in fact, achieved much better fibre orientation than Elsayed et al

[21] due to the distributed wires in the rotating collector of the present work. The present

study has also surpassed our previous study [18] in improving fibre orientation by increasing

the collector rotation speed from 2 rpm [18] to 30 rpm and also in improving productivity rate

by increasing the feed rate from 1.5 ml/h [18] to 5 ml/h.

FTIR spectra indicated the strong presence of phosphate groups in the composite fibre

scaffolds. The presence of weak CO3 bands in the spectra of composite samples also

indicates the substitution of a few of the phosphates in the HA particles by CO 3 and confirms

the presence of carbonate-containing apatites. There were slight shifts in the gelatin

characteristic bands in the composite FTIR spectra compared to the pure gelatin spectra. This

signified the presence of some nanoscale interactions between the gelatin matrix and HA

particles, most probably between Ca2+ ions of the HA and the RCOO- ions of the gelatin

matrix whereby the cations interfered with the surrounding electron clouds to cause peak

shifts. The XRD analyses indicated the formation of nanocrystalline but poorly crystallised

20

HA particles. The AFM force maps illustrated that the HA solid nanoparticles were

concentrated in the central core of the electrospun fibres which can be attributed to the

elongational flow during electrospinning under simultaneous solvent evaporation at the fibre

surface. However, HA nanoparticles are still expected to be well dispersed in the outer gelatin

shell of each fibre offering a large surface area of HA and apatite groups to the cells in the

phase of cell attachment and growth. Dynamic water contact angle measurements (Table 2)

revealed that all scaffolds were hydrophilic with a decreasing contact angle upon spreading of

water, with paricularly high hydrophilicity of the 25 wt% HA-gelatin scaffolds; to our

knowledge, no CAG tests have been performed for gelatin-HA scaffolds of past studies.

Gelatin is a key component for the improvement of hydroplilicity, in fact in the past it was

added to PCL increasing the hydrophilicity of PCL/gelatin electrospun scaffolds versus

scaffolds made of PCL only, leading to an increase in NIH3T3 fibroblast cell proliferation

[22].

The microstructural data of all scaffolds presented in Table 2 (for wet scaffolds) were

assembled in the graphs of Figure 11 and it was possible to draw two master curves

indicating the relation between fibre diameter and pore size as well as the relation between

fibre diameter and porosity. The results show clearly that scaffolds with smaller fibre

diameters were fabricated by increasing the applied electrospinning voltage or at higher HA

concentrations. As the feed rate was kept the same for all electrospinning runs, the same mass

of material was deposited on the collector which means that thinner fibres would be packed

more closely resulting in smaller pore size for all scaffolds as is demonstrated in the master

curve of dp,wet versus df,wet in Figure 11. On the other hand, the porosity increased with fibre

diameter up to about 60 % for a fibre diameter of about 2 m and stayed approximately the

same for tested fibre diameters up to 5.5 m. Crosslinked scaffolds were fabricated with

21

average wet pore diameters ranging from 7 to 32 m and corresponding wet porosity in the

range 40 to 60%.

The microstructure of the scaffolds has a direct effect on their mechanical properties with the

scaffold having the lowest porosity and pore size (25 wt% HA-gelatin, electrospun at 30 kV)

exhibiting the highest Young’s modulus (925 MPa) and tensile strength (9.8 MPa) in the

main fibre direction. These values are the highest for any gelatin-HA or collagen-HA fibre

scaffolds previously reported in the literature [13,18], more than double the values reported

by Kim et al [13], and can be attributed to the high degree of fibre orientation achieved in this

study, surpassing even that of our previous study on gelatin-HA nanocomposite fibre

scaffolds [18] by increasing the collector rotation speed by 15 times in the present study. The

effect of fibre orientation has been also recognised in the conclusion drawn by Fee et al [23]

that there is a linear relationship between the degree of fibre orientation of PCL/gelatin

scaffolds and their tensile properties: In another study, fibre orientation of electrospun

PCL/HA/poly(vinyl alcohol) scaffolds is reported to have increased the Young’s modulus

two-fold compared to random fibre scaffolds [24].

The next phase involved cell seeding on scaffolds and biological characterisation as a

function of cell culture time. As seen in Table 2, cell seeding efficiency was observed to be

over 90% after 3 hours of seeding. There were small improvements in seeding efficiency for

scaffolds with increased HA concentration compared to pure gelatin scaffolds although the

differences were not statistically significant (p > 0.05). There was also higher cell growth for

all scaffolds electrospun at lower applied voltage. This can be explained by the larger pores

of scaffolds electrospun at lower voltage, which favours migration of the attached large

hFOB cells the size of which was determined to be about 18 m. Increased cell migration and

homogeneous population of the scaffold by cells allows for enhanced growth of cells as there

22

are no spatial constraints from pore saturation with cells and the culture medium has access

though the scaffold to provide nutrients across the whole scaffold during the tested maximum

incubation period. This is consistent with the results of previous studies on the effect of the

fibre size and pore size of electrospun PLLA-based scaffolds [8] and electrospun gelatin [10]

on the growth of mouse pre-osteoblast cells that indicated that cellular proliferation was

greater on larger fibres and larger pores than on smaller ones.

Osteoblasts, like many other cells, contain integrins on their surfaces that enable them to

attach to extracellular proteins such as collagen, fibronectin [25] and vitronectin [26] on the

surface of the scaffold at their arginine-glycine-aspartate (RGD) sites to facilitate cell

attachment to the scaffold [26]. Gelatin, a collagen derivative, has RGD sites and favours cell

attachment. Serum protein adsorption on scaffolds has also been found to promote osteoblast

cell attachment [26], most probably due to fibronectin and vitronectin as previously reported

for, mouse osteoblast cells on porous bioactive glass and hydroxyapatite [26] and human

osteoblast cells on nanoporous titanium [27]. The scaffolds in this study were saturated with

serum-based culture medium and the adsorbed fibronectin and vitronectin were probably

bound by integrins on the surface of hFOB cells, thereby improving cell attachment and

seeding efficiency.

The presence of either or both of Ca2+ and Mg2+ ions in a scaffold upregulates the binding

affinity and specificity of integrins to ligands [25]. Thus, the presence of Ca2+ ions in the HA-

gelatin scaffolds could have contributed to improvement in cell attachment. The total surface

area of the scaffold available to cells (specific pore surface area, SSA) has an inverse

relationship with pore size. A decrease in pore size of collagen-glycosaminoglycan scaffolds,

i.e. increased SSA, resulted in increased attachment of mouse pre-osteoblast cells [28-29]. It

is clear from Table 2, that as the HA content is increased in the electrospun scaffolds from 0

23

to 25 wt%, fibre diameter and pore size decrease but the estimated specific pore surface area,

SSA, increases 3 to 6 times, which is expected to have contributed to higher seeding

efficiency.

Overall, increasing the HA concentration in the scaffolds also increased cell proliferation,

despite the corresponding pore size reduction, due to the increased cell attachment which is

an important factor for cell survival and proliferation. The beneficial effect of HA on

osteoblast cell growth and osteogenesis concluded here is in agreement with previous studies

[9,11,13-14]. It is also consistent with the findings of Zhou et al. [30] that the presence of HA

in collagen-HA electrospun scaffolds increased human myeloma cell viability and

differentiation (ALP activity). Thus, cells seeded on the 25 wt% HA 20 kV scaffold in this

study had the highest proliferation rates.

The graphs of collagen production follow the corresponding graphs of cell proliferation in

Figures 7 as expected; they produced linear positive correlation between collagen production

and fluorescence intensity with the same collagen productivity factor for all scaffolds of

0.002 (g/L)/FU, where a correlation between the cell number and their emitted fluorescence

may be considered from the initial cell adherence and fluorescence data which was calculated

as 4.5 cells/FU for the 20 kV gelatin scaffold and 4.3 cells/FU for the 20 kV 25 wt% HA

scaffold.

ECM mineralisation is the last stage of osteoblast differentiation starting after day 7 of cell

culture in Figures 7(j)-(l) where the increments of the produced calcium amount are

consistent with the increased expression of ALP activity by the hFOB cells during that period

as presented in Figures 7(g)-(i). During that period between days 7 and 18, calcium

productivity with respect to cell fluorescence unit was higher for scaffolds electrospun at 20

kV varying for different scaffold compositions as follows: 8x10-6 mM/FU for pure gelatin

24

scaffolds, 10x10-6 mM/FU for 10 wt% HA-gelatin scaffolds, 7x10-6 mM/FU for 20 wt% HA-

gelatin scaffolds and 6x10-6 mM/FU for 25 wt% HA-gelatin scaffolds. On the other hand, an

average ALP activity of about 4x10-5 (IU/L)/FU between days 7 and 18 can be derived for all

scaffolds from Figures 7(g)-(i).

The presence of HA at 10 wt% in the HA-gelatin scaffold seems to increase the calcium

productivity factor with respect to cell concentration but further increase of HA concentration

brings this factor down. We believe that as increased HA concentration in the scaffold

enhances cell proliferation and, hence, overall calcium production as seen in Figure 7(l), the

scaffold fills up with calcium quickly which probably contributes to reduce calcium

productivity per cell due to spatial constraints. The cell culture duration, the scaffold type (in

terms of electrospinning voltage and HA concentration) and their interactions had significant

effects (p < 0.05) on cell proliferation, alkaline phosphatase activity and collagen production

and mineralization. Overall, the 20 kV electrospun 25% HA-gelatin scaffolds contained the

highest amount of mineralised collagen matrix, outperforming all the other scaffolds.

The rate of synthesis and mineralisation of the ECM on the scaffolds depends on the level of

cell attachment, proliferation and differentiation of the hFOBs, which are influenced by the

HA concentration, as highlighted above, but also the degree of fibre orientation of the

scaffolds. The latter has been shown by some researchers to provide topographical cues to

regulate cell morphology and orientation [22, 31], proliferation and differentiation [24]

compared to random fibre scaffolds. It was also reported that PCL/gelatin scaffolds with

oriented fibres upregulated actin and focal adhesion related genes in NIH3T3 fibroblast cells

[22] that probably led to cell orientation and proliferation parallel to the direction of fibre

orientation. As there were only small variations in the degrees of fibre orientation (within

±15°) between the scaffolds developed in this study, as shown in Figure 2, it can be suggested

25

that the HA concentration played the major role in the observed differences in cell

proliferation, differentiation and ECM production among the scaffolds of the present study.

5. Conclusions

As expected, the gelatin matrix of the crosslinked electrospun aligned fibre scaffolds proved

an excellent protein ligand for cell attachment. This led to 90% seeding efficiency for gelatin

scaffolds rising to 96% for the 25 wt% HA-gelatin scaffolds. Scaffolds electrospun at 20 kV

or with higher HA concentration favoured cell proliferation. Cell viability was high, generally

above 90% during cell culture. ECM production was closely related to cell proliferation such

that collagen productivity amounted to about 0.002 (g/L)/FU for all scaffolds.

Mineralisation took place from day 7, with measured ALP activity between days 7 and 18

averaging at about 4x10-5 (IU/L)/FU with corresponding increased calcium concentration.

The high degree of fibre alignment in the scaffolds (rather than random fibre orientation of

past studies and even better orientation than in our previous study [18]) and the formation of

HA-gelatin nanocomposites (rather than microcomposites of past studies) reinforced the

mechanical properties where the 25 wt% HA-gelatin scaffolds exhibited the highest

mechanical performance, a Young’s modulus of 0.9 GPa and tensile strength of 10 MPa for

scaffolds electrospun at 30 kV, and the best cell proliferation, ECM production and

mineralisation after 18 days for scaffolds electrospun at 20 kV. As a result of these

conclusions, the superior 25 wt% HA-gelatin scaffolds of this study have been selected to be

further investigated in the fabrication of multilayer cellular scaffold stacks for bone tissue

engineering [32].

6. References

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30

Table 1. Rheology of solutions used for the electrospinning: power law parameters fitted

according to experimental data (standard errors have been determined from the deviation of

the experimental data from the power-law fit lines)

Solutions of 10

%w/v solid in TFE;

Solid composition: 100 % Gelatin 10 wt% HA-

90 wt% gelatin

20 wt% HA-

80 wt% gelatin

25 wt% HA-

75 wt% gelatin

Shear flow:

o (mPa s)

n1

6070 ±25

-0.62

4770 ±25

-0.56

4710 ±20

-0.59

5290 ±25

-0.54

Elongational flow:

el,o (mPa s)

n2

44 ±5

-1.1

12 ±6

-1.4

17 ±2

-1.4

13±2

-1.4

Relaxation time:

trel,o (s)

n3

9512±20

-2.24

42±25

-1.34

5244±20

-1.92

120±20

-1.69

31

Table 2. Microstructure parameters of the wet, crosslinked, electrospun scaffolds: average

fibre diameter df,wet (±0.08m), pore size dp,wet(±16%) and porosity calculated

from measurements from 25 images from 5 samples, estimated specific pore surface area

(SSA), water contact angle goniometry (CAG) (from 5 dynamic CAG tests of different

samples, ), mechanical tensile properties in the fibre direction calculated from 6 tests of

different samples, and also cell seeding efficiency for cells seeded on scaffolds electrospun at

20 kV.

Scaffold

composition

Pure Gelatin 10 %wt HA -

Gelatin

20 %wt HA -

Gelatin

25 %wt HA -

Gelatin

Voltage (kV) for

electrospinning

20 25 30 20 25 30 20 25 30 20 25 30

df,wet(m) 5.2 4 3.4 1.8 1.6 1.4 1.5 1.1 1.0 1.2 1 0.7

dp,wet(m) 31.7 25.4 24.5 9.5 8.8 8.2 8.8 8.2 7.8 7.8 7.5 7.2

wet 0.59 0.59 0.55 0.61 0.55 0.52 0.60 0.52 0.50 0.49 0.45 0.41

SSA (m2/g) 0.74 0.94 1.09 2.00 2.23 2.53 2.31 3.10 3.36 2.83 3.35 4.67

o (o)

t (o)

tspread (s)

30

12

17

20

20

2

22

12

1

70

28

2

31

10

1

25

10

0.6

40

27

1

27

26

1

29

28

1

20

5

0.7

18

5

0.7

16

4

0.5

Young’s modulus

(MPa)

255

±70

278

±50

282

±60

280

±70

286

±120

404

±100

327

±30

400

±110

452

±110

490

±120

625

±200

925

±100

Ultimate tensile

strength (MPa)

3

±1.7

4

±1.5

4.4

±1.2

1.9

±1.2

2.8

±0.3

3.4

±1.0

4.5

±1.5

4.3

±2.5

4.3

±2.0

4.1

±0.4

6.4

±3

9.8

±2.6

Ultimate tensile

strain

0.10

±0.02

0.10

±0.02

0.11

±0.03

0.04

±0.01

0.04

±0.01

0.02

±0.01

0.08

±0.01

0.05

±0.01

0.06

±0.01

0.05

±0.01

0.05

±0.02

0.032

±0.005

Cell seeding

efficiency (%)

90±4 93±5 94±2 96±2

32

Table 3: The hFOB cell viability on the 20 kV scaffolds at various stages in static culture

obtained from analysis of the fluorescence images using JMicroVision toolbox (n = 3). No

significant differences between pure gelatin and 25 wt% HA cell viability (p < 0.05).

Days in

Culture

Cell Viability (%) (Mean ± Standard Deviation)

Pure Gelatin 20 kV

Scaffold

25 wt% HA 20 kV

Scaffold

1 90 ± 4 87 ± 3

4 92 ± 4 90 ± 3

7 89 ± 2 92 ± 6

11 92 ± 5 94 ± 5

14 93 ± 1 97 ± 1

18 91 ± 5 95 ± 3

33

FIGURES

Figure 1. Proton NMR spectra of (10 %w/v solid in TFE) solutions in CDCl3 NMR solvent,

for four different solid compositions: 100% gelatin; 90 wt% gelatin – 10 wt% HA; 80 wt%

gelatin – 20 wt% HA; and 75 wt% gelatin – 25 wt% HA.

34

Figure 2. Fibre orientation distribution for crosslinked fibre scaffolds electrospun at 20 kV.

35

Figure 3. AFM micrographs of the 25 wt% HA scaffold electrospun at 20 kV: (left)

topography and (right) force map of the surface of the scaffold.

36

Figure 4. X-ray diffractograms of the gelatin–HA composites; (a) pure gelatin reference (b)

10 wt% HA (c) 20 wt% HA and (d) 25 wt% HA. The asterisk (*) symbols show the HA

characteristic peaks accompanied by the corresponding lattice plane numbers.

37

Figure 5. FTIR spectra of pure gelatin and gelatin–HA nanocomposites showing the

characteristic peaks for gelatin matrix (**) and hydroxyapatite particles (*) and their

corresponding band assignments.

38

Figure 6. Stress-strain curves from tensile tests of crosslinked electrospun scaffolds (a)

parallel to the main fibre direction (b) perpendicular to the main fibre direction. Scaffold type

(with respect to electrospinning voltage, HA concentration and fibre direction) had significant

effects on the mechanical properties of scaffolds (p < 0.05).

39

Figure 7: Graphs of hFOB cell growth and ECM production as a function of cell incubation

time: (a), (d), (g), (j) (a) in scaffolds of pure gelatin and (b), (e), (h), (k) in 25 wt% HA-

gelatin scaffolds, produced by electrospinning at different voltages; (c), (f), (i), (l) in 20 kV

electrospun gelatin scaffolds with various HA concentrations. (a), (b), (c) Fluorescence

intensity graphs (excitation/emission: 540/590 nm) of hFOB cells (representing amount of

cells). (d), (e), (f) Graphs of the amounts of collagen synthesised by the hFOB cells and

40

secreted onto the scaffold during new collagen matrix synthesis. (g), (h), (i) Graphs of the

expression of alkaline phosphatase activity by the hFOB cells growing on different scaffolds.

Both cell culture duration and scaffold type (with respect to electrospinning voltage and HA

concentration) had significant effects on cell proliferation, alkaline phosphatase activity,

collagen and calcium production (p < 0.05).

41

Figure 8: SEM micrographs at different magnifications of the mineralised bone nodules after

18 days of cell incubation on the 25 w% HA scaffold electrospun at 20 kV.

42

Figure 9: Fluorescence images of live (green) and dead (red) hFOB cells on (a)-(e) pure

gelatin 20 kV scaffolds and (f)-(j) 25 wt% HA-gelatin 20 kV scaffolds, as a function of cell

incubation time at (a),(f): day 1 in culture; (b),(g) day 4 in culture; (c),(h) day 7 in culture;

(d),(i) day 11 in culture; (e),(j) day 14 in culture. Scale bar = 100 m.

43

Figure 10: SEM micrographs of the hFOB cells attached to 20 kV electrospun scaffolds of

(a)-(d) pure gelatin and (e)-(h) 25 wt% HA-gelatin, as a function of cell incubation time at

(a),(e): day 1 in culture; (b),(f) day 4 in culture; (c),(g) day 7 in culture; (d),(h) day 14 in

culture. Scale bar = 200 m.

44

Figure 11. Master curves fitting the average values of the microstructural parameters of all

wet, crosslinked, electrospun scaffolds investigated in this study: solid symbols and solid line

are associated with the left hand side vertical axis (dp,wet-pore size of wet scaffold); open

symbols and broken line are associated with the right hand side vertical axis (porosity of wet

scaffold).

45