Influence of Nonsulfated Polysaccharides on the Properties of

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The influence of non-sulphated polysaccharides on the properties of electro-spun poly(lactic-co-glycolic acid) fibres Ayesha Azeem, Lucia Marani, Kieran Patrick Fuller, Kyriakos Spanoudes, Abhay Pandit, and Dimitrios I Zeugolis ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00206 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 11, 2016

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ACS Biomaterials Science & Engineering

Title The influence of non-sulphated polysaccharides on the properties of electro-spun poly(lactic-coglycolic acid) fibres

Authors A Azeem * (1, 2), L Marani * (1, 2), K Fuller (1, 2), K Spanoudes (1, 2), A Pandit (2), DI Zeugolis † (1, 2)

Affiliations (1) Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland (2) Science Foundation Ireland (SFI) Centre for Research in Medical Devices (CÚRAM), Biomedical Sciences Building, National University of Ireland Galway (NUI Galway), Galway, Ireland

*

AZ and LM share first authorship

†

Corresponding Author: Email: [email protected]; Tel: +353 (0) 9149 3166; Fax:

+353 (0) 9156 3991

ACS Paragon Plus Environment


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Abstract Biomimetic tissue engineering aspires to develop bio-inspired implantable devices that would positively interact with the host. Given that glycosaminoglycans are involved in many physiological processes, whilst their deprivation is associated with pathophysiologies, functionalisation of implantable devices with natural and/or synthetic carbohydrate moieties is at the forefront of scientific research and industrial innovation. Herein, we venture to assess the influence of various concentrations (0.01 %, 0.1 %, 1 %) of hyaluronic acid and Ficoll™ on the structural, thermal, biomechanical and biological (human osteoblasts) properties of electro-spun poly(lactic-co-glycolic acid) fibres. The addition of hyaluronic acid and Ficoll™ reduced the fibre diameter, with the 1 % hyaluronic acid exhibiting the smallest fibres diameter (p < 0.001). Neither the addition of hyaluronic acid nor the addition Ficoll™ significantly affected the onset and peak temperatures (p > 0.05). All hyaluronic acid and Ficoll™ treatments significantly reduced stress at break, strain at break and elastic modulus values (p < 0.001). Hyaluronic acid and Ficoll™ did not affect temperatures (p > 0.05) osteoblast viability and metabolic activity; the 1 % hyaluronic acid and Ficoll™ significantly increased (p < 0.001) osteoblast proliferation at day 21. By day 21, the 1 % hyaluronic acid and 1 % Ficoll™ fibres showed the highest alkaline phosphatase activity and calcium deposition. At day 21, osteocalcin was not detected, whilst osteopontin was detected on all samples. Collectively, our data clearly illustrate the biological benefit of non-sulphated polysaccharides as functionalisation molecules.

Keywords Electro-spinning; Functionalised scaffolds; Glycosaminoglycans; Hyaluronic acid; Ficoll™; Osteoblast response


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Introduction Advancements in engineering, materials science, polymer chemistry and biology have made available numerous nano- to micro- fabrication processes and functionalised osteoinductive biomaterials to meet the increasing worldwide demand for functional implantable devices for bone repair and regeneration 1. Among the various fabrication technologies, electro-spinning has been favoured for biomedical applications, as it produces two- and three- dimensional scaffolds that closely imitate the structural and physical properties of native bone tissue and offers the opportunity to deliver therapeutics, biologics and viable cell populations at the site of interest 2. To further enhance biomimicry, hydroxyapatite has been primarily incorporated into these scaffolds to improve mineralisation, osteogenesis and cell recruitment, adhesion and proliferation 3. Growth factors have also been incorporated into electro-spun meshes and the resultant scaffolds have been shown to significantly improve osteogenesis in vitro and in vivo. Specifically, electro-spun meshes loaded with FGF2-FGF18 have been shown to significantly stimulate in vitro rat mesenchymal stem cell proliferation, induction of alkaline phosphate activity and mineralisation and to significantly enhance bone-forming ability, in terms of bone volume and density, in vivo 4. PDGF-BB loaded scaffolds have been shown to induce increased expression of osteogenic markers of human mesenchymal stem cells 5. VEGF loaded scaffolds promoted attachment, proliferation and alkaline phosphatase production of human osteoblasts, whilst histological analysis revealed new bone matrix formation after 10 weeks of implantation 6. BMP-2 / dexamethasone loaded scaffolds promoted osteoblast differentiation in vitro and enhanced osteogenesis in vivo in a critical sized rat calvarial defect model 7. Stromal cell derived factor-1α induced stimulated chemotactic migration of stem cells in vitro and increased the amount of bone formed in vivo 8. However, the short half-life of growth factors 9, along with the literally infinite number of permutations (e.g. concentration, combination and timing) has triggered investigation into alternative functionalisation molecules. Glycosaminoglycans (GAGs) are crucial in numerous physiological processes, whilst their absence or down-regulation is associated with pathophysiologies

10

. Specifically to bone tissue, heparin



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sulphate is key regulator of endochondral ossification and osteochondroma development

11

. Of

significant importance is in vivo data demonstrating the superiority of heparin sulphate over BMP-2 in promoting critical size bone defect healing 12, possibly due to the recruitment and sequestering of host growth factors at the side of injury 13. Further, chondroitin sulphate and glucosamine have been used extensively in clinic for osteoarthritis management

14

. Heparin has been shown to increase

mRNA expression of alkaline phosphatase and BMP-4, to promote mineralisation and overall to support osteogenic differentiation of stem cells in vitro 15. A heparin / bFGF / poly-ε-caprolactone scaffold

16

and a heparin / methacrylamide / BMP-2 scaffold

17

enhanced cell attachment and

proliferation and induced high alkaline phosphate activity. A heparin / BMP-2 / titanium scaffold improved osteoblast function in vitro and new bone formation in vivo

18

. A heparin / collagen /

hydroxyapatite / VEGF scaffold improve mineralisation and stimulated vascularisation 19. Although hyaluronic acid (HA), the sole non-sulphated GAG, has extensively been studied in tendon repair and regeneration 20, its potential in bone repair and regeneration has only recently started taking off. Specifically, chitosan / HA containing calcium phosphate cements have been shown to support growth and enhance alkaline phosphatase activity in osteoblast culture 21. HA inhibited expression of MMP and ADAMTS in murine osteoblast culture 22. A HA / gelatin / biphasic calcium phosphate scaffold resulted in a significant increase in stem cell proliferation and high alkaline phosphatase activity in vitro, whilst in vivo, it induced rapid new bone formation and a high rate of collagen mineralisation

23

. Although low molecular weight HA has been shown to effectively maintain

murine embryonic stem cells in a viable and undifferentiated state 24, high molecular weight HA has been shown to decrease cell adhesion, as a function of increasing the concentration of HA, to increase mRNA expression of alkaline phosphatase, osteocalcin and RUNX-2 and to promote bone formation

25

. Of significant importance is recent data demonstrating the superiority of HA, over

chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate and heparin, in osteogenic differentiation of human bone marrow mesenchymal stem cells and bone matrix synthesis

26

.

Further, although increasing data demonstrate the suitability of GAG-derivatives in the design of


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functional biomaterials

27

, only the potential of sulphated polysaccharides has been assessed for

bone repair 28. Thus, herein we ventured to assess the influence of HA (high molecular weight nonsulphated polysaccharide with an unbranched backbone composed of alternating sequences of β-(14)-glucoronic acid and β-(1-3)-N-acetyl-glucosamine moieties) and Ficoll™ (FC; high molecular weight

non-sulphated

sucrose-polymer

formed

by

copolymerisation

of

sucrose

with

epichlorohydrin) on the structural, thermal, physical and biological properties of electro-spun scaffolds. FC is a neutral non-sulphated polysaccharide that has been shown to enhance ECM deposition, when it is used as macromolecular crowder

29, 30

and to enhance cell attachment and

growth, when it is used as functionalisation molecule 31.



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Materials and Methods

Fabrication of electro-spun scaffolds Typical protocols for electro-spinning

32-35

and functionalisation

36-40

protocols were utilised.

Briefly, 8 % w/v (80 mg/ml) 85/15 poly(lactic-co-glycolic acid) (PLGA, Purac, The Netherlands) was vortexed until dissolved in 2,2,2-trifluoroethanol (TFE, Sigma-Aldrich, Ireland) and was extruded at 10 µl/min through an 18 G stainless steel needle (BD, Ireland). Upon application of high voltage (~12 KV) between the needle and the rotating (1,400 revolutions per minute) collector (~18 cm distance), the solvent was evaporated and the fibres were collected on the aluminium collector. 0.01 %, 0.1 % and 1.0 % w/v (0.1, 1 and 10 mg/ml) solutions of HA (851,000 – 1,190,000 Da; Lifecore Biomedical, Chaska, MN, USA) and FC (1,000,000 Da; TdB Consultancy, Sweden) were suspended in TFE using sonication amplitude of 40 % for 30 min. An 8 % w/v PLGA was added to these solutions and following vortexing, they were electro-spun as described above. All electrospinning experiments were carried out at room temperature (22 to 26 °C) and 55 to 73 % relative humidity.

Fibre morphology assessment Electro-spun mats were mounted onto a carbon tape, gold-sputter coated and imaged using a Hitachi Scanning Electron Microscope S-4700 (Hitachi High-Technologies Europe GmbH, Germany). Fibre diameter analysis was conducted using NIH ImageJ software.

Differential scanning calorimetry (DSC) assessment Thermal analysis was carried out using the DSC-60 (Shimadzu Corp, Japan) equipment, based on well-established protocols 41. Briefly, all scaffolds were rehydrated in phosphate buffer saline (PBS) overnight at room temperature. Using tissue paper excess PBS was removed and subsequently heating was applied at a constant temperature ramp of 10 °C / min in the temperature range of 25


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°C and 100 °C. The endothermic transition was recorded as a peak. Onset (temperature at which the tangent to the initial power versus temperature line crosses the baseline) and peak (temperature of maximum power absorption) temperatures were recorded.

Mechanical assessment Mechanical properties were assessed under uniaxial tension, using a Zwick/Roell (Leominster, Herefordshire, UK) Z005 testing machine, loaded with a 10 N load cell, as has been described previously

42, 43

. The samples were pre-cut into a dog-bone shape, as per ASTM D882-2010

guidelines. Prior to testing, all samples were incubated overnight at room temperature in PBS and tissue paper was used to remove excess PBS. The samples thickness was measured using digital callipers (Scienceware®, Digi-Max™, Sigma-Aldrich, Ireland). The samples were hand-tightened between the vertical grips, which were set at 5 cm gauge length. Scaffolds that broke at contact points with the grips were rejected from the analysis. The extension rate was set at 10 mm/min. The following definitions were used to calculate mechanical data: stress at break was defined as the load at failure divided by the original cross-sectional area (engineering stress), strain at break was defined as the increase in scaffold length required to cause failure divided by the original length and modulus was defined as the stress at 0.02 strain divided by 0.02.

Osteoblast culture Primary human osteoblasts were derived from femoral head biopsies of male subjects (Lonza, USA and PromoCell, Germany). The cells were cultured in Dulbecco’s modified Eagle’s medium (1000 mg glucose, Sigma Aldrich, Ireland) supplemented with 10 % foetal bovine serum and 1 % penicillin/streptomycin (Sigma Aldrich, Ireland). The osteoblast culture was maintained at 37 °C in a humidified 5 % CO2 incubator, until the cells were approximately 80 % confluent. The culture media was changed every 4 days. The cells (passage 4 to 5) were seeded for 1, 11 and 21 or 7, 14 and 21 days on the functionalised substrates in 24-well plates (Lab-Tek®, UK) at a cell density of 4



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x 104 / 1.9 cm2. For mineralisation studies, the osteoblast growth medium was supplemented with 50 µg/ml ascorbic acid, 10 nM dexamethasone and 5 mM β-glycerophosphate.

Osteoblast metabolic activity, viability and proliferation assessment Cell metabolic activity was determined on days 1, 11 and 21 using alamarBlue® assay (Thermo Scientific, UK). Briefly, alamarBlue® dye was diluted with Hank’s balance salt solution (HBSS, Sigma Aldrich, Ireland) to make a 10 % (v/v) alamarBlue® solution. Media was removed from each well and 0.2 ml alamarBlue® solution was added to each well. The cells were incubated for 4 h at 37 °C and then absorbance was measured at wavelengths of 570 nm and 600 nm using a microplate reader (Varioskan Flash, Thermo Scientific, UK). The level of metabolic activity was calculated using the simplified method of calculating % reduction, according to the supplier’s protocol. Cell viability was assessed at days 1, 11 and 21, using the Pierce lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (CytoTex 96®, Promega), as per manufacturer’s protocol. Briefly, the LDH released into the medium was transferred to a new plate and mixed with Reaction Mixture. After 30 min at 37 °C, reactions were stopped using the Stop Solution. Absorbance at 490 nm and 680 nm was measured using a microplate reader (Varioskan Flash, Thermo Scientific, UK) to determine LDH activity. To assess osteoblast proliferation, the total viable cell number was calculated by counting the DAPI stained nuclei (see section 2.9) from six randomly selected images from each group at a given time point.

Osteoblast alkaline phosphatase (ALP) activity assessment ALP activity of osteoblasts was measured on days 7, 14 and 21. At each time point, cells were washed x2 with HBSS to remove media serum proteins. The cell layers were then lysed (0.1 % Triton-X in 10mM Trizma-Base, pH 7.4; Sigma Aldrich, Ireland) at 4-8 °C under agitation for 1 h. 250 µl alkaline buffer (1.5 M, at pH 10.3, Sigma Aldrich, Ireland) and 50 µl substrate solution (4nitrophenyl phosphate, Sigma Aldrich, Ireland) were dissolved in 1 M diethanolamine buffer (in 0.5


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mM MgCl2; Sigma Aldrich, Ireland) and 100 µl of cell lysate and 100 µl of H2O were added. Following incubation at 37 °C for 30 min, the reaction was stopped using 500 µl of 0.1 M NaOH. Using p-nitrophenol (10 mM, Sigma Aldrich, Ireland) as standard, the optical density (OD) was measured at 405 nm (Varioskan Flash, Thermo Scientific, UK). The activity of ALP was expressed as the OD divided by the incubation time and the total protein content was assayed by the Bio-Rad Laboratories (UK) protein assay. All materials for this assay were bought from Sigma Aldrich (Ireland).

Osteoblast mineralisation assessment Osteoblasts mineralisation was assessed using alizarin red staining at days 7, 14 and 21. Cells were fixed in 10 % formalin (Sigma Aldrich, Ireland) for 30 min at room temperature and then stained with 40 mM (pH 4.1) alizarin red (Sigma Aldrich, Ireland) for 30 min, before washed with distilled H2O. Stained monolayers were visualised by bright-field microscopy, using an inverted microscope (Leica S 40/0.45, 10X). Alizarin red was extracted and quantified by adding 10 % v/v acetic acid to each sample for 30 min. The slurry was overlaid with mineral oil (Sigma Aldrich, Ireland) and heated to 85 °C for 10 min, then transferred to ice for 5 min, followed by centrifugation at 20,000 g for 15 min. 10 % v/v ammonium hydroxide was added to the supernatant to neutralise the acid, the pH was confirmed to be between 4.1 and 4.5. Aliquots of the supernatant were read at 405 nm in 96-well plate using a microplate reader (Thermo Scientific, Varioskan Flash). The results were normalised to total cellular protein values, measured in cell lysates by the Bradford method (BioRad Laboratories, UK).

Immunofluorescence assessment of osteocalcin (OCN) and osteopontin (OPN) At the end of the culture time, the cells were fixed with 4 % paraformaldehyde for 30 min at room temperature. The cells were then washed x3 with phosphate buffered saline (PBS, Sigma Aldrich, Ireland) and permeabilised with 0.2 % TritonX-100 (Sigma Aldrich, Ireland) for 10 min at room



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temperature, followed by x3 5 min washes with PBS. The cytoskeleton of the cells was stained with rhodamine-conjugated phalloidin (Molecular Probes, Ireland) for 1 h and the nuclei were then stained with 4,0,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Ireland) for 5 min. Three 5 min washes with PBS were carried out between and after these stains. Following permeabilisation with Triton X (see above), all samples were incubated for 1 hour at room temperature in 1 % BSA / PBS followed by addition of anti-OCN antibody (1:100, v-19, sc-18319, Santa Cruz Biotechnology, USA) / anti-OPN antibody (1:100, k-20, sc-10591, Santa Cruz Biotechnology, USA) and incubated overnight at 4 °C. The cells were then washed x3 for 5 min in PBS. Images were captured at 10X magnification using an inverted BX51 Olympus (Japan) fluorescence microscope.

Statistical analysis Data were analysed using SPSS 21.0 software for Mac (SPSS Inc., Chicago, IL, USA). Normal distribution was assessed using Kolmogorov-Smirnov test, whilst equal variance was assessed using Levine test. For parametric statistics, one-way analysis of variance (ANOVA) was employed for multiple comparisons, whilst for non-parametric statistics Kruskal-Wallis was used for multiple comparisons. Statistical significant was accepted at p < 0.05. Data are expressed as mean ± standard deviation.


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Results

Fibre morphology assessment SEM micrographs (Supplementary Figure S1) revealed that all scaffolds exhibited a bead-free morphology. Fibre diameter distribution analysis (Supplementary Figure S2) showed that all treatments had an average fibre diameter in the range of 350 to 480 nm. The addition of HA and FC appear to decrease fibre diameter; this trend became significant (p < 0.001) for the 0.1 % and 1 % HA and FC concentrations (Table 1).

Thermal and mechanical assessment Table 1 summarises the thermal and mechanical properties of the scaffolds produced in this study. Neither the addition of HA nor the addition of FC significantly affected the onset and peak temperature of the electro-spun scaffolds (p > 0.05). All HA and FC treatments significantly reduced stress at break, strain at break and modulus values (p < 0.001). With respect to HA treatments, the mechanical properties were reduced, as the HA concentration was increased. As such, among the HA treatments, the 1 % HA exhibited the lowest stress at break, strain at break and modulus values (p < 0.001). This trend was not observed with the FC treatments. The 0.1 % FC and 1 % FC treatments exhibited the lowest stress at break values (p < 0.001); the 0.01 % and 1 % FC treatments exhibited the lowest strain at break values (p < 0.001) and the 1 % FC treatment exhibited the lowest modulus values (p < 0.001). Between the HA and FC treatments, no statistical difference was observed for the stress at break values between the 0.01 % and 0.1 % treatments (p > 0.05), whilst the 1 % HA treatment exhibited significantly lower values than the 1 % FC treatment (p < 0.001). With respect to stain at break values, no statistical difference was observed between the 0.1 % and 1 % treatments (p > 0.05), whilst the 0.01 % HA treatment exhibited significantly higher values than the 0.01 % FC treatment (p < 0.001). All HA treatments exhibited significantly higher modulus values to their FC counterparts (p < 0.001).



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Osteoblast morphology, metabolic activity, viability and proliferation assessment Immunofluorescence staining demonstrated that all preparations supported osteoblast growth and gross qualitative analysis indicated that the addition of HA and FC restricted cytoskeleton spreading (Supplementary Figure S3). Osteoblast viability (Supplementary Figure S4) and metabolic activity (Supplementary Figure S5) were increased as a function of time in culture, but no significant difference (p > 0.05) was detected at a given time point between the groups. Osteoblast proliferation (Supplementary Figure S6) was also increased as a function of time in culture and at day 21, the 1 % HA and 1 % FC functionalised scaffolds exhibited significantly higher (p < 0.001) osteoblast proliferation to non-functionalised PLGA scaffolds.

Osteoblasts’ ALP activity and mineralisation assessment At day 7, no significant difference (p > 0.05) was observed in alkaline phosphatase activity between the groups (Figure 1a). At day 14 and 21, 1 % HA and 1 % FC exhibited significantly higher (p < 0.05) alkaline phosphatase activity than the PLGA alone scaffolds (Figure 1a). In general, an increase in alkaline phosphatase activity was observed at a given time point as the concentration of HA or FC were increased (Figure 1a). At day 7 and day 14, the 1 % HA electro-spun scaffolds induced the highest (p < 0.05) calcium deposition (Figure 1b). At day 21, the 0.1 % and 1 % HA and the 1 % FC exhibited significantly higher (p < 0.05) calcium deposition than PLGA alone scaffolds (Figure 1b). In general, an increase in calcium deposition was observed at a given time point as the concentration of HA or FC were increased (Figure 1b). Visual assessment of alizarin red stained samples (Figure 2) did not reveal any noteworthy differences in matrix mineralisation between the groups (tissue culture plastic, PLGA alone, PLGA with various concentrations of HA or FC).

Expression of Osteonectin and Osteopontin


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At day 21, immunofluorescent staining for osteocalcin (Figure 3) was negative for all samples (tissue culture plastic, PLGA alone and PLGA with various concentrations of HA and FC), whilst osteopontin (Figure 4) was positive for all samples (tissue culture plastic, PLGA alone and PLGA with various concentrations of HA and FC).



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Discussion Given that imprinting (e.g. creation of, usually, anisotropic nano- to micro-grooves on polymeric substrates) has failed to direct functional regeneration in vivo

44, 45

, electro-spinning has remained

the sole nano-fabrication technology that can produce functionalised with bioactive / therapeutic cargos three-dimensional substrates for regenerative medicine purposes

46

. Specifically to bone

tissue engineering, electro-spun scaffolds, loaded with a growth factor 47 or a viable cell population 48

or with both

49

, have been shown to promote functional repair and regeneration in various

preclinical models. Despite these promising preliminary results, none of these technologies has been clinically translated. We attribute this limited clinical translation to the complexity of the proposed devices. Although scaffolds loaded with sulphated GAGs and carbohydrates have been shown to direct stem cell lineage commitment 50 and to constitute a suitable biomimetic device for various clinical indications, including skin

51

, cartilage

52

, bone

53

, scaffolds loaded with non-

sulphated polysaccharides (e.g. HA) have primarily been studied as adhesion prevention devices 54. Herein, as first, we ventured to assess the influence of non-sulphated polysaccharides (HA and FC) on the biophysical and biomechanical properties of electro-spun scaffolds and to assess their ability to promote in vitro osteogenesis. Starting with structural characterisation, SEM micrographs revealed bead-free scaffold morphology, with mean fibre diameter in the range of 350 to 480 nm. This fibre diameter distribution is in accordance to previously produced electro-spun meshes out of, for example, gelatin and collagen 55, PLGA-gelatin-elastin 56, low concentration PLLA 57, PLLA-collagen-nano-hydroxyapatite 58 and 80 % HA – 20 % collagen

59

. It is worth pointing out that similar to ours in diameter silk fibroin

electro-spun constructs supported significantly higher cell proliferation and expression of extracellular matrix genes than electro-spun fibres with average fibre diameter of ~ 1 µm 60. The addition of 0.01 % (0.1 mg/ml) HA and FC to 8 % (80 mg/ml) PLGA did not appear to affect significantly fibre diameter, which is in accordance to previous studies, where poly-ε-caprolactone fibres were functionalised with various peptides

61

. The addition of higher concentrations (0.1 %


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and 1 % / 1 mg/ml and 10 mg/ml) of HA and FC significantly reduced fibre diameter. This is in agreement with previous studies, where the average fibre diameter of electro-spun fibres was reduced as the concentration of: heparin (poly-ε-caprolactone alone: 810 nm; 0.05 % w/v heparin: 680 nm; 0.5 % w/v heparin: 550 nm)

40

and aniline pentamer-graft-gelatin (poly-L-lactide alone:

900 nm; 0.95 % w/v aniline pentamer-graft-gelatin: 300 nm; 2.4 % w/v aniline pentamer-graftgelatin: 270 nm; 3.5 % w/v aniline pentamer-graft-gelatin: 200 nm) 62 were increased. The addition of high concentrations of HA and FC may have increased the charge density on the surface of the electro-spinning jet, stimulating higher electrostatic forces to draw the fibres and ultimately resulting in fibres with smaller diameters

63

. It is interesting to note that the addition of HA in

collagen resulted in increased fibre diameter up to ratio of 8,000 mg/ml collagen / 1,500 mg/ml HA; however at ratio 8,000 mg/ml collagen / 2,000 mg/ml HA, the fibre diameter was decreased 64, as it was observed herein with the high concentrations of HA and FC. Unfortunately, lower / higher ratios of HA to collagen were not assessed to see whether this trend would have continued. Intermediate PEG and NaCl concentrations (20 % as opposed to 5 % and 40 %) have also been shown to reduce the diameter of extruded collagen fibres due to salting-in / salting-out effect 42, 65. Further, physiologically speaking, it is of interest to note that excessive HA inhibits lateral growth of collagen fibrils

66

, which again may explain this reduction in fibre diameter as a function of

increased HA and FC concentration. Neither the addition of HA nor the addition of FC affected the onset and peak temperatures of the produced scaffolds. This is in accordance to previous publication, where the addition of FC did not affect the thermal properties of collagen films 31. With respect to HA, it is worth pointing out that HA functionalised scaffolds have been reported twice to exhibit higher melting temperature than the non-functionalised scaffolds. However, the first study provided only the absolute mean values of two replicates and, as such, it is inconclusive 67 and, in the second study, only the 15 % HA loaded scaffold exhibited higher melting temperature than the non-functionalised scaffold and the 7.5 % HA functionalised scaffold

68

. In any case, all produced scaffolds herein exhibited melting



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Page 16 of 41

temperature well-above body temperature, which clearly indicates their suitability for biomedical applications. With respect to the biomechanical properties, all treatments, but the 0.01 % FC, resulted in electrospun fibres with reduced stress at break, strain at break and modulus values, as compared to the non-functionalised PLGA electro-spun fibres. Previous studies have also shown that increasing concentrations of PEG, NaCl and dermatan sulphate were associated with reduction in the biomechanical properties of reconstituted collagen

42, 65, 69

. It is worth pointing out that the fibres

produced herein had similar or superior mechanical properties to previously produced PLGA collagen type I 70, collagen type II 71, gelatin 72, PCL

73, 74

, hydroxyapatite-PCL

32

,

75, 76

, peptide-PCL

61

, gelatin-PCL 77 and pullulan / dextran 78 electro-spun scaffolds, to mention only a few.

With respect to biological analysis, all scaffolds, independently of the presence or absence of HA and FC, supported osteoblast growth with no apparent differences in viability, metabolic activity and proliferation. This is in accordance to previous publications, where: increasing concentrations of HA in collagen electro-spun collagen fibres did not affect viability of human foreskin fibroblasts 64

; increasing concentrations of HA in collagen scaffolds did not affect pre-adipocyte proliferation

68

; increasing concentrations of hydroxyapatite in PCL/PLA electro-spun fibres did not affect

MC3T3-E1 osteoblast-like cell proliferation

79

; and incorporation of hydroxyapatite in

PLACL/gelatin electro-spun scaffolds did not affect proliferation of human foetal osteoblast cells 80. An increased osteoblast viability and proliferation has been reported previously when high concentrations of peptides were used to functionalise electro-spun PCL fibres 61. Although FC has been shown to increase the metabolic activity and proliferation of lung fibroblasts 31, it has no effect in viability, proliferation and metabolic activity of skin, lung and corneal fibroblasts, when it is used as a macromolecular crowding agent 29, 30. The addition of HA and FC in PLGA electro-spun fibres appeared to reduce osteoblast spreading. This is in accordance to previous work, where GAGs were shown to inhibit both spreading and adherence of osteoclasts and their precursors, illustrating that way the therapeutic potential of


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GAGs in osteolytic diseases

81

. Further, the addition of HA and FC in PLGA electro-spun fibres

appeared to enhance osteoblast mineralisation. This is a very interesting outcome, given that previous studies have shown that the addition of hydroxyapatite in: PLACL/gelatin electro-spun scaffolds did not affect osteoblast mineral deposition, not even after 30 days in culture 80; PCL/PLA electro-spun scaffolds did not affect MC3T3-E1 osteoblast-like cell mineralisation for up to 15 days in culture 79; PCL electro-spun scaffolds did not affect osteoblast mineralisation for up to 5 days in culture 76. This observation further enhances previous studies, where sulphated glycosaminoglycans increased up to 28-fold alkaline phosphatase, osteoprotegerin and osteocalcin gene expression and up to 4-fold calcium deposition in osteoblast culture

82

and promoted osteoclastogenesis through

interference with the NF-κB ligand / osteoprotegerin complex formation 83. In addition, GAGs have been shown to enhance osteoblastic differentiation of mesenchymal stem cells 84, with HA to be the most potent GAG

26

. It is also worth noting that electro-spun GAG-mimetic peptide nano-fibres

have been shown to promote osteogenic activity and mineralisation of osteoblastic cells through interaction with BMP-2 85. Osteopontin, middle to late stage bone formation marker, was evidenced in all scaffolds, as was observed previously on collagen-GAG scaffolds

86

. The rather limited

osteocalcin (late bone formation marker) expression observed herein is not surprising, given that the cells were not extensively mineralised 87. Further, similar to our data, osteocalcin expression was not altered on artificial matrices comprised of collagen and different GAG derivatives 28.



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Conclusion Herein, we demonstrated that functionalisation of PLGA electrospun fibres with HA and FC did not affect onset and peak temperatures, whilst reduced fibre diameter, stress at break, strain at break and modulus. Osteoblast viability and metabolic activity were not affected as a function of HA and FC functionalisation, whilst the 1 % HA and 1 % FC significantly increased osteoblast proliferation at day 21. By day 21, osteoblasts seeded on 1 % hyaluronic acid and 1 % Ficoll™ fibres showed the highest alkaline phosphatase activity, the highest calcium deposition and expressed osteopontin. This study further supports the biological benefits of carbohydrate functionalised scaffolds.

Acknowledgements and Competing Interests This work is supported by: the EU FP7/2007-2013, NMP award, Green Nano Mesh Project (Grant Agreement Number: 263289) to DZ; the Health Research Board, Health Research Awards Programme (Grant Agreement Number: HRA_POR/2011/84) to DZ; and the Irish Research Council, Government of Ireland Postgraduate Scholarship Scheme (Grant Agreement Number: GOIPG/2014/385) to KS and DZ; and the Enterprise Partnership Scheme, Irish Research Council (Project Number: EPSPG/2011/64) to KF and DZ. This publication has also been supported from Science Foundation Ireland and the European Regional Development Fund (Grant Agreement Number: 13/RC/2073). The authors also acknowledge the Electron Microscopy Unit within the Centre for Microscopy & Imaging and the Histology Core Facility of the National Centre for Biomedical Engineering Science (NCBES) at NUI Galway, which are funded by NUI Galway and the Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007-2013. The authors have no competing interests.


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Tables Table 1: Thermal, structural and mechanical properties of the scaffolds produced in this study. The addition of HA and FC did not significantly affect the onset and peak temperatures of the produced scaffolds (p > 0.05). The addition of HA and FC decreased fibre diameter; the 1 % HA treatment yielded the thinnest fibres (p < 0.001). The addition of HA and FC significantly reduced the stress at break, strain at break and modulus values (p < 0.001).

Onset (°C)

Fibre Diameter

Stress at Break

Strain at Break

Modulus at 2 %

(µm)

(MPa)

(%)

Strain (MPa)

Peak (°C)

PLGA

44.32 ± 3.05

50.45 ± 1.76

0.48 ± 0.12

3.40 ± 1.24

23.72 ± 2.35

166.85 ± 90.46

0.01 % HA

43.31 ± 7.04

48.15 ± 3.29

0.44 ± 0.10

2.04 ± 0.52

20.42 ± 3.44

145.90 ± 20.01

0.1 % HA

46.09 ± 1.11

50.01 ± 0.39

0.35 ± 0.11

1.67 ± 0.79

17.59 ± 6.54

96.56 ± 48.80

1 % HA

40.40 ± 8.79

51.78 ± 0.76

0.35 ± 0.07

0.27 ± 0.11

11.47 ± 6.38

10.26 ± 4.58

0.01 % FC

48.07 ± 0.77

50.03 ± 4.45

0.41 ± 0.15

2.15 ± 1.08

12.02 ± 1.30

66.59 ± 34.43

0.1 % FC

45.58 ± 1.53

49.92 ± 0.68

0.44 ± 0.07

1.62 ± 0.32

18.52 ± 4.81

81.90 ± 27.20



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1 % FC

45.84 ± 0.9

50.7 ± 0.12

0.37 ± 0.09

1.70 ± 0.79


Page 20 of 41

11.53 ± 2.99

1.14 ± 0.51

Page 21 of 41

Figures Figure 1: At day 21, 1 % HA and 1 % FC exhibited the highest (p < 0.001) alkaline phosphatase activity between the groups (a). At day 21, 0.1 % HA, 1 % HA and 1 % FC exhibited significantly higher (p < 0.001) calcium deposition than PLGA alone scaffolds and tissue culture plastic (b). Note: (*) indicates significant difference to PLGA alone scaffolds. TCP

PLGA

PLGA + FC 0.01%

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + FC 0.1%

PLGA + FC 1%

PLGA + HA 1%

(b) 0.7 * *

5

*

*

4 3 2 1 0

Ctrl

HA

Day 7

FC

Ctrl

HA

Day 14

FC

Ctrl

HA

FC

Calcium/Total Protein (µg/ml)

(a) 6 ALP/Total protein (µg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


0.6

*

**

0.5

*

*

0.4 0.3 0.2 0.1 0.0

Ctrl

Day 21


HA

Day 7

FC

Ctrl

HA

Day 14

FC

Ctrl

HA

Day 21

FC


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 22 of 41

Figure 2: Visual assessment of alizarin red stained samples did not reveal any significant differences in matrix mineralisation between the groups. Note: Scale bar: 100 µm for all micrographs. TCP

PLGA

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + HA 1%

Day 7

Day 14

Day 21


PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + FC 1%

Page 23 of 41

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Figure 3: Immunofluorescent staining for osteocalcin was negative for all samples. Note: Staining at day 21. Note: Scale bar: 100 µm for all micrographs.

TCP

PLGA

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + HA 1%

PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + FC 1%

Red; Actin fibres (Rhodamine Phalloidin), Blue; Nuclei (DAPI), Green; Osteocalcin staining



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Page 24 of 41

Figure 4: Immunofluorescent staining for osteopontin was positive for all samples. Note: Staining at day 21. Note: Scale bar: 100 µm for all micrographs.

TCP

PLGA

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + HA 1%

PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + FC 1%

Red; Actin fibres (Rhodamine Phalloidin), Blue; Nuclei (DAPI), Green; Osteopontin staining


Page 25 of 41

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For Table of Contents Use Only

Title The influence of non-sulphated polysaccharides on the properties of electro-spun poly(lactic-coglycolic acid) fibres

Authors A Azeem, L Marani, K Fuller, K Spanoudes, A Pandit, DI Zeugolis

Brief Synopsis The biological activity of electro-spun poly(lactic-co-glycolic acid) fibres is enhanced through the addition of hyaluronic acid or Ficoll™.

PLGA + Ficoll™ Electro-spun Fibres

Osteopontin Expression of Human Osteoblasts on PLGA + Ficoll™ Electro-spun Fibres



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TCP

ALP/Total protein (µg/ml)

(a) 6


PLGA

PLGA + FC 0.01%

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + FC 0.1%

PLGA + FC 1%

(b) 0.7

*

*

5

PLGA + HA 1%

*

*

4 3 2 1 0

Ctrl

HA

Day 7

FC

Ctrl

HA

Day 14

FC

Ctrl

HA

FC

Calcium/Total Protein (µg/ml)

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*

0.6

**

0.5

*

*

0.4 0.3 0.2 0.1 0.0

Ctrl

Day 21


HA

Day 7

FC

Ctrl

HA

Day 14

FC

Ctrl

HA

Day 21

FC

TCP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

PLGA

PLGA + HA 0.01%

PLGA + HA 0.1% ACS Biomaterials Science & Engineering PLGA + HA 1%

Day 7

Day 14

Day 21 ACS Paragon Plus Environment

PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + Page FC 38 1% of 41

Page 39 of 41

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TCP


PLGA

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + HA 1%

PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + FC 1%


Red; Actin fibres (Rhodamine Phalloidin), Blue; Nuclei (DAPI), Green; Osteocalcin staining

TCP


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

PLGA

PLGA + HA 0.01%

PLGA + HA 0.1%

PLGA + HA 1%

PLGA + FC 0.01%

PLGA + FC 0.1%

PLGA + FC 1%

Page 40 of 41


Red; Actin fibres (Rhodamine Phalloidin), Blue; Nuclei (DAPI), Green; Osteopontin staining

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PLGA + Ficoll™ Electro-spun Fibres

Osteopontin Expression of Human Osteoblasts on PLGA + Ficoll™ Electro-spun Fibres


Influence of Nonsulfated Polysaccharides on the Properties of

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