Acta Biomaterialia 10 (2014) 4410–4418

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Composite pullulan–dextran polysaccharide scaffold with interfacial polyelectrolyte complexation fibers: A platform with enhanced cell interaction and spatial distribution Marie Francene Arnobit Cutiongco a,1, Ming Hao Tan a,1, Martin Yoke Kuang Ng a, Catherine Le Visage b, Evelyn King Fai Yim a,c,d,⇑ a

Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore INSERM, U698, Cardiovascular Bioengineering, Paris, France c Mechanobiology Institute of Singapore, National University of Singapore, Singapore d Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore b

a r t i c l e

i n f o

Article history: Received 26 January 2014 Received in revised form 12 June 2014 Accepted 20 June 2014 Available online 28 June 2014 Keywords: Hydrogel Chemical cross linking Hybrid scaffold

a b s t r a c t Hydrogels are highly preferred in soft tissue engineering because they recapitulate the hydrated extracellular matrix. Naturally derived polysaccharides, like pullulan and dextran, are attractive materials with which to form hydrophilic polymeric networks due to their non-immunogenic and non-antigenic properties. However, their inherent hydrophilicity prevents adherent cell growth. In this study, we modified pullulan–dextran scaffolds with interfacial polyelectrolyte complexation (IPC) fibers to improve their ability to support adherent cell growth. We showed that the pullulan–dextran–IPC fiber composite scaffold laden with extracellular matrix protein has improved cell adhesion and proliferation compared to the plain polysaccharide scaffold. We also demonstrated the zero-order release kinetics of the biologics bovine serum albumin and vascular endothelial growth factor (VEGF) incorporated in the composite scaffold. Lastly, we showed that the VEGF released from the composite scaffold retained its capacity to stimulate endothelial cell growth. The incorporation of IPC fibers in the pullulan–dextran hydrogel scaffold improved its functionality and biological activity, thus enhancing its potential in tissue engineering applications. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The development of diverse biomaterials has paved the way for breakthroughs in tissue engineering. Specifically, hydrogels are being extensively developed for use in soft tissue engineering. Hydrogels are polymeric networks that swell in water, mimicking the extracellular matrix of various soft tissues [1]. For clinical utilization, hydrogels must have the same mechanical property as the target tissue and the ability to interact with cells to provide a sustained regenerative milieu. Naturally derived polysaccharides are widely used as hydrogels in tissue engineering. For instance, the sugars pullulan and dextran have been explored for their possible use in vascular tissue ⇑ Corresponding author. Postal address: Regenerative Nanomedicine Laboratory, Department of Biomedical Engineering, EA-03-12, 9 Engineering Drive 1, National University of Singapore, Singapore 117575, Singapore. Tel.: +65 6516 7322; fax: +65 6872 3069. E-mail address: [email protected] (E.K.F. Yim). 1 These authors contributed equally to the work. http://dx.doi.org/10.1016/j.actbio.2014.06.029 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

engineering [1,2]. The advantages of using these polysaccharide biomaterials are their biocompatible, non-immunogenic and non-antigenic properties, along with their capacity to imbibe water and other biological fluids. Various studies have shown the in vitro capacity of pullulan-based polysaccharides for culture and proliferation of mesenchymal stem cells [3] and endothelial progenitor cells [4]. However, these cells were shown to grow in clumps rather than spreading across the surface of the scaffolds, implying the limited ability of the scaffolds to support adherent cell growth. The hydrophilic nature of the polysaccharide scaffold may prevent the adsorption of proteins, limiting support for cellular attachment and spreading [5]. The use of interfacial polyelectrolyte complexation (IPC) fibers has been touted as a stable and easy way to protect and deliver biological agents. IPC fibers are formed at the interface of two oppositely charged polyelectrolyte solutions under ambient conditions, thus enabling the incorporation of proteins and cells [6]. The addition of a biological component into the polyelectrolyte solution will result in its incorporation in the resultant fiber.

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Various studies have demonstrated the incorporation of growth factors [7] and even cells [8,9] for spatiotemporally controlled delivery. Aside from its utility in the containment and delivery of biochemical cues, IPC fibers contain inherent biophysical cues within their fibrous construct, which may mimic native extracellular matrix (ECM) structures [10–12]. Biophysical cues are also responsible for eliciting cell specific behaviors such as proliferation, differentiation and migration. Fibrous scaffolds are often used in various regenerative medicine applications, such as improving endothelial cell adhesion in vascular grafts and differentiation of human mesenchymal stem cells into osteoblasts for bone tissue engineering [13]. In addition to providing support for adherent cells, the spatial control of cell distribution is also important for tissue regeneration. Physiologically, cells are arranged in an ordered manner that leads to a fully functioning tissue. Successful tissue engineered constructs require spatial control of cell adhesion and the necessary orientation. For example, studies have shown that surface modification with a ligand requires a certain ligand orientation and spatial geometry and distribution to produce desirable interactions with cells [14]. IPC fibers have been shown to control cell distribution either by direct incorporation of cells [8] or restriction of cell shape and orientation along fibers [15]. Therefore, IPC fibers are a promising tool to improve the functionality of polysaccharide scaffolds through the presentation of specific ligands, biophysical cue for contact guidance and regulation of cell orientation and 3-D spatial distribution. In this study, we hypothesized that IPC fibers could be combined with a pullulan–dextran (PD) hydrogel to form a gel–fiber composite scaffold. First, we characterized IPC fiber integration into the PD scaffold, in which spatial distribution of IPC fibers could be constructed in a controlled manner. The composite scaffold was also incorporated with ECM or growth factors through IPC fibers. Thereafter, we tested cell seeding on the composite PD–IPC fiber scaffold. The incorporation of fibronectin improved cell attachment and proliferation on the composite scaffold. Interestingly, we observed near-zero-order release of bovine serum albumin (BSA) over a period of 2 months and the controlled release of vascular endothelial growth factor (VEGF) over 7 days from the composite scaffold. We also observed the retained biological activity of VEGF released from the composite scaffold. The incorporation of IPC fibers in the PD hydrogel scaffold enhanced biological functionality, implying refined utility in diverse tissue engineering applications. 2. Materials and methods

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(Fig. 1A). Chitosan (190–310 kDa, 75–85% deacetylation, Sigma Aldrich) was purified following the procedure detailed by Liao et al. [7]. Purified chitosan was dissolved in 0.15 M acetic acid solution at 1% w/v and subsequently adjusted to pH 6.0 with 0.5 M NaOH. Alginic acid (sodium salt from brown algae, low viscosity, Sigma Aldrich) was dissolved in DDI water to obtain a 1% (w/v) solution. Table 1 presents a summary of the properties of all molecules used in IPC fiber formation. The zeta potential of electrolytes was measured using the Malvern Zeta Sizer 2000. The theoretical charge of proteins was calculated using The Protein Calculator (The Scripps Research Institute, La Jolla, CA). Fig. 1A shows IPC fiber drawn from the interface of two polyelectrolytes. IPC fiber was drawn from the interface of 10 ll of chitosan and 10 ll of alginate solution placed on a polystyrene dish surface. A continuous strand of fiber was drawn vertically from the solution interface by a speed-controlled motor at an approximate speed of 10 mm s1. Fibers were collected on a pair of collecting needles affixed to a linear motor (Fig. 1B) or a sacrificial pullulan frame, as necessary. To create a sacrificial pullulan frame, a 20% (w/v) aqueous pullulan solution was prepared. For this, 15 g of pullulan solution was cast onto a polystyrene dish and allowed to evaporate at 37 °C overnight. The dried pullulan film was cut into 7 mm  7 mm square frames. 2.2.2. Fibronectin incorporation into IPC fibers Human fibronectin (Biological Industries) was added to 1% w/v chitosan solution to a final concentration of 2 lg ml1 and drawn against 1% w/v alginate solution. To visualize incorporation of fibronectin into IPC fibers, fluorescently labeled fibronectin (HiLyte Fluor 488, Cytoskeleton Inc.) was added to 1% w/v chitosan at the same concentration and visualized using a Leica DMIRB inverted fluorescence microscope. 2.2.3. BSA incorporation into IPC fibers BSA (Sinopharm Chemical Reagent, Shenyang, China) was used as a molecule model. BSA was incorporated at a final concentration of 2.5 mg ml1 into 1% alginate. A total of 250 lg of BSA was incorporated into 100 mg of IPC fibers. The fibers were drawn from 20 ll of each polyelectrolyte solution and a continuous strand of fiber was drawn vertically onto a pair of collecting needles at an approximate speed of 1 mm s1. Upon completion of the fiber drawing, 500 ll of phosphate-buffered saline (PBS) was used to collect the remaining pool of polyelectrolyte solution. The BSA concentration was determined using a BCA assay (Pierce) to assess the incorporation efficiency.

2.1. Activation of PD pre-gel mixture and fabrication of PD scaffold PD scaffolds were prepared according to Le Visage et al. [3] and Fricain et al. [16]. Briefly, a pre-gel mixture of 75:25 pullulan (200 kDa; Hayashibara Inc., Okayama, Japan) and dextran (500 kDa; Sigma Aldrich) was prepared with a total concentration of 30% (w/v) in distilled deionized (DDI) water. To create pores in the polysaccharide scaffolds, sodium bicarbonate (Sigma Aldrich) was added to a final concentration of 20% (w/v). The mixture was either used immediately after preparation or stored in sealed containers at 4 °C for later use. One gram of the pre-gel mixture was mixed with 100 ll of 10 M sodium hydroxide (Sigma Aldrich) and 100 ll of 11% (w/v) sodium trimetaphosphate (STMP; Sigma Aldrich). The activated PD mixture was gelled at 37 °C for 30 min to create a PD scaffold.

2.2.4. VEGF incorporation into IPC fibers VEGF (R&D Systems) was added to 1% chitosan at a final concentration of 2.5 lg ml1. A total of 100 ng of VEGF was incorporated into 40 mg of IPC fibers. Fibers were drawn from 20 ll of each polyelectrolyte solution. The polycation solution was drawn against 1% (w/v) polyanion solution, composed of 1% alginate and 1% heparin at varying ratios (Table 2). Heparin was previously demonstrated to attenuate the initial burst release of plateletderived growth factor (PDGF) from IPC fibers [7]. The remaining polyelectrolyte solution was collected using 500 ll PBS and the remaining VEGF was measured using Duo-Set ELISA kit (R&D Systems). 2.3. Fabrication of composite PD–IPC scaffold to visualize cell attachment

2.2. IPC fiber fabrication 2.2.1. IPC fiber fabrication for cell seeding For this study, chitosan and alginate were the chosen polycations and polyanions, respectively, for IPC fiber fabrication

The fabrication of the PD–IPC composite scaffold is summarized in Fig. 1C. After drying IPC fibers at 4 °C overnight, the fibers were immersed in the activated PD mixture. The scaffold was gelled by incubating at 37 °C for 30 min. The sacrificial pullulan frame

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B

A ii

i

iii Incorporated molecule Alginate

Chitosan

1% chitosan

1% alginate

D

C Activa te Pullula d n Dextr an

Freeze and lyohilize Crosslink pullulan-dextran at 37°C for 30 minutes

Induce pore formation for 15 minutes in 20% acetic acid

Wash in PBS

Bottom view

IPC fibers on collecting needle

500 µm

Fig. 1. Creation of interfacial polyelectrolyte complexation fibers and subsequent incorporation into the polysaccharide scaffold. (A) Formation of IPC fibers by drawing at the interface of oppositely charged polyelectrolytes. (B) Schematic diagram showing an IPC fiber collected using a pair of collecting needles. (C) Incorporation of the IPC fibers into PD to create a composite scaffold. (D) Lyophilized polysaccharide scaffold.

Table 1 Properties of the various biomolecules used in the study. Molecule

Molecular weight (kDa)

Isoelectric point

Charge at IPC fiber formation

Zeta potential (mV)

Theoretical charge at given pH

Chitosan Alginate BSA Heparin VEGF

190–310 240 69 12–15 43

6.3 [16] 5.4 [17] 4.1 [7] ND 8.5 [18]

Positive Negative Negative Negative Positive

+35.2 ± 3.51 44.8 ± 5.16 ND 10.9 ± 1.69 ND

ND ND 14.4 ND +17.7

ND: not determined.

Table 2 Summary of different alginate and heparin content used to incorporate VEGF. Sample name

Ratio of 1% alginate:1% heparin (A:H)

VEGF incorporation efficiency (%)

VEGF1 VEGF2 VEGF3

9:1 8:2 1:1

71.5 ± 0.8 75.5 ± 2.7 97.0 ± 3.8

containing fibers (Section 2.1) was dissolved during the process, allowing full integration of the IPC fibers into the crosslinked scaffold. The composite scaffolds were then immersed in 20% acetic acid (J.T. Baker) for 1 h to induce pore formation. The gels were immediately washed in PBS to neutralize and to remove unreacted reagents. The scaffolds were further washed in DI water five times to further remove the salt from the PBS used in the previous washing. The physical structure of the obtained pullulan-based hydrogel with incorporated IPC fibers was observed using scanning electron microscopy (Section 2.6). Immediately after washing in PBS, scaffolds were frozen for at least 1 day at 80 °C. Scaffolds were then freeze dried for 24 h at 106 to 110 °C and approximately 0.15 mbar. Lyophilized scaffolds (Fig. 1D) were UV-irradiated for 1 h immediately before

cell culture. L929 mouse fibroblasts (ATCC, passage 20-23) were grown with Dulbecco’s modified Eagle’s medium (Sigma Aldrich) supplemented with 10% fetal bovine serum (Hyclone, Invitrogen) and 1% penicillin/streptomycin (Sigma Aldrich). Cells were maintained at 37 °C with 5% CO2. Fibroblasts were washed with sterile PBS and harvested using 0.1% trypsin (Gibco) to create a single-cell suspension. Cell suspension containing 2  105 cells in 200 ll of medium was added gradually to the scaffolds. Cells were allowed to adhere and scaffolds were allowed to swell for 20 min, after which each scaffold-containing well was topped up with 1 ml of fresh culture medium. The cell-seeded scaffolds were maintained in standard culture conditions. 2.4. Scanning electron microscopy (SEM) of PD-IPC fiber composite scaffolds SEM studies were carried out to assess the structure of the polysaccharide scaffolds and the morphology of the L929 fibroblasts seeded on the scaffolds. After 7 days of culture, cell-seeded scaffolds were fixed using 2% v/v glutaraldehyde (Sigma Aldrich) solution in cacodylate buffer (0.1 M sodium cacodylate (Sigma Aldrich) and 3 mM CaCl2 (Sigma Aldrich) in DI water, pH 7. Scaffolds were then washed thoroughly in cacodylate buffer

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solution. Ethanol dehydration was carried out by immersing scaffolds in solutions of increasing ethanol concentrations (25, 50, 75, 90 and 100%). Scaffolds were then dried with hexamethyldisilazane (Sigma Aldrich), followed by sputter-coating with gold (JEOL JFC 1600 Fine Gold Coater, 10 mA, 90 s). The surface structure and morphology of the scaffolds and cells, respectively, were then observed with an FEI Quanta 200F scanning electron microscope. 2.5. Measurement of cell viability and cell number in PD–IPC fiber composite scaffolds PD–IPC fiber composite scaffolds were fabricated using IPC fibers collected on a pair of needles (Fig. 1C). Briefly, the needles with the IPC fibers were stuck onto a polystyrene container of 10 mm diameter. Activated PD solution (0.3 mg) was poured into the container and allowed to crosslink at 37 °C for 30 min. The needles were removed before the scaffold was immersed in 20% (v/v) acetic acid for 20 min. Scaffolds were washed in PBS five times before lyophilization, as described in Section 2.3. A cell suspension of 3  104 L929 fibroblast cells in 100 ll of culture medium was seeded onto each scaffold, as described in Section 2.3. An alamarBlueÒ assay (Molecular Probes, Invitrogen) was used to assess the metabolic activity at various time points. alamarBlueÒ was mixed with cell culture medium at a 1:10 ratio and incubated for 4 h under standard cell culture conditions (Section 2.3). The fluorescence intensity of the supernatant was measured at excitation and emission wavelengths of 544 and 590 nm, respectively. Cell viability is reported as the percent reduction of alamarBlueÒ. Each scaffold type was assessed in quadruplicate. A CyQuant cell proliferation assay (Life Technologies) was also used to determine the total number of cells on each scaffold. Scaffolds with L929 fibroblasts were digested at different time points to collect cells [19]. In brief, scaffolds were washed in PBS and moved to a clean well plate. Next, scaffolds were incubated in DMEM containing pullanase (10 U ml1, Sigma Aldrich) and dextranase (20 U ml1, Sigma Aldrich). After overnight incubation at 37 °C, cells were separated by centrifugation at 1000 rpm for 5 min and the supernatant was removed. The cell pellet was then frozen at 80 °C until the cell pellets were available for measurement using the CyQuant cell proliferation assay. Known cell numbers were used as a standard for determining the cell number from each scaffold. Each scaffold type was assessed in triplicate. 2.6. Measurement of cell viability A LIVE/DEADÒ assay (Invitrogen Life Science) was carried out 7 days after the cell-seeding experiment. A dye mixture containing 4 lM ethidium homodimer and 2 lM calcein-AM was prepared in PBS. A 100 ll of this dye mixture was then added to each gel sample and subsequently incubated at 37 °C for 45 min. Afterwards, the dye mixture was aspirated from the gel samples. The samples were washed with PBS and viewed using a fluorescence microscope. 2.7. Measurement of controlled release of biologics from PD–IPC fiber composite scaffolds To determine the controlled release profile of BSA, scaffolds in a 24-well plate were immersed in 1 ml of sterile PBS at 37 °C. In the case of VEGF controlled release studies, 1% (w/v) BSA in PBS was used as the release solution. At each indicated time point, the release solution was collected completely and replaced with 1 ml of fresh release solution. Release solutions were stored in 80 °C for subsequent testing. BSA concentrations were quantified using a BCA assay kit while VEGF concentrations were assessed with the Duo-Set ELISA kit (R&D Systems). A total of three samples per scaffold type were used in the experiments.

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2.8. Cell seeding on PD-IPC composite scaffolds with VEGF Similar to Section 2.5, composite scaffolds containing VEGF were used for cell culture studies to determine bioactivity of released VEGF. Human umbilical vein endothelial cells (HUVECs, Lonza, passages 4-5) were grown in Endothelial Growth Medium2MV (EGM-2MV, Lonza) at 37 °C and 5% CO2. HUVECs were washed with HEPES-buffered saline solution (10 mM HEPES, 151 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 7.8 mM glucose, pH 7.4) and trypsinized with 0.1% trypsin (Gibco) to create a single-cell suspension. A cell suspension with 2  105 cells in 200 ll of medium was added gradually to the scaffolds. Cells were allowed to adhere to the swelling scaffold for 20 min. Afterwards, 1 ml of EGM-2MV was added to each scaffold. Cell-seeded scaffolds were maintained in standard culture conditions for 8 days, with total replacement of the culture medium every 2 days. alamarBlueÒ assay (Section 2.5) was used to determine cell viability every 2 days. The experiment was performed in triplicate. Cells were stained with 40 ,6-diamidino-2-phenylindole and Alexa-Fluor Phalloidin (Life Technologies) 24 h after seeding on various scaffolds and viewed under a Leica SP5 confocal microscope. 2.9. Statistical analysis Statistical analysis was performed using one-way analysis of variance coupled with Tukey’s post-test using GraphPad Prism v6.0 and p < 0.05 was considered significant. 3. Results 3.1. Characterization of porous PD scaffold and PD–IPC fibers composite scaffold In this study, the ultrastructure of the porous PD scaffold and composite scaffold with plain IPC fibers (PD–IPC) were examined. Fig. 2A shows the porous PD scaffold with high uniformity and porosity. A higher magnification image (Fig. 2A, inset) also shows the smooth texture of the hydrogel and pore interconnectivity. Fig. 2B shows a bundle of bare IPC fibers, with individual strands having an average diameter of 11.0 ± 2.2 lm. The image of the freeze-fractured scaffold in Fig. 2C shows that the integration of these two constructs into a composite scaffold did not disrupt the integrity of both structures, keeping them separate from each other. The polysaccharide component retained its texture and high pore interconnectivity, in which the IPC fibers intercalated and integrated. However, the pore size was smaller compared to that of the plain PD scaffold. Moreover, the composite scaffold exhibited excellent integration, with clear demarcation and identification of each component (Fig. 2D). This demonstrates the ability of IPC fibers to withstand both the harsh alkaline and acidic conditions used during the polysaccharide gelling process. Additionally, we showed that the fibers could be spatially oriented with respect to the polysaccharide platform. Aside from the parallel orientation of fibers (Fig. 2E), we also created fibers that were aligned orthogonally to each other (Fig. 2F). Composite scaffolds subsequently used in the study contained parallel-aligned IPC fibers. 3.2. PD–IPC fiber composite scaffold as a platform for cell attachment The ability of the composite scaffold to support cell attachment and cell proliferation was examined. In addition to studying plain PD scaffolds and composite scaffolds with plain IPC fibers (PD– IPC), we also examined scaffolds with the ECM protein fibronectin incorporated in IPC fibers (fibronectin composite scaffold). Fig. S1

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A

B

C

D

E

F

Fig. 2. Characterization of PD hydrogel scaffold integrated with IPC fibers. SEM micrographs show a porous plain PD hydrogel (A) and plain IPC fibers (B). (C) The cross-section of a composite scaffold composed of aligned IPC fibers embedded in the PD hydrogel. (D) Higher-magnification image showing the longitudinal section of the composite scaffold, with IPC fibers intercalated into the polysaccharide scaffold. Both the polysaccharide scaffold and the IPC fibers have retained their structural integrity. Light microscopy images of the composite scaffold with IPC fibers incorporated in either a parallel (E) or an orthogonal orientation (F).

shows the successful incorporation of fluorescently labeled fibronectin into the IPC fibers. Fig. 3A and D show poor attachment of cells on the plain PD scaffold, where a small number of rounded cells remained clumped. On the other hand, there was an increased number of cells seeded on the PD–IPC composite scaffolds compared to the plain PD scaffold (Fig. 3B and E). The cells appeared attached to the IPC fibers, although they were still rounded in shape and grown in clumps, implying loose adhesion of the cells. The incorporation of fibronectin markedly increased the number of cells adhered to the IPC fibers (Fig. 3C and F). Furthermore, the cells had a more spread and elongated morphology along the IPC fibers as compared to on the plain PD or PD–IPC fiber scaffolds, denoting good adherence to the IPC fibers. Putative ECM deposits were found on the fibronectin composite scaffold (Fig. 3G). Additionally, a LIVE/DEADÒ assay was carried out to ascertain the numbers of viable cells distributed in the different scaffolds (Fig. 3H, I and J). Cells were found in clumps in the plain PD scaffold, reiterating the lack of cell attachment thereto. In contrast, cells appeared to adhere and distribute more evenly in fibronectin composite scaffolds compared to the plain PD scaffolds, further demonstrating improved cell attachment on this scaffold compared to composite scaffold with plain IPC fibers. An alamarBlueÒ assay was also carried out to determine cell metabolic activity and estimate cell number at different time points after seeding on various scaffolds (Fig. 4A). Fibroblast cells grown on PD scaffolds showed a significantly higher reduction in alamarBlueÒ at day 1, while fibronectin composite scaffolds had a significantly higher percent reduction only in comparison to PD–IPC scaffolds. The percent reduction of PD–IPC scaffolds drastically decreased at day 3, after which the level was sustained until day 7. On the other hand, at days 3 and 5, fibronectin composite scaffolds exhibited the highest alamarBlueÒ percent reduction compared to the other two scaffolds. PD–IPC composite scaffolds showed the lowest level of alamarBlueÒ reduction at day 2. At day 7, the percent reduction on the fibronectin composite scaffolds continued to be significantly higher, while the PD–IPC scaffold showed a significantly lower percent reduction compared to the

other two scaffolds. Cells on the fibronectin composite scaffolds showed a stable trend of alamarBlueÒ reduction throughout the experiment. Additionally, the alamarBlueÒ reduction on the fibronectin composite scaffolds was significantly higher than on the PD-IPC composite scaffolds at all time points. A CyQuant assay was then carried out to accurately determine the cell number. The total cell count of the plain PD scaffold showed that it contained the lowest cell numbers at days 1 and 7 (Fig. 4B). While PD–IPC scaffolds contained the highest cell number at day 1, it decreased significantly to contain the lowest cell number at day 5, with a marginally increased cell number at day 7. In contrast, fibronectin composite scaffolds showed an increasing trend of total cell number from day 1 to day 7. At day 7, the composite scaffold with fibronectin showed a significantly higher total cell count than the plain PD and PD–IPC composite scaffolds. Our data suggests that composite scaffolds with fibronectin supported long-term growth and viability of fibroblast cells better than plain polysaccharide scaffolds.

3.3. PD–IPC composite scaffold for controlled release of growth factor Another way to functionalize the polysaccharide scaffold is to turn it into a reservoir for growth factors. We first incorporated BSA as a model molecule for VEGF. Fig. 5 shows the controlled release profile of BSA from bare IPC fibers and from the PD-IPC composite scaffold. The overall incorporation efficiency for BSA was 45 ± 0.97%. BSA from IPC fibers alone showed a high initial burst release that was stabilized after 1 day and reached a plateau at 20 days. In contrast, the release of BSA from the PD–IPC composite scaffolds showed an attenuated burst release and a steady release profile. Notably, BSA was released in a sustained manner from the porous composite scaffold, following zero-order release kinetics. Overall, it was promising to note that total BSA release reached 90–97% after 2 months, indicating that there was no loss of BSA during the gelling process or in the process to create the porous gel.

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A

150 µm

B

J

I

H

150 µm

G

F

E

D

C

150 µm

Fig. 3. Improving cell adhesion to PD scaffolds with IPC fibers and fibronectin-incorporated IPC fibers. (A–C) Microscopic image of a plain PD hydrogel (A) showing clumps of cells, as highlighted in the red circles. The PD-IPC composite scaffold (B) have rounded cells minimally attached to the IPC fibers (red arrows). Fibronectin composite scaffold (C) show elongated cells attached to the IPC fibers (red arrows). (D–G) SEM images of cells seeded on various scaffolds. (D) A clump of cells found sandwiched between two planes of the PD scaffold. The cells were mainly rounded and clumped. (E, F) Increased numbers of cells, albeit with rounded morphology, attached to the PD-IPC scaffold. (F) The high number of elongated cells attached to fibronectin composite scaffold. These cells may have secreted ECM, highlighted in the red circle in (G). Insets show highmagnification images. (H–J) LIVE/DEADÒ assay performed on cells 7 days after seeding on scaffolds. Cells were shown to be viable after seeding on the plain PD scaffold (H), PD-IPC scaffold (I) or fibronectin composite scaffold (J).

B *

Plain PD PD-IPC composite Fibronectin composite

150

*

* *

*

* *

*

100

*

50 0

Total cell number 40000

Plain PD PD-IPC composite Fibronectin composite

30000

*

*

* 20000

*

*

10000

Days

7

5

7

5

3

1

0

1

200

Cell number/scaffold

Alamar blue reduction (%)

A

Days

Fig. 4. Assessment of cell viability and number 7 days after seeding on different scaffolds. (A) Cell metabolism as measured by the alamarBlueÒ assay after seeding on various samples. (B) Total cell number on different scaffolds after days 1, 5 and 7. Cells seeded on fibronectin composite scaffolds showed the highest proliferation rate at both day 5 and day 7 post-seeding. Plain PD denotes plain PD hydrogel. PD–IPC composite represents the composite scaffold with plain IPC fibers. Fibronectin composite represents scaffolds with fibronectin incorporated in the IPC fibers. ⁄Statistically significant difference (p < 0.05) in cell proliferation rate between two groups denoted by the bar.

We proceeded to test the incorporation of a growth factor for tissue engineering applications. We selected VEGF, an important biochemical cue that would be useful for the application of the PD scaffold for vascularization of biomaterial implants. VEGF was incorporated together with heparin in the composite scaffolds. Heparin is known to bind, stabilize, localize and augment the activity of VEGF [20]. Various ratios of alginate and heparin were used

to determine the best composition that results in maximum VEGF incorporation and the best release prolife. Table 2 shows the various incorporation efficiencies using different ratios of alginate and heparin (A:H). As expected, VEGF3 (A:H = 1:1) scaffolds resulted in the most efficient incorporation due to the increased heparin content that binds VEGF molecules. Furthermore, this polyanion mixture resulted in the best release profile from the composite

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Cumulative BSA release (%)

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100 80 60 40

PD-IPC composite Bare IPC fiber

20 0 0

20

40

60

80

Time (Days) Fig. 5. Controlled release of BSA from PD-IPC composite scaffold and IPC fibers alone. BSA was incorporated in both porous composite scaffold and IPC fibers. The release of BSA was measured at various time points and measured as the total amount of BSA released, starting from the first measured time point.

scaffolds, with VEGF having minimal burst release at day 1 and a sustained release rate for at least 1 week (Fig. 6A). On the other hand, VEGF1 (A:H = 9:1) and VEGF2 (A:H = 8:2) scaffolds did not significantly differ in incorporation efficiency. Nevertheless, their controlled release profiles were vastly different from each other. VEGF2 scaffolds exhibited a sustained release profile comparable to VEGF3. VEGF1 scaffolds, containing the least amount of heparin, were found to have the poorest release profile, without any noticeable increase in VEGF release after day 1. To test the bioactivity of the released VEGF, HUVECs were seeded onto composite scaffolds. alamarBlueÒ reduction was used to estimate HUVEC metabolic activity. Fig. 6B shows the alamarBlueÒ percent reduction of HUVECs at days 1, 3 and 6 after seeding. At day 1, VEGF2 showed the highest alamarBlueÒ reduction compared to plain PD scaffolds, PD–IPC and VEGF1 composite scaffolds. At days 3 and 6, the alamarBlueÒ percent reduction demonstrated by the VEGF2 scaffold decreased, becoming comparable with the other scaffolds. Cell morphology analysis showed that predominantly rounded HUVECs attached to the polysaccharide portion of the scaffolds, with some cells showing a spread morphology on VEGF scaffolds (Fig. 6C). Together, our results demonstrated the potential of using a porous PD–IPC fiber composite scaffold for the sustained release of proteins and growth factors.

4. Discussion Though naturally biocompatible and non-immunogenic, the inherent hydrophilic nature of polysaccharide-derived hydrogels limits their application for adherent cell culture and tissue engineering. In this study, we enhanced the bioactivity of PD scaffold through the fabrication of a composite scaffold with IPC fibers. First, we demonstrated improved cell adhesion and proliferation of cells seeded on PD–IPC fiber composite scaffolds. Composite scaffolds incorporating the ECM protein fibronectin showed further improvement in cell adhesion, leading to putative ECM deposition as a possible precursor to tissue remodeling. In addition, we showed that the composite scaffold could be turned into a reservoir for the sustained delivery of a model protein and the growth factor VEGF. Lastly, we showed the ability of the PD–IPC fiber composite scaffolds to retain the bioactivity of incorporated VEGF. IPC fibers have been used for protection and delivery of various biological agents, such as cells and proteins. ECM components such as gelatin, hyaluronic acid [21] and methylated collagen [16] were previously shown to provide cues for cell attachment and subsequently facilitate contact guidance of cells on IPC fibers. We also showed in this study that fibronectin incorporation in composite

scaffolds could also provide an attachment cue, leading to enhanced cell proliferation and sustained cell metabolic activity, as shown by consistent alamarBlueÒ reduction. Indeed, fibronectin in the composite scaffold provided the cues for adhesion, thereby allowing cells to grow and proliferate in the scaffolds. Most notably, fibronectin remained bioactive after exposure to the harsh environment of pullulan–dextran crosslinking. Possible ECM deposits on the fibronectin-incorporated composite scaffold were also noted, suggesting favorable cell interaction and a possible foundation for stimulating tissue regeneration. The composite scaffold has presumably created a favorable cellular milieu, thus inducing cells to start recreating the native tissue by secreting the ECM on the polysaccharide compartment, which in turn supported even more cell growth until 7 days. This may eventually lead to further cell repopulation to create an organized, functional tissue. Meanwhile, cell adherence on the composite scaffold with plain IPC fibers was also notable. Aside from the delivery of biochemical cues, IPC fibers contain inherent biophysical cues based on its fibrous construct, with each primary IPC fiber being shown to be an aggregate of fine, submicron fibers [6,21]. The topological features of the IPC fibers may elicit cell-specific functions and behaviors such as proliferation, differentiation and migration. The effect was most marked in the preference of cells to attach to the IPC fibers and not the polysaccharide area of the composite scaffold. Yet it seemed that the biophysical cue by itself is inadequate to maintain cells over the long term, since the total cell number at day 7 was only comparable to that of plain PD scaffolds. In contrast, plain PD scaffolds without any kind of adhesion cue did not provide an environment to sustain cell proliferation. Nevertheless, the plain PD scaffolds had the highest cell activity at day 1 while showing lowest adherent cell numbers. The discrepancy between the two assays may indicate a difference in cell seeding efficiency, with the larger pores and higher tortuosity of the plain PD scaffold compared to the PD–IPC composite scaffolds possibly allowing a higher number of cells to infiltrate during seeding. Since the cells remained round and clumped, washing the plain PD scaffolds before digestion would remove all cells that were temporarily and loosely attached to the hydrophilic scaffold, giving the lowest number of adherent cells at day 1. Provision of both the attachment factor and an appropriate biophysical cue provides the best combination for sustained and long-term maintenance of fibroblast growth on PD–IPC composite scaffolds. The mechanism of IPC fiber formation ensures that any polyelectrolyte added, such as fibronectin, BSA and VEGF, will be incorporated in the IPC fiber during complexation of alginate and chitosan [6]. IPC fiber formation thus indicates controlled release of various biological agents in a spatial and temporal manner. Sustained release of BSA was observed even with the plain IPC fiber, which showed burst release and diminishing release after 20 days, further supporting the incorporation of biologics within the IPC fibers. While mere surface deposition of BSA on IPC fiber is possible, this would be expected to yield an even faster release kinetics, with complete BSA release within a few days. We showed the almost linear release of BSA from the PD–IPC composite scaffold in 9 weeks. Zero-order or linear release kinetics is ideal for the sustained delivery of various biologics, such as therapeutic drugs, to ensure a constant and therapeutic level of drugs in the blood and tissues [22]. Theoretically, zero-order release can be achieved when a matrix is completely governed by polymer relaxation mechanisms [22]. For example, scaffolds with a homogeneous distribution of therapeutic drugs, a rate-limiting impermeable surface and a small orifice for release can exhibit near-zero release kinetics [23]. Lee [22] showed that a nonuniform, sigmoidal drug distribution, where the highest concentration is at the middle of the scaffold, resulted in zero-order release

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Fig. 6. Controlled release and bioactivity of VEGF from PD–IPC fiber composite scaffold. (A) Alginate:heparin (A:H) ratios of 9:1, 8:2 and 1:1 (with a total concentration of 1% w/v) were tested for the controlled release of VEGF. (B) Cell viability of HUVECs grown on PD-IPC composite scaffolds with VEGF. (C) Morphological analysis of HUVECs grown on PD-IPC composite scaffolds with VEGF. White arrows denote spread cells. Scale bar = 50 lm. Plain PD denotes plain PD hydrogel. PD–IPC composite represents scaffold with plain IPC fibers. VEGF1 and VEGF2 represent composite scaffolds with VEGF incorporated into the IPC fibers.

kinetics over time. This may be relevant for our case, where the concentration of the incorporated biologics would be highest within the IPC fibers, decreasing outwards to the outer edges of the polysaccharide matrix. The release of biologics from the IPC fibers would be through Fickian diffusion release kinetics, which is primarily determined by the diffusion characteristics of the molecule and characterized by diminishing release over time [24], as observed in the bare IPC fibers. After release from IPC fibers, diffusion from the polysaccharide hydrogel to the release solution may be governed by the relaxation of polymer chains and penetration of the release medium into the hydrogel [22,24,25]. Additionally, movement of the biologics may be hindered by the macromolecular mesh and tortuosity of the hydrogel [22]. Though the proposed mechanism of controlled release must be further evaluated, the use of the composite scaffold for creating a linear release of biologics is beneficial for drug delivery and growth factor therapy. For example, the angiogenic factor VEGF requires a therapeutic dose to be delivered over a minimum period of 7 days to stimulate endothelial progenitor cell recruitment and infiltration into the diseased site [26], and blood vessel expansion and remodeling for the treatment of ischemia [27]. In this study, a linear release of VEGF, similar to the BSA release profile, was achieved. However, the VEGF release from the composite scaffold was suboptimal and the cumulative release remained low at 5% after 7 days. We speculate that the positively charged VEGF may be trapped in the IPC fibers due to its interaction with heparin [28]. Liao et al. [7] observed the same effect, with less than 5% of PDGF being released from IPC fibers after 25 days. By removing heparin during incorporation, PDGF achieved a cumulative release of 50%. The elimination of heparin during the incorporation process may have an adverse effect on growth factor stability, release and bioactivity after release. In fact, we showed that, with higher heparin content, the release of VEGF was more sustained in VEGF2 than VEGF1 scaffolds. The crosslinking of pullulan and dextran using STMP is hypothesized to occur through the formation of intermolecular phosphate linkages [29,30], which can also trap the positively

charged VEGF. Yet this may be unlikely to occur, since a recent study by Purnama et al. showed that plain PD scaffolds loaded with VEGF before freeze-drying exhibited rapid release within 24 h without any marked release thereafter for the next 2 days [31]. We also observed that the HUVEC metabolic activity on various scaffolds was highly correlated with the VEGF release profile. VEGF is a well-known mitogenic and chemotactic factor for endothelial cells [20]. The sustained release of VEGF from VEGF2 scaffolds resulted in the highest HUVEC metabolic activity at day 1. From day 3, when the VEGF release rate was starting to diminish, the HUVEC metabolic activity was also marginally affected. Similarly, the lack of any VEGF released from VEGF1 scaffolds was reflected in the stable alamarBlueÒ reduction across time, showing a correlation between the amount of VEGF released and the bioactivity retained. We demonstrated that growth factor release from the PD–IPC fiber composite scaffold was shown to be both feasible and sustainable. The HUVEC morphology was primarily rounded, which was expected from the high compliance and low stiffness of the scaffolds. Yet the presence of HUVECs with a spread morphology on VEGF scaffolds further corroborates the retained bioactivity of the VEGF scaffolds. Moreover, we showed the ability of the PD–IPC composite scaffold to retain the bioactivity of VEGF, thus also emphasizing the protective function of IPC fibers for biological agents. The diverse uses of IPC fibers, as illustrated above, can be used to create composite scaffolds with multiple fibers containing either ECM proteins or growth factors, or both. For example, both VEGF and PDGF can be incorporated into separate fibers. Sustained, linear release of both angiogenic factors has been shown to enhance the durability of newly formed vessels and thus to be more effective in therapeutic angiogenesis in comparison with treatment using either growth factor singularly [32–34]. Davies et al. [32] in fact used a porous polyurethane scaffold for this purpose. Unsurprisingly, VEGF release was complete in as little as 30 h, while PDGF release exhibited diminishing kinetics after 100 h. The use of our composite scaffold instead may be more beneficial for therapeutic applications.

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Combining ECM protein and growth factors in IPC fibers confers greater advantages for directed spatial organization of cells within a scaffold. This may be advantageous for in vitro co-culture, such as endothelial cells with endothelial progenitor cells or mesenchymal stem cells for various therapeutic applications [32,35]. Moreover, the composite scaffold can be used to recreate organization inherent in tissues. Preferential adhesion of cells to IPC fiber with specific biological cues, coupled with the ability to easily incorporate multiple types of fibers in a wide range of orientations within the scaffold, facilitates mimicking of the 3-D construct with organized cell types. The possibility of using different cell-specific ligands may aid proper cell organization and tissue regeneration in situ. 5. Conclusion The PD–IPC fiber composite scaffold demonstrated improved capacity for cell interaction and an excellent capability for the sustained release of biologics. Cell attachment and proliferation were significantly enhanced compared to the plain polysaccharide scaffold, showing possible ECM deposition on composite scaffolds incorporating fibronectin. Controlled release of the biologics model of BSA and VEGF showed a near-linear profile over the long term, enabling delivery of a constant and therapeutic biologics dose. VEGF released from PD–IPC composite scaffolds also retained bioactivity, as demonstrated by sustained HUVEC metabolic activity. The modification of PD scaffolds using IPC fibers is a novel and effective way to further improve the functionality of an otherwise biologically compatible scaffold. Acknowledgements This research is supported by the Singapore National Research Foundation via the Exploratory/Developmental Grant funding scheme (grant number NMRC/EDG/0068/2009), administered by the Singapore Ministry of Health’s National Medical Research Council, and partially supported by the MERLION Program 5.03.08. MFAC is supported by the Agency for Science, Technology and Research (Singapore) and National Agency for Research (France) joint program under project number 1122703037. MHT thanks the Ministry of Education and Department of Biomedical Engineering for her scholarship. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–3 and 6 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:http://dx.doi.org/ 10.1016/j.actbio.2014.06.029). Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014. 06.029. References [1] Kirschner CM, Anseth KS. Hydrogels in healthcare: from static to dynamic material microenvironments. Acta Mater 2013;61:931–44. [2] Chaouat M, Le Visage C, Autissier A, Chaubet F, Letourneur D. The evaluation of a small-diameter polysaccharide-based arterial graft in rats. Biomaterials 2006;27:5546–53. [3] Le Visage C, Gournay O, Benguirat N, Hamidi S, Chaussumier L, Mougenot N, et al. Mesenchymal stem cell delivery into rat infarcted myocardium using a porous polysaccharide-based scaffold: a quantitative comparison with endocardial injection. Tissue Eng Part A 2012;18:35–44. [4] Lavergne M, Derkaoui M, Delmau C, Letourneur D, Uzan G, Le Visage C. Porous polysaccharide-based scaffolds for human endothelial progenitor cells. Macromol Biosci 2012;12:901–10.

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