Îº-Carrageenan Enhances the Biomineralization ... - ACS Publications

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#–Carrageenan enhances the biomineralization and osteogenic differentiation of electrospun PHB and PHBV fibers Nowsheen Goonoo, Behnam khanbabaee, Marc Steuber, Archana BhawLuximon, Ulrich Jonas, Ullrich Pietsch, Dhanjay Jhurry, and Holger Schönherr Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00150 • Publication Date (Web): 27 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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κ–Carrageenan enhances the biomineralization and osteogenic differentiation of electrospun PHB and PHBV fibers Nowsheen Goonoo1,2*, Behnam Khanbabaee3, Marc Steuber1, Archana Bhaw-Luximon2, Ulrich Jonas4, Ullrich Pietsch3, Dhanjay Jhurry2, and Holger Schönherr1* 1

Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cµ), University of Siegen, 57076 Siegen, Germany

2

Centre for Biomedical and Biomaterials Research, MSIRI Building, University of Mauritius, Réduit, Mauritius

3

Solid State Physics, Department of Physics, University of Siegen, 57076 Siegen, Germany 4

Macromolecular Chemistry, Department of Chemistry and Biology, University of Siegen, 57076 Siegen, Germany *

Corresponding authors: [email protected] and [email protected]

KEYWORDS: Biomineralization, κ–Carrageenan, osteogenic differentiation, PHB and PHBV

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ACS Paragon Plus Environment

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Abstract

Novel electrospun materials for bone tissue engineering were obtained by blending biodegradable polyhydroxybutyrate (PHB) or polyhydroxybutyrate valerate (PHBV) with the anionic sulfated polysaccharide κ–carrageenan (κ–CG) in varying ratios. In both systems, the two components phase separated as shown by FTIR, DSC and TGA. According to the contact angle data, κ–CG was localized preferentially at the fiber surface in PHBV/ κ–CG blends in contrast to PHB/ κ–CG, where the biopolymer was mostly found within the fiber. In contrast to the neat polyester fibers, the blends led to the formation of much smaller apatite crystals (7 µm vs 800 nm). According to the MTT assay, NIH3T3 cells grew in higher density on the blend mats in comparison to neat polyester mats. The osteogenic differentiation potential of the fibers was determined by SaOS-2 cell culture for 2 weeks. Alizarin red-S staining suggested an improved mineralization on the blend fibers. Thus PHBV/ κ–CG fibers resulted in more pronounced bioactive and osteogenic properties, including fast apatite-forming ability and deposition of nano-sized apatite crystals.

Introduction One of the major goals of bone tissue engineering (BTE) is the development of biomedical scaffolds to repair/bridge large bone defects caused by trauma, tumor and infection. Scaffolds play a crucial role in BTE and one of their primary functions is to act as an appropriate template facilitating cell growth and differentiation within the bone defect as well as providing structural and functional support for newly formed bone.1,2 The scaffold should mimic the extracellular matrix (ECM), provide mechanical support3, be biocompatible, be osteoconductive, 2


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osteoinductive, osseointegrative4,5, and possess high porosity as well as large interconnected pores6,7,8. As the new bone forms, the biodegradation of the scaffold should not interfere with the production of ECM by cells and the rate of biodegradation of the scaffold should be carefully matched to create space for the new bone to grow9,10. Realistically, a broad spectrum of material properties that work in concert within the engineered tissue is required to fulfil all the above mentioned criteria. Polymers of natural origin are one class of biomaterials with significant potential to contribute to the engineering of tissues, mainly due to their resemblance to the ECM, their high chemical versatility, and their inherent interaction with biological systems. Polysaccharides, such as gellan gum11, alginate12, gelatin13, hyaluronic acid14, chitosan15, chondroitin sulfate16, dextran17,18 or pullulan17 have been successfully photo-crosslinked to form hydrogels that maintain good cell viability. Blending with synthetic polymers has also been shown to offer the possibility to obtain unique and often synergistic property combinations for related biomedical applications19. Recently, κ–CG was proposed as a potential candidate for TE applications, due to its gelation properties, mechanical strength and its innate resemblance to natural glycosaminoglycans (GAGs) present in native ECM20. Furthermore, its inherent thixotropic behavior allowed its use as an injectable matrix for the delivery of macromolecules and cells for minimally invasive therapies21. However, despite encouraging results22,23, there is still inadequate control over the swelling properties, degradation characteristics and mechanical properties of ionically crosslinked κ-CG hydrogels due to the uncontrollable exchange of ions with other cations from the surrounding physiological environment11. Carrageenans were reported to trigger the production of pro-inflammatory cytokines depending on the dose and on the structure of the

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carrageenans.24,25 However, compared to lambda-carrageenan, κ–CG induced the highest production of the anti-inflammatory interleukin-10 (IL-10)26 (120 % vs 100 %). To address these potential shortcomings, we recently demonstrated the possibility to tailor the degradation behavior and mechanical performance of PHB and PHBV films by blending the biodegradable polyesters with κ-CG without compromising the cell viability27. Although PHB and PHBV possess good biodegradability and biocompatibility, they are more hydrophobic compared to natural biopolymers, such as collagen and silk fibroin. PHB is highly crystalline, very brittle and relatively hydrophobic. On the other hand, copolymers of PHB with polyhydroxyvalerate (PHV) are less crystalline and more readily processable. However, like most synthetic polymers, polyhydroxyalkanoates (PHAs) display, in particular, poor osteoinductivity28. Therefore, as a means to overcome the limitations of κ-CG hydrogels and to enhance the biological properties of PHB and PHBV, the hydrophilic sulfated polysaccharide κCG was blended with either PHB or PHBV. Interestingly, PHBV/ κ-CG blend films, which showed least miscibility, displayed the highest biomineralization activity and fibroblast cell response due to the higher amount of biopolymer on the surface27. Following these encouraging results, we investigated, as reported here, the physico-chemical and biological properties of these polymeric blends as electrospun nanofibrous scaffold materials, which could enhance cellular response. Several processing techniques have been used before to fabricate scaffolds with the ability to mimic natural bone ECM niches. Amongst the processing techniques reported29,30,31, electrospinning exhibits several advantages due to its simplicity and cost-effectiveness as well as the possibility to process a wide range of materials21 coupled with the option to incorporate bioactive species, such as enzymes, DNAs, and growth factors allowing for better control over cell proliferation and differentiation32. Moreover, the diameter of 4


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electrospun fibers can be modulated in size to mimic fibrils of the native ECM. This property is fundamental, as it has been previously demonstrated that the topography of electrospun constructs play an important role in cell attachment and proliferation33,34. Furthermore, nanofibrous mats were found to be ideal for cell adhesion due to the higher surface area available for cell interaction35,36. It has also been reported that nanofiber scaffolds can direct osteoblastic differentiation and mineralization of osteoprogenitor cells more effectively than a solid-wall scaffold37. In addition, the porosity and interconnectivity of pores in electrospun constructs ease nutrient transport for cells. More importantly, electrospun mats have been successfully used for in vivo bone reconstruction (Table 1).

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Figure 1. Schematic of blend electrospinning approach, including the molecular structures of PHB, PHBV and κ–CG.

Hence, electrospun scaffold materials of blends of PHB or PHBV with κ–CG were fabricated (Figure 1) and characterized, to compare the interaction with cells as well as their biomineralization potential and human osteosarcoma (SaOS-2) cell differentiation ability to the homopolymer nanofibers on one hand and the blend fiber (surface) composition, hydrophilicity, and morphology on the other hand.

Table 1. Summary of in vivo studies using electrospun scaffolds for bone regeneration Polymer nature

Animal Model

Defect size

Silk38

Rats

7 mm calvarial critical sized defects

PLGA/ tricalcium

Rabbits

6 mm calvarial non-critical size

PLLA/BMP-240

Rats

5 mm calvarial critical size defects

PLLA/HA41

Rats

5 mm calvarial critical size

PCL/PMMA42

Rats

5 mm cranial defects

Gelatin/TCP43

Rats

5 mm calvarial critical sized defects

phosphate (TCP)39

2.0 Experimental Section 2.1 Materials

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PHB, PHBV (HV content 12 mol %), and κ–CG were bought from Sigma-Aldrich. 1,1,1,3,3,3hexafluoroisopropanol (HFIP) and chloroform (CHCl3) purchased from FluoroChem and SigmaAldrich, respectively, were used as received. The Milli-Q water used in this study was drawn from a Millipore Direct Q8 system (Millipore, Schwalbach, with Millimark Express 40 filter, Merck, Germany) with a resistivity of 18.0 MΩ cm. Phosphate buffer tablets (PBS) was purchased from VWR Life Sciences and PBS solution was prepared by dissolving one tablet in 100 mL of Milli-Q water.

2.2 Electrospinning Blends of PHB/ κ–CG and PHBV/ κ–CG were prepared in the following ratios: 100/0, 90/10, 80/20 and 70/30 w/w%. PHB/ κ–CG blend solutions at a concentration of 50 mg/mL were prepared by dissolving PHB and κ–CG into a 8/2(v/v) HFIP/CHCl3 solution. Similarly, PHBV/ κ–CG blend solutions with a concentration of 150 mg/mL were prepared by dissolving PHBV and κ–CG into a 8/2(v/v) HFIP/CHCl3 solution. PHBV/ κ–CG blend solutions were used at a higher concentration due to low solution viscosity at a concentration of 50 mg/mL (probably due to lower molar mass compared to the PHB). All polymer blend solutions were left on a shaker plate overnight before electrospinning. The polymer solutions were then loaded into a 1 mL plastic syringe (BD PlastipakTM) and dispensed at a constant rate of 3.5 and 3.0 mL/hr for PHB/ κ–CG and PHBV/ κ–CG blend solutions respectively. Electrospinning was conducted using a homebuilt electrospinning setup comprising a high voltage power supply (FuG Electronik GmbH, HCN 35-35000), a programmable double syringe pump (AL4000, WPI Inc.) and a piece of aluminum foil as static collector (Figure S1). The electrospinning parameters (Table 2) were optimized to produce continuous fibers, which were collected as non-woven fiber mats on the 7


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statically grounded rectangular aluminum target. After electrospinning, the scaffold materials were removed from the collecting target and stored in a desiccation chamber until further analysis.

Table 2. Electrospinning parameters used for PHB/ κ–CG and PHBV/ κ–CG solutions Blend solution PHB/ κ–CG PHBV/ κ–CG

Concentration / (mg/mL) 50 150

Flow rate / (mL/hour) 3.5 3.0

Voltage / (kV) +20 +20

Air-gap distance / (cm) 15 15

2.3 Characterization of electrospun mats 2.3.1 Fiber diameter and pore size The average fiber diameter was determined by measuring the diameter of 50 different fibers from scanning electron microscopy (SEM) images. Data analysis was carried out using ImageJ software.

2.3.2 SEM and FE-SEM To analyze the surface morphology of gold sputtered electrospun mats, SEM images were taken using a CamScan microscope (CS24, USA). High resolution field emission (FE)-SEM images were taken using a Quanta 450 ﬁeld-emission-scanning electron microscope with a solid state secondary ion detector (accelerating voltage: 30 keV). Before imaging, all samples were mounted on an aluminum stub and sputter coated with gold (8-10 nm). Measurements were taken for at least three independent samples.

2.3.3 Contact angle 8


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The contact angles of the fiber mats were measured using Milli-Q water as a probe liquid with an OCA 15plus instrument (Data Physics Instruments GmbH, Germany). Static contact angle data based on the sessile drop method were acquired immediately after deposition of a 2 µL drop on at least three positions for each sample and are stated as the arithmetic mean.

2.4 In vitro biomineralization potential Electrospun blend mats were incubated in simulated body fluid (c-SBF)44 under static conditions in a tissue culture grade 24-well plate containing 3 mL of corresponding SBF in an incubator at 37 °C in 5% CO2 atmosphere for 14 days. The c-SBF solution was prepared according to a previously published protocol44. The c-SBF solutions were renewed every 3 days. After 14 days, the blend mats were removed from the c-SBF solutions and dried under vacuum before taking SEM images.

2.5 In vitro hydrolytic degradation studies Electrospun blend mats were cut into specimens with size 1 cm × 1 cm, soaked in 15 mL PBS (pH 7.3) in closed vials and incubated at 37 °C for up to 5 weeks. After each week, the specimens were withdrawn from the PBS and dried under vacuum at room temperature until a constant weight was obtained. The weight after complete drying (final mass) was noted and used to calculate the mass loss according to Equation 1. Measurements were taken for at least three independent samples.

% mass loss =

Initial Mass - Final Mass x 100 Initial Mass

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Equation 1

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2.6 Preliminary in vitro cell studies (NIH3T3 mouse fibroblast cells) The NIH3T3 fibroblast cell line was obtained from Dr. Jürgen Schnekenburger (Biomedical Technology Center of the Medical Faculty Münster, Germany). The cells were cultured at standard conditions (37 °C, 5% CO2) as reported earlier45. Triplicates of 1 ×1 cm2 of each blend mat were disinfected (30 min ethanol followed by three 10 min PBS washes) and transferred to a 96 well-plate. Samples, seeded with 50 µL cell suspension containing 20,000 NIH3T3 cells, were incubated for 20 minutes to allow the cells to attach to the blend fibers. Afterwards, 100 µL of cell medium was added and the cells were cultured for 3 and 7 days. The cell medium was replaced every 3 days. After each time point, cell seeded scaffold materials were taken out and prepared for SEM analysis and MTT assay45. Cell-seeded samples were fixed for SEM analysis as reported in Lilge et al. 45.

2.6.1 MTT Assay The MTT assay was conducted to quantify the number of cells on the blend mats on days 3 and 7. After 3 and 7 days, the cell medium was removed and replaced with fresh one. 10 µL of 12 mM MTT solution was added to each well and the plate incubated at 37 °C for 4 hours. After 4 hours, 75 µL of the cell medium was removed and 50 µL of dimethyl sulfoxide (DMSO) was added to each well and the plate was incubated for an additional 10 minutes. The solutions were mixed well and the absorbance at 540 nm was measured in a Tecan Safire microplate reader. The wells containing only the cell medium and MTT solution were considered as the blank references. Measurements were taken for at least three independent samples. Statistical analyses were done with the one-way analysis of variance (ANOVA) test (Graph Pad Software, San 10


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Diego, CA, USA) and a Bonferroni post-test was used. A value of p < 0.05 was considered statistically significant.

2.7 SaOS-2 cell culture The SaOS-2 cells (human osteosarcoma cell line) were obtained from Dr. Ulrike Ritz (University Medical Center of the Johannes Gutenberg University Mainz, Germany). The cells were cultured in 75 cm2 flasks at 37 °C in a humidified incubator and supplied with 5% CO2 using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL; Gibco, Life Technologies), streptomycin (100 µg/mL; Gibco, Life Technologies) and 2 mM L-glutamine. The culture medium was changed every 3–4 days.

2.7.1 Mineralization of SaOS-2 cells by Alizarin red-S staining To induce mineralization of the cells, the culture medium was replaced with osteogenic medium; Mesencult-Medium supplemented with 10 mM ß-glycerophosphate, 50 µg/mL ascorbic acid and 0.1 µg/mL dexamethasone. The ability for mineralized nodule formation and calcium deposition by SaOS-2 cells on PHB and PHBV blend fibers was assessed using Alizarin red-S staining. Cell growth was not investigated during the mineralization process. Briefly, cells were seeded on the electrospun mats in a 24-well plate (50,000 cells/well). After reaching 70% confluency (2 days), the cell medium was replaced with osteogenic medium and the cells were incubated for up to 14 days. After 7 and 14 days in culture, the cells were washed with ice-cold PBS and fixed in PFA for 20 minutes at room temperature. Afterwards, the fixed cells were first washed with MilliQ water and then stained with 40 mM Alizarin red-S solution (pH 4.2) for 30 11


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min at room temperature. The scaffold materials were then washed four or five times with MilliQ water to remove any unbound stain. Photographs of the stained scaffolds were then taken. To quantify the amount of deposited calcium, the stained scaffold materials were transferred to a 2 mL microcentrifuge tube containing 1.5 mL of 50% acetic acid to destain for 1 h at room temperature. 500 µL of the solubilized stain was added to 600 µL of 1 M NaOH to adjust the pH to 4.1. 200 µL of this solution was pipetted into a 96-well plate and absorbance read at 550 nm using a Tecan Safire microplate reader. Measurements were taken for at least three independent samples. Statistical analyses were done with the one-way analysis of variance (ANOVA) test (Graph Pad Software, San Diego, CA, USA) and a Bonferroni post-test was used. A value of p < 0.05 was considered statistically significant.

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3.0 Results & Discussion 3.1 Fiber characterization Blends of semi-crystalline PHB or PHBV with amorphous κ–CG in varying weight ratios (100/0, 90/10, 80/20 and 70/30) were electrospun at concentrations of 50 and 150 mg/mL respectively, with an air gap of 15 cm and an applied voltage of 20 kV. Random fibrous, nonwoven mats were obtained, as shown in SEM images (Figure 2 and Figure S1). Table 3 summarizes the fiber diameter values determined from the SEM images. No significant change in fiber diameter was noted with increasing κ–CG content in the blends. Moreover, electrospun fibers could not be formed, when the κ–CG content was > 30 wt % due to increasing repulsive forces between the charged sulfate groups, which inhibited continuous fiber formation. High resolution FE-SEM analyses were carried out to get better insight into the surface morphologies of the electrospun blend fibers (Figures 2E & F and Figure S1E & F). All electrospun PHB/ κ–CG and PHBV/ κ–CG fibers exhibited cylindrical morphologies irrespective of the blend composition. While the neat PHBV fibers possessed a smooth surface, PHBV/ κ– CG 70/30 fibers showed a heterogeneous surface. Indeed, the presence of nanoscale protrusions with widths varying between 130-180 nm was noted on the surface of PHBV/ κ–CG 70/30 fibers. These nanoscale features on the fiber surface was possibly due to kinetic entrapment of κ– CG within PHBV/ κ–CG fibers. On the other hand, electrospun PHB/ κ–CG 70/30 fibers were smooth and did not exhibit the presence of protrusions (Figure S1E & F). This may be explained by the difference in solution properties of the PHB/ κ–CG and the PHBV/ κ–CG blends. The higher molar mass of PHB polymer (higher viscosity) led to faster jet transformation and thereby forming smooth fibers. 13


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B E

A

50 µm

50 µm

D

C

50 µm

E

50 µm

F

200 nm

200 nm

Figure 2. SEM images of electrospun PHB/ κ–CG (A) 100/0 (B) 70/30 and PHBV/ κ–CG (C) 100/0, (D) 70/30 mats; FE-SEM images of PHBV/ κ–CG (E) 100/0 and (F) 70/30 fibers. (SEM images of 90/10 & 80/20 blend fibers and FE-SEM images of 100/0 and 70/30 PHB/ κ–CG

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fibers are depicted in Figure S1; Fiber diameter distribution of electrospun PHB/ κ–CG (A) 100/0 (B) 70/30 and PHBV/ κ–CG (C) 100/0, (D) 70/30 mats are given in Figure S2).

Table 3. Summary of fiber diameter of PHB/ κ–CG and PHBV/ κ–CG mats. Blend Fiber diameter/ composition µm PHB/ κ–CG mats 100/0 2.0 ± 0.5 90/10 1.9 ± 0.6 80/20 1.8 ± 0.6 70/30 1.8 ± 0.5 PHBV/ κ–CG mats 100/0 2.0 ± 0.5 90/10 1.9 ± 0.5 80/20 1.8 ± 0.5 70/30 1.6 ± 0.5

In general, five characteristic structures have been reported in the literature during blend nanofiber evolution in electrospinning, namely columns, plates, micropores, bumps and smooth surface46. Initially, the jet is accelerated and elongated in the direction of the electric field. Then, column-like features are formed due to fluctuations enhanced by the electric field. The latter are then stretched into plates followed by the formation of a porous surface due to solvent evaporation and phase separation. The micropores formed become denser and smaller with time and their depth also decreases. Finally, these micropores disappear due to extensive stretching of the jet and bumps are extruded out of the surface. Afterwards, the jet is stretched and bent into a smooth one. Here, the absence of bumps on the surface of the pure PHBV fibers indicate that the latter are stretched more in comparison to the 70/30 blend fibers.

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To have better insight on the distribution of κ–CG within the blend fibers, static contact angles were measured. As noted from Table 4, a decrease in contact angles was noted for both PHB/ κ– CG and PHBV/ κ–CG blend mats with increasing κ–CG content. However, this decrease was more pronounced for the PHBV/ κ–CG blend system. Indeed, comparing similar blend compositions of PHB/ κ–CG and PHBV/ κ–CG blends, it can be observed that contact angles of PHBV/ κ–CG is smaller than that of the corresponding PHBV/ κ–CG. It was therefore concluded that the biopolymer κ–CG was mostly located on the fiber surface in PHBV/ κ–CG fibers while in PHB/ κ–CG systems, the majority of the biopolymer was distributed within the fiber as schematically illustrated in Figure 3. In addition, the rougher blend fiber morphology observed by SEM (Figure 2F) should result in increased water contact angle for the PHB/ κ–CG blends and decreased values for the PHBV/ κ–CG blends.

Table 4. Summary of contact angle (CA) data. Blend composition CA/ º (wt/wt %) (Mean ± SD) PHB/ κ–CG 100/0 126 ± 1 90/10 120 ± 2 80/20 107 ± 1 70/30 104 ± 1 PHBV/ κ–CG 100/0 107 ± 2 90/10 74 ± 3 80/20 62 ± 1 70/30 58 ± 2

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Figure 3. κ–CG localization in PHB/ κ–CG and PHBV/ κ–CG blend fibers. Electrospun PHB/ κ–CG and PHBV/ κ–CG mats were further characterized by FTIR, DSC, XRD and TGA to investigate the influence of κ–CG incorporation on the resulting physicochemical properties of the blend fibers. FTIR analysis revealed a more pronounced shift of the carbonyl band towards lower wavenumbers for PHBV/ κ–CG (1727 to 1721 cm-1) compared to PHB/ κ–CG (1723 to 1721 cm-1) (Figure S3 and Table S1). In addition, no change in melting transitions was noted in PHB/ κ–CG and PHBV/ κ–CG blends (Table S2), suggesting that the biopolymer κ–CG was immiscible with the crystalline phase of PHB and PHBV. From the plots of crystallinity against polyester content, it can be concluded that κ–CG interfered with the crystallization of PHB and PHBV in the blends (Figure S4). The absence of significant peak shifts with increasing κ–CG content in the XRD spectra of the blend fibers implied unaltered crystal structure (Table S3 and Figure S5). Thermogravimetric profiles of electrospun PHB/ κ– CG mats showed 2, 3 or 4 stages of thermal degradation in contrast to PHBV/ κ–CG blends whereby the number of degradation steps remained unchanged irrespective of blend composition (Figure S6, Tables S4 and S5). This result indirectly suggests a higher degree in immiscibility in PHB/ κ–CG mats compared to PHBV/ κ–CG mats. Hydrolytic degradation studies of electrospun PHB/κ–CG and PHBV/κ–CG blend mats were conducted to ensure that the structural integrity is maintained throughout the cell culture period. Increasing addition of hydrophilic κ–CG to both PHB and PHBV favored hydrolytic 17


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degradation, as can be noted from the higher mass loss values (Figure S7). Indeed, contact angle values indicated increased surface hydrophilicity with higher κ–CG contents. Mass loss profiles of all PHB/κ–CG and PHBV/ κ–CG blends were non-linear. The apparent rate constants of degradation (K) were calculated by fitting the degradation curves to the Equation 2. M= M0 (1- ݁ ି௞௧ )

Equation 2

Whereby M : mass loss at time = 0 M0 : mass loss at time = t K: apparent rate constant The apparent rate constants of degradation (K) were dependent on the blend ratio and nature of polymer blend (Table 5). For instance, comparing mass loss values of the 70/30 blend mats, it can be concluded the PHBV/κ–CG underwent a higher extent of degradation due to increased surface hydrophilicity and reduced crystallinity (Figure S7). Similarly, PHBV/ κ–CG films degraded slightly faster compared to the corresponding PHB/ κ–CG films due to enhanced surface hydrophilicity and reduced crystallinity27. Furthermore, a decrease in fiber diameters of the electrospun mats was noted after 5 weeks degradation, suggesting a surface erosion mechanism (Table 6). Pure PHB and PHBV mats exhibited only few regions of fiber melting, as marked by red arrows while both 70/30 blends displayed significantly more areas of fiber melting (Figure 4). This can be attributed to PBS uptake, which results in the swelling of individual ﬁbers that fuse together during the degradation process. In summary, hydrolytic degradation studies indicated that all blend fibers maintained a fibrous morphology throughout the degradation period investigated.

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Table 5. Summary of apparent rate constant of degradation for PHB/ κ–CG mats and PHBV/ κ–CG mats.

Blend composition 100/0 90/10 80/20 70/30 100/0 90/10 80/20 70/30

Apparent rate constant of degradation / K PHB/ κ–CG mats 0.099 ±0.006 0.151 ± 0.009 0.053 ± 0.004 0.119 ± 0.007 PHBV/ κ–CG mats 0.073 ± 0.004 0.104 ± 0.006 0.227 ± 0.014 0.211 ± 0.013

Table 6. Summary of fiber diameter of electrospun mats before and after degradation. Time/ (weeks)

Fiber diameter (Mean ± SD) PHB

PHB/ κ –CG 70/30

PHBV

PHBV/ κ –CG 70/30

0

2.0 ± 0.5

1.8 ± 0.5

2.0 ± 0.5

1.6 ± 0.5

5

0.5 ± 0.1

0.5 ± 0.1

0.4 ± 0.1

0.3 ± 0.1

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B

50 µm

D

C

Figure 4. SEM images of (A) PHB, (B) PHB/ κ –CG 70/30 (C) PHBV and (D) PHBV/ κ –CG 70/30 fibers following 5 week hydrolytic degradation. The red arrows and circles denote areas of regions of fiber melting.

3.2 Biomineralization potential The bone-bonding ability (bioactivity) of the electrospun blend fibers (i.e., the potential to bind with hard tissues) is an important aspect to consider for bone tissue engineering scaffold materials. The ability of a material to form a hydroxyapatite (HA)-like surface layer when immersed in a simulated body fluid (SBF) in vitro is often taken as an indication of its bioactivity47. Ionic exchange phenomena occurring between the scaffold material and SBF solution determine the bone bonding potential of the material. It is well established that the formation of apatite depends on the reactivity (dissolution–precipitation) of the substrate48,49. The 20


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mineralized interface formed during the biomineralization process ensures proper physicochemical and mechanical cohesion between the scaffold and the host bone and accelerates bone apposition47. In this study, the apatite forming ability of the scaffold materials was examined by incubating them in SBF solution with ion concentrations nearly equal to those of human blood plasma for 2 weeks. The surface of the blend mats was then investigated by SEM (Figure 5 and Figure S8) and the morphology of the apatite deposits was assessed by FE-SEM (Figure 6). The apatite deposited on the surface of electrospun PHB and PHBV scaffold materials was cube-shaped with a typical size of 7 µm. By contrast, finer and smaller apatite crystals of approximate size of 800 nm were formed on the blend surfaces (Figure 6B). As reported previously, the helical structure of κ–CG probably leads to specific binding to the (002) face of hydroxyapatite50. However, in this study, the peak due to 3,6 anhydro galactose, which is crucial for the formation of helical structure in κ–CG could not be observed in the FTIR spectra of the blend mats, presumably due to the low content of k-CG in the blend fibers. The apatite particles formed on the blend scaffold materials (Figure 6B) were closer in size to the ones in native bone (approx. 10 nm)51 compared to those formed on PHB and PHBV surfaces. Subsequent energy dispersive X-ray (EDX) analysis confirmed that the deposits consisted mainly of Ca and P (Figure 7). Minor peaks due to Na, Mg and Cl observed in the EDX spectra originate from the SBF solution. The atomic ratio of Ca/P of the particles formed during the biomineralization process was 1.60 which was close to that of stoichiometric hydroxyapatite (1.67).

It was recently reported that polysaccharides possess the ability to induce the heterogeneous nucleation of apatite from SBF, which impacts on crystal growth and hence on the shape and size 21


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of the crystals52. In fact, the stronger the interaction between the polysaccharides and HA, the smaller is the HA crystal size according to Fang et al. 52. We noted that the blending of anionic κ–CG with PHB or PHBV promoted the precipitation of apatite from SBF, indicating improved biomineralization. Apatite crystals were initially formed on the fiber surface and later in between the fibers (Figure S8 E). The presence of κ–CG on the fiber surface and hence sulfate groups generate non-homogenously distributed local charges. Due to differences in the local charge on the blend fibers, a local Ca2+ supersaturation is generated, leading to apatite formation. The formation of an interfacial mineralized layer between the scaffold and bone tissue ensures their cohesion53. As mentioned before, the particle dimension of apatite formed on the blend scaffold material (Figure 6B) during the biomineralization process was nano-sized, which is close to the dimension of that present in natural bone (approx. 10 nm)51,54. This may be rationalized by the stronger interaction between κ–CG and HA compared to the polyester and HA. The nanometer size of both collagen fibers and apatite crystals as well as the organization of the composite contribute significantly to the excellent mechanical properties and functionality of bone55.

50 µm

50 µm

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20 µm

20 µm

Figure 5. SEM images of PHB/ κ–CG mats (A) 100/0, (B) 70/30, and of PHBV/ κ–CG mats (C) 100/0 and (D) 70/30 following incubation in SBF after 2 weeks.

A

B

1 µm

Figure 6. FE-SEM images of PHB/ κ–CG mats (A) 100/0, (B) 70/30 following incubation in SBF after 2 weeks (Micron-sized apatite was false colored in red using GNU Image Manipulation Program software; GIMP2 Inc.).

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Ca/P = 1.60

Figure 7. EDX data (from FE-SEM) of crystals formed during the biomineralization process.

3.3 Cell studies using NIH3T3 cells The response of NIH3T3 cells to PHB/ κ–CG and PHBV/ κ–CG fibers was also investigated. The fibroblast cells were observed to be elongated and adopted a spindle-shaped morphology on all electrospun scaffold materials indicating effective cell attachment to the matrix (Figure S9). Furthermore, the surface of the blend scaffolds was almost completely covered with cells on day 7 (Figure S9). Interestingly, the number and length of filopodia was larger on the blend scaffold materials (Figure 8). Filopodia are needle-like, actin-rich protrusions from the cell surface. They are highly dynamic structures capable of extending and retracting over a timeframe of 10 seconds56. Cells use filopodia extensions to probe substrate rigidity from a distance and regulate their responses based on the stiffness of the substrate. The mechanical signal determines the stability of the extension and the rate and efficiency of subsequent cell spreading. Hence, the observations indicate that the NIH3T3 cells formed more stable filopodia on the blend mats compared to pure PHB and PHBV due to favorable surface properties. The well-developed 24


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filopodia of cells on the blend scaffold materials facilitated cell proliferation, since filopodia are not only the initiation sites for adhesion, but also act as a sensing organelle producing guidance cues and traction force to move the cell body57,58. The MTT assay (Figure 9 and Figure S10) showed that both PHB/ κ–CG and PHBV/ κ–CG scaffold materials exhibited higher cell viability than pure PHB and PHBV after 3 and 7 days of culture. Improved cell proliferation can be ascribed mainly to enhanced surface hydrophilicity of blend fibers due to the presence of κ–CG near or at the surface of the fiber. Indeed, previous studies have demonstrated that compared to hydrophobic surfaces, hydrophilic ones led to enhanced cellular adhesion and proliferation due to higher vitronectin and fibronectin absorption, which are essential proteins for binding with cell surface receptors59,60.

B

A

2 µm

Figure 8. FE-SEM images of NIH3T3 cell seeded PHBV/ κ–CG mats (A) 100/0, (B) 70/30 after 7 days showing filopodia of cells.

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0.8

**

0.7 *

0.6 Absorbance

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ns

** *

PHB/KCG PHBV/KCG

*

0.5 0.4 0.3 0.2 0.1 0.0 0

10 20 κ–CG content/ (wt %)

30

Figure 9. MTT assay results following NIH3T3 culture on PHB/ κ–CG and PHBV/ κ–CG fibers on day 7. All measured absorbance from the blend fibers were compared with the corresponding pure polyester and were found to be significantly higher than pure PHB or PHBV except where it is indicated: * p < 0.05; ** p< 0.0001 and (ns) not significant.

3.4 Mineralization using SaOS-2 cells Under certain conditions, SaOS-2 cells differentiate into premature osteoblasts and further into mature osteoblasts61. This process is marked by the formation of mineralized calcium nodules, which is an important indicator for the ability of a material to promote bone formation61. In the present study, Alizarin Red-S staining was used to indirectly study the differentiation of SaOS-2 cells. Figures 10 A & B show the corresponding quantification of the minerals that originated from calcium deposition of SaOS-2 cell culture. Compared to the pure PHB scaffold material, higher absorbance values were noted for PHB/ κ–CG blends, suggesting higher SaOS-2 mineralization. Similar observations were made in the case of PHBV/ κ–CG scaffold materials.

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Moreover, comparing similar blend composition scaffold materials, it was found that PHBV/ κ– CG fibrous mats led to better SaOS-2 mineralization (Figure 10 C). Overall, addition of κ–CG to both PHB and PHBV led to better in vitro apatite formation, enhanced NIH3T3 proliferation and SaOS-2 cell mineralization. In addition, the blend scaffold materials exhibited higher potential for SaOS-2 differentiation as demonstrated by Alizarin Red– S staining. However, electrospun blends of PHBV and κ–CG were found to be the best candidates for BTE applications due to better NIH3T3 proliferation, SaOS-2 differentiation and mineralization and also due to the formation of apatite crystals close in size to native bone. This can be rationalized by variations in physico-chemical properties and differences in miscibility of the polymers as a result of blending.

0.3 0.3 Absorbance at 550 nm

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 49 50 51 52 53 54 55 56 57 58 59 60

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**

** **

0.2

** **

*

Day 7 Day 14

0.2 0.1 0.1 0.0 0


(A)

27


30

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0.6 0.5 Absorbance at 550 nm

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 49 50 51 52 53 54 55 56 57 58 59 60

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**

** **

0.4

**

0.3

**

*

Day 7

0.2

Day 14

0.1 0.0 0


30

(B)

(C)

Figure 10. Absorbance of Alizarin-Red S staining (for apatite deposition by SaOS-2 cells) of (A) PHB/ κ–CG, (B) PHBV/ κ–CG after 7 & 14 days and (C) photographs of Alizarin Red-s stained scaffold materials after 14 days. All measured absorbance from the blend fibers were 28


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compared with the corresponding pure polyester and were found to be significantly higher than pure PHB or PHBV except where it is indicated: * p < 0.001 and ** p < 0.0001.

4.0 Conclusion PHB/ κ–CG and PHBV/ κ–CG electrospun fibers developed in this study were shown to possess enhanced bioactivity properties for consideration as scaffold materials in bone tissue engineering applications. FTIR, DSC, XRD, TGA and contact angle measurements indicated that κ–CG incorporation in PHB and PHBV led to varying degrees of miscibility, which in turn influenced fiber morphology and surface properties. In particular, a higher degree of interaction occurring between PHBV and κ–CG resulted in enrichment of the biopolymer at the fiber surface in contrast to PHB/ κ–CG blend systems whereby most of the biopolymer was located within the fibers. The degree of crystallinity and hence the hydrolytic degradation rate of the blend fibers could be controlled by variation of κ–CG content without compromising their apatite-forming ability. Due to favorable surface properties of the blend fibers, longer and more mature welldeveloped filopodia from fibroblast cells were formed on the surface of the blend fibers which facilitated cell proliferation. The presence of κ–CG at the blend fiber surface resulted in the formation of nano-sized apatite crystals compared to micron-sized ones formed on the surface of the pure polyesters. In addition, κ–CG incorporation within PHB and PHBV fibers resulted in improved SaOS-2 differentiation and mineralization in vitro. These results taken together suggest that the use of a carefully chosen minority biopolymer phase with an optimized ratio can substantially improve the in vitro cell response towards PHAs, which originates in particular from the blend miscibility, nanoscale texture/mechanics and chemistry of the fiber surface.

Supporting Information 29


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Figure S1. SEM images of electrospun PHB/ κ -CG (A) 90/10, (B) 80/20 and PHBV/ κ -CG (C) 90/10, (D) 80/20 mats, FE-SEM images of PHB/ κ–CG (E) 100/0 and (F) 70/30 fibers; Figure S2: Fiber diameter distributions of electrospun PHB/ κ–CG (A) 100/0 (B) 70/30 and PHBV/ κ– CG (C) 100/0, (D) 70/30 mats; Figure S3: FTIR spectra of PHB, PHBV, 70/30 PHB/ κ–CG and 70/30 PHBV/ κ–CG fibers; Table S1: Summary of C=O peak maxima for PHB/ κ–CG and PHBV/ κ–CG blend mats; Table S2: Summary of thermal properties of PHB/ κ–CG and PHBV/ κ–CG blend mats; Figure S4: Graph of crystallinity v/s polyester content for PHB/ κ–CG and PHBV/ κ–CG fibers; Table S3: Interplanar distances (d) of the (130) and (040) planes in PHB or PHBV blend fibers; Figure S5: X-ray diffractograms of PHB/ κ–CG (A) and PHBV/ κ–CG (B) fibers with varying κ–CG content; Figure S6: TGA profiles for electrospun PHB/ κ–CG and PHBV/ κ–CG fibers fibers; Table S4: Summary of results obtained for PHB/ κ–CG fibers from TGA analysis; Table S5: Summary of results obtained for PHBV/ κ–CG fibers from TGA analysis; Figure S7: Mass loss profiles for electrospun (A) PHB/ κ–CG and (B) PHBV/ κ–CG mats during a 5-week degradation period; Figure S8: SEM images of PHB/ κ –CG mats (A) 100/0, (B) 90/10, and PHBV/ κ –CG (C) 80/20 and (D) 70/30 following incubation in SBF for 2 weeks; FE-SEM images of PHB/ κ–CG (E) 100/0 fibers following incubation in SBF for 2 weeks; Figure S9: SEM images of NIH3T3 cell seeded PHB/ κ -CG mats (A) 100/0, (B) 70/30 and PHBV/ κ -CG (C) 100/0 and (D) 70/30 after 7 days; Figure S10: MTT assay results following NIH3T3 culture on PHB/ κ–CG and PHBV/ κ–CG fibers on day 3.

AUTHOR INFORMATION Corresponding Authors

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* Dr. Nowsheen Goonoo Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cµ), University of Siegen, 57076 Siegen, Germany * Prof. Dr. Holger Schönherr Physical Chemistry I, Department of Chemistry and Biology & Research Center of Micro and Nanochemistry and Engineering (Cµ), University of Siegen, 57076 Siegen, Germany

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources The authors acknowledge generous financial support from the Alexander von Humboldt Foundation (Georg Forster postdoc stipend to NG), the European Research Council (ERC project ASMIDIAS, Grant no. 279202) and the University of Siegen.

Acknowledgements We thank Dr. Yvonne Voß, Dipl.-Ing. Gregor Schulte, Dipl.-Chem. Ing. Petra Frank for their support and helpful advice, as well as Dr. Ulrike Ritz (University Medical Center of the Johannes Gutenberg University Mainz, Germany) and Dr. Jürgen Schnekenburger (Biomedical 31


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Technology Center of the Medical Faculty Münster, Germany), who kindly provided the cell lines. We are also grateful to DELTA (TU-Dortmund) for providing beamtime.

ABBREVIATIONS ATR-FTIR, attenuated total reflectance-Fourier transform infra-red spectroscopy; BTE, bone tissue engineering; CHCl3, chloroform; c-SBF, conventional simulated body fluid; DSC, differential scanning calorimetry; DMSO, dimethyl sulphoxide; DMEM, dulbecco’s modified Eagle’s media; ECM, extracellular matrix; GAG, glycosaminoglycans; HFIP, 1,1,1,3,3,3hexafluuoroisopropanol; κ–CG, kappa-carrageenan; PBS, phosphate buffer solution; PHAs, polyhydroxyalkanoates; PHB, polyhydroxybutyrate; PHBV, polyhydroxybutyrate-co-valerate; TGA, thermogravimetric analysis; TE, tissue engineering; XRD, X-ray diffraction

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Îº-Carrageenan Enhances the Biomineralization ... - ACS Publications

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