Goonoo, N, Bhaw-luximon, A, Passanha, P, Esteves, S, Schönherr, H & Jhurry, D 2017, 'Biomineralization potential and cellular response of PHB and PHBV blends with natural anionic polysaccharides' Materials Science and Engineering: C, vol 76, pp. 13-24. DOI: 10.1016/j.msec.2017.02.156

This is an Accepted Manuscript of an article published by Elsevier in Materials Science and Engineering: C on 01/07/2017, available online: http://dx.doi.org/10.1016/j.msec.2017.02.156

© 2016. This accepted manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

1

Biomineralization potential and cellular response of

PHB and PHBV blends with natural anionic

polysaccharides

Nowsheen Goonoo1,3, Archana Bhaw-Luximon1, Pearl Passanha2, Sandra Esteves2, Holger

Schönherr3, Dhanjay Jhurry1*

1Centre for Biomedical and Biomaterials Research (CBBR), MSIRI Building, University of

Mauritius, Réduit, Mauritius

2University of South Wales, Sustainable Environment Research Centre, Upper Glyntaff,

Pontypridd CF37 4AT, Wales, UK

3Physical Chemistry I and Research Center of Micro and Nanochemistry and Engineering (Cμ),

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

* Corresponding Author

Abstract

In this paper, the biomineralization potential and cellular response of novel blend films of the

anionic sulfated polysaccharides kappa-carrageenan (KCG) and fucoidan (FUC) derived from

seaweeds with semi-crystalline polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-

2

valerate (PHBV), respectively, were analyzed. The incorporation of KCG and FUC into PHB

and PHBV, which has been studied here for the first time, led to an overall decrease in

crystallinity, enhanced surface hydrophilicity, reduced brittleness and faster degradation of the

polymer blend films. All PHB/KCG, PHBV/KCG and PHBV/FUC films exhibited a two-stage

mass loss profiles with pH stabilization. PHBV/KCG film showed the highest biomineralization

activity due the presence of sulfate groups on the surface of the films. NIH3T3 cells attached and

proliferated well on all blend films on account of enhanced surface hydrophilicity and improved

flexibility. PHBV/KCG led to a promoted cellular activity compared to PHBV/FUC, presumably

due to phase separation and higher amount of biopolymer on the film surface that was a

consequence of the immiscibility of the polymers in the blend films.

KEYWORDS: polyhydroxyalkanoates, anionic polysaccharides, k-carrageean, fucoidan, blend

films, biological response

1.0 Introduction

Due to their biodegradability and biocompatibility, polyhydroxyalkanoates (PHAs) have

attracted much attention with potential applications in various fields ranging from bioplastics

through drug delivery [1,2,3] and anti-bacterial applications [4] to bone tissue engineering. These

biopolyesters are synthesized by microorganisms as intracellular carbon and energy storage

compounds under unbalanced growth conditions [5]. Amongst the members of the PHA family,

poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are

the most widely studied due to their biodegradability, processing versatility and excellent

biocompatibility. PHB consisting of 3-hydroxybutyrate (HB) monomer units is a semi-crystalline

3

polymer with a high melting temperature (176-178 °C) and a rather high degree of crystallinity

(32-53 %) [6]. The synthesis of PHBV via incorporation of HV units in PHB through

copolymerization was found to be an effective way to increase flexibility of PHB as a result of a

decrease in crystallinity.

To overcome the limitations associated with PHAs such as poor mechanical properties, high

production cost, limited functionalities, incompatibility with conventional thermal processing

techniques, their susceptibility to thermal degradation as well as to tailor unique property

combinations required for biomedical applications, PHB and PHBV have been blended with

synthetic and natural polymers [7]. The mechanical and thermal properties were modified which

in turn, impacted on degradation rate. Blending PHB and PHBV with synthetic polyesters such

as poly(L-lactic acid) (PLLA) resulted in mostly immiscible blends with improved toughness and

ductility [8,9,10], decreased degradation rate [11,12], enhanced thermal stability [8] and in one

case better cell growth [13]. Blends of PHB with poly(ethylene oxide) (PEO), were miscible in

both amorphous and crystalline phases [14], had increased flexibility [15,16], decreased

hydrophobicity [15,16] and improved cell growth of Chinese hamster lung cells [17]. In addition,

the hydrophilicity of electrospun PHB mats as well as the in vitro blood clotting rate were

significantly improved by blending with Pluronic [18]. Furthermore, the mechanical performance

of PHAs could be reinforced by blending with hydroxyapatite [19]. All these blends exhibited

reduced degradation rates compared to pure PHAs but still lack chemical functionalities such as

hydroxyl and amine groups which would enhance biological activity. Blending of PHAs with

natural polymers, neutral or ionic, may help overcome some of these limitations. Natural

polymers are known to have inherent biocompatibility and in some cases bioactivity, for

4

instance, chitosan shows antimicrobial activity arising from its cationic character [20]. They are

hydrophilic in nature and due to presence of OH, NH2 or CONH groups, natural polymers have

high chemical versatility. PHB and PHBV can behave differently in terms of miscibility with

some natural polymers. For instance, PHB resulted in partially miscible blends with starch [18]

and lignin [21] whereas PHBV resulted in immiscible blends with both [22,23]. However, both

PHB and PHBV were immiscible with ethyl cellulose [24,25] and silk fibroin [26,27]. Overall

from the results obtained, immiscibility reduced thermal stability, tensile strength but improved

flexibility. PHB blended with chitosan resulted in miscible blend with improved thermal stability,

tensile strength, flexibility and swelling capacity [28,29,30].

In general, blends of PHB and PHBV are immiscible leading to reduced mechanical properties

due to poor interfacial adhesion and phase boundaries [21,26]. Degradation rates varied

depending on the hydrophobic-hydrophilic nature of the polymer blended with the PHA [24].

Furthermore, immiscibility does not necessarily translate into poor biological response [31]. On

the other hand, nano-morphological features arising due to immiscibility or changes in physico-

chemical properties had a positive influence on cell growth [11]. Miscible blends were formed

due to favorable hydrogen bonding occurring between the CO groups of PHAs and amide or

hydroxyl or ether groups in chitosan, lignin or PEO respectively. Similar changes in physico-

chemical properties were noted in both films and electrospun mats [30,32]. Blends of PHAs with

natural polymers also led to materials with enhanced hydrophilicity, reduced brittleness, faster

degradation, which in turn resulted in better biological response. For instance, thermal analysis,

in particular, melting point depression and the presence of a single glass transition temperature in

PHB/chitosan and PHBV/chitosan blends indicated that chitosan is miscible with either PHB or

PHBV at all compositions [33]. PHB/chitosan films prepared by an emulsion blending technique

5

displayed improved tensile strength and elongation at break with increasing chitosan content

[33]. Electrospun blend mats of PHBV/chitosan were prepared by mixing appropriate volumes of

solutions of PHBV in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chitosan in trifluoroacetic

acid (TFA)/dichloromethane (DCM). The brittleness of electrospun PHBV mats (Young`s

modulus 151 MPa) was reduced via incorporation of chitosan (Young`s modulus 105 MPa for

90/10 blend mat) [32] and cellulose acetate [34]. As a result of enhanced hydrophilicity, higher

NIH3T3 cell proliferation was noted on electrospun PHB/cellulose acetate scaffolds in contrast

to PHB mats. Moreover, better hFOBs response was noted on PHBV/Chitosan/hydroxyapatite

(HA) scaffolds compared to PHBV scaffold [32]. In comparison to electrospun PHBV mats,

PHBV/chitosan mats displayed enhanced hydrophilicity suggesting presence of the biopolymer

on the fiber surface [32]. Similarly, compared to electrospun PHBV, electrospun PHBV/gelatin

nanofibers fabricated from a solvent mixture of 2,2,2-trifluoroethanol/ formic acid led to

improved hydrophilicity. Furthermore, cytocompatibility tests showed that NIH3T3 cell

proliferation on the nanofibrous scaffolds increased with increasing gelatin content [34].

Enhanced Schwann cell proliferation and faster differentiation were noted on electrospun

PHB/PHBV/collagen mats compared to PHB/PHBV mats due to enhanced surface wettability

and presence of amino groups on the mat surface [36]. Electrospun PHB/chitosan mats degraded

faster than PHB mats due to enhanced hydrophilicity [37].

Plant-derived natural polymers such as kappa-carrageenan (KCG) and fucoidan (FUC) have

enormous potential in tissue engineering (TE) applications. Indeed, both carrageenan and

fucoidan have been shown to possess anti-thrombogenic, anti-viral, anti-tumor and immune-

modulatory properties [38,39]. Carrageenan has been investigated for drug delivery systems [40],

6

immobilization of enzymes [41] and encapsulation of cells and growth factors [42,43] targeting

cartilage and bone regeneration [44] respectively. Fucoidan has also been used in many

biomedical-related fields. For instance, microspheres and nanoparticles consisting of FUC in

combination with chitosan were developed for drug delivery applications [45,46]. In addition,

blends of FUC with polymers such as chitosan [47], alginates [48] and polycaprolactone (PCL)

[49,50] have been processed into hydrogels, films or nanofibers for TE applications.

The main objective of the research reported in this paper was therefore to investigate whether

and to which extent blending of PHB and PHBV with the natural anionic polymers KCG and

FUC could improve the biological response. In particular, the impact of the blend surface

properties and degradation behavior on the one hand and the biomineralization potential and

cellular response of blend films on the other hand were unraveled to provide a basis for potential

application of the blends in tissue engineering and cell culture applications.

2. Experimental Section

2.1. Materials

PHB (Mw = 145,000 g/mol) and PHBV (Mw = 300,000 g/mol; Đ = 8.3) were prepared at the

University of South Wales, UK. Molar masses were determined using GPC (chloroform as

eluent) equipped with a refractive index detector using polystyrene standards. Monomodal and

bimodal distributions were obtained for PHB and PHBV respectively. From the 1H-NMR

spectra, the % HV content in PHBV was estimated to be around 14.4 % (Equation 1).

7

% HV content = Area of CH 3(HV )

Area of CH 3 ( HV )+ Areaof CH 3 (HB)

Equation 1

KCG (Mw = 268,000 g/mol) was provided by Shemberg Marketing Corporation, Philippines

Lot no. Z0701-1 and FUC (Mw = 80,000 g/mol) was purchased from Lyphar Company, China.

Molar masses of the biopolymers were determined by light scattering. The sulfate content of

KCG and FUC was determined using the barium sulfate turbidity method [51]. Briefly, the

polysaccharides were subjected to acid hydrolysis followed by the addition of barium chloride.

The absorbance of the barium sulfate precipitate was then measured at 420 nm. The KCG used

had a sulfate content of 26.3 % while FUC had a sulfate content of 12.8 %.

1,1,1,3,3,3 hexafluoroisopropanol (HFIP) from Apollo Scientific Limited was used as

received.

2.2. Methods

2.2.1. Preparation of PHB/KCG, PHBV/KCG and PHB/FUC blend films

Blends of PHB/KCG with varying weight percentages were prepared by solvent-casting at

room temperature. For instance, blend (80/20) (10 % w/v) was prepared by dissolving 0.38 g

PHB and 0.02 g KCG in 2 ml HFIP until completely homogenized. The mixture was then poured

in a Teflon mold (40 mm x 10 mm x 4 mm) and the solvent allowed to evaporate in a fume hood

for 24 hours.

2.2.2. Hydrolytic Degradation

8

Blend films with size 1 × 1 cm2 were cut and placed in 15 ml of phosphate buffer solution

(PBS) at a temperature of 37 ⁰C for five weeks. % mass loss of the blend films was calculated

according to Equation 2.

% mass loss = Initial Mass - Final MassInitial Mass

x 100 Equation 2

2.3. Measurements

Differential Scanning Calorimetry (DSC) analysis was carried out using a DSC 200F3 Maia®

thermal analyzer. All blend samples were heated from 30 to 200 °C, cooled to -20 °C and

reheated to 200 °C at 10 °C /min. Surface morphology characterization (SEM) was accomplished

using a CamScan microscope (CS24, USA) at an accelerating voltage of 25 kV. Blend samples

were mounted on an aluminum stub and sputter coated with gold for imaging. The chemical

compositions of the blend films were analyzed by using energy dispersive X-ray (EDX)

spectrometer attached to the SEM. 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: 5 keV). The chemical compositions of biomineralized films as

well as the Ca/P ratio were determined by using energy dispersive X-ray (EDX) spectrometer

attached to the FE-SEM. Tensile tests were carried out as reported previously [52]. Fluorescence

microscopy images were taken using an Evos FL Auto Fluorescence microscope. Static contact

angle measurements were measured on the films using the sessile drop method with an OCA

15plus instrument (Data Physics Instruments GmbH, Filderstadt, Germany) with Milli-Q water

at ambient conditions. For determination of surface free energies of the films, the static contact

angles were also measured using diiodomethane (99%, Alfa Aesar) and glycerol (99.6%, Acros

Organics). The surface free energy parameters were calculated according to reference [53]. The

Lifshitz-van der Waals interaction parameter comprising dispersion, orientation and induction

9

forces, γs+ (acidic term) and γs

- (basic term) was used to characterize the film surface. In eqs 1-3, θ

denotes the measured contact angles, and the subscripts D, G, and W denote diiodomethane,

glycerol, and water, respectively.

γsLW = γD

(1+cosθ )4

2

Equation 3

γG (1 + cosθG) - 2 √γ s

LW γGLW = 2 √γ s

+¿γ G−¿ ¿¿ + 2 √γ s

−¿ γG+¿¿¿

Equation 4

γW (1 + cosθW) - 2 √γ s

LW γWLW = 2 √γ s

+¿γ W−¿ ¿¿ + 2 √γ s

−¿ γW+¿¿¿ Equation 5

The acid-base component of the surface free energy, γsAB may be determined using Equation 6

and the total surface energy is given by Equation 7.

γsAB = 2 √γ s

+¿γ s−¿ ¿¿ Equation 6

γ = γsLW + γs

AB Equation 7

2.4 In vitro biomineralization studies

Blend films were incubated in conventional simulated body fluid (SBF) 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 protocol [54]. The c-SBF solutions were renewed every 3 days. After 14

days, the blend films were removed from the c-SBF solutions and dried under vacuum before

taking SEM images. The samples were mounted on aluminium stubs and sputter-coated with

gold (60 seconds) for SEM imaging. Apatite formation was confirmed by carrying out EDX

analysis of the biomineralized films after 2 weeks.

10

2.5 NIH3T3 cell seeding

NIH3T3 cells were obtained as a kind gift from Dr. Jurgen Schnekenburger (Biomedical

Technology Center of the Medical Faculty Munster, Germany). The NIH3T3 fibroblast cells

were cultured at standard conditions (37 °C, 5% CO2) as reported previously [55]. 1 ×1 cm2 of

each blend film were disinfected (30 min ethanol followed by three 10 min 1x PBS washes) and

transferred to a 96 well-plate. Blend films were seeded with 50 µL cell suspension containing

20,000 NIH3T3 cells. They were then incubated for 20 minutes to allow the cells to attach to the

blend films. After 20 minutes, 100 µL of cell medium was added and cells cultured for up to 7

days. The cell medium was replaced every 3 days. After 7 days, the cell-seeded samples were

taken out and prepared for SEM analysis. The scaffolds were treated with 3% glutaraldehyde in

PBS (30 minutes at room temperature), followed by subsequent dehydration with 30 %, 50 %, 70

%, 90 % and 100 % ethanol. They were then washed with a mixture of ethanol/

hexamethyldisilazane (HMDS) (1/1 v/v) and finally with pure HMDS. The samples were

prepared for SEM as described before.

2.6 MTT Assay

The MTT assay was conducted to quantify the number of cells on the blend films on days 3

and 7 respectively. 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 DMSO added to

each well and the plate incubated for an additional 10 minutes. The solutions were mixed well

and the absorbance read at 540 nm. The well containing only the cell medium and MTT solution

was considered as the blank reference.

11

2.7 Statistical Analysis

Data are presented as mean ± standard error of mean. 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.

3.0 Results and Discussions

We recently reviewed the key polymeric properties of biopolymers, synthetic polymers and

their blends for bone and vascular scaffold eligibility. The success of scaffolds is highly

dependent on striking the right balance of physico-chemical, biological and mechanical

properties of polymers [56]. PHB and PHBV homopolymers (Figures 1 A & B) offer the basic

advantages of being biodegradable and biocompatible. However, to be successful materials as

tissue engineering scaffolds, they require enhancement or modification of some of their physico-

chemical and biological properties. One such strategy is through blending with polymers which

can complement these lacunas. We have chosen two seaweed-derived sulfated polysaccharides

namely k-carrageenan (KCG) and fucoidan (FUC) (Figures 1 C & D). KCG contains one sulfate

group per repeat unit, while FUC has three sulfate groups per repeat unit.

(A) (B)

12

O

CH3 O y

(C)

(D)

Figure 1. Repeat units of (A) PHB, (B) PHBV, (C) KCG and (D) FUC

3.1 Blend miscibility

Blends films of PHB or PHBV were prepared with KCG and FUC using the solvent casting

method. Miscibility of the blend films was first assessed using FTIR and thermal analysis.

FTIR Spectroscopic analysis

PHB/KCG, PHBV/KCG and PHBV/FUC blend films displayed peaks characteristic of the

corresponding polymers (Figure S1). The OH, CO and S-O regions were analyzed in depth to

investigate any interactions between the homopolymers. No shift was observed in the OH band

of KCG (OH= 3379 cm-1) after blending with either PHB or PHBV. The broadness of the band

doesn’t allow a conclusion on the presence or absence of interaction between the OH of KCG

and CO of the polyesters. However, a change in the shape of the OH band from broad to sharp

13

was noted with increasing KCG content for PHBV/KCG films. Usually such a band change

results from inter H-bonding shifting to intra H-bonding. The latter may correspond to the intra

H-bonding present in the helical conformation of KCG. The double helix in KCG is known to be

stabilized by three inter-chain hydrogen bonds [57]. The OH band in FUC showed a clear shift to

higher wavenumbers in PHBV/FUC blend films (from 3381 to 3436 cm-1) indicating possible

interaction. No carbonyl peak shift was noted in any of the blends. Analysis of the second

derivative spectra of PHB/KCG, PHBV/KCG and PHBV/FUC blend films showed the presence

of only two peaks at 1720 and 1746 cm-1 showing that that CO groups in the polyesters were not

involved in the formation of H-bonding with the OH groups in KCG or FUC.

Bands in the region of 840-850 cm-1 in KCG were attributed to the presence of C-O-SO3- group

on C4 of the 3-linked -D-galactopyranosyl unit [58]. Both signals showed a shift in blends with

PHB and PHBV (Figure S1), this is also accompanied by a shift in the C-C (825 cm−1) stretching

peak of PHB and PHBV. These observations indicate interactions involving sulfate groups. FUC

displayed a broad band at 1183-1280 cm−1 with a maximum at 1216 cm−1 (S-O stretching). The

broad band at 830 cm−1 (C–S–O) suggest a complex pattern of substitution [59]. The 50/50

PHBV/FUC blend presents a single band at 794 cm-1 with a slight shouldering at 818 cm-1,

(Figure S1), which tends to indicate some degree of interaction between PHBV and FUC.

Based on peak shift corresponding to C-O-SO3- bond, it can be deduced that the extent of

interaction between the polymers varied in the following order:

PHBV/FUC > PHB/KCG > PHBV/KCG

A lower molar mass of FUC compared to KCG coupled with higher sulfate content might

explain the higher interaction with PHBV and FUC.

14

Thermal Analysis of pure polymers and blends

PHB and PHBV homopolymers showed glass transition temperatures, Tg at -2.7 and -0.9 °C

respectively. PHB displayed a melting transition at 160.3 °C and PHBV possessed two melting

transitions at 150.4 and 162.7 °C confirming the broad molar mass distribution of PHBV sample

(Đ = 8.3) noted by GPC.

KCG can exist as random coil or can adopt a helical conformation in the solid state. During the

first heating, a broad band is observed centered at 90.5 °C corresponding to the dehydration peak

of KCG and possibly to the break-down of the helical conformation [60]. The second heating

curve indicates a Tg at 105 °C [61] which may be attributed to the random coils. FUC showed a

similar peak at 123.9 °C during the first heating corresponding to dehydration; no Tg was

detected in the second heating scan.

Thermal analysis results of PHB/KCG and PHBV/KCG & PHBV/FUC films are summarized

in Tables 1 and 2 respectively. The degree of crystallinity of the blend films (Xblend) was

calculated as reported previously [52]. For that purpose, the enthalpy of melting for 100 %

crystalline PHB and PHBV were taken from the literature as 146 J/g and 109 J/g respectively

[62,63].

The presence of two glass transition temperatures (Tg) for PHB/KCG and PHBV/KCG films

indicates that the polymers are not miscible with each other. The lower glass transition

temperature (Tg1) was assigned to PHB or PHBV rich phases while the higher temperature one

(Tg2) was attributed to KCG rich phase.

For all PHBV/FUC films, only one Tg was noted. Furthermore, the shift in Tg noted for 70/30

and 50/50 films suggest higher degree of interaction between the polymers in these films.

15

Overall, the presence of KCG in PHB/KCG and PHBV/KCG blend films led to an increase in

the crystallization temperature (Tc) and a decrease in the enthalpy of crystallization. A much

more significant decrease was observed in the enthalpy of crystallization upon addition of KCG

to PHBV. The presence of recrystallization peaks (Tcc) in all PHBV/KCG and PHBV/FUC

(except 90/10) films suggests that crystallization was not complete during the cooling step

possibly due to slowed crystallization of PHBV.

In general, the melting temperatures of PHB in the PHB/KCG blend films increased with

KCG. However, for 80/20 blend, a bimodal melting peak was observed most probably as a result

of difference in crystallization rates. The lower temperature one was attributed to the melting of

the “as-formed” PHB crystallites during processing, and the highest temperature peak was due to

the melting of the PHB crystals formed from the recrystallization during the heating scan [63].

Two melting points were noted (shoulder to a major peak) for PHBV/KCG and PHBV/FUC

films (Tables 1 and 2), with a main melting transition accompanied by a slight shouldering. The

two melting transitions for PHBV/KCG films remained practically unchanged irrespective of

KCG content and remained close to those of pure PHBV, thereby suggesting immiscibility of the

polymers in the crystalline region. On the other hand, the two melting transitions noted for

PHBV/FUC films remained close to those of pure PHBV up to 20 wt % FUC. Blend films with

higher FUC content displayed lower melting transitions (for instance 146 °C vs 163 °C and 126

°C vs 150 °C for a 50/50 film). This indicates enhanced degree of miscibility with increasing

FUC contents.

The degree of crystallinity of the blend was calculated according to Equation 8. The presence

of KCG or FUC in PHB/KCG, PHBV/KCG and PHBV/FUC blend films decreased the overall

crystallinity. However, a sharper drop in crystallinity was noted for PHB/KCG and PHBV/KCG

16

films compared to PHBV/FUC films. This indicates that KCG had a higher influence on the

crystallization kinetics of the semi-crystalline polymers and inhibited crystallization process to a

higher extent compared to FUC.

χblend = ∆ Hm

∆ Hm0 × 100 % Equation 8

where ∆Hm is the enthalpy of melting per gram of blend and ∆Hm0 represents the enthalpy of

melting for 100 % crystalline PHB or PHBV.

In conclusion, the thermal analysis of the blend films showed that both PHB/KCG and

PHBV/KCG films are partially miscible in the amorphous regions and, as expected, immiscible

in the crystalline regions. At low FUC contents (<20 wt%), PHBV/FUC films were partially

miscible in the amorphous regions. At higher FUC contents, PHBV and FUC were miscible as

indicated by the presence of a single Tg and more drastic decrease in Tm values.

The negatively charged sulfate groups enhance intramolecular repulsion and steric hindrance

between segments in the molecular chain of KCG and FUC and thus prevent the chain from

adapting a compact conformation [63]. There are more sulfate groups present on FUC than KCG.

This may be responsible for the ability of FUC chains to intermix with PHBV in the amorphous

regions and thus influence the thermal properties.

Moreover, FUC had a less significant influence on the crystallization process of the films

compared to KCG, due to higher extent of interaction between FUC and PHBV.

Table 1. Summary of thermal properties of PHB/KCG blend films

Sample Tg1/ (°C)

Tg2/(°C)

Second heating scan Tc/ (°C)Tm/ (°C) Xblend (%)

PHB -2.7 160.3 37.5 67.3KCG 105.790/10 -3.4 99.0 171.1 28.9 96.780/20 -3.0 98.2 162.8, 151.7 18.6 75.7

17

70/30 -2.9 103.1 171.9 14.8 92.550/50 -3.3 103.9 174.6 14.8 110.9

Table 2. Summary of thermal properties of PHBV/KCG and PHBV/FUC blend films

Sample Tg1/ (°C)

Tg2/(°C)

Second heating scan Tc / (°C)Tm/ (°C) Xblend

(%)PHBV/KCG

PHBV -0.9 162.7, 150.4 38.4 -KCG 105.790/10 -1.4 97.8 163.0, 150.7 35.1 45.280/20 -1.3 107.5 163.7, 153.2 29.1 44.370/30 -1.7 105.4 164.3, 151.1 25.3 61.450/50 -2.1 98.6 164.6, 153.8 13.6 72.3

PHBV/FUCFUC90/10 -3.2 161.7, 150.5 42.4 38.9 45.480/20 -2.3 161.9, 150.2 36.0 33.070/30 -8.2 150.0, 131.8 29.7 27.250/50 -9.6 146.0, 125.5 23.9 21.9

3.2 Mechanical properties

Blend miscibility is known to impact on mechanical performance [64]. Overall, addition of

KCG led to a decrease in mechanical properties of the films with more drastic effects noted in

PHB/KCG films (Tables 3 & 4). A decrease in Young`s modulus values was noted for

PHB/KCG films with increasing KCG content. As indicated by thermal properties, KCG inhibits

crystallization of PHB, leading to lower Young`s modulus. Mechanical properties of an

immiscible polymer blend depend also on phase morphology, and molecular interactions.

Immiscibility in PHB/KCG films resulted in enhanced crystallization rates (Figure S2) leading to

much higher degree of disruption in the fibrous structure of PHB in PHB/KCG films. On the

other hand, KCG acts as anti-nucleating agent in PHBV/KCG films (Figure S3), slowing

crystallization rates and allowing for much better polymer chain arrangements. This rationalises

18

the much sharper drop in extension at break noted for PHB/KCG films as opposed to

PHBV/KCG films. In comparison to PHB/KCG films, addition of KCG resulted in an increase in

Young`s modulus values of all PHBV/KCG films except for the 50/50 film. The Young’s

modulus is related to the degree of crystallinity, Xblend. In general, higher crystallinity leads to

higher Young`s modulus [65]. This may be explained by the extensiveness of secondary

bonding, which is present when molecular chains are closely packed together as a result of a

higher miscibility of chains. In amorphous regions, secondary bonding is weaker due to chain

misalignment and thus lower crystallinity and modulus are observed. On the other hand,

incorporation of FUC within PHBV led to increased flexibility as noted by a significant decrease

in Young`s modulus values (Table 5). In line with the conclusions derived from thermal analysis,

70/30 and 50/50 PHBV/FUC films displayed almost similar Young`s modulus values which

were smaller compared to the remaining compositions suggesting reduced brittleness due to

better miscibility. Based on Young`s modulus values of cancellous bone (20-500 MPa),

PHB/KCG, PHBV/KCG and PHBV/FUC films with low biopolymer content may be suitable for

bone tissue engineering applications [66].

Table 3. Mechanical properties of solution cast PHB/KCG films

Blend compositionPHB/KCG

Extension at break (mm/mm)

Tensile stress at break (MPa)

Tensile strain at break (MPa)

Modulus (MPa)

100/0 11.10 0.22 1.11 88.395/5 6.42 0.68 0.64 83.990/10 1.16 4.38 0.12 67.480/20 0.51 1.77 0.05 97.670/30 3.08 0.44 0.31 75.550/50 1.57 0.14 0.16 37.3

Table 4. Mechanical properties of solution cast PHBV/KCG films

19

Blend compositionPHBV/KCG

Extension at break (mm/mm)

Tensile stress at break (MPa)

Tensile strain at break (MPa)

Modulus (MPa)

100/0 3.43 0.01 0.34 43.395/5 3.53 0.44 0.35 69.890/10 2.43 - 0.24 149.780/20 3.35 - 0.34 53.770/30 1.29 1.96 0.13 64.050/50 3.35 0.04 0.34 12.7

Table 5. Mechanical properties of solution cast PHBV/FUC films

Blend compositionPHBV/FUC

Extension at break (mm/mm)

Tensile stress at break (MPa)

Tensile strain at break (MPa)

Modulus (MPa)

100/0 2.06 0.13 0.21 156.095/5 1.72 1.51 0.17 52.590/10 2.65 0.10 0.26 56.880/20 8.46 0.92 0.85 69.270/30 2.82 0.14 0.28 37.450/50 2.30 0.24 0.23 35.8

3.3 Surface Properties & Degradation Behaviors

Addition of KCG and FUC to PHB and PHBV leads to changes in surface morphology of the

blend films (Figures 2-3). Incorporation of KCG disrupted the surface morphology of PHB to a

much greater extent compared to PHBV films, thereby supporting the sharper decrease in

mechanical properties of PHB/KCG films. Addition of 10 wt % KCG led to the formation of a

porous structure, while spherical protrusions were observed in the 50/50 PHB/KCG film (Figure

2). Furthermore, indication of phase separation was noted in the fluorescence microscopy images

of 70/30 PHB/KCG (Figure 3). PHB and KCG-rich phases appearing as green and grey regions

respectively can be clearly distinguished in fluorescence microscopy images unlike in

PHBV/KCG films due to higher degree of miscibility in the latter.

20

Fucoidan was homogeneously distributed within the PHBV matrix as noted by the presence of

round structures (Figure 3). Moreover, no drastic perturbation in surface morphology of

PHBV/FUC was noted with increasing addition of the biopolymer in contrast to PHB/KCG and

PHBV/FUC (Figure 2). In summary, surface morphology analysis supports the fact that the

extent of miscibility varies in the order:

PHBV/FUC > PHB/KCG > PHBV/KCG

21

50 m50 m

50 m50 m

50 m50 m50 m

50 m

G

C

H

BA

D E F

Figure 2. SEM images of PHB/KCG (A) 100/0, (B) 90/10, (C) 50/50 film, (D) PHBV, (E)

PHBV/KCG 90/10, (F) PHBV/KCG 50/50, (G) PHBV/FUC 90/10, (H) PHBV/FUC 50/50. All

scale bars are 50 m.

22

DC

20 m

E

20 m

BA

100 m 100 m

Figure 3. Fluorescence microscopy images of (A) PHB/KCG 90/10 (B) PHB/KCG 70/30, (C)

PHBV/KCG 90/10, (D) PHBV/KCG 70/30, (E) PHBV/FUC 90/10 and (F) PHBV/FUC 70/30

Magnification 40X (PHB and PHBV were stained green using Nile Red as hydrophobic probe)

To confirm the blend surface observations, contact angles (CA) of all the blend films were

measured (Table 6). Addition of both KCG and FUC to PHB and PHBV led to increased

hydrophilicity as shown by the drop in contact angles. PHBV/KCG film surfaces were slightly

more hydrophilic than PHB/KCG surfaces. Almost similar contact angle values were obtained

for PHBV/KCG and PHBV/FUC films (biopolymer content < 30 wt %). For 70/30 and 50/50

films, incorporation of KCG led to more hydrophilic surfaces due possibly to partial miscibility

of PHBV/KCG. Pure PHB or PHBV have lower surface free energy than the blend films which

show their low ability to take part in polar interactions due probably to their lack of polar

functional groups [67]. An increase in surface energy is first observed upon the addition of 10%

of KCG to PHB or PHBV. This increase indicates a change in the surface component of the

blend with the presence of hydrophilic hydroxyl and sulphate groups of KCG taking part in polar

interactions thus enhancing surface energy. As the content of polysaccharide increases, a general

decrease in surface free energy is observed. The surface free energy gives an indication of the

phase separation for polymer blends and is thus an indication of miscibility. Phase separation

23

F

20 m20 m

occurs when the blend can lower its total free energy. A decrease in interface energy indicates

rearrangements of polymer molecules at the blend surface and interfacial segregation creating a

more homogenous system. This again suggests that KCG is present at the surface of the blend

films and as its content increases, phase segregation occurs [68].

Table 6. Summary of static water contact angle and surface free energy measurements for

PHB/KCG, PHBV/KCG and PHBV/FUC films

Blend composition (wt/wt %) CA (Mean ± SD) Surface free energy (mN/m)

PHB/KCG100/0 92 ± 1.5 2890/10 88 ± 5.9 5380/20 84 ± 5.0 4470/30 75 ± 4.1 3250/50 69± 5.1 NA

PHBV/KCG100/0 88 ± 1.2 5390/10 81 ± 2.2 7680/20 73 ± 2.6 4470/30 63 ± 2.8 5750/50 53 ± 1.5 NA

PHBV/FUC100/0 88 ± 1.2 5390/10 80 ± 1.5 NA80/20 74 ± 3.1 NA70/30 68 ± 2.0 NA50/50 64 ± 1.6 NA

NA: Not applicable since the diiodomethane droplet was absorbed too quickly on the film surface

24

Analysis of the blend films by EDX also confirmed the presence of the biopolymers KCG and

FUC on the surface. Figure 4 depicts the EDX spectra of PHB/KCG, PHBV/KCG and

PHBV/FUC 70/30 films which reveal the characteristic peaks of S at 2.33 keV for all 3 samples

[69].

In addition, the presence of K (3.3 keV and 3.68 keV) was also noted for KCG-containing

blend films. KCG is known to contain ions, which are responsible for its helical conformation.

The shoulder at 2.12 keV was attributed to Au. The weak signal at 1.1 keV was assigned to Na in

KCG containing blends [69].

0 0.5 1 1.5 2 2.5 3 3.5 40

200400600800

100012001400160018002000

PHBKCGPHBVKCGPHBVFUC

Energy/ KeV

CPS

Figure 4. EDX spectra of PHB/KCG, PHBV/KCG and PHBV/FUC 70/30 films

In vitro hydrolytic degradation of PHB/KCG, PHBV/KCG and PHBV/FUC blend films was

investigated at 37°C in PBS. Figures 5-7 depict mass loss profiles for PHB/KCG, PHBV/KCG

and PHBV/FUC blend films during the hydrolytic degradation period. Both PHB and PHBV

films showed almost similar degradation profiles and lost approximately 20 % of their mass after

5 weeks. As noted from Table 6, PHB and PHBV displayed contact angle values close to each

25

K

K

S

Au

Na

other indicating that the extent of hydrophobicity was almost same for both polymer films. Due

to enhanced hydrophilicity and decreased crystallinity, addition of KCG and FUC to PHB and

PHBV increases hydrolytic degradation as can be observed by higher mass loss values. The

blend films showed two-phase degradation profiles during the 5 week degradation period

investigated with an initial slow degradation followed by faster degradation. All PHB/KCG,

PHBV/KCG and PHBV/FUC films maintained mechanical integrity during the first two weeks with

relatively low mass loss values (less than 20 wt %) except for 50/50 PHBV/KCG and PHBV/FUC

films.

This is interesting for bone scaffolds given that mechanical integrity of the scaffolds should be

maintained during the first few weeks (slow degradation) to allow for new blood vessel

formation.

In summary, PHB/KCG, PHBV/KCG and PHBV/FUC films show appropriate two-stage mass

loss profiles with pH stabilization. Furthermore, low degradation rates during the first few weeks

will allow for in vivo angiogenesis within the scaffolds.

0 1 2 3 4 5 60

10

20

30

40

50

60

70

50/5070/3090/10PHB

Time/ (Weeks)

Mas

s Los

s/ (%

)

26

Figure 5. Mass loss of PHB/KCG blend films as a function of hydrolysis time in PBS at 37 °C

0 1 2 3 4 5 60

1020304050607080

50/5070/3090/10PHBV

Time/ (Weeks)

Mas

s Los

s/ (%

)

Figure 6. Mass loss of PHBV/KCG blend films as a function of hydrolysis time in PBS at 37°C

0 1 2 3 4 5 60

1020304050607080

50/5070/3090/10PHBV

Time/ (Weeks)

Mas

s Los

s/ (%

)

Figure 7. Mass loss of PHBV/FUC blend films as a function of hydrolysis time in PBS at 37°C

3.4 Biomineralization potential and in vitro biocompatibility studies

27

The formation of a bone-like apatite layer on biomaterials is considered to be the precondition

for their osteoinduction to induce bone formation. The biomineralization potential of the blend

films was investigated by incubating the films in simulated body fluid (SBF) at 37°C for 14 days.

The surface of the blend films was investigated by SEM (Figure 8). Almost no inorganic deposits

were found on the surface of PHB and PHBV films. On the other hand, significant calcium

phosphate deposition was noted on the surface of blend films as confirmed by EDX analysis

(Figure 9) confirming the presence of Ca2+ and phosphate. Cl- is considered as an impurity, but

importantly, no Na+ is observed in the EDX spectra, which further supports the assignment to

calcium phosphate. Interestingly, the morphology of the calcium phosphate crystals was different

in PHB/KCG, PHBV/KCG and PHBV/FUC films. Numerous tiny granular calcium phosphate

particles were present on the surface of PHB/KCG films (Figure 8C). These were in fact

assemblies of tiny crystals formed on the surfaces of the blend film. Similar apatite-like deposits

was formed following 7-day incubation of gelatin/LDPE composite film in SBF [70]. In contrast,

the calcium phosphate particles were worm-like on the surface of PHBV/KCG films (Figure 8D)

and the film surface was completely covered by the rough deposit layer. The surface of

PHBV/FUC films were covered with both worm like particles and spherical ones (Figure 8E).

Subsequent energy dispersive X-ray (EDX) analysis revealed that the deposits consisted mainly

of Ca and P (Figure 9). 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 varied between 1.52-1.60. These values were lower than the Ca/P ratio

reported in the literature for stoichiometric hydroxyapatite (1.67). According to the literature

[71], this may indicate the formation of an amorphous phase of Ca-poor calcium phosphate on

the surface of the films during the biomineralization process.

28

The biomineralization activity varied as follows: PHBV/KCG > PHB/KCG> PHBV/FUC. This

trend may be correlated to the hydrophilicity of the film surface due to the presence of KCG. The

presence of KCG on the surface with its negative charge attracted the positive calcium ions in

SBF to form rich calcium surface, which then binds with negative phosphate ions in SBF

inducing the precipitation of calcium phosphate [72,73].The nucleation occurs from an aqueous

solution, thus adsorption of water on the surface or penetration of water inside the porous

structure are important factors for biomineralization. PHBV/KCG having the most hydrophilic

surface also showed the highest biomineralization potential. Both PHBV/KCG and PHB/KCG

have porous surfaces, which is according to the literature a requirement for initiation of apatite

nucleation [74]. However, the deposits observed in this study could not be assigned

unequivocally to (pure) hydroxyapatite, since the peaks observed in the XRD measurements

were broad (no data shown).

29

A B

200 nm

Figure 8. Qualitative analysis of mineral growth by FE-SEM of (A) PHB (B) PHBV, (C)

PHB/KCG 70/30, (D) PHBV/KCG70/30 and (E) PHBV/FUC 70/30 blend films after incubation

in c-SBF for 2 weeks

30

C D

E

31

B

Ca/P = 1.52

A

Ca/P = 1.55

Figure 9. EDX spectra of biomineralized (A) PHB/KCG, (B) PHBV/KCG and (C) PHBV/FUC

70/30 films

One of the primary scaffold requirements is that the latter should be biocompatible and

should not elicit any undesirable responses to cells in vivo. In the present study, the preliminary

in vitro biocompatibility of the blend films was investigated by culturing NIH3T3 fibroblast cells

to confirm the better performance of films of PHB or PHBV blended with KCG and FUC. These

cell studies are intended to guide us into further biological studies. The results of the MTT assay

for 3 and 7 days are summarized in Figures 10-11 respectively. NIH3T3 cells attached and

proliferated well on all blend films. However, the incorporation of polysaccharides in PHB and

PHBV led to enhanced cellular response as can be deduced from higher number of cells. This

can be rationalized by higher surface hydrophilicity and also improved flexibility. However,

compared to PHBV/FUC, PHBV/KCG led to much better cell responses possibly due to

immiscibility of polymers, which led to phase separation and higher amount of hydrophilic

biopolymer on the film surface leading to higher water uptake [75,76]. Water uptake translates in

32

C

Ca/P = 1.60

the ability of the scaffold to absorb body fluid and transfer cell nutrients, thus supporting cell

growth.

0 10 20 300

100002000030000400005000060000700008000090000

100000

PHBKCGPHBVKCGPHBVFUC

Polysaccharide content/ (wt %)

Num

ber

of c

ells

Figure 10. Summary of MTT assay results on day 3. Cell numbers on the blend films were

compared with the corresponding pure polyester and were found to be significantly lower than

pure PHB or PHBV except where it is indicated: * p < 0.05, **p < 0.001, and *** p < 0.0001

0 10 20 300

20000

40000

60000

80000

100000

120000

PHBKCGPHBVKCGPHBVFUC

Polysaccharide content/ (wt %)

Num

ber

of c

ells

33

*** ***

***

*

******

* *

***

*

***

***

***

******

ns **

Figure 11. Summary of MTT assay results on day 7. All cell numbers from the blend films were

compared with the corresponding pure polyester and were found to be significantly lower than

pure PHB or PHBV except where it is indicated: * p < 0.05, **p < 0.001, *** p < 0.0001 and

(ns) not significant

Conclusions

Blending of natural anionic polysaccharides κ-carrageenan and fucoidan with PHB and PHBV

resulted in partially miscible blends. FTIR data and thermal analysis indicated that the

PHBV/FUC showed the highest interaction due to backbone flexibility of PHBV, lower sulfate

content of FUC compared to KCG and larger difference in molar mass which allowed a higher

degree of dispersion of FUC and better phase interaction. Contact angle and EDX data indicated

that the polysaccharides are mostly present on the surface of the blends. The blends exhibited an

initial phase of slow degradation followed by a faster degradation profile, which is fully

compatible with the requirements for bone scaffolds. The Young’s modulus values obtained with

polysaccharide content up to 30 wt % fall within the range exhibited by cancellous bone. The

miscibility characteristics of the different blends was also shown to impact on their biological

behavior. Indeed, the PHBV/KCG blend, which showed the least miscibility, displayed the

highest biomineralization activity and fibroblast cell response, which is attributed to the higher

amount of hydrophilic biopolymer on the surface. In summary, a higher surface hydrophilicity,

higher water uptake and immiscibility of polymer components gave rise to an improved

biological response of PHBV/KCG blends towards fibroblast growth.

ACKNOWLEDGMENTS

34

We thank Dr. Yvonne Voß and Dipl.-Ing. Gregor Schulte for their support and helpful advice as

well as Clemens Padberg from the University of Twente (Netherlands) for GPC analysis of the

polymers. We are also grateful to Qasim Alhusaini for FE-SEM & EDX analysis.

Funding Sources

We are indebted to the Mauritius Research Council for funding biomaterials and drug delivery

research at CBBR. Nowsheen Goonoo thanks the Alexander von Humboldt Foundation for

financial support in the Georg Forster Postdoctoral Fellowship program. Holger Schönherr

acknowledges financial support from the European Research Council (ERC project ASMIDIAS,

Grant no. 279202) and the University of Siegen.

Appendix A: Supplementary data

FTIR spectra of hompolymers and corresponding blends; Plots of relative crystallinity versus

crystallization time for PHB/KCG blend films; Plots of relative crystallinity versus

crystallization for PHBV/KCG.

ABBREVIATIONS

EDX, energy dispersive X-ray; FTIR, fourier transform infra-red; FUC, fucoidan; HA,

hydroxyapatite; HFIP, 1,1,1,3,3,3 hexafluoroisopropanol, hFOB, human fetal osteoblasts; KCG,

kappa-carrageenan; PBS, phosphate buffer saline; PEO, poly(ethylene oxide); PHA,

polyhydroxyalkanoate; PHB, polyhydroxybutyrate; PHBV, polyhydroxybutratevalerate; PLLA,

poly(L-lactic acid); SBF, simulated body fluid; SF, silk fibroin.

35

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