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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/
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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-
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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
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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
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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
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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],
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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).
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% 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
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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
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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.
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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.
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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
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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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