J Mater Sci: Mater Med DOI 10.1007/s10856-013-5105-0
Properties and in vitro characterization of polyhydroxybutyrate– chitosan scaffolds prepared by modified precipitation method Lubomir Medvecky • Maria Giretova Radoslava Stulajterova
•
Received: 30 May 2013 / Accepted: 22 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Porous polyhydroxybutyrate (PHB)–chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends. Keywords Chitosan Polyhydroxybutyrate Propylene carbonate Cytotoxicity Microstructure Blend
L. Medvecky (&) M. Giretova R. Stulajterova Institute of Materials Research, Slovak Academy of Science, Watsonova 47, 040 01 Kosice, Slovak Republic e-mail: [email protected]
1 Introduction Polyhydroxybutyrate (PHB) is a member of the group of hydrophobic biodegradable biopolymers and it can actively have an affect on the hydrophilic/hydrophobic ratio in composites, where it is one of the components. It is known that cell adhesion to the implant and cell activity are influenced by properties of the implant surface such as e.g. surface tension, roughness, etc. [1, 2]. PHB and its composites exhibited excellent cell proliferation [3]. Doyle et al. [4] established that implants with the PHB addition produced a consistent favorable bone tissue adaptation response without an undesirable chronic inflammatory response. The chitosan (chit) represents polysaccharide biopolymers with inductive and stimulative activities on connective tissue rebuilding [5]. Both biopolymers are successfully utilized as one of the composite components e.g. in blends based on poly(3-hydroxy)butyrate [6], composites with other natural biopolymers or fibers [7–9]. Deacatylated chitosan is easily soluble in acid aqueous solutions (acetic or hydrochloric acid). The fully N- and O-acetylated (stearoyl, hexanoyl or lauroyl) chitosan derivates were soluble in non-polar organic solvents [10, 11]. The organic carbonates (ethylene, propylene carbonates) were applied as plasticizers of chitosan [12, 13]. Rajan et al. [14] studied the thermal and mechanical properties of PHB–chit composites prepared by mechanical mixing in a microcompounder. A decrease in polyhydroxybutyrate crystallinity with a chitosan content in polyhydroxybutyrate–chitosan blends has been observed, obtained by mixing the biopolymer solutions dissolved in co-solvent (1,1,1,3,3,3-hexafluoro-2-propanol (HFP)). The miscibility of polymers and intermolecular interaction between polyhydroxybutyrate and chitosan chains were verified by by
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Cheung et al. [15] and Ikejima et al. [16]. The nanofibrous PHBV–chit scaffolds prepared by electrospinning from the HFP solutions had good fibroblast proliferation activity and a superior ability for improving fibroblast adhesion [17]. Organic soluble (in chloroform) PHB–chit composites and fibers exhibited non-toxic behavior [18]. Wang et al. [19] carried out direct chitin grafting with PHBV via the intermediate product synthesized by the chlorination of PHBV [19]. In this paper, the physico-chemical properties, microstructure and cytotoxicity of porous polyhydroxybutyrate– chitosan blended scaffolds (PHB–chit) prepared by the precipitation of dissolved biopolymers in miscible aqueous or low toxic organic solvents were evaluated. This method has not been used as of yet. The presented preparation procedure of blends is very simple and no special requirements were given, miscibility or other physicochemical properties being examples. Our aim was to apply a simple method without complicated working procedures or chemical modifications of biopolymers, and with environmentally friendly biopolymer solvents.
of molecular weights of PHB–chitosan in blends was determined by gel permeation chromatography (GPC, Watrex, RI detector), with chloroform as a mobile phase for the PHB characterization and 0.5 M NaCl ? 0.05 M NaH2PO4 (pH2) mobile phase for the chitosan analysis. The separation was carried out on the PL gel mixed C 5 lm column (PHB) and PL gel mixed OH 8 lm (chitosan) at 1 mL/min flow rate of mobile phase. The calibrations of molecular weights for the calculation of molecular weight distribution in polymers were carried out using dextrans (standards for chitosan) and polystyrene (standards for PHB) (American polymer standard corporation) with various average molecular weights. The chitosan degradation in scaffolds by lysozyme solution (2 mg/mL, PBS) at 37 °C was characterized by the amount of reducing sugars created during hydrolysis at selected times. The reducing sugars were determined by the potassium ferricyanide method described by Ni et al. [20]. N-Glucosamine (Sigma-Aldrich) solutions were used as standards, and determination was carried out at 65 °C by the kinetic measurement of absorbance at 420 nm after 10 min from reagent addition and sample mixing.
2 Materials and methods
2.3 Cell cultivation and viability testing
2.1 Preparation of blends
L 929 mouse fibroblasts (ECACC, Salisbury, UK) were cultured in culture flasks with surface areas of 75 cm2 (SPL Life Sciences, Korea) in MEM (Minimum essential medium) with Earles balanced salts, 2 mM L-glutamine (SAFC Biosciences, Hampshire, UK), 10 % fetal bovine serum (Sigma-Aldrich) and ATB-Antimycotic (Penicillin, Streptomycin, Amphotericin) solution (Sigma-Aldrich). Cells were maintained at 37 8C in 5 % CO2 atmosphere with 95 % humidity in an incubator (Memmert). The medium was changed every 2 days. After the cells reached about 80 % confluence, they were harvested by trypsinization using 0.25 % Trypsin–EDTA (Sigma-Aldrich) solution followed by the addition of fresh medium to create a cell suspension and the cell numbers were calculated using a Neubauer hemacytometer. All sides of the scaffolds were sterilized by UV radiation in a laminar flow box for 30 min before their immersion into culture plates. The cell proliferation was examined using an MTS test (Cell titer 96 aqueous one solution cell proliferation assay, Promega, Madison, USA). The sterilized scaffolds were placed into the 48-well suspension plate, seeded with 2.0 9 104 cells in 500 ll of complete medium and cultured at 37 °C in atmosphere containing 5 vol % CO2 and 95 % humidity in an incubator. The cell proliferation on scaffolds was evaluated for 24 h, 5 and 10 days after cell seeding. Immediately before the assay, the culture medium was removed from the wells and replaced with fresh medium and the MTS reagent was
The blends with various polyhydroxybutyrate (GoodFellow) to chitosan (SigmaAldrich, middle) ratios (3:1, 1:1, 1:3) were prepared by mixing of PHB (dissolved in propylene carbonate) and chitosan (in 1 % acetic acid) solutions. The same volumes of differently concentrated biopolymer solutions were used for the precipitation. Mixing was carried out using a magnetic stirrer at 400 rpm. After 10 min mixing, the acetone was slowly added to suspensions for the complete precipitation of biopolymers. Final blends were filtered, washed with distilled water and molded into block form (4 mm 9 25 mm 9 3 mm), cut to smaller parts with the final dimensions (4 9 4 9 3 mm) and lyophilized (Ilshin) for 6 h. 2.2 Characterization methods The thermal degradation and melting of blends were analyzed by differential scanning calorimetry (DSC) and thermogravimetry (TG) (Mettler, 2000C). The chemical interaction and crystallinity were evaluated by FTIR spectroscopy (Shimadzu, IRAffinity1, KBR method). The microstructure of scaffolds was observed by scanning electron microscopy (FE SEM JEOL7000). Optical fluorescence microscopy (inverted optical microscope Leica DM IL LED) with phase modulation contrast was used for the observation of biopolymer precipitates. The distribution
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added. After 2 h of incubation, the intensities of coloring, which characterize the formazan concentration (produced by proliferating cells) in culture medium, were evaluated using a UVVIS spectrophotometer (Shimadzu) at a wavelength of 490 nm. The measured absorbances of medium from wells with cell seeded substrates were compared with the ones from wells free of scaffolds in the tissue culture polystyrene plate (a treated 48-well tissue culture plate, Santa Cruz Biotechnology, Santa Cruz, USA) seeded with cells (2.0 9 104 cells in 500 ll of complete medium). The calculated ratios were normalized to the same substrate mass (scaffold with a maximum mass). The pure complete culture medium was used as a blank. Acridine orange (AO) (Sigma-Aldrich) was used to stain the L929 cells proliferated on films prepared by the pressing of porous scaffolds. After 5 days of cultivation, the samples were rinsed with phosphate buffered saline solution (PBS); cells were fixed in 96 % ethanol for 20 min and stained with 0.01 % AO solution for 2 min in the dark. The cells on substrates were observed using a fluorescence microscope (Leica DM IL LED, blue filter) for investigating morphology and distribution of cells.
3 Results and discussion 3.1 Morphology of biopolymer precipitates and scaffold microstructures The suspensions of precipitated biopolymers and blends are shown in Fig. 1. The long chitosan fibers (up to a few hundred lm) and 1–5 lm thickness are visible in Fig. 1a. The addition of PHB to chitosan caused a strong change in morphology of precipitates in mixture with the ratio of PHB:chit = 1:3, where very fine spherical and short thin fibrous precipitates with diameters not exceeding 1 lm were observed (Fig. 1b). Larger globular particles in fine matrix were found in the biopolymer suspensions at higher PHB contents (Fig. 1c, d). No particles of this shape were observed in pure PHB suspension (Fig. 1e), where homogeneous fine grained precipitates with unresolved inner structure were visible. Organ et al. [21] observed precipitation of the needle-like shaped PHB crystallites from propylene carbonate biopolymer solutions. Iwata et al. [22] found a similar morphology of the PHB–co-valerate copolymer particles precipitated from chloroform-ethanol solutions. Chitin nanocrystals grafted with PHB had needle-like morphology. The microstructure of pure chitosan scaffolds corresponds with the fibrous morphology of the precipitated biopolymer. The inner chitosan surfaces in the scaffold were smooth without visible sharper edges or the presence of separated smaller particles. Three different pore size
fractions were present in the microstructure of pure chitosan scaffolds—the large rounded almost spherical pores (50–100 lm size); the high number of pores with dimensions around 10 lm and micropores up to 1 lm in size (Fig. 2a). A different image can be visible in the case of the pure PHB biopolymer scaffolds (Fig. 2b), where the large pores were not found and the microstructure was uniform with three clearly distinguished particle types—the globular particles (\1 lm); coarser agglomerates (up to 5 lm) and very thin short mutually interconnected fibers. Apart from the scaffold microstructure, irregularly shaped pores with maximum dimensions around 20 lm and a high fraction of micropores were found. The blended scaffolds with PHB:chit = 1:1 were composed of separated and two morphologically different polymer forms—the honeycomb or sponge-like particles with a wide pore size distribution and some fraction of larger pores (about 60 lm) (Fig. 2c); the fine microporous agglomerates with both the fibrous and more granular morphologies. The fraction of fine granular agglomerates rose with the PHB content in blended scaffolds, and these ones can be related to the pure PHB or mutually bounded fine structured PHB–chitosan polymers in blends. The microstructure of scaffolds with the PHB:chit biopolymer ratio equal to 1:3 was composed of sponge-like objects with smooth surfaces and larger irregular pores (up to 40 lm in size) (Fig. 2d). Besides this, the thin plate-like biopolymers were tightly bounded to the surface of these objects. Contrary to the above, more compact microporous microstructure with a low fraction of longer fibers and a high density of short thin fibers or biopolymer plates were observed in scaffolds with PHB:chit = 3:1 (Fig. 2e). It results from the above, that the separation of biopolymers into individual specific forms was strongly reduced with the rise in PHB content and the microstructures obtain a more uniform character. 3.2 TG, DSC and GPC analysis of blends Results of TG and DSC measurements of samples are shown in Fig. 3. Three endo effects with maxima at 149, 184 and 305 °C were found on the DSC curve of pure PHB (Fig. 3a), which correspond to the melting temperature of the amorphous (or low molecular), crystalline PHB fractions and the PHB decomposition (with a mass loss on the TG curve), respectively. The pure chitosan decomposes in three steps, which verify mass losses on the TG curve (Fig. 3b, c) (the water releases up to 150 °C, the mass loss between 220 and 320 °C characterizes the amine units decomposition, saccharide chains are degraded above 300 °C) and the decomposition finishes at 600 °C [23]. Note that a single wide exo-effect at 300 °C is observable on the chitosan DSC curve. On the DSC curve of the PHB:chit (1:1) blend, two endo-effects at 180 and 290 °C
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Fig. 1 Morphology of biopolymer precipitates in suspensions (a chit, b PHB:chit (1:3), c PHB:chit (1:1), d PHB:chit (3:1), e PHB)
arise from the melting and decomposition of PHB, a doublet of exo-effects represents the chitosan thermal decomposition with maxima at 310 and 330 °C., Similar endoeffects with maxima at 181 and 290 °C as above were observed in the case of PHB:chit (1:3) blends. Besides this, the chitosan decomposition in this sample characterizes both the sharp exo-effect at 305 °C mutually mixed with a wide exo-effect. From the comparison of DSC and TG curves of pure biopolymers and blends, it clearly resulted that the rate of chitosan decomposition was strongly affected by the thermal degradation of PHB. A more detailed analysis of exo-effects related to the chitosan decomposition in blends verify the existence of a doublet at around 300 °C in DSC curves, where one peak is expanded to the wide temperature interval between 245 and 400 °C. The second peak is sharper and narrow (the width of temperature interval about 40 °C), which is comparable
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with this one found during pure chitosan decomposition (Fig. 3c, curve a). We believe that the presence of these doublets can be related to two structurally different forms of chitosan in the microstructures of blends. Thus, in blends, phases with more fibrous character represent almost pure chitosan and regions with the fine structured plate-like forms located on surfaces of fibers (or tightly bounded to their surface) and separated fine fibrous biopolymer agglomerates characterize interconnected chitosan and PHB chains. It is clear that the fibrous (plate- or spongelike) chitosan objects in blends with compact structure and longer, mutually crosslinked polysaccharide chains will be more stable toward thermal decomposition than the fine structured fibrous chitosan particles. The finer objects of various morphologies began to decompose very rapidly after PHB thermal degradation, from which results the direct mutual bounding between chitosan and PHB
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Fig. 2 Microstructures of scaffolds (a chit, b PHB, c PHB:chit (1:1), d PHB:chit (1:3), e PHB:chit (3:1))
biopolymer chains. After decomposition of the PHB polymer, the fine chitosan chains bounded with the origin PHB particles have surfaces opened to an air oxygen attack and they are more susceptible to oxidation and thermal degradation. In the case of PHB, a reduction in melting points and decomposition temperatures after the chitosan addition were found. These effects can be caused by the decrease in lamellar thickness of the PHB crystallites in blends because of intramolecular interactions between the PHB and chitosan polymers which suppress the PHB crystallization [16, 24, 25]. Another reason for reduction in the PHB melting point can be the decrease in average molecular weight of the PHB during the preparation of blends [25]. The comparison of degradation TG curves of native and precipitated chitosans (Fig. 3c) clearly shows
that the precipitated chitosan starts to decompose about 25 °C earlier than its native form, and the exo-effect at 300 °C is tailored to the side of lower temperatures. Besides, more gradual thermal decomposition of polysaccharide chains was found than in the case of native chitosan without visible exo-effect at 550 °C, which confirms the structural changes in precipitated chitosan. The molecular weights of PHB in blends determined by the GPC analysis confirmed the reduction in average Mw and Mn and the rise in polydispersity degrees (PD) at higher chitosan contents from 108 to 57 kDa (PD = 1.9) in pure PHB to 81 and 22 kDa (3.7) in PHB:chit (1:1) blend (Table 1). The dissolution of PHB in propylene carbonate was carried out about at 120 °C for 20 min. According to the study of propylene carbonate effect on the PHB
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chains after the chitosan addition. In the case of chitosan, no interaction between chitosan and organic carbonates was found after mixing various carbonates with chitosan [12, 13]. The carbonates act as chitosan plasticizers, which cause a decrease in Tg temperature of chitosan. Thus the chitosan chains are more flexible after addition of the organic carbonate. No effect of carbonates on the distribution of chitosan molecular weights was studied to this point. In comparison of native chitosan and precipitated chitosan Mw in Table 1 there were seen strong reductions in Mw and PD values of chitosan precipitated from acetic acid solution by the propylene carbonate addition. It is clear that the mole fraction of low molecular chitosan chains rose mainly by the depolymerization of high molecular chains. A further small decrease in PD was found after addition of the PHB to blends, which confirms the opinion that the possible weak mutual interactions between biopolymers cause a shortening of their chains. It has been shown that chitosan can be thermally degraded (e.g. during drying) at moderate temperatures but no such rapid degradation and reduction in Mw were observed at 105 °C [27]. Similarly, no decrease of Mw can be related to acid chitosan hydrolysis because very soft experimental conditions were used during chitosan dissolution only [28]. Note that results of the GPC analysis are in accordance with the ones of thermal analysis. 3.3 FTIR analysis blends and chitosan degradation with lysozyme
Fig. 3 DSC (a) and TG (b) analysis of scaffolds (chit precip (underline), PHB:chit (1:3) (dotted line), PHB:chit (1:1) (hyphenated line), PHB (dotted hyphenated line)), c comparison of DSC and TG curves of native (a) and precipitate (b) chitosans
degradation [26] at 120 °C, the average molecular weight Mn of PHB falls down approx. to 90 % of the origin Mn whereas the PD were not changed during dissolution. Note that the temperature of the PHB solution during mixing with chitosan solution decreased to about 80 °C and the possible influence of temperature or propylene carbonate on the PHB degradation was not significant in this case. It results from the above that the decrease in average molecular weights (Mw and Mn) and the rise of polydispersity in the PHB:chit (1:1) blend can be related to the effect of a higher chitosan amount in the blend. The reason for increase of the PD in the sample is mainly the decrease of Mn due to a much higher mole fraction of low molecular PHB chains. Note that the Mn distribution curve of PHB in this blend is changed to a more asymmetric (tailored) shape on the side of low molecular weights, which verifies the preferential depolymerization of original shorter polymer
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The FTIR spectra of PHB, chitosan and PHB–chit blends are shown in Figs.4, 5. In the case of pure chitosan samples (Fig. 4), small changes in the spectra of native and precipitated chitosans were found—amide I band had an unresolved doublet at 1,650 cm-1, amide II band was shifted about 6–1,561 cm-1 and band arises from stretching C–N vibrations at 1,324 cm-1 was shifted to lower frequencies (about 8 cm-1) and split, which is a characteristic attribute of the formation of complexes in precipitated chitosan. Besides this, a rise in intensity of bands corresponding to free hydroxyl groups at wave numbers 1,000–1,200 cm-1 [29] was observed. All these differences verify the increase in number of hydroxyl groups, because of the chitosan depolymerization, and the presence of weak intramolecular interactions between precipitated polysaccharide chains. In a spectrum of pure polyhydroxybutyrate (Fig. 5b), were found bands from C=O stretching vibrations at 1,724 and 1,746 cm-1, as well as bands in the region between 1,120 and 1,300 cm-1 ascribing the stretching vibrations of the C–O–C ester groups and bending CH2-, CH3-group vibrations under 1,000 cm-1 [30]. Small shifts were observed in stretching vibrations of the PHB carbonyl group in blends. Note that the peak at
J Mater Sci: Mater Med Table 1 Average molecular weights (Mw and Mn) and polydispersity degrees (PD) of biopolymers in scaffolds PHB:CHIT
MwCHIT [kDa]
MnCHIT
PD
Chit native
363
9
44
Chit precip
60
6
10
1:1
41
5
8
MwPHB [kDa]
MnPHB
PD
81
22
3.7
3:1
110
53
2.1
PHB
110
57
1.9
around 1,745 cm-1 represents a low ordered or amorphous part of PHB, whereas the band at 1,720 cm-1 characterizes the crystalline fraction in PHB. The amide I chitosan band in blends (vibration of C=O in acetylated amino group) is shifted about 6 cm-1 from the one in precipitated chitosan, whereas an insignificant shift was observed for amide II band (NH deformation vibrations) [31–34]. The small shift of amide I band in blends confirms the mutual interaction between PHB and chitosan biopolymers. No bands from vibrations of groups in propylene carbonate were found in spectra. Ikejima et al. [34] confirmed by 13C NMR spectroscopy the intermolecular bonding between PHB carbonyl and chitosan amide groups, and showed an increase of the amorphous PHB component with the chitosan amount in blends prepared from homogeneous polymer solutions. Cheung et al. [15] using 1H NMR spectroscopy verified the intermolecular interaction between biopolymers. These facts are in accordance with our results. Small differences in intensities of bands characterize the ether bonds (–C–O–C–), due to the glycosidic linkage between chitosan monomer units, were found in FTIR spectra of carboxymethylated chitosans with various average molecular weights [28]. In this case, the chitosans were
precipitated from a strong acid solution after an addition of 1 M NaOH, which can affect the aggregation of chitosan chains because of the high ionic concentration. NaCl concentration has been found to have significant effect on the content of aggregates in chitosan solutions [35]. It is very hard to analyze any changes in intensities of ether and ester bands in blends as a result of overlapping the PHB and chitosan bands in spectra. Lysozyme is one of the enzymes [36, 37], which are able to depolymerize biopolymers based on N-acetylated -glucosamine units bonded via b-(1-4)-glycosidic linkage. The hydrolysis rate of chitosan by lysozyme is strongly affected by the degree of chitosan deacetylation [38]. More detailed analysis of the mechanism of chitosan degradation by lysozyme is shown in Ref. [39]. Lysozyme is very little active in PHB depolymerization, which can be effectively carried out by another enzyme—PHB depolymerase [40]. Besides this, PHB hydrolyzes to crotonic and 3-hydroxybutyric acids in strong acid or alkaline conditions (deep degradation in a relatively short time) [41], but in neutral media the degradation rate is low. Note that the insignificant PHB degradation in scaffolds at 37 °C in PBS was found during 14 days of soaking (measured by the HPLC method, results not shown). The influence of blend composition on the degradation of chitosan with lysozyme is shown in Fig. 6. The increase in content of reducing sugars (calculated as the amount of glucosamine) in solutions was observed after 14 days from the chitosan addition to lysozyme solution, whereas small differences in its amount were found after 1 and 5 days in all samples. Note that around 16 wt% of the original chitosan amount was degraded after 14 days in blended scaffolds independently of their composition. The degradation rates of pure chitosan and PHB:chit (1:3) scaffolds were slower than in samples with a higher PHB content during 1–5 days. This
Fig. 4 FTIR spectra of native (a) and precipitated chitosan (b)
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J Mater Sci: Mater Med Fig. 5 FTIR spectra of PHB:chit (1:1) (a), (1:3) (c) and PHB (b)
effect can be related to significantly reduced microporosities of individual polymer components in these scaffolds, which was confirmed by the lower values of specific surface (Fig. 7). Besides this, as resulting from SEM observations, more uniform microstructures without separated coarser polymer agglomerates were found in samples with a higher PHB amount which verifies their better mutual mixing. Cunha-Reis et al. [42] showed statistically significant dependence of the chitosan fiber degradation rate on fiber microporosity, and this influence was more ratedetermined than an effect of the fiber specific surface areas. Very low degradation of chitosan with the degree of N-acetylation (DA) equal 0.2 was observed [36] at similar conditions as in our paper, but no mention about chitosan Mw is given in this work. On the other hand, approximately 20 wt % losses were found after 3 week chitosan degradation in DMEM containing 0.5 mg/mL lysozyme, where chitosan samples were highly porous and Mw was around 300 kDa [43]. The degradation degree of chitosan fibers with DA equals 21 % and Mw = 30 kDa after 10 days was 29 % [38]. Chitosan oligomers enhanced wound healing acceleration and activated fibroblasts more intensively than chitosans with the high average molecular weight [44]. It has been shown that chitosan has antimicrobial effect and it is enhanced for gram-negative bacteria with a decrease in chitosan molecular weight [45]. Hu et al. [46] verified that PHBV grafting with chitosan exhibited antibacterial activity against gram-negative and gram-positive bacteria. 3.4 Analysis of initial cell attachment and proliferation The initial cell attachments of fibroblasts on different scaffolds after 3 h of seeding are shown in Fig. 7. The
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highest amount of cells was attached to the pure chitosan scaffolds and the number of cells gradually decreased with the content of PHB in blended scaffolds. It is clear from SEM observation and the above analysis that the PHB was deposited in scaffolds on surfaces of chitosan fibers or in the form of separated fine fibrous or globular agglomerates. These cause the significant morphological and surface roughness changes of biopolymers from the smoother fiber texture with open microstructure in pure chitosan scaffolds to the microporous microstructure in pure PHB scaffolds. Besides the important surface characteristics of blends, from the point of view of cell attachment—the hydrophobicity—it increases with the PHB content [2, 47]. After 1 day of cell culture, the number of viable cells on scaffolds was the lowest in chitosan scaffold (Fig. 8). The fibroblasts rapidly proliferated on pure PHB scaffolds and their numbers were almost double those in blended scaffolds after 5 days of cultivation. The relative cell densities on the surface of the chitosan scaffold were practically the same during the first 10 days of fibroblast culture and did not exceed 30 % of the control sample. The cells proliferated very well on scaffolds prepared from blends with approximately 60–80 % viability related to the control sample at a given time. The fibroblast population on substrates related to their population after 24 h of culture per unit, scaffold mass rose with time (Fig. 9). There is visible a rapid increase in cell numbers after 5 days of cultivation in pure PHB [29] and blended scaffolds (approx. 4-fold), whereas the cell population was double in the pure chitosan sample only. After 10 days, a stronger population growth was found in samples with higher chitosan content despite PHB:chit (3:1), (1:1) and pure PHB scaffolds having still higher cell numbers. From the comparison of specific surface areas in scaffolds, it resulted that scaffolds with a
J Mater Sci: Mater Med Fig. 6 Degradation of chitosan in lysozyme solution (2 mg/mL, PBS) at 37 °C characterized as the amount of reducing sugars to the starting chitosan amount in samples (mean ± SD, n = 4)
Fig. 7 Initial cell attachment of fibroblasts on different substrates (seeding density of 5 9 104) determined by the relative number of cells adhered from seeding number (mean ± SD, n = 4). Numbers in bars represent values of specific surface pore areas of samples
larger pore area per mass unit with micropores in microstructure showed faster cell growth. Shown in Fig. 10 are the total cell densities in scaffolds, calculated as the ratio of MTS absorbance (at 490 nm) to the value of the specific surface of each scaffold. It resulted from comparison that higher cell densities were observed in PHB:chit (1:3) and pure chitosan scaffolds after 24 h of culture, but the rapid increase in cell densities was visible on pore surfaces in pure PHB and PHB:chit (1:3) scaffolds after 5 days. Note that despite the large difference between pore surface area in pure PHB and PHB:chit (1:3) scaffolds, the pore surface population densities were comparable. A small rise in surface population densities was found in pure PHB and PHB:chit (3:1, 1:1) scaffolds, whereas the cell density in the PHB:chit (1:3) sample decreased after 10 days of fibroblast proliferation. We believe that the cell growth could reach confluence in this sample because of the small pore surface area. The distributions of fibroblasts on
biopolymer films after 5 days of proliferation prepared by pressing the original scaffolds are demonstrated in Fig. 11. This method makes it possible to eliminate the effects of varied porosity on cell proliferation. No significant differences in the number of cells on biopolymer films are visible in the figure. The fibroblasts are well spread mainly on pure PHB, PHB:chit (1:1) and (1:3) films with observable interconnections via filopodia. The cytotoxicity of biopolymer samples was very low. The results verified good fibroblast attachment and excellent proliferation on PHB– chit scaffolds. It has been shown that the amount of attached L929 cells rose after PHBV grafting with chitosan but the cell proliferation was similar on pure and grafted chitosan [46]. A low number of attached fibroblasts were found on chitosan membranes after 1 h incubation and the amount of cells significantly increased after 24 h of culture [48]. A much higher fibroblast attachment was observed on chitin–PHBV composites than on the pure chitin [49]. Note
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J Mater Sci: Mater Med Fig. 8 Relative proliferation of fibroblasts on scaffolds (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) expressed as the ratio of formazan absorbance (490 nm) in the sample to control (mean ± SD, n = 4, statistical significant differences with P \ 0.05)
Fig. 9 Growth of fibroblast population on substrates related to their population after 24 h of culture per unit scaffold mass (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) calculated as the ratio of formazan absorbance (490 nm) in sample to sample absorbance after 24 h of culture (mean ± SD, n = 4, statistically significant differences with P \ 0.05)
Fig. 10 Total cell densities on scaffold pore surfaces, calculated as the ratio of formazan absorbance (at 490 nm) to the value of the specific surface of each scaffold (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) (mean ± SD, n = 4, statistically significant differences with P \ 0.05)
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Fig. 11 Distributions of fibroblasts on biopolymer films after 5 days of proliferation at 37 °C in 5 % CO2 and 90 % humidity, prepared by pressing the original scaffolds (optical fluorescence micrographs
(acridine orange staining), magnification 9200; chitosan (a), PHB (b), PHB:chit (3:1) (c), PHB:chit (1:1), PHB:chit (3:1) scaffolds) (Color figure online)
that the PHB–chitosan blended scaffolds could be effectively utilized in regeneration or reconstruction of connective tissues or cartilage because both biopolymers have been individually (or in blends with various biopolymers) tested for these applications [50–52].
precipitates obtain a globular shape after the addition of more concentrated PHB solutions. The prepared scaffolds differ by porosity, morphology of biopolymer particles and agglomerates in microstructures. The average molecular weight (Mw) of chitosan in pure chitosan scaffolds decreased after precipitation in propylene carbonate solution, and Mw of both biopolymers were reduced in blends. The mutual biopolymer interactions were confirmed in blended scaffolds and between chitosan polysaccharide chains in the pure chitosan scaffold. A faster lysozyme degradation was shown in samples with a higher PHB content during 1–5 days because of different scaffold microporosities and around 16 wt % of chitosan was degraded in blended scaffolds after 14 days irregardless of blend composition. Good initial cell attachments (even in
4 Conclusions The PHB–chitosan biopolymer scaffolds that were prepared by precipitation after mixing of biopolymer solutions contain miscible low toxic solvents. The different type of surface physicochemical properties of co-precipitated biopolymers had a strong effect on the morphology of precipitates in suspensions, where the separated chitosan
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pure chitosan) on all scaffolds were found with excellent proliferation. Acknowledgments This work was supported by the Slovak Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, Project No. 2/0026/11 and projected within the framework of COST MP 1005.
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17. Veleirinho B, Coelho DS, Dias PF, Maraschin M, Ribeiro-doValle RM, Lopes-da-Silva JA. Nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/chitosan scaffolds for skin regeneration. Int J Biol Macromol. 2012;51:343–50. 18. Ma G, Yang D, Wang K, Han J, Ding S, Song G, Nie J. OrganicSoluble Chitosan/Polyhydroxybutyrate Ultrafine Fibers as Skin Regeneration Prepared by Electrospinning. J Appl Polym Sci. 2010;118:3619–24. 19. Wang J, Wang Z, Li J, Wang B, Liu J, Chen P, Miao M, Gu Q. Chitin nanocrystals grafted with poly(3-hydroxybutyrate-co-3hydroxyvalerate) and their effects on thermal behavior of PHBV. Carbohydr. Polym. 2012;87:784–9. 20. Ni Y, Huang Ch, Kokot S. A kinetic spectrophotometric method for the determination of ternary mixtures of reducing sugars with the aid of artificial neural networks and multivariate calibration. Anal Chim Acta. 2003;480:53–65. 21. Organ SJ, Li J, Terry AJ, Hobbs JK, Barham PJ. Crystallization of hydroxybutyrate oligomers. Part 2. Growth and thickening of solution grown crystals observed in situ using synchrotron radiation. Polymer. 2004;45:8925–36. 22. Iwata T, Doi Y, Nakayama SI, Sasatsuki H, Teramachi S. Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Int J Biol Macromol. 1999;25:169–76. 23. Choe S, Cha YJ, Lee HS, Yoon JS, Choi HJ. Miscibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(vinyl chloride) blends. Polymer. 1995;36:4977–82. 24. Sudesh K, Abe H, Doi Y. Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci. 2000;25:1503–55. 25. Organ SJ, Barham PJ. On the equilibrium melting temperature of polyhydroxybutyrate. Polymer. 1993;34:2169–74. 26. McChalicher ChWJ, Srienc F. Solubility and Degradation of Polyhydroxyalkanoate Biopolymers in Propylene Carbonate. AIChE J. 2010;56:1616–25. 27. Holme HK, Foros H, Pettersen H, Dornish M, Smidsrod O. Thermal depolymerization of chitosan chloride. Carbohydr Polym. 2001;46:287–94. 28. Tsao CT, Chang CH, Lin YY, Wu MF, Han JL, Hsieh KH. Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation. Carbohydr Res. 2011;346:94–102. 29. Liu H, Du Y, Wang X, Hu Y, Kennedy JF. Interaction between chitosan and alkyl b-D-glucopyranoside and its effect on their antimicrobial activity. Carbohydr Polym. 2004;56:243–50. 30. Padermshoke A, Katsumoto Y, Sato H, Ekgasit S, Noda I, Ozaki Y. Melting behavior of poly(3-hydroxybutyrate) investigated by two-dimensional infrared correlation spectroscopy. Spectrochim Acta A. 2005;61:541–50. 31. Li Z, Ramay HR, Hauch KD, Xiao D, Zhang M. Chitosan– alginate hybrid scaffolds for bone tissue engineering. Biomaterials. 2005;26:3919–28. 32. Kim SS, Kim SH, Lee YM. Preparation, characterization, and properties of b-Chitin and N-acetylated b-Chitin. J Polym Sci B Polym Phys. 1996;34:2367–74. 33. Van de Velde K, Kiekens P. Structure analysis and degree of substitution of chitin, chitosan and dibutyrylchitin by FT-IR spectroscopy and solid state 13C NMR. Carbohydr Polym. 2004;58:409–16. 34. Ikejima T, Yagi K, Inoue Y. Thermal properties and crystallization behavior of poly(3-hydroxybutyric acid) in blends with chitin and chitosan. Macromol Chem Phys. 1999;200:413–21. 35. Chen LY, Du Y. Aggregation behavior of 3,6-O-carboxymethylated chitin in aqueous solutions. J Appl Polym Sci. 2002;86: 1838–43.
J Mater Sci: Mater Med 36. Hirano S, Tsuchida H, Nagao N. N-acetylation in chitosan and the rate of its enzymatic hydrolysis. Biomaterials. 1989;10: 574–6. 37. Lin SB, Lin YCh, Chen HH. Low molecular weight chitosan prepared with the aid of cellulase, lysozyme and chitinase: characterisation and antibacterial activity. Food Chem. 2009; 116:47–53. 38. Yang YM, Hu W, Wang XD, Gu XS. The controlling biodegradation of chitosan fibers by N-acetylation in vitro and in vivo. J Mater Sci Mater Med. 2007;18:2117–21. 39. Varum KM, Holme HK, Izume M, Stokke BT, Smidsrod O. Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans. Biochim Biophy Acta. 1996;1291:5–15. 40. Abe H, Doi Y. Structural effects on enzymatic degradabilities for poly[(R)-3-hydroxybutyric acid] and its copolymers. Int J Biol Macromol. 1999;25:185–92. 41. Yua J, Plackett D, Chen LXL. Kinetics and mechanism of the monomeric products from abiotic hydrolysis of poly[(R)-3hydroxybutyrate] under acidic and alkaline conditions. Polym Degrad Stab. 2005;89:289–99. 42. Cunha-Reis C, Tuzla-Koglu K, Baas E, Yang Y, Haj EA, Reis RL. Influence of porosity and fibre diameter on the degradation of chitosan fibre-mesh scaffolds and cell adhesion. J Mater Sci Mater Med. 2007;18:195–200. 43. Tigli RS, Karakecili A, Gu¨mu¨sderelioglu M. In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree. J Mater Sci Mater Med. 2007;18:1665–74. 44. Minagawa T, Okamura Y, Shigemasa Y, Minami S, Okamoto Y. Effects of molecular weight and deacetylation degree of chitin/ chitosan on wound healing. Carbohydr Polym. 2007;67:640–4.
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Properties and in vitro characterization of polyhydroxybutyrate– chitosan scaffolds prepared by modified precipitation method Lubomir Medvecky • Maria Giretova Radoslava Stulajterova
•
Received: 30 May 2013 / Accepted: 22 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Porous polyhydroxybutyrate (PHB)–chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends. Keywords Chitosan Polyhydroxybutyrate Propylene carbonate Cytotoxicity Microstructure Blend
L. Medvecky (&) M. Giretova R. Stulajterova Institute of Materials Research, Slovak Academy of Science, Watsonova 47, 040 01 Kosice, Slovak Republic e-mail: [email protected]
1 Introduction Polyhydroxybutyrate (PHB) is a member of the group of hydrophobic biodegradable biopolymers and it can actively have an affect on the hydrophilic/hydrophobic ratio in composites, where it is one of the components. It is known that cell adhesion to the implant and cell activity are influenced by properties of the implant surface such as e.g. surface tension, roughness, etc. [1, 2]. PHB and its composites exhibited excellent cell proliferation [3]. Doyle et al. [4] established that implants with the PHB addition produced a consistent favorable bone tissue adaptation response without an undesirable chronic inflammatory response. The chitosan (chit) represents polysaccharide biopolymers with inductive and stimulative activities on connective tissue rebuilding [5]. Both biopolymers are successfully utilized as one of the composite components e.g. in blends based on poly(3-hydroxy)butyrate [6], composites with other natural biopolymers or fibers [7–9]. Deacatylated chitosan is easily soluble in acid aqueous solutions (acetic or hydrochloric acid). The fully N- and O-acetylated (stearoyl, hexanoyl or lauroyl) chitosan derivates were soluble in non-polar organic solvents [10, 11]. The organic carbonates (ethylene, propylene carbonates) were applied as plasticizers of chitosan [12, 13]. Rajan et al. [14] studied the thermal and mechanical properties of PHB–chit composites prepared by mechanical mixing in a microcompounder. A decrease in polyhydroxybutyrate crystallinity with a chitosan content in polyhydroxybutyrate–chitosan blends has been observed, obtained by mixing the biopolymer solutions dissolved in co-solvent (1,1,1,3,3,3-hexafluoro-2-propanol (HFP)). The miscibility of polymers and intermolecular interaction between polyhydroxybutyrate and chitosan chains were verified by by
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Cheung et al. [15] and Ikejima et al. [16]. The nanofibrous PHBV–chit scaffolds prepared by electrospinning from the HFP solutions had good fibroblast proliferation activity and a superior ability for improving fibroblast adhesion [17]. Organic soluble (in chloroform) PHB–chit composites and fibers exhibited non-toxic behavior [18]. Wang et al. [19] carried out direct chitin grafting with PHBV via the intermediate product synthesized by the chlorination of PHBV [19]. In this paper, the physico-chemical properties, microstructure and cytotoxicity of porous polyhydroxybutyrate– chitosan blended scaffolds (PHB–chit) prepared by the precipitation of dissolved biopolymers in miscible aqueous or low toxic organic solvents were evaluated. This method has not been used as of yet. The presented preparation procedure of blends is very simple and no special requirements were given, miscibility or other physicochemical properties being examples. Our aim was to apply a simple method without complicated working procedures or chemical modifications of biopolymers, and with environmentally friendly biopolymer solvents.
of molecular weights of PHB–chitosan in blends was determined by gel permeation chromatography (GPC, Watrex, RI detector), with chloroform as a mobile phase for the PHB characterization and 0.5 M NaCl ? 0.05 M NaH2PO4 (pH2) mobile phase for the chitosan analysis. The separation was carried out on the PL gel mixed C 5 lm column (PHB) and PL gel mixed OH 8 lm (chitosan) at 1 mL/min flow rate of mobile phase. The calibrations of molecular weights for the calculation of molecular weight distribution in polymers were carried out using dextrans (standards for chitosan) and polystyrene (standards for PHB) (American polymer standard corporation) with various average molecular weights. The chitosan degradation in scaffolds by lysozyme solution (2 mg/mL, PBS) at 37 °C was characterized by the amount of reducing sugars created during hydrolysis at selected times. The reducing sugars were determined by the potassium ferricyanide method described by Ni et al. [20]. N-Glucosamine (Sigma-Aldrich) solutions were used as standards, and determination was carried out at 65 °C by the kinetic measurement of absorbance at 420 nm after 10 min from reagent addition and sample mixing.
2 Materials and methods
2.3 Cell cultivation and viability testing
2.1 Preparation of blends
L 929 mouse fibroblasts (ECACC, Salisbury, UK) were cultured in culture flasks with surface areas of 75 cm2 (SPL Life Sciences, Korea) in MEM (Minimum essential medium) with Earles balanced salts, 2 mM L-glutamine (SAFC Biosciences, Hampshire, UK), 10 % fetal bovine serum (Sigma-Aldrich) and ATB-Antimycotic (Penicillin, Streptomycin, Amphotericin) solution (Sigma-Aldrich). Cells were maintained at 37 8C in 5 % CO2 atmosphere with 95 % humidity in an incubator (Memmert). The medium was changed every 2 days. After the cells reached about 80 % confluence, they were harvested by trypsinization using 0.25 % Trypsin–EDTA (Sigma-Aldrich) solution followed by the addition of fresh medium to create a cell suspension and the cell numbers were calculated using a Neubauer hemacytometer. All sides of the scaffolds were sterilized by UV radiation in a laminar flow box for 30 min before their immersion into culture plates. The cell proliferation was examined using an MTS test (Cell titer 96 aqueous one solution cell proliferation assay, Promega, Madison, USA). The sterilized scaffolds were placed into the 48-well suspension plate, seeded with 2.0 9 104 cells in 500 ll of complete medium and cultured at 37 °C in atmosphere containing 5 vol % CO2 and 95 % humidity in an incubator. The cell proliferation on scaffolds was evaluated for 24 h, 5 and 10 days after cell seeding. Immediately before the assay, the culture medium was removed from the wells and replaced with fresh medium and the MTS reagent was
The blends with various polyhydroxybutyrate (GoodFellow) to chitosan (SigmaAldrich, middle) ratios (3:1, 1:1, 1:3) were prepared by mixing of PHB (dissolved in propylene carbonate) and chitosan (in 1 % acetic acid) solutions. The same volumes of differently concentrated biopolymer solutions were used for the precipitation. Mixing was carried out using a magnetic stirrer at 400 rpm. After 10 min mixing, the acetone was slowly added to suspensions for the complete precipitation of biopolymers. Final blends were filtered, washed with distilled water and molded into block form (4 mm 9 25 mm 9 3 mm), cut to smaller parts with the final dimensions (4 9 4 9 3 mm) and lyophilized (Ilshin) for 6 h. 2.2 Characterization methods The thermal degradation and melting of blends were analyzed by differential scanning calorimetry (DSC) and thermogravimetry (TG) (Mettler, 2000C). The chemical interaction and crystallinity were evaluated by FTIR spectroscopy (Shimadzu, IRAffinity1, KBR method). The microstructure of scaffolds was observed by scanning electron microscopy (FE SEM JEOL7000). Optical fluorescence microscopy (inverted optical microscope Leica DM IL LED) with phase modulation contrast was used for the observation of biopolymer precipitates. The distribution
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added. After 2 h of incubation, the intensities of coloring, which characterize the formazan concentration (produced by proliferating cells) in culture medium, were evaluated using a UVVIS spectrophotometer (Shimadzu) at a wavelength of 490 nm. The measured absorbances of medium from wells with cell seeded substrates were compared with the ones from wells free of scaffolds in the tissue culture polystyrene plate (a treated 48-well tissue culture plate, Santa Cruz Biotechnology, Santa Cruz, USA) seeded with cells (2.0 9 104 cells in 500 ll of complete medium). The calculated ratios were normalized to the same substrate mass (scaffold with a maximum mass). The pure complete culture medium was used as a blank. Acridine orange (AO) (Sigma-Aldrich) was used to stain the L929 cells proliferated on films prepared by the pressing of porous scaffolds. After 5 days of cultivation, the samples were rinsed with phosphate buffered saline solution (PBS); cells were fixed in 96 % ethanol for 20 min and stained with 0.01 % AO solution for 2 min in the dark. The cells on substrates were observed using a fluorescence microscope (Leica DM IL LED, blue filter) for investigating morphology and distribution of cells.
3 Results and discussion 3.1 Morphology of biopolymer precipitates and scaffold microstructures The suspensions of precipitated biopolymers and blends are shown in Fig. 1. The long chitosan fibers (up to a few hundred lm) and 1–5 lm thickness are visible in Fig. 1a. The addition of PHB to chitosan caused a strong change in morphology of precipitates in mixture with the ratio of PHB:chit = 1:3, where very fine spherical and short thin fibrous precipitates with diameters not exceeding 1 lm were observed (Fig. 1b). Larger globular particles in fine matrix were found in the biopolymer suspensions at higher PHB contents (Fig. 1c, d). No particles of this shape were observed in pure PHB suspension (Fig. 1e), where homogeneous fine grained precipitates with unresolved inner structure were visible. Organ et al. [21] observed precipitation of the needle-like shaped PHB crystallites from propylene carbonate biopolymer solutions. Iwata et al. [22] found a similar morphology of the PHB–co-valerate copolymer particles precipitated from chloroform-ethanol solutions. Chitin nanocrystals grafted with PHB had needle-like morphology. The microstructure of pure chitosan scaffolds corresponds with the fibrous morphology of the precipitated biopolymer. The inner chitosan surfaces in the scaffold were smooth without visible sharper edges or the presence of separated smaller particles. Three different pore size
fractions were present in the microstructure of pure chitosan scaffolds—the large rounded almost spherical pores (50–100 lm size); the high number of pores with dimensions around 10 lm and micropores up to 1 lm in size (Fig. 2a). A different image can be visible in the case of the pure PHB biopolymer scaffolds (Fig. 2b), where the large pores were not found and the microstructure was uniform with three clearly distinguished particle types—the globular particles (\1 lm); coarser agglomerates (up to 5 lm) and very thin short mutually interconnected fibers. Apart from the scaffold microstructure, irregularly shaped pores with maximum dimensions around 20 lm and a high fraction of micropores were found. The blended scaffolds with PHB:chit = 1:1 were composed of separated and two morphologically different polymer forms—the honeycomb or sponge-like particles with a wide pore size distribution and some fraction of larger pores (about 60 lm) (Fig. 2c); the fine microporous agglomerates with both the fibrous and more granular morphologies. The fraction of fine granular agglomerates rose with the PHB content in blended scaffolds, and these ones can be related to the pure PHB or mutually bounded fine structured PHB–chitosan polymers in blends. The microstructure of scaffolds with the PHB:chit biopolymer ratio equal to 1:3 was composed of sponge-like objects with smooth surfaces and larger irregular pores (up to 40 lm in size) (Fig. 2d). Besides this, the thin plate-like biopolymers were tightly bounded to the surface of these objects. Contrary to the above, more compact microporous microstructure with a low fraction of longer fibers and a high density of short thin fibers or biopolymer plates were observed in scaffolds with PHB:chit = 3:1 (Fig. 2e). It results from the above, that the separation of biopolymers into individual specific forms was strongly reduced with the rise in PHB content and the microstructures obtain a more uniform character. 3.2 TG, DSC and GPC analysis of blends Results of TG and DSC measurements of samples are shown in Fig. 3. Three endo effects with maxima at 149, 184 and 305 °C were found on the DSC curve of pure PHB (Fig. 3a), which correspond to the melting temperature of the amorphous (or low molecular), crystalline PHB fractions and the PHB decomposition (with a mass loss on the TG curve), respectively. The pure chitosan decomposes in three steps, which verify mass losses on the TG curve (Fig. 3b, c) (the water releases up to 150 °C, the mass loss between 220 and 320 °C characterizes the amine units decomposition, saccharide chains are degraded above 300 °C) and the decomposition finishes at 600 °C [23]. Note that a single wide exo-effect at 300 °C is observable on the chitosan DSC curve. On the DSC curve of the PHB:chit (1:1) blend, two endo-effects at 180 and 290 °C
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Fig. 1 Morphology of biopolymer precipitates in suspensions (a chit, b PHB:chit (1:3), c PHB:chit (1:1), d PHB:chit (3:1), e PHB)
arise from the melting and decomposition of PHB, a doublet of exo-effects represents the chitosan thermal decomposition with maxima at 310 and 330 °C., Similar endoeffects with maxima at 181 and 290 °C as above were observed in the case of PHB:chit (1:3) blends. Besides this, the chitosan decomposition in this sample characterizes both the sharp exo-effect at 305 °C mutually mixed with a wide exo-effect. From the comparison of DSC and TG curves of pure biopolymers and blends, it clearly resulted that the rate of chitosan decomposition was strongly affected by the thermal degradation of PHB. A more detailed analysis of exo-effects related to the chitosan decomposition in blends verify the existence of a doublet at around 300 °C in DSC curves, where one peak is expanded to the wide temperature interval between 245 and 400 °C. The second peak is sharper and narrow (the width of temperature interval about 40 °C), which is comparable
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with this one found during pure chitosan decomposition (Fig. 3c, curve a). We believe that the presence of these doublets can be related to two structurally different forms of chitosan in the microstructures of blends. Thus, in blends, phases with more fibrous character represent almost pure chitosan and regions with the fine structured plate-like forms located on surfaces of fibers (or tightly bounded to their surface) and separated fine fibrous biopolymer agglomerates characterize interconnected chitosan and PHB chains. It is clear that the fibrous (plate- or spongelike) chitosan objects in blends with compact structure and longer, mutually crosslinked polysaccharide chains will be more stable toward thermal decomposition than the fine structured fibrous chitosan particles. The finer objects of various morphologies began to decompose very rapidly after PHB thermal degradation, from which results the direct mutual bounding between chitosan and PHB
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Fig. 2 Microstructures of scaffolds (a chit, b PHB, c PHB:chit (1:1), d PHB:chit (1:3), e PHB:chit (3:1))
biopolymer chains. After decomposition of the PHB polymer, the fine chitosan chains bounded with the origin PHB particles have surfaces opened to an air oxygen attack and they are more susceptible to oxidation and thermal degradation. In the case of PHB, a reduction in melting points and decomposition temperatures after the chitosan addition were found. These effects can be caused by the decrease in lamellar thickness of the PHB crystallites in blends because of intramolecular interactions between the PHB and chitosan polymers which suppress the PHB crystallization [16, 24, 25]. Another reason for reduction in the PHB melting point can be the decrease in average molecular weight of the PHB during the preparation of blends [25]. The comparison of degradation TG curves of native and precipitated chitosans (Fig. 3c) clearly shows
that the precipitated chitosan starts to decompose about 25 °C earlier than its native form, and the exo-effect at 300 °C is tailored to the side of lower temperatures. Besides, more gradual thermal decomposition of polysaccharide chains was found than in the case of native chitosan without visible exo-effect at 550 °C, which confirms the structural changes in precipitated chitosan. The molecular weights of PHB in blends determined by the GPC analysis confirmed the reduction in average Mw and Mn and the rise in polydispersity degrees (PD) at higher chitosan contents from 108 to 57 kDa (PD = 1.9) in pure PHB to 81 and 22 kDa (3.7) in PHB:chit (1:1) blend (Table 1). The dissolution of PHB in propylene carbonate was carried out about at 120 °C for 20 min. According to the study of propylene carbonate effect on the PHB
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chains after the chitosan addition. In the case of chitosan, no interaction between chitosan and organic carbonates was found after mixing various carbonates with chitosan [12, 13]. The carbonates act as chitosan plasticizers, which cause a decrease in Tg temperature of chitosan. Thus the chitosan chains are more flexible after addition of the organic carbonate. No effect of carbonates on the distribution of chitosan molecular weights was studied to this point. In comparison of native chitosan and precipitated chitosan Mw in Table 1 there were seen strong reductions in Mw and PD values of chitosan precipitated from acetic acid solution by the propylene carbonate addition. It is clear that the mole fraction of low molecular chitosan chains rose mainly by the depolymerization of high molecular chains. A further small decrease in PD was found after addition of the PHB to blends, which confirms the opinion that the possible weak mutual interactions between biopolymers cause a shortening of their chains. It has been shown that chitosan can be thermally degraded (e.g. during drying) at moderate temperatures but no such rapid degradation and reduction in Mw were observed at 105 °C [27]. Similarly, no decrease of Mw can be related to acid chitosan hydrolysis because very soft experimental conditions were used during chitosan dissolution only [28]. Note that results of the GPC analysis are in accordance with the ones of thermal analysis. 3.3 FTIR analysis blends and chitosan degradation with lysozyme
Fig. 3 DSC (a) and TG (b) analysis of scaffolds (chit precip (underline), PHB:chit (1:3) (dotted line), PHB:chit (1:1) (hyphenated line), PHB (dotted hyphenated line)), c comparison of DSC and TG curves of native (a) and precipitate (b) chitosans
degradation [26] at 120 °C, the average molecular weight Mn of PHB falls down approx. to 90 % of the origin Mn whereas the PD were not changed during dissolution. Note that the temperature of the PHB solution during mixing with chitosan solution decreased to about 80 °C and the possible influence of temperature or propylene carbonate on the PHB degradation was not significant in this case. It results from the above that the decrease in average molecular weights (Mw and Mn) and the rise of polydispersity in the PHB:chit (1:1) blend can be related to the effect of a higher chitosan amount in the blend. The reason for increase of the PD in the sample is mainly the decrease of Mn due to a much higher mole fraction of low molecular PHB chains. Note that the Mn distribution curve of PHB in this blend is changed to a more asymmetric (tailored) shape on the side of low molecular weights, which verifies the preferential depolymerization of original shorter polymer
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The FTIR spectra of PHB, chitosan and PHB–chit blends are shown in Figs.4, 5. In the case of pure chitosan samples (Fig. 4), small changes in the spectra of native and precipitated chitosans were found—amide I band had an unresolved doublet at 1,650 cm-1, amide II band was shifted about 6–1,561 cm-1 and band arises from stretching C–N vibrations at 1,324 cm-1 was shifted to lower frequencies (about 8 cm-1) and split, which is a characteristic attribute of the formation of complexes in precipitated chitosan. Besides this, a rise in intensity of bands corresponding to free hydroxyl groups at wave numbers 1,000–1,200 cm-1 [29] was observed. All these differences verify the increase in number of hydroxyl groups, because of the chitosan depolymerization, and the presence of weak intramolecular interactions between precipitated polysaccharide chains. In a spectrum of pure polyhydroxybutyrate (Fig. 5b), were found bands from C=O stretching vibrations at 1,724 and 1,746 cm-1, as well as bands in the region between 1,120 and 1,300 cm-1 ascribing the stretching vibrations of the C–O–C ester groups and bending CH2-, CH3-group vibrations under 1,000 cm-1 [30]. Small shifts were observed in stretching vibrations of the PHB carbonyl group in blends. Note that the peak at
J Mater Sci: Mater Med Table 1 Average molecular weights (Mw and Mn) and polydispersity degrees (PD) of biopolymers in scaffolds PHB:CHIT
MwCHIT [kDa]
MnCHIT
PD
Chit native
363
9
44
Chit precip
60
6
10
1:1
41
5
8
MwPHB [kDa]
MnPHB
PD
81
22
3.7
3:1
110
53
2.1
PHB
110
57
1.9
around 1,745 cm-1 represents a low ordered or amorphous part of PHB, whereas the band at 1,720 cm-1 characterizes the crystalline fraction in PHB. The amide I chitosan band in blends (vibration of C=O in acetylated amino group) is shifted about 6 cm-1 from the one in precipitated chitosan, whereas an insignificant shift was observed for amide II band (NH deformation vibrations) [31–34]. The small shift of amide I band in blends confirms the mutual interaction between PHB and chitosan biopolymers. No bands from vibrations of groups in propylene carbonate were found in spectra. Ikejima et al. [34] confirmed by 13C NMR spectroscopy the intermolecular bonding between PHB carbonyl and chitosan amide groups, and showed an increase of the amorphous PHB component with the chitosan amount in blends prepared from homogeneous polymer solutions. Cheung et al. [15] using 1H NMR spectroscopy verified the intermolecular interaction between biopolymers. These facts are in accordance with our results. Small differences in intensities of bands characterize the ether bonds (–C–O–C–), due to the glycosidic linkage between chitosan monomer units, were found in FTIR spectra of carboxymethylated chitosans with various average molecular weights [28]. In this case, the chitosans were
precipitated from a strong acid solution after an addition of 1 M NaOH, which can affect the aggregation of chitosan chains because of the high ionic concentration. NaCl concentration has been found to have significant effect on the content of aggregates in chitosan solutions [35]. It is very hard to analyze any changes in intensities of ether and ester bands in blends as a result of overlapping the PHB and chitosan bands in spectra. Lysozyme is one of the enzymes [36, 37], which are able to depolymerize biopolymers based on N-acetylated -glucosamine units bonded via b-(1-4)-glycosidic linkage. The hydrolysis rate of chitosan by lysozyme is strongly affected by the degree of chitosan deacetylation [38]. More detailed analysis of the mechanism of chitosan degradation by lysozyme is shown in Ref. [39]. Lysozyme is very little active in PHB depolymerization, which can be effectively carried out by another enzyme—PHB depolymerase [40]. Besides this, PHB hydrolyzes to crotonic and 3-hydroxybutyric acids in strong acid or alkaline conditions (deep degradation in a relatively short time) [41], but in neutral media the degradation rate is low. Note that the insignificant PHB degradation in scaffolds at 37 °C in PBS was found during 14 days of soaking (measured by the HPLC method, results not shown). The influence of blend composition on the degradation of chitosan with lysozyme is shown in Fig. 6. The increase in content of reducing sugars (calculated as the amount of glucosamine) in solutions was observed after 14 days from the chitosan addition to lysozyme solution, whereas small differences in its amount were found after 1 and 5 days in all samples. Note that around 16 wt% of the original chitosan amount was degraded after 14 days in blended scaffolds independently of their composition. The degradation rates of pure chitosan and PHB:chit (1:3) scaffolds were slower than in samples with a higher PHB content during 1–5 days. This
Fig. 4 FTIR spectra of native (a) and precipitated chitosan (b)
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J Mater Sci: Mater Med Fig. 5 FTIR spectra of PHB:chit (1:1) (a), (1:3) (c) and PHB (b)
effect can be related to significantly reduced microporosities of individual polymer components in these scaffolds, which was confirmed by the lower values of specific surface (Fig. 7). Besides this, as resulting from SEM observations, more uniform microstructures without separated coarser polymer agglomerates were found in samples with a higher PHB amount which verifies their better mutual mixing. Cunha-Reis et al. [42] showed statistically significant dependence of the chitosan fiber degradation rate on fiber microporosity, and this influence was more ratedetermined than an effect of the fiber specific surface areas. Very low degradation of chitosan with the degree of N-acetylation (DA) equal 0.2 was observed [36] at similar conditions as in our paper, but no mention about chitosan Mw is given in this work. On the other hand, approximately 20 wt % losses were found after 3 week chitosan degradation in DMEM containing 0.5 mg/mL lysozyme, where chitosan samples were highly porous and Mw was around 300 kDa [43]. The degradation degree of chitosan fibers with DA equals 21 % and Mw = 30 kDa after 10 days was 29 % [38]. Chitosan oligomers enhanced wound healing acceleration and activated fibroblasts more intensively than chitosans with the high average molecular weight [44]. It has been shown that chitosan has antimicrobial effect and it is enhanced for gram-negative bacteria with a decrease in chitosan molecular weight [45]. Hu et al. [46] verified that PHBV grafting with chitosan exhibited antibacterial activity against gram-negative and gram-positive bacteria. 3.4 Analysis of initial cell attachment and proliferation The initial cell attachments of fibroblasts on different scaffolds after 3 h of seeding are shown in Fig. 7. The
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highest amount of cells was attached to the pure chitosan scaffolds and the number of cells gradually decreased with the content of PHB in blended scaffolds. It is clear from SEM observation and the above analysis that the PHB was deposited in scaffolds on surfaces of chitosan fibers or in the form of separated fine fibrous or globular agglomerates. These cause the significant morphological and surface roughness changes of biopolymers from the smoother fiber texture with open microstructure in pure chitosan scaffolds to the microporous microstructure in pure PHB scaffolds. Besides the important surface characteristics of blends, from the point of view of cell attachment—the hydrophobicity—it increases with the PHB content [2, 47]. After 1 day of cell culture, the number of viable cells on scaffolds was the lowest in chitosan scaffold (Fig. 8). The fibroblasts rapidly proliferated on pure PHB scaffolds and their numbers were almost double those in blended scaffolds after 5 days of cultivation. The relative cell densities on the surface of the chitosan scaffold were practically the same during the first 10 days of fibroblast culture and did not exceed 30 % of the control sample. The cells proliferated very well on scaffolds prepared from blends with approximately 60–80 % viability related to the control sample at a given time. The fibroblast population on substrates related to their population after 24 h of culture per unit, scaffold mass rose with time (Fig. 9). There is visible a rapid increase in cell numbers after 5 days of cultivation in pure PHB [29] and blended scaffolds (approx. 4-fold), whereas the cell population was double in the pure chitosan sample only. After 10 days, a stronger population growth was found in samples with higher chitosan content despite PHB:chit (3:1), (1:1) and pure PHB scaffolds having still higher cell numbers. From the comparison of specific surface areas in scaffolds, it resulted that scaffolds with a
J Mater Sci: Mater Med Fig. 6 Degradation of chitosan in lysozyme solution (2 mg/mL, PBS) at 37 °C characterized as the amount of reducing sugars to the starting chitosan amount in samples (mean ± SD, n = 4)
Fig. 7 Initial cell attachment of fibroblasts on different substrates (seeding density of 5 9 104) determined by the relative number of cells adhered from seeding number (mean ± SD, n = 4). Numbers in bars represent values of specific surface pore areas of samples
larger pore area per mass unit with micropores in microstructure showed faster cell growth. Shown in Fig. 10 are the total cell densities in scaffolds, calculated as the ratio of MTS absorbance (at 490 nm) to the value of the specific surface of each scaffold. It resulted from comparison that higher cell densities were observed in PHB:chit (1:3) and pure chitosan scaffolds after 24 h of culture, but the rapid increase in cell densities was visible on pore surfaces in pure PHB and PHB:chit (1:3) scaffolds after 5 days. Note that despite the large difference between pore surface area in pure PHB and PHB:chit (1:3) scaffolds, the pore surface population densities were comparable. A small rise in surface population densities was found in pure PHB and PHB:chit (3:1, 1:1) scaffolds, whereas the cell density in the PHB:chit (1:3) sample decreased after 10 days of fibroblast proliferation. We believe that the cell growth could reach confluence in this sample because of the small pore surface area. The distributions of fibroblasts on
biopolymer films after 5 days of proliferation prepared by pressing the original scaffolds are demonstrated in Fig. 11. This method makes it possible to eliminate the effects of varied porosity on cell proliferation. No significant differences in the number of cells on biopolymer films are visible in the figure. The fibroblasts are well spread mainly on pure PHB, PHB:chit (1:1) and (1:3) films with observable interconnections via filopodia. The cytotoxicity of biopolymer samples was very low. The results verified good fibroblast attachment and excellent proliferation on PHB– chit scaffolds. It has been shown that the amount of attached L929 cells rose after PHBV grafting with chitosan but the cell proliferation was similar on pure and grafted chitosan [46]. A low number of attached fibroblasts were found on chitosan membranes after 1 h incubation and the amount of cells significantly increased after 24 h of culture [48]. A much higher fibroblast attachment was observed on chitin–PHBV composites than on the pure chitin [49]. Note
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J Mater Sci: Mater Med Fig. 8 Relative proliferation of fibroblasts on scaffolds (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) expressed as the ratio of formazan absorbance (490 nm) in the sample to control (mean ± SD, n = 4, statistical significant differences with P \ 0.05)
Fig. 9 Growth of fibroblast population on substrates related to their population after 24 h of culture per unit scaffold mass (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) calculated as the ratio of formazan absorbance (490 nm) in sample to sample absorbance after 24 h of culture (mean ± SD, n = 4, statistically significant differences with P \ 0.05)
Fig. 10 Total cell densities on scaffold pore surfaces, calculated as the ratio of formazan absorbance (at 490 nm) to the value of the specific surface of each scaffold (PHB(P), PHB:chit (3:1) (1CH3P), PHB:chit (1:1) (CHP), PHB:chit (3:1) (3CHP), chit (ch)) (mean ± SD, n = 4, statistically significant differences with P \ 0.05)
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Fig. 11 Distributions of fibroblasts on biopolymer films after 5 days of proliferation at 37 °C in 5 % CO2 and 90 % humidity, prepared by pressing the original scaffolds (optical fluorescence micrographs
(acridine orange staining), magnification 9200; chitosan (a), PHB (b), PHB:chit (3:1) (c), PHB:chit (1:1), PHB:chit (3:1) scaffolds) (Color figure online)
that the PHB–chitosan blended scaffolds could be effectively utilized in regeneration or reconstruction of connective tissues or cartilage because both biopolymers have been individually (or in blends with various biopolymers) tested for these applications [50–52].
precipitates obtain a globular shape after the addition of more concentrated PHB solutions. The prepared scaffolds differ by porosity, morphology of biopolymer particles and agglomerates in microstructures. The average molecular weight (Mw) of chitosan in pure chitosan scaffolds decreased after precipitation in propylene carbonate solution, and Mw of both biopolymers were reduced in blends. The mutual biopolymer interactions were confirmed in blended scaffolds and between chitosan polysaccharide chains in the pure chitosan scaffold. A faster lysozyme degradation was shown in samples with a higher PHB content during 1–5 days because of different scaffold microporosities and around 16 wt % of chitosan was degraded in blended scaffolds after 14 days irregardless of blend composition. Good initial cell attachments (even in
4 Conclusions The PHB–chitosan biopolymer scaffolds that were prepared by precipitation after mixing of biopolymer solutions contain miscible low toxic solvents. The different type of surface physicochemical properties of co-precipitated biopolymers had a strong effect on the morphology of precipitates in suspensions, where the separated chitosan
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pure chitosan) on all scaffolds were found with excellent proliferation. Acknowledgments This work was supported by the Slovak Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, Project No. 2/0026/11 and projected within the framework of COST MP 1005.
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