Article pubs.acs.org/Biomac
Surfactant as a Critical Factor When Tuning the Hydrophilicity in Three-Dimensional Polyester-Based Scaffolds: Impact of Hydrophilicity on Their Mechanical Properties and the Cellular Response of Human Osteoblast-Like Cells Yang Sun,†,‡ Zhe Xing,†,§ Ying Xue,§ Kamal Mustafa,§ Anna Finne-Wistrand,‡ and Ann-Christine Albertsson*,‡ ‡
Department of Fibre and Polymer Technology, KTH Royal Institute of Technology, Stockholm, Sweden Department of Clinical Dentistry-Center for Clinical Dental Research, Faculty of Medicine and Dentistry, University of Bergen, Norway.
§
S Supporting Information *
ABSTRACT: In tissue engineering, the hydrophilicity of porous scaffolds is essential and influences protein and cell adhesion as well as nutrient diffusion into the scaffold. The relative low hydrophilicity of degradable polyesters, which limits diffusion of nutrients, is a major drawback in large porous scaffolds of these materials when used for bone tissue engineering and repair of critical size defects. Designing porous biodegradable polymer scaffolds with improved hydrophilicity, while maintaining their mechanical, thermal, and degradation properties is therefore of clinical interest. Here, surfactants were used to tune the hydrophilicity and material properties. A total of 3−20% (w/w) of surfactant, polysorbate 80 (Tween 80), was used as an additive in poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] and poly(L-lactide)-co-(ε-caprolactone) [poly(LLA-coCL)] scaffolds. A significantly decreased water contact angle was recorded for all the blends and the crystallinity, glass transition temperature and crystallization temperature were reduced with increased amounts of surfactant. Copolymers with the addition of 3% Tween 80 had comparable mechanical properties as the pristine copolymers. However, the E-modulus and tensile stress of copolymers decreased significantly with the addition of 10 and 20% Tween 80. Initial cell response of the material was evaluated by seeding human osteoblast-like cells (HOB) on the scaffolds. The addition of 3% Tween 80 did not significantly influence cell attachment or proliferation, while 20% Tween 80 significantly inhibited osteoblast proliferation. RT-PCR results showed that 3% Tween 80 stimulated mRNA expression of alkaline phosphatase (ALP), osteoprotegerin (OPG), and bone morphogenetic protein-2 (BMP-2).
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INTRODUCTION Degradable polymers have been shown to have promise as bone defect scaffolding materials, able to support cell proliferation and differentiation. We have previously synthesized copolymers of L-lactide and 1,5-dioxepan-2-one or εcaprolactone, and these have shown greater potential for bone tissue engineering than their homopolymers poly(L-lactide) (PLLA) and poly(ε-caprolactone) (PCL).1,2 The degradation profiles of the copolymers poly(L-lactide)-co-(1,5-dioxepan-2one) [poly(LLA-co-DXO)] and poly(L-lactide)-co-(ε-caprolactone) [poly(LLA-co-CL)] have also been investigated in vitro and in vivo, showing the potential for these aliphatic polymers to be used in tailor-made clinical applications.3−5 For successful bone regeneration, it is important to optimize both the material and the 3D scaffold design. The ideal scaffold for bone tissue engineering would not only act as the supporting material for autologous or xenogenous cell trans© 2014 American Chemical Society
plantation, that is, have good mechanical and degradation properties, but would also support cell adhesion, proliferation, and differentiation. A number of publications have reported important relationships between surface properties of the material such as surface roughness,6 surface chemistry,7,8 surface charge,9,10 and hydrophilicity,11−13 on cell growth and differentiation. High hydrophilicity is a key factor for water diffusion through the scaffold during cell culture. Unlimited water diffusion improves medium transfer together with the exchange of nutrients, oxygen, ions, and proteins that impact cell survival in scaffolds. This is a critical parameter, especially when the scaffolds are needed to repair large defects (a critical size defect in tibia and long bone sheep model, for example, Received: December 11, 2013 Revised: February 12, 2014 Published: February 21, 2014 1259
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and differentiation of HOB, homogeneous films, and porous scaffolds samples were prepared
usually requires a scaffold thicker than 3 cm).14,15 Adequate flow through a scaffold may be achieved in culture by using a dynamic bioreactor system, where the flow of medium is maintained by a circulation pump.16,17 Alternatively, flow may be improved by a careful design for the scaffold.18 Here, hydrophilic scaffolds have been designed to attract water for nutrient exchange. There are different methods for improving hydrophilicity, for example adjusting the monomer ratio within the copolymer19−21 or by producing polymer blends.22 The disadvantage of these methods is that they also influence the thermal and/or mechanical properties of the polymer. Another option is to graft hydrophilic polymers/monomers to the surface.23,24 This method is very useful for introducing functional groups to surfaces. However, surface-grafted functional groups are quantitively limited, and the hydrophilic effect becomes attenuated along over time. Other methods to vary the hydrophilicity have also been evaluated, such as sodium hydroxide hydrolysis11,25 or using other hydrophilic segments binding.26,27 Together, the published results show that material hydrophilicity affects cell adhesion and proliferation, principally as an effect on protein adhesion.12,28,29 An interesting alternative is to use surfactants. Tween 80 is a commercialized nonionic surfactant and emulsifier widely used as an additive in food and pharmaceutical applications.30 Its hydrophilicity/lipophilicity balance (HLB) value is 15 and the molecular structure includes 3 hydrophilic polyethylene glycol side chains with 20 ethylene oxide units that compose the hydrophilic segments (Figure 1).
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MATERIALS AND METHODS
Sample Fabrication. Poly(LLA-co-DXO) and poly(LLA-co-CL) were synthesized as previously described.1 Bulk polymerizations were performed at 110 °C over 72 h and stannous octoate (Sn(Oct)2) was used as catalyst with a monomer/Sn(Oct)2 ratio of 10000:1. Copolymers were characterized by proton nuclear magnetic resonance (1H NMR, Bruker Avance 400) for monomer conversion, copolymer composition, and the amount of Tween 80. Molecular weights and polydispersity index (PDI) were determined by size exclusion chromatography (SEC, Polymer Laboratories, U.K.) using tetrahydrofuran (THF) as solvent and polystyrene as standard. Tween 80 (Sigma-Aldrich, Germany) was used as received. In the fabrication of porous scaffolds, sodium chloride was used as porogen using a previously described leaching method.1 The size of the porogen ranged from 90 to 500 μm. Tween 80 was added and mixed overnight together with the dissolved polymer solution at ratios of 3, 10, and 20% (w/w). Eight different material groups were set up (see Table 1).
Table 1. Groups Evaluated and Components of Each Group components
abbreviation
poly(LLA-co-DXO) poly(LLA-co-DXO)+3% Tween 80 poly(LLA-co-DXO)+10% Tween 80 poly(LLA-co-DXO)+20% Tween 80 poly(LLA-co-CL) poly(LLA-co-CL)+3% Tween 80 poly(LLA-co-CL)+10% Tween 80 poly(LLA-co-CL)+20% Tween 80
DXO DXO+3% Tween DXO+10% Tween DXO+10% Tween CL CL+3% Tween CL+10% Tween CL+20% Tween
When producing scaffolds containing Tween 80, considering the water solubility of Tween 80, an extra feeding ratio (data not shown) was used to ensure the Tween 80 composition after salt-leaching. Copolymer without Tween 80 was used as control. After solvent evaporation, scaffold samples were cut into small cylinder pieces with diameter of 1.2 cm and thickness of 1 mm. Then salt particles were leached using deionized water and the porous scaffolds were then dried under vacuum. The film samples were fabricated using single step solvent casting method. Sterilization was accomplished by electron beam radiation (25 kGy dose) using the pulsed electron accelerator (Mikrotron, Acceleratorteknik, Stockholm) under an inert environment at 6.5 MeV. Contact Angle Test. Water contact angle testing was completed on film samples using the CAM 200 optical tensiometer system (KSV instruments Ltd., Finland). A drop of 5 μL Milli-Q water was added on to the surface and the measurements were recorded by optical camera. The average angles were calculated from five different locations. KSV software was used to analyze the frames and measurements. Thermal Properties. The thermal properties of scaffolds were measured using a differential scanning calorimetry (DSC) system. A Mettler-Toledo DSC instrument with DSC 820 module was used under a nitrogen atmosphere with the gas flow rate of 50 mL/min and a heating/cooling rate of 10 °C/min. Specimens were heated from 25 to 175 °C to erase the thermal history during the first scan. The second heating scan ranged from −60 to 175 °C and was used to measure the heat of fusion (ΔHf), glass transition temperature (Tg), crystallization temperature (Tc), and melting temperature (Tm). The degree of crystallinity (χc) was calculated according to the equation:
Figure 1. Chemical structure of Tween 80.
Besides the hydrophilic arms, a hydrophobic chain of about 16 carbons is available to interact and entangle with the hydrophobic polymer. These interactions decrease also the extraction during the scaffolding process. Compared with other surfactants such as Tween 60, Texapon K12, and Triton X100, Tween 80 showed the lowest cytotoxicity in neutral red test and MTT assay using human fibroblasts.31 The use of Tween 80 as an additive to increase polymer hydrophilicity has previously been reported with poly(DL-lactide-co-glycolic acid) (PLGA).32 The PLGA/Tween 80 film was prepared by solvent casting using 10% Tween 80, which resulted in a rapid decrease of hydrophobicity and increased human chondrocyte proliferation in cultures up to 3 weeks. Here, surfactants are used to design hydrophilic porous polyester-based scaffolds that permit efficient diffusion of nutrients and thereby function well as bone tissue engineering scaffolds in critical size defects. Our hypothesis was that the addition of Tween 80 to polyester-based scaffolds would generate a more hydrophilic surface, without negatively influencing the mechanical properties of the scaffold, nor decreasing cell attachment, proliferation, and differentiation. To evaluate the effect of surfactant on surface hydrophilicity, mechanical and thermal properties, attachment, proliferation
χc = (ΔHf /ΔHf ∗ ) × 100% where ΔHf* is the heat of fusion for 100% crystalline polymer. The ΔHf of copolymer poly(LLA-co-DXO) was assumed to be contributed by poly-L-lactide (PLLA) segments. The ΔHf* of PLLA is 93 J/g.33 Tensile Testing. The mechanical properties of the dried polymer films and scaffolds were tested using Instron 5566 tensile equipment 1260
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Statistical Analysis. Experiment was repeated with HOB from two different donors. Sigmastat 3.1 software (Systat Software Inc., Point Richmond, U.S.A.) was applied for statistical processing and analysis. One-way ANOVA test was performed for the analysis and p < 0.05 was considered to be a significant difference between means. All values in bar diagrams are presented as mean ± standard deviation.
(Instron, U.K.). Specimens were cut to strips with 30 mm length, 5 mm width, and about 1 mm thickness. All specimens were placed in 23 °C, 50% humidity for 24 h before measurements were made. The crosshead speed was set up to 50 mm/min with load cell of 100 N. For each type of material, five parallel tests were made to ensure adequate statistics. Scanning Electron Microscopy. An ultra-high resolution scanning electron microscope (SEM, Hitachi S-4300) was used to visualize scaffold topographies. SEM samples were randomly chosen and cut in half to expose the cross-section. All samples were coated with 5 nm thick platinum layers using an automatic Sputter Coater (Agar Scientific, Stansted, U.K.). An accelerating voltage of 15 kV with ×100 magnification was used to acquire overview images. Cellular Response to Medium Containing Different Ratios of Tween 80. Human alveolar bone specimens were obtained from patients undergoing routine oral surgery at the Section for Oral and Maxillofacial Surgery, Department of Clinical Dentistry, University of Bergen and Haukeland University Hospital, Bergen, Norway.34 The isolation of human osteoblast-like cells (HOB) from bone specimens and the culture procedure has been described previously.35,36 HOB were expanded, and passage 2−4 were used in the experiments. For studying direct response of HOB to Tween 80, complete medium containing different concentrations of Tween 80 were used to replace the normal culture medium for HOB. After 24 h incubation with the new medium (containing 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 4, 10, 20% (w/w) Tween 80), 2-D culture plates were rinsed with phosphate buffered saline (PBS), and the WST-1 assay was used to determine the activity37 of HOB in response to the new medium. The ratio of Tween 80 was calculated based on the average weight of the porous scaffolds used in cell culture test. Cell Attachment and Proliferation Assay. To test the cell attachment on different materials, 2 × 105 cells suspended in 500 μL culture medium were placed on top of the scaffolds. The cells/scaffold constructs were harvested 1 and 3 h after seeding, rinsed in phosphate buffered saline (PBS), and the WST-1 assay was used to determine cellularity. To test HOB cell proliferation, scaffolds from the eight groups were prewetted with α-MEM complete medium (Sigma-Aldrich, Germany) and then seeded with 1 × 105 HOB per scaffold. The cells/scaffold constructs were cultured under static culture condition in culture plates overnight and then transferred to spinner flasks which have been described previously.38 The constructs were cultured for 1, 3, 7, and 14 days then rinsed in PBS and moved to medium contained WST-1 reagent, shaken briefly and then incubated for 1 h before absorbance was measured as described above. Quantitative Real-Time RT-PCR Analysis. Based on proliferation results, the following four groups were used for evaluation of bone marker expression: DXO, DXO+3% Tween, CL, and CL+3% Tween. Constructs were cultured in a spinner flask for 2 weeks before harvest. Total RNA was isolated and purified using a Maxwell 16 RNA purification kit (Promega Corporation, U.S.). RNA purity and quantification were determined using a Nanodrop spectrophotometer, as previously described.39 The reverse transcription reaction was carried out with a High Capacity cDNA Archive Kit (Applied Biosystems): 1000 ng total RNA dissolved in 50 μL nuclease-free water was collected and mixed with reverse transcriptase (RT) buffer, random primers, dNTPs, and MultiScribe RT. Real time RT-PCR was run under standard enzyme and cycling conditions on a StepOnePlus real time PCR system, using TaqMan gene expression assays (Applied Biosystems) for proliferating cell nuclear antigen (PCNA), osteocalcin (OC), calcitonin receptor-like (CALCRL), vascular endothelial growth factor A (VEGFa), osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), bone morphogenetic protein-2 receptors (BMP-2R), osterix (Osx), integrin alpha-1 (ITGA1), and the Taqman Pre-Developed Assay GAPDH. cDNA corresponding to 10 ng of mRNA was used in each PCR reaction and mixtures were made up in 10 μL triplicates for each target cDNA. Amplification was performed in 96-well thermal cycler plates using a StepOnePlus real time PCR system. The data were analyzed using a comparative delta Ct method with StepOnePlus software.
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RESULTS AND DISCUSSION Material Synthesis. Two different copolymers, poly(LLAco-CL) and poly(LLA-co-DXO) were synthesized by ringopening polymerization. The number average molecular weights (Mn) ranged from 80000−100000 g/mol and the polydispersity index (PDI) was around 1.4 for all the materials. The copolymer contained 75 mol % LLA and 25 mol % CL or DXO, verified by 1H NMR. Blends of Tween 80 and copolymers were used to prepare homogeneous films and porous scaffold samples. Compared to direct blending for preparing film samples, scaffold samples required another feeding ratio to ensure the final composition of Tween 80 due to the process of salt-leaching (Figures S1 and S2). Different Tween 80 compositions were evaluated by 1H NMR (Table 2). The extraction of Tween 80 in deionized water showed an increased Tween 80 extraction when the amount of Tween 80 was raised in the samples. Table 2. Composition of the Homogeneous Films and Porous Scaffolds groups
Tween 80 composition in films (%)
Tween 80 composition in scaffolds (%)a
CL CL+3% Tween 80 CL+10% Tween 80 CL+20% Tween 80 DXO DXO+3% Tween 80 DXO+10% Tween 80 DXO+20% Tween 80
0 3 10 20 0 3 10 20
0 2.6 8.4 19.4 0 2.9 10.7 18.6
a
Tween 80 composition was calculated and evaluated based on 1H NMR spectra.
Contact Angle Test. Contact angle was used to measure the surface hydrophilicity of the homogeneous films. The average value and standard deviation results are shown below (Figure 2a) together with corresponding images taken from the scaffolds (Figure 2b). With the addition of 3% Tween 80, the contact angle of poly(LLA-co-CL) was reduced by 60% from about 85 to 35° and by 70% for poly(LLA-co-DXO) films from about 74 to 19°. With the addition of higher amounts of Tween 80, the contact angles became lower and the surface became very hydrophilic. Contact angle measurements of porous scaffolds alone are not adequate to evaluate hydrophilicity. To better evaluate the hydrophilicity of porous scaffolds and also evaluate diffusion throughout the scaffolds, a water absorption test was done on the porous scaffolds. Rapid water absorption was observed from most of the Tween 80 scaffolds, and the results correlated with high hydrophilicity (Figure 2b). We assume that the scaffolds have the same degree of hydrophilicity as corresponding films with the same Tween 80 composition. To confirm this, all the scaffolds were cut horizontally and characterized for homogeneity by 1H NMR, and no difference in Tween 80 composition between top surface and bottom were seen, which excludes gravity-induced phase separation. The hypothesis that Tween 1261
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Figure 2. Values of water contact angle from homogeneous films (a) and corresponding images (b) at different time point of copolymer poly(LLAco-CL) and poly(LLA-co-DXO) porous scaffolds blended with 0, 3, 10, and 20% Tween 80.
Figure 3. Scaffold cross-section morphology (surface up) as shown by scanning electron microscopy.
microsurface of pristine scaffolds and 3% Tween 80 scaffolds was observed. The microsurface of the scaffolds including 10 and 20% Tween 80 might be different compared to the surface of the pristine scaffolds and scaffolds with 3% Tween 80 (Figure 3). This bumpy surface was likely caused by the removal of surface aggregated Tween 80. It was also observed that it was easier to blend 10% Tween 80 in to poly(LLA-co-
80 would enhance hydrophilicity of our scaffolds was therefore found to be correct. Our results show that only a small amount of Tween 80 is needed to obtain a surface with high hydrophilicity. Scaffold Topography. The porous scaffolds made here were visualized by SEM. All the scaffolds maintained high porosity and average pore size around 300 μm. A smooth 1262
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Figure 4. Typical thermograms of poly(LLA-co-CL) (left), poly(LLA-co-DXO) (right), and their blends with different amounts of Tween 80. Detailed values are presented in Table 3 and the values are presented as an average value ± standard deviation based on five replicates.
Table 3. Thermal Properties of Poly(LLA-co-CL), poly(LLA-co-DXO), and Corresponding Polymer/Tween 80 Blend Scaffolds groups Tween 80 CL CL+3% Tween 80 CL+10% Tween 80 CL+20% Tween 80 DXO DXO+3% Tween 80 DXO+10% Tween 80 DXO+20% Tween 80
χc (%)
Tg (°C)
Tc (°C)
Tm (°C) −10.5 ± 0.1
4.1 ± 0.2 −0.2 ± 0.7 −4.0 ± 1.2 −9.15 ± 0.1 10.3 ± 2.4 5.4 ± 1.3 0.7 ± 1.0 −2.4 ± 4.4
18.6 17.9 16.3 16.2
± ± ± ±
0.1 1.2 0.6 1.8
98.1 92.9 88.1 83.8
± ± ± ±
4.7 4.5 5.1 6.1
143.4 142.6 141.8 141.7
± ± ± ±
0.3 0.3 0.7 0.9
Figure 5. Tensile property at break point of poly(LLA-co-CL), poly(LLA-co-DXO), and its blends with corresponding amounts of Tween 80.
CL) compared to poly(LLA-co-DXO). The reason could be due to the discrepancy of hydrophobicity, as poly(LLA-co-CL) is more hydrophobic than poly(LLA-co-DXO) and therefore easier to entangle and blend with Tween 80. This may also be related to the fact that different feeding ratio was needed to obtain the scaffold compositions presented in Table 1, as more
Tween 80 was added to poly(LLA-co-DXO) than to poly(LLAco-CL) blends. Thermal Properties. Thermal properties including glass transition temperature (Tg), crystallization temperature (Tc) and melting temperature (Tm) were recorded for each sample. Degree of crystallinity (χc) was calculated according to the equation presented in the experimental section. The thermal 1263
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properties including Tg, Tc, χc, and Tm decreased when the amount of Tween 80 increased (Figure 4, Table 3). The alteration in thermal properties verifies effective entanglement between Tween 80 and the polymer chains. Tween 80 likely acts as to soften the scaffold, increase the chain movement and decrease the energy required for the movement. We know that the degree of crystallinity of poly(LLA-co-DXO) is mainly dependent on the length of LLA segments and the decreased χc implies therefore that crystalline segments were disrupted by Tween 80. One possible course of event is that, when the polymers crystallize during solvent casting, redundant Tween 80 is squeezed out by the crystals forming aggregates on the surface which then dissolve to create the surface morphology presented in Figure 3. Thus, the thermal properties seen here likely reflect the changes in thermal properties induced by Tween 80 and not the free Tween 80, since the free Tween 80 was dissolved during the scaffold fabrication process. This reduction of Tg, Tc, and Tm agree with what has been reported for other Tween 80 blend systems.40,41 Tensile Testing. Tensile testing was implemented to evaluate the mechanical properties of the materials, including the effect of different amounts of Tween 80. E-modulus and stress decreased with an increased amount of Tween 80 (Figure 5). However, a constant strain value was observed. The addition of 3% Tween 80 did not influence tensile properties and the largest E-modulus decrease was seen when 20% Tween 80 was added. Poly(LLA-co-CL) is an amorphous copolymer that is elastic and could gain high tensile strain. The ductility of the two copolymers is something we want to maintain, as it reduces the probability of damage when deformed. The significant decrease of E-modulus for the scaffolds with 10 and 20% Tween 80 indicates that Tween 80 acts as a softener and weakens the chain entanglements and hydrophobic interactions between the Tween 80 molecules and the polymer chains. These interactions correspond well also with the thermal analysis, in which Tween 80 showed the tendency to decrease Tg and crystallinity (Table 3). Importantly, the scaffolds with 3% Tween 80 had mechanical properties similar to the pristine copolymer scaffolds. Cellular Response to Culture Medium Containing Different Ratios of Tween 80. The cell viability in response to culture medium with different amounts of Tween 80 was evaluated. While absorbance values from medium containing 0.25−2% Tween 80 were slightly elevated (Figure 6), indicating
a possible low stimulation effect of Tween 80 on cell proliferation, the differences were not statistically significant. There was, however, significantly less cellular activity by this criterion from medium containing 10−20% Tween 80, suggesting a possible negative effect on celluar activity. The toxic effect was observed when Tween 80 composition was higher than 10%. This result could be considered as a dosedependent toxic effect of Tween 80. Tween 80 is well-known as a stabilizer when storage various proteins for preventing denaturalization.42 In addition, Tween 80 is a surface-active surfactant that could selectively change cell membrane morphology and permeability of various compounds to enhance the intracellular accumulation of specific compounds in the cell.43−45 Another possible explanation for the toxic effect seen here is the influence of impurities such as peroxide compounds that can be derived from photoinduced oxidation of Tween 80.46,47 Thus, the dose-dependent toxic effect of Tween 80 may result from a combination of a protein stabilization effect and a change in cell membrane fluidity. When cells are cultured with low dose Tween 80, it may protect proteins from unfolding or aggregating to allow proper cell attachment. At high dose, there is a risk to have higher peroxide production that may cause toxicity. This direct toxic effect from Tween 80 has been observed in other studies.48 However, we evaluated the issue of whether Tween 80 was dissolved in water during the salt leaching process and as there was no significant decrease of Tween 80 in the scaffolds after 7 days in deionized water, as measured by 1H NMR, we conclude that the amount of leached Tween 80, if any, is too small to cause toxicity. Cell Attachment. The numbers of cells attached after 1 and 3 h incubation on different scaffolds as determined by WST-1 assay are shown in Figure 7. Unattached cells were gently rinsed away before adding the WST-1 assay reagent. No significant increase in absorbance value was observed when comparing scaffolds with different hydrophilicity and amount of Tween 80. Thus, the addition of Tween 80 (3−20%) to the scaffolds did not alter the initial cell attachment of HOB. Based on WST-1 results after 1 and 3 h, hydrophilicity did not show a significant effect on initial cell attachment. Cell attachment is a complicated process influenced by many factors such as protein absorption, surface properties, and cell types. These may have different characteristics and sensitivities depending on the surface hydrophilicity. From our results it was obvious that HOB adhesion was dependent on protein absorption to the scaffold surface. The surface proteins offer specific cell binding and adhesion sites, and it has been reported that proteins have unique active sites that respond and adhere to different types of surfaces. Proteins also have different degrees of hydrophilicity and the structure of the protein is sometimes altered when touching surfactants or very hydrophilic surfaces.49−51 In addition, surface topography has been shown to influence the cell attachment and differentiation. The SEM images (Figure 3) showed that high concentration of Tween 80 might influence the topography of the material. However, a detailed evaluation of the surface roughness of the material was precluded. HOB Proliferation on 3D Porous Scaffolds. WST-1 assay was used to determine cellular proliferation on the different scaffolds after dynamic culture of HOB in spinner flasks. The results did not indicate significant difference between the samples with 3% Tween 80 and the pristine groups (DXO, CL; Figure 8). Samples with 20% Tween 80 showed a significantly lower absorbance compared to pristine
Figure 6. WST-1 results after 24 h incubation of cells with medium containing different concentrations of Tween 80 (*p < 0.05). 1264
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Figure 7. WST-1 results after 1 and 3 h show no significant difference in HOB attachment to the surface of poly(LLA-co-CL) and poly(LLA-coDXO) with different ratios of Tween 80.
Figure 8. WST-1 results after 1, 3, 7, and 14 days show the proliferation of HOB to the surface of poly(LLA-co-CL) and poly(LLA-co-DXO) with different ratios of Tween 80 (*p < 0.05).
proliferation. In addition, scaffolds with higher amounts of Tween 80 might have released more Tween 80. However, instead of cultured in the independent environment in culture plate, all scaffolds were cultured in the same environment in spinner flasks, which partly eliminate the imbalance of Tween 80 release from different scaffolds. In addition, from the pure Tween 80 cell viability test in Figure 6, we found that low Tween 80 concentrations did not inhibit cell viability. It is therefore most likely that the low HOB proliferation seen in samples with high amounts of Tween 80 result from the different levels of hydrophilicity or from specific protein absorption on the hydrophilic surfaces.
copolymers after 1 week, as well as significantly lower absorbance from scaffolds with 10 and 20% Tween 80 after 2 weeks. In conclusion, cell viability was inversely proportional to the amount of Tween 80. WST-1 results after 1 and 2 weeks of culture showed low proliferation activity from HOB on samples with 20% Tween 80. There was a tendency toward lower proliferation activity in the samples with 10% Tween 80. For the samples with 3% Tween 80, HOB proliferation was similar to that with the pristine copolymers. The culture time in this study was up to two weeks, and it is possible that a small amount of Tween 80 might have been released into the culture medium and influenced HOB 1265
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Figure 9. Real time RT-PCR results after 2 weeks show the effect on differentiation of HOB to the surface of poly(LLA-co-CL) and poly(LLA-coDXO) with different ratios of Tween 80 (*p < 0.05).
Quantitative Real-Time RT-PCR Analysis. Pristine copolymers and copolymers with 3% Tween 80 were evaluated by RT-PCR to determine the influence of Tween 80 on cell differentiation after 2 weeks. Increased expression of OPG, BMP-2, and ALP were seen with 3% Tween 80 compared to the original unmodified materials, and in most cases, these increases were statistically significant. The data demonstrated lower expression of VEGFa, OC, CALCRL, and Osterix in the samples with 3% Tween 80, and again, most of these were statistically significantly different (Figure 9). Furthermore, the cells grown on the 3% Tween scaffolds had similar mRNA expression of PCNA, BMP-2R, and ITGA1, as on the pristine polymers. In the samples with Tween 80, key biomarkers, including osteogenic markers, were examined by real time RT-PCR. The results were essentially similar between the two groups with Tween 80 and between the two control materials, the results were not very dependent on the copolymer composition. The higher expression of OPG, BMP-2, and ALP by the cells grown on the scaffolds modified with 3% Tween 80 might indicate the hydrophilicity of the material is significantly influenced by the osteogenic potential of osteoblasts. This was not supported by
the results related to the expression of osteocalcin mRNA which is a specific marker of mineralization. However, the study design precludes using several time points or kinetic curve to determine the peak of osteocalcin expression.
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CONCLUSIONS
Using a surfactant, hydrophilic polyester-based porous scaffolds with similar thermal properties and mechanical properties as scaffolds from the pristine copolymers were produced. With 3% (w/w) Tween 80 we obtained highly hydrophilic scaffolds. The increased hydrophilicity did not show significant influence on attachment of HOB and the scaffolds with 3% Tween 80 showed similar HOB proliferation as the pristine copolymers. There was an indication that 3% Tween 80 might influence differentiation of HOB and sustain the stemness of HOB. In addition, it was obvious that the porous degradable scaffolds with 3% Tween absorbed water quickly, which is important for efficient diffusion of nutrients. Our results, in combination with the fact that this method of designing hydrophilic scaffolds is easily scaled up, confirm that Tween 80 is a good and efficient additive in large porous polyester-based scaffolds. Such large 1266
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scaffolds are attractive for use in bone tissue engineering of critical size defects.
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ASSOCIATED CONTENT
S Supporting Information *
Calculations of the amount of Tween 80 in the samples. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +46-8-790-82 74. Fax: +46-8-20 84 77. E-mail: aila@ polymer.kth.se. Author Contributions †
These authors contributed equally to this work (Y.S. and Z.X.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Research Council of Norway [(FRIMED, 17734/V50)] and the European Union, the 7th Frame Program, Vascubone Project No. 242175.
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REFERENCES
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