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Nanocomposite scaffold with enhanced stability by hydrogen bonds between collagen, polyvinyl pyrrolidone and titanium dioxide
Colloids and Surfaces B: Biointerfaces 140 (2016) 287–296
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Nanocomposite scaffold with enhanced stability by hydrogen bonds between collagen, polyvinyl pyrrolidone and titanium dioxide Na Li a , Xialian Fan a , Keyong Tang a,∗ , Xuejing Zheng a , Jie Liu a , Baoshi Wang b a b
School of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001, PR China Luoyang Petrochemical Engineering Corporation, SINOPEC, Luoyang 471003, PR China
a r t i c l e
i n f o
Article history: Received 14 August 2015 Received in revised form 30 November 2015 Accepted 1 December 2015 Available online 24 December 2015 Keywords: Type I collagen Polyvinyl pyrrolidone Titanium dioxide Nanocomposite scaffold Stability
a b s t r a c t In this study, three-dimensional (3D) nanocomposite scaffolds, as potential substrates for skin tissue engineering, were fabricated by freeze drying the mixture of type I collagen extracted from porcine skin and polyvinyl pyrrolidone (PVP)-coated titanium dioxide (TiO2 ) nanoparticles. This procedure was performed without any cross-linker or toxic reagents to generate porosity in the scaffold. Both morphology and thermal stability of the nanocomposite scaffold were examined. The swelling behavior, mechanical properties and hydrolytic degradation of the composite scaffolds were carefully investigated. Our results revealed that collagen, PVP and TiO2 are bonded together by four main hydrogen bonds, which is an essential action for the formation of nanocomposite scaffold. Using Coasts–Redfern model, we were able to calculate the thermal degradation apparent activation energy and demonstrated that the thermal stability of nanocomposites is dependent on amount of PVP incorporated. Furthermore, SEM images showed that the collagen fibers are wrapped and stabilized on scaffolds by PVP molecules, which improve the ultimate tensile strength (UTS). The UTS of PVP-contained scaffold is four times higher than that of scaffold without PVP, whereas ultimate percentage of elongation (UPE) is decreased, and PVP can enhance the degradation resistance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Due to its nontoxic and biocompatible properties, collagen has been widely used as a versatile basic material for various applications in biomedical and biotechnological fields. However, major limitations of collagen-based materials, such as poor thermal instability, poor mechanical properties, and possible contamination by pathogenic substances [1], hamper their further applications. Therefore, various crossing linkers, both natural and synthetic ones, such as glutaraldehyde [2], epoxy compounds [3] and EDC (1ethyl-3-(3-dimethyl aminopropyl) carbodiimide) [4] are employed to enhance the stability of collagen-based materials for in vivo applications. Blending modification is an effective, convenient and economical method which has been widely used to improve the properties of collagen-based materials. Blending of collagen with organic or inorganic polymers is done by means of facilitating covalent bond formation between the abundant OH, COOH, NH2 functional groups of collagen and functional groups on polymers and metal ions [5]. Thus, this shows the advantages of using blend-
∗ Corresponding author. E-mail address: [email protected] (K. Tang). http://dx.doi.org/10.1016/j.colsurfb.2015.12.005 0927-7765/© 2015 Elsevier B.V. All rights reserved.
ing modification over cross linking method in enhancing collagen stability on nanocomposites. Polyvinyl pyrrolidone (PVP) has been used as an additive in collagen-based biomaterials because of its excellent water solubility, excellent adsorption and complexing ability, film-forming property, and cohesiveness. PVP is also known to be a good stabilizing agent for transition metal particles. The excellent miscibility of collagen and PVP has been demonstrated previously [6–9]. Collagen–PVP based composite exhibits good tolerance, antiinflammatory and anti-fibrotic properties [10–12]. Collagen–PVP blend can be safely used for biomedical materials because of its non-genotoxicity and diminutive localized hypersensitivity reaction [13]. Excellent biocompatibility of titanium dioxide (TiO2 ) combined with anticancer and antimicrobial property [14] makes TiO2 one of the most promising nanoparticles for biomedical applications. For example, Grassian et al. reported that inhalation exposure of TiO2 in mice, showed minimal inflammatory response and pulmonary toxicity [15]. Li et al. demonstrated that TiO2 nanoparticles can be utilized to shield protein against photo degradation and photo-oxidation [16]. The feasibility of using TiO2 in biological tissue engineering [17–19] and using TiO2 nanoparticles in collagen-based composite to make nanocomposite scaffolds were
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also reported. Sol–gel method is one of the most widely used techniques for preparing nano-sized metallic oxide materials with photocatalytic activities [20], and controlling material’s porosity and shape [21]. However, consumption of reaction medium, such as ethanol and ethylene glycol, and cost are some of the limitations of the sol–gel method. Herein, we improved a three-dimensional nanocomposite material by blending collagen with PVP coated TiO2 particles to obtain collagen-based composite. The goal is to fabricate a potentially simple, but effective skin scaffold for wound healing. Conglomeration of nanoparticles is deemed as one of the main obstacles in the preparation of nanomaterials. To reduce the agglomeration, PVP was added as a coating agent for nano TiO2 particles, which enhances the stability of nanoparticle colloid. The extracted collagen, nano TiO2 particles and resultant freeze-dried ternary composite scaffolds were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), X-ray diffraction (XRD), dynamic light scattering technique (DLS), scanning electron microscope (SEM), attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis-differential scanning calorimetry (TGA-DSC). The formation of four hydrogen bonds between collagen, PVP and TiO2 particles in the uniform mixture is based on the changes in particle size and analyzed by ATR-FTIR. The stability of TiO2 nanoparticles, such as thermal stability, swelling properties, mechanical stability and degradation properties, in ternary blending solution were also investigated.
2. Experimental 2.1. Materials Fresh porcine skin was obtained from local wet market. Sodium sulfide (Na2 S), hydrochloric acid (HCl) and acetone were purchased from Tian-li Chemicals Ltd., Tianjin, China. Pepsinum and ethylenediamine tetraacetic acid (EDTA) were provided by Sigma, USA. Calcium hydroxide (Ca(OH)2 ), sodium hydroxide (NaOH), tetrabutyl titanate (TBT), absolute ethanol (EtOH) and glacial acetic acid (HAc) were all purchased from Feng-chuan Chemicals Science and Technology Ltd., Tianjin, China. Polyvinyl pyrrolidone (PVP) K-30 was supplied by Sinopharm, China. Double-distilled water was used as solvent throughout the experiment. 2.2. Extraction of collagen from porcine skin The collagen was extracted as previously reported in the literature [22]. Briefly, depilated porcine skin was minced, and then dipped into a degreasing agent for 16 h and followed by acetone for 16 h. After suspending in ethanoic acid solution with pepsin at 4 ◦ C, the resulting suspension was centrifuged to yield the supernatant liquor. The pH was then adjusted to 8 using NaOH solution. Collagen was then repeatedly purified by salt precipitation, centrifugation and ethanoic acid solubilization. The final collagen solution was dialyzed and stored at 4 ◦ C until use.
Table 1 Mass ratios and names of samples. Scaffold (PVP–TiO2 )1 (PVP–TiO2 )2 (PVP–TiO2 )3 (PVP–TiO2 )4
TBT with a purity of 99% was used as precursor, which was dissolved in the mixture of EtOH and HCl, and was dropped slowly into solution of HCl in distilled water under magnetic stirring for two hours. The mixture was stirred at a constant temperature for another 2 h. The molar ratio of the reactants was TBT:H2 O:EtOH:HCl = 1:200:16:0.8. The mixture gave a light blue transparent TiO2 sol. A portion of as-prepared titanium hydrosol
(Col–TiO2 )
1:0.5
(Col–PVP–TiO2 )1 (Col–PVP–TiO2 )2 (Col–PVP–TiO2 )3 (Col–PVP–TiO2 )4
1:5:0.5 1:10:0.5 1:15:0.5 1:20:0.5
was kept at 4 ◦ C for subsequent use, and a fraction was dried at 120 ◦ C for 12 h, and then grinded. 2.4. Preparation of collagen–PVP–TiO2 ternary nanocomposite scaffold Different amount of PVP was dissolved in 10 ml distilled water separately to obtain PVP solutions with various concentrations. TiO2 was added to above PVP solution in a certain stirring speed at room temperature, and resultant mixtures were named as (PVP–TiO2 )1–4 . To the above mixtures, collagen solution (2 mg/ml) was added and continuously stirred at room temperature for 2 h to give a homogeneous mixture. The gel-like solutions with the same quality were poured onto the equally-sized glass culture dishes. The samples were vacuum freeze-dried at −50 ◦ C, and then corresponding porous nanocomposite scaffold were obtained. The mass ratios and appropriate names are listed in Table 1. 2.5. Characterization SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Mini-Protean electrophoresis apparatus. The particle size of TiO2 and micelle particles were studied by dynamic light scattering technique (DLS) using a Malvern Zetasizer Nano-ZS90 at 25 ◦ C. The attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) of the extracted collagen and ternary nanocomposites were performed using EQUINX55 in the range of 600–4000 cm−1 . The thermograms were recorded on TGA/DSC 1 synchronized thermal analyzer (METTLER TOLEDO) at the temperature range from 50 to 650 ◦ C with a heating rate 10 ◦ C/min in nitrogen atmosphere. The surface morphology of the collagen and nanocomposites was investigated using the scanning electron microscopy (SEM, FEI Quanta 200, US). 2.6. Swelling property The pre-weighed dry scaffolds were immersed in phosphate buffer saline (PBS) (pH 7.4) solution at room temperature. The weights of swollen composites were measured after the surface excess water was filtered and drained until no free water remained. The procedure was repeated every thirty minutes until the weight was stable. The degree of swelling (DS) for each sample was calculated by the following equation [23]: DS =
2.3. Preparation of TiO2 sol–gel
5:0.5 10:0.5 15: 0.5 20: 0.5
Wt − W0 W0
(1)
where Wt and W0 are the weight of sample at time t and in the dry state, respectively. Each sample was measured in quadruplicate. 2.7. Mechanical property All specimens were tensile-tested at a loading rate of 10 mm/min using an Instron Testing Machine Model 1026, after being rehydrated by immersion in neutral PBS for 0.5 or 1.5 h at
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Fig. 1. SDS-PAGE test of collagen extracted from porcine skin by pepsin. (a) Protein electrophoresis band of extract (lane 1 contains standard protein marker, lane 2 contains porcine skin type I collagen). (b) The relation between lg Mr and relative mobility (MR ) of protein molecular weight marker. (c) Parameters determination of each collagen component. Error bars represent mean ± standard deviation with n-3.
room temperature. Prior to testing, samples were cut and a polymer mesh was incorporated into the sample, for ease of handling as well as providing more accurate measure of the strength. The area between the grips was free of mesh so that only the sample was tested. Five independent measurements were performed for each sample, and the average is reported.
2.8. Hydrolytic degradation Hydrolytic degradation assay at 37.0 ± 0.5 ◦ C and pH of 7.4 was carried out in Simulated Body Fluid (SBF). SBF was prepared by dissolving 7.996 g NaCl, 0.350 g NaHCO3 , 0.224 g KCl, 0.228 g K2 HPO4 ·3H2 O, 0.305 g MgCl2 ·6H2 O, 0.278 g CaCl2 , 0.071 g Na2 SO4 , 40 ml HCl 1 N, and 6.057 g NH2 C(CH2 OH)3 (TRIS) in 1 l distilled water. A certain size of each scaffold sample was immersed in SBF until equilibrium state, then free water was removed as described in Section 2.6 and was weighed as W0 . Each sample was then immersed in SBF and degradation measurement was made every 24 h for a period of 10 days. The measured weight is Wt after eliminating the free water. Each scaffold was assayed in quadruplicate. The degradation was assessed by measuring the wet weight loss which was defined as the following expression:
Wet weight loss% =
Wt − W0 × 100% W0
(2)
3. Results and discussion 3.1. Characterization 3.1.1. Gel electrophoresis patterns and relative molecular mass of collagen Collagen has a triple helix as the basic structure motif and is a major structural protein found in the extracellular matrix [24]. Thus type I collagen is widely used in clinics for skin and tissue repairing purposes. As is the case, we have incorporated this structural protein into our TiO2 nanocomposite scaffold, with the goal of fabricating next stage skin healing scaffold. The purity of the prepared collagen was confirmed by SDS-PAGE, and the collagen concentration in the solution was determined by measuring the hydroxyproline content, according to the method reported by Woessner [25]. The two prominent bands in the electrophoretogram (Fig. 1(a)) with blurred boundary around 116 kDa is assigned to ˛1 -chain and ˛2 -chain [26], which are the major components of type I collagen [27]. The bands around and above 200 kDa belonged to ˇ-chain and -chain, respectively. So, the extracted collagen from porcine skin has its intact triple helical structure. No low molecular weight protein can be seen on the SDS-PAGE gel further indicating that there was no degradation of the pigskin collagen during the extraction process and a high purity of type I collagen has been obtained. Fig. 1(b) is the standard curve obtained by plotting relative mobility (MR ) against log of molecular weight (lg Mr ), from which the linear equation lg Mr =−1.24 MR + 5.36 was derived. The linear regression coefficient (R2 ) for the equation is 1.24, indicating a per-
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Fig. 2. Particle size analysis of TiO2 particle and micelle particle. (a) The list of average particle diameters and PDI in different solutions. (b) The bar chart of average particle diameter.
fect fit. The MR value obtained from SDS-PAGE electrophoresis was inserted into the above equation and the molecular weight of each protein components was calculated (Fig. 1(c)). 3.1.2. Particle size analysis Prior to the preparation of nanocomposite scaffold, i.e., the mixture before freeze drying comprised of water, minuscule amount of ethanol, and PVP, which is non-ionic surfactant that fulfill the basic requirement to form a microemulsion. In the binary liquid system (PVP and TiO2 ) and ternary liquid system (PVP, TiO2 and collagen), micelle particles can be formed with TiO2 as core. The hydrodynamic sizes of TiO2 particles and micelle particles in different mixtures liquid were compared and analyzed. The particle sizes in different solutions and their corresponding polydispersity indexes (PDI) are shown in Fig. 2. All samples have wide range of size distributions and differ from each other based on PDI. As shown in Fig. 2(b), the particle size of prepared TiO2 in the sol is 24.4 nm. For (PVP–TiO2 )1–4 samples, with the increase in the concentration of PVP, the particle size increased significantly and (PVP-TiO2 )4 has a final size of 916.6 nm. The adsorption of PVP onto TiO2 is achieved by the hydrogen bonding between the two reactants through acid–base mechanism, as shown in Scheme 1(a). The adsorption amount of PVP is independent of its molecular weight, but dependent on the concentration, which is in agreement with previous reports [28–30]. In this system, water and ethanol were added during the preparation of titania solution. The driving force for the adsorption of PVP on TiO2 is the interaction between carbonyl groups of pyrene ring in PVP macromolecule and hydroxyl groups on the surface of TiO2 . It can be also
observed from Fig. 2(a) that PVP can facilitate the concentrated distribution of particle size which is indicated by lower PDI. However, in the case of (Col–PVP–TiO2 )1–4 solution, compared with (PVP–TiO2 ), particle sizes are not markedly increased by increasing the amount of PVP. The incorporation of type I collagen into the solution mixture may have decreased the PVP adsorption onto TiO2 surface, which results in no significant change to particle sizes. The competing adsorption between collagen–PVP and TiO2 –PVP is another plausible explanation to the reduced particle size. In addition to the PVP–TiO2 hydrogen bond as seen with (PVP–TiO2 ) only solution, three other hydrogen bonds exist in the (Col–PVP–TiO2 ) solution. They are formed between collagen and PVP, collagen and collagen [6–8], and collagen and TiO2 [31,32]. These hydrogen-bondings have been reported and are shown in Scheme 1(b and c). Hydrogen-bonding between collagen and PVP could be studied by considering the interaction between proton-accepting carbonyl moiety in pyrrolidone rings of PVP and amino/hydroxyl side groups on collagen. Collagen–PVP shows stronger hydrogen interactions compared to both PVP–PVP and collagen–collagen, which can be proved by increased mutual miscibility. Due to the stronger hydrogen bonding formed between collagen to PVP than PVP to TiO2, the PVP adsorption amount onto the surface of TiO2 was decreased. Thus, it can be why the particle size of (Col–PVP–TiO2 ) solutions is smaller than that of (PVP–TiO2 ). It is noteworthy that the particle size obtained from (Col–TiO2 ) solution is bigger than that of TiO2 solution alone. This is a result of the enhanced adsorption of collagen onto TiO2 owing to the coordination ability of carboxylic and hydroxyl group with metal ions.
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Scheme 1. Hydrogen bonds of TiO2 –PVP (a), collagen–PVP (b), collagen–collagen (c) and collagen–TiO2 (d).
These carboxylic and hydroxyl groups probably act as the nucleation site for nanoparticle formation [33]. 3.1.3. Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) analysis Infrared spectroscopy can be widely used to monitor protein structure in films and solution. In the FTIR spectrum of the extracted pigskin collagen (Fig. 3), 3315 cm−1 and 3086 cm−1 are assigned to N H stretching vibration for amide band A and amide band B of collagen, respectively. The peak at 1652 cm−1 is due to C O stretching vibration in amide I bands, and the band in the region of 1557 cm−1 is assigned to N H bending vibration of amide II [34]. The FTIR peaks at 1402 cm−1 and 1239 cm−1 are due to COO symmetrical stretching and C N stretching of amide III, respectively [35]. The characteristic peak at 1454 cm−1 is due to pyrrolidine block for proline and hydroxyproline, which shows a complete triple helical structure of the extracted collagen [36]. The absorption intensity ratio of 1239 cm−1 to 1454 cm−1 is 1.02, which indicates that the secondary structure of collagen is undamaged [37,38]. The extent of intermolecular hydrogen bonding can be studied by determining the frequency shifts. Collagen shows a well-defined peak with maxima at 3315 cm−1 and a weak shoulder at 3423 cm−1 , due to H-bonded N H and H-bonded free N H, respectively. It can
Fig. 3. ART-FTIR spectrum of (a) extracted collagen, (b) scaffold (Col–TiO2 ) and (c) scaffold (Col–PVP–TiO2 )4 after freeze drying.
be observed that, compared to spectra of collagen, N H peak of scaffold (Col–PVP–TiO2 )4 has shifted from 3315 cm−1 to 3408 cm−1 , and peak is broadened. These indicate a very strong hydrogen bonding in this compound, which is as expected. Similarly, the scaffold (Col–TiO2 ) also shows one broad peak with peak maximum at 3316 cm−1 indicating the presence of hydrogen bonding of N H in this composite. However, the shift is much less than that of scaffold (Col–PVP–TiO2 )4 . This may be due to two different types of hydrogen bonding energies in these two scaffolds, namely N H· · ·O C for scaffold (Col–PVP–TiO2 )4 and N H· · ·O for (Col–TiO2 ). This change in hydrogen bonding energies can be understood that the interaction between collagen and PVP via hydrogen bond is stronger than that between collagen and TiO2 . For amide band B and amide band II, the peaks of (Col–TiO2 ) show somewhat shifts of 3 cm−1 and 9 cm−1 to low frequency separately, whereas these peaks of (Col–PVP–TiO2 )4 disappear and may be because of the peaks overlapping when amide band peaks shift to lower frequency caused by strong hydrogen bonding. In the FTIR spectra the shifts of the bands derived from groups taking part in the hydrogen bonds suggest that the interactions between collagen and PVP occur by hydrogen bonds [8], which is quite limited between collagen and TiO2 . It is obvious that the spectrum of (Col–PVP–TiO2 )4 is totally dominated by the spectrum of collagen. But it is also noteworthy that, probably because of the formation of hydrogen bonding between the N H and the oxygen atom on TiO2 surface, the peak at 1557 cm−1 shifted to 1548 cm−1 and the absorption intensity is weakened. 3.1.4. Thermal stability The thermal stability of nanocomposite scaffold was investigated by means of TGA and DSC. Fig. 4 shows that when more stable moiety, like PVP, is used, the thermal stability of the nanocomposite scaffold is improved. Three main temperature regions of weight loss appear in all TGA curves (Fig. 4(a)). Initial weight loss below 200 ◦ C can be attributed to the evaporation of physically absorbed water, or residual solvent in the sample. Corresponding smaller endothermic peak in DSC curve (Fig. 4(b)) represents the unfolding of triple helical structure of collagen. The significant weight loss observed between 200 and 500 ◦ C can be due to thermal decomposition of collagen and PVP [8,39], TiO2 OH [40] and the fracture of intermolecular hydrogen bonds as mentioned in Section 3.1.2. The subsequent endothermic transitions around 425 ◦ C can be observed in DSC. The pyrolysis onset temperature of (Col–PVP–TiO2 )1–4 nanocomposite slightly decreases with
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Fig. 4. (a) TGA and (b) DSC curves of (Col–TiO2 ) and (Col–PVP–TiO2 )1–4 nanocomposite scaffolds. (c) Linear plots of ln [G(ɑ)/T2 ] versus 1/T for nanocomposite scaffolds. (b) Thermal degradation apparent activation energy of nanocomposite scaffolds.
decreasing the PVP content, while its melting temperature slightly increases oppositely. It was found that the weight loss above 500 ◦ C can be neglected as only TiO2 remained. The obvious exothermic peak around 620 ◦ C in DSC curves might be due to the change of TiO2 crystal structure from anatase to rutile [41,42]. Similar TGA and DSC curves for the (Col–PVP–TiO2 ) samples suggest that increasing PVP concentration did not have a significant impact on thermal stability. However, the (Col–TiO2 ) sample has a much lower thermal stability as the scaffolds degrades at much earlier temperature range and at a faster rate. The corresponding endothermic peak around 325 ◦ C in DSC curve indicated that PVP is high in thermal stability. Thus the thermal stability of collagen-based nanocomposite scaffold was improved. The apparent activation energy is normally used to quantitatively characterize the thermal stability of scaffold. Coats-Redfern method has been widely accepted in calculating the apparent activation energy. Coats-Redfern integral formula is usually described as [43]: ln
G(˛) AR E = ln( )− RT ˇE T2
(3)
The meaning of the symbols is shown in Supporting information Table S1. This integral method lead to –Ea /R from the slope of the line as determined by plotting ln[G(˛)/T2 ] against 1/T at any certain conversion rate.
From both the TGA and DSC analyzes, temperature ranging from 200 ◦ C to 500 ◦ C has been shown as the main thermal decomposition factor of more than 70% of the polymer composite, and the decomposition conversion thereafter is less meaningful due to too high temperature and tiny sample weight-loss. Thus, the emphasis was placed on a conversion range from 0.4 to 0.9, instead of the entire process. This would offer a simplified and more meaningful way to modeling thermal decomposition behavior of nanocomposite scaffolds. Because G(˛) has thirty integral forms, the function G(˛) = −ln (1 − ˛) was most reasonable for this system and is used for comparing and evaluating the linear fitting. Fig. 4(c) shows the linear plots of ln [G(˛)/T2 ] versus 1/T˛ for both (Col–TiO2 ) and (Col–PVP–TiO2 )1–4 scaffolds. Fig. 4(d) shows the thermal degradation apparent activation energy values for certain conversion rates. As observed from the two figures, the thermal degradation apparent activation energies of scaffolds (Col–PVP–TiO2 )1–4 are higher than that of scaffold without PVP (scaffold (Col–TiO2 )). The activation energy increases with increasing the PVP content. That is suggesting that the thermo-stability for nanocomposite increases with increasing PVP content in the study, which further supports the aforementioned results. 3.1.5. Scanning electron microscopy (SEM) analysis The types of matrix used, or more precisely the exact structures of the matrix, directly affect properties of polymer products, including swelling behavior, mechanical property, cell growth and tissue formation. The surface morphologies of collagen (Fig. 5a),
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Fig. 5. SEM images of collagen (a), scaffold (Col–TiO2 ) (b) and (Col–PVP–TiO2 )4 (c), EBSD image of scaffold (Col–PVP–TiO2 )4 with EDAX.
(Col–TiO2 ) (Fig. 5b), and (Col–PVP–TiO2 )4 (Fig. 5c) were observed and compared using scanning electron microscopy (SEM). As shown in Fig. 5a (1), pure collagen after freeze drying presents with a combination of fiber and sheet morphology, with the majority of it being sheet formation. Fracture in the sheet is not due to an integrated plane, as wrinkles and grooves exist on the sheet surface. Normal collagen fibers have smooth surface but do intertwine with one another, as this phenomenon is seen in Fig. 5a (2). With the addition of nano-TiO2 particles (Fig. 5b), instead of dense sheet layer, discontinuous flakes form a tri-dimensional fluffy structure and gives a more porous nature than pure collagen. The exposed fibers become finer in structure and exhibit a more independent nature to each other. Lastly, from the SEM analysis, scaffold (Col–PVP–TiO2 )4 , stand in marked contrast to pure collagen and scaffold (Col–TiO2 ). The collagen fibers from (Col–PVP–TiO2 )4 are cemented together by PVP to achieve good cohesiveness. This leads to fewer exposed fibers on surface and affects the porous structure of original collagen. Furthermore, because of the good film-forming property of PVP, there are less flakes in the ternary system and more continuous interface than scaffold (Col–TiO2 ), along with some wrinkles and less grooves (Fig. 5c(1)). It should also be noted that, due to the enwrapping with PVP, the collagen fibers become larger in diameter and the scaffold surface become less smooth (Fig. 5c(2)). Some TiO2 particles with large diameter, which attach to the surface of the enwrapped fiber can also be detected. In a higher magnification Fig. 5c(3), irregularly-shaped TiO2 can be observed on the sheet surface of the (Col–PVP–TiO2 )4 scaffold,
which reveals that there is possible agglomerative phenomenon accompanying wrapping for nanoparticles. Fig. 5c(4) is the scanning back scattering image of the same area of Fig. 5c(3), and the EDAX spectrum recorded in the spot profile mode form one of the bright regions with the binding energy region of 0–20 keV is shown in Fig. 5c(5). The peak from the spectrum revealed the presence of Ti and O at 4.5 and 0.5 keV, respectively. The combination of Fig. 5c(4) and c(3) indicate the crystal phase uniform distribution of TiO2 in organic phase.
3.2. Swelling property Water adsorption of biomaterial is important for cell uptake, as it affects the distribution of cell suspension throughout the material and the transfer efficiency of oxygen and nutrient [44]. Thus, a faster rate can promote faster absorption of wound surface effusion. The swelling property of ternary nanocomposite scaffolds in PBS (pH 7.4) is therefore studied and is shown in Fig. 6(a) and (b). All scaffold samples reached equilibrium after being soaked in PBS for 1.5 h. The equilibrium water absorbency in PBS is in the order (Col–TiO2 ) > (Col–PVP–TiO2 )1 > (Col–PVP–TiO2 )2 > (Col–PVP–TiO2 )3 > (Col–PVP–TiO2 )4 . Some parameters may affect the swelling ratio, such as hydrophilicity, stiffness and porous structure of the matrix. The carboxyl group on collagen chain is an anionic hydrophilic group which can be ionized. Thus the space for absorbing and holding water increased due to anion–anion electrostatic repulsion [45]. Furthermore, in this ternary system, three-dimensional network of the scaffold was the
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Fig. 6. The equilibrium swelling degree (a) and time-dependent changes in DS (b) of prepared scaffolds in PBS. (c–d) Tensile properties of nanocomposite scaffolds with different PVP contents. (e) Wet weight loss of scaffolds vs. degradation time in SBF. Error bars represent mean ± standard deviation (n-4 for (a, b, e); n-5 for (c, d)).
result of interaction of collagen, PVP and TiO2 . The collagen fibers adhered to each other by the effect of PVP, and its individual pore structure was destroyed. Besides, with increasing the PVP content, the decrease in collagen relative content resulted in decreased numbers of carboxyl group with strong hydrophilicity, which is the reason why (Col–PVP–TiO2 )4 had the weakest absorbency. It is interesting to note that PVP is non-ionic polymer, and therefore electrostatic repulsion of polymeric chains is small. Hence, the DS
of scaffold (Col–TiO2 ) without PVP is the biggest, and it decreases with increasing PVP content. 3.3. Mechanical property Scaffold biomaterials are supposed to support desired cellular, process or can develop appropriate mechanical characteristics. This scaffold is designed as skin scaffold for wound healing and sup-
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posed to use under moist condition. All scaffold samples can reach equilibrium soon as shown in the swelling study. Therefore, the results of UTS and UPE depending on percentage of PVP and scaffold soaking time in PBS are graphically represented in Fig. 6c and d, respectively. As shown in Fig. 6c and d, the plasticizing action of water was obvious in all cases, independently of scaffold type. The wet strength reduced slightly when soaking time is extended from 0.5 h to 1.5 h, and scaffold with more PVP had a larger decrease. However, the ultimate elongation increased to varying degree with the increase of soaking time. Water is known to be an effective plasticizer for most biopolymers [46] and its plasticizing action is reflected in lowering of the fracture strength, elastic modulus and increasing of flexibility of the material [47]. So, the addition of more water as the plasticizer can decrease intermolecular forces that permit smaller UTS but greater UPE of the scaffold before break. Moreover, PVP could leach out more with a longer soaking time, which may cause unconsolidated structure, and as a result the strength of scaffolds reduced. For UTS, scaffolds after a soaking time of 0.5 h showed a increasing tendency from (Col–TiO2 ) to (Col–PVP–TiO2 )4 . Scaffolds with PVP exhibited higher values of ultimate tensile strength (1491 ± 20 kPa–945 ± 16 kPa), which is about 3–5 times higher than that of scaffold without PVP (304 ± 15 kPa). It suggests that (Col–TiO2 ) have lower wet strength, and the increase of UTS with PVP incorporated into the scaffolds can be due to cementation of collagen fiber with PVP increasing the joining force among collagen fibers and the H-bond between different molecules. However, the change of elongation property (Fig. 6d) with increasing PVP content did not follow the same pattern as the UTS. It showed lower values of UPE for higher concentration of PVP in the scaffold. So it is indicating that removal of the cohesive action of PVP on collagen fibers enhanced the tensile strength of scaffolds but decreased their distensibility. It should be noted that, scaffold (Col–TiO2 ) with soaking time 0.5 h showed slightly lower UPE than (Col–PVP–TiO2 )4 . Thus it can be interpreted that excess PVP can cause compact structure with poor elongation and appropriate amount of PVP can facilitate the structural continuity, which contributes to the elongation.
3.4. Hydrolytic degradation behavior The analysis of wet weight loss of scaffold in SBF (Fig. 6(e)) could reflect the degradation condition in vitro. In the early stage of analysis, it was the degradation of small molecules. It is interesting to note that during the early stage, scaffold (Col–PVP–TiO2 ) with higher PVP content has the largest wet weight loss percentage. However, after the fourth day, the degradation rates for (Col–PVP–TiO2 )4 declined and the ternary system with the lowest PVP content has the largest final overall wet weight loss. This may ascribe to the multi-sites binding of collagen, PVP and TiO2 , such as H-bond between PVP, collagen and TiO2 . This indicates indirectly the hydrogen bond can protect collagen from the quick decomposition. By the eighth day, fine cracks appeared on all four samples, but were slightly smaller and less in number in the scaffold with the higher concentration of PVP. On the tenth day, all four scaffolds fragmented. Scaffold (Col–TiO2 ) always degraded faster in SBF with cracks appearing on the fourth day of analysis, and it fragmented into pieces on the sixth day. By the eighth day, the material disintegrated completely. Therefore, the addition of PVP may increase the binding of different molecules, and then effectively maintain the integrate structure and slow the degradation of collagen-based scaffold.
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4. Conclusions A new nanocomposite scaffold composing of type I collagen, PVP and nanoparticle TiO2 in anatase form is fabricated to serve as an appropriate skin scaffold for wound healing. Based on existing experimental data, we interpret there are four main hydrogen bonds formed between collagen, PVP and titanium dioxide. It is inferred that the interaction between collagen and PVP, PVP and TiO2 are stronger than others, and this hydrogen bonding plays an important role in determining the overall properties and performance. The nanocomposites exhibit adequate mechanical properties to serve as a promising wound healing dressing. The addition of PVP can form strong interaction in this ternary system by increasing the tensile strength and improving the resistance of scaffolds in SBF, while decreasing the degree of swelling in PBS and the percentage of elongation of collagen-based scaffold. However, further studies are needed to address the nanocomposites efficacy.
Acknowledgements We would like to thank Dr. Xiaoqiang Yang, Benjamin Fook Lun Lai and Dr. Bo Deng of the Centre for Blood Research (CBR) at the University of British Columbia at Vancouver for good advices to this paper. This research was funded by the National Natural Science Foundation of China (No. 51373158).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.12. 005.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Nanocomposite scaffold with enhanced stability by hydrogen bonds between collagen, polyvinyl pyrrolidone and titanium dioxide Na Li a , Xialian Fan a , Keyong Tang a,∗ , Xuejing Zheng a , Jie Liu a , Baoshi Wang b a b
School of Materials Science and Engineering, Zhengzhou University, Zhengzhou 450001, PR China Luoyang Petrochemical Engineering Corporation, SINOPEC, Luoyang 471003, PR China
a r t i c l e
i n f o
Article history: Received 14 August 2015 Received in revised form 30 November 2015 Accepted 1 December 2015 Available online 24 December 2015 Keywords: Type I collagen Polyvinyl pyrrolidone Titanium dioxide Nanocomposite scaffold Stability
a b s t r a c t In this study, three-dimensional (3D) nanocomposite scaffolds, as potential substrates for skin tissue engineering, were fabricated by freeze drying the mixture of type I collagen extracted from porcine skin and polyvinyl pyrrolidone (PVP)-coated titanium dioxide (TiO2 ) nanoparticles. This procedure was performed without any cross-linker or toxic reagents to generate porosity in the scaffold. Both morphology and thermal stability of the nanocomposite scaffold were examined. The swelling behavior, mechanical properties and hydrolytic degradation of the composite scaffolds were carefully investigated. Our results revealed that collagen, PVP and TiO2 are bonded together by four main hydrogen bonds, which is an essential action for the formation of nanocomposite scaffold. Using Coasts–Redfern model, we were able to calculate the thermal degradation apparent activation energy and demonstrated that the thermal stability of nanocomposites is dependent on amount of PVP incorporated. Furthermore, SEM images showed that the collagen fibers are wrapped and stabilized on scaffolds by PVP molecules, which improve the ultimate tensile strength (UTS). The UTS of PVP-contained scaffold is four times higher than that of scaffold without PVP, whereas ultimate percentage of elongation (UPE) is decreased, and PVP can enhance the degradation resistance. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Due to its nontoxic and biocompatible properties, collagen has been widely used as a versatile basic material for various applications in biomedical and biotechnological fields. However, major limitations of collagen-based materials, such as poor thermal instability, poor mechanical properties, and possible contamination by pathogenic substances [1], hamper their further applications. Therefore, various crossing linkers, both natural and synthetic ones, such as glutaraldehyde [2], epoxy compounds [3] and EDC (1ethyl-3-(3-dimethyl aminopropyl) carbodiimide) [4] are employed to enhance the stability of collagen-based materials for in vivo applications. Blending modification is an effective, convenient and economical method which has been widely used to improve the properties of collagen-based materials. Blending of collagen with organic or inorganic polymers is done by means of facilitating covalent bond formation between the abundant OH, COOH, NH2 functional groups of collagen and functional groups on polymers and metal ions [5]. Thus, this shows the advantages of using blend-
∗ Corresponding author. E-mail address: [email protected] (K. Tang). http://dx.doi.org/10.1016/j.colsurfb.2015.12.005 0927-7765/© 2015 Elsevier B.V. All rights reserved.
ing modification over cross linking method in enhancing collagen stability on nanocomposites. Polyvinyl pyrrolidone (PVP) has been used as an additive in collagen-based biomaterials because of its excellent water solubility, excellent adsorption and complexing ability, film-forming property, and cohesiveness. PVP is also known to be a good stabilizing agent for transition metal particles. The excellent miscibility of collagen and PVP has been demonstrated previously [6–9]. Collagen–PVP based composite exhibits good tolerance, antiinflammatory and anti-fibrotic properties [10–12]. Collagen–PVP blend can be safely used for biomedical materials because of its non-genotoxicity and diminutive localized hypersensitivity reaction [13]. Excellent biocompatibility of titanium dioxide (TiO2 ) combined with anticancer and antimicrobial property [14] makes TiO2 one of the most promising nanoparticles for biomedical applications. For example, Grassian et al. reported that inhalation exposure of TiO2 in mice, showed minimal inflammatory response and pulmonary toxicity [15]. Li et al. demonstrated that TiO2 nanoparticles can be utilized to shield protein against photo degradation and photo-oxidation [16]. The feasibility of using TiO2 in biological tissue engineering [17–19] and using TiO2 nanoparticles in collagen-based composite to make nanocomposite scaffolds were
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also reported. Sol–gel method is one of the most widely used techniques for preparing nano-sized metallic oxide materials with photocatalytic activities [20], and controlling material’s porosity and shape [21]. However, consumption of reaction medium, such as ethanol and ethylene glycol, and cost are some of the limitations of the sol–gel method. Herein, we improved a three-dimensional nanocomposite material by blending collagen with PVP coated TiO2 particles to obtain collagen-based composite. The goal is to fabricate a potentially simple, but effective skin scaffold for wound healing. Conglomeration of nanoparticles is deemed as one of the main obstacles in the preparation of nanomaterials. To reduce the agglomeration, PVP was added as a coating agent for nano TiO2 particles, which enhances the stability of nanoparticle colloid. The extracted collagen, nano TiO2 particles and resultant freeze-dried ternary composite scaffolds were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), X-ray diffraction (XRD), dynamic light scattering technique (DLS), scanning electron microscope (SEM), attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) and thermogravimetric analysis-differential scanning calorimetry (TGA-DSC). The formation of four hydrogen bonds between collagen, PVP and TiO2 particles in the uniform mixture is based on the changes in particle size and analyzed by ATR-FTIR. The stability of TiO2 nanoparticles, such as thermal stability, swelling properties, mechanical stability and degradation properties, in ternary blending solution were also investigated.
2. Experimental 2.1. Materials Fresh porcine skin was obtained from local wet market. Sodium sulfide (Na2 S), hydrochloric acid (HCl) and acetone were purchased from Tian-li Chemicals Ltd., Tianjin, China. Pepsinum and ethylenediamine tetraacetic acid (EDTA) were provided by Sigma, USA. Calcium hydroxide (Ca(OH)2 ), sodium hydroxide (NaOH), tetrabutyl titanate (TBT), absolute ethanol (EtOH) and glacial acetic acid (HAc) were all purchased from Feng-chuan Chemicals Science and Technology Ltd., Tianjin, China. Polyvinyl pyrrolidone (PVP) K-30 was supplied by Sinopharm, China. Double-distilled water was used as solvent throughout the experiment. 2.2. Extraction of collagen from porcine skin The collagen was extracted as previously reported in the literature [22]. Briefly, depilated porcine skin was minced, and then dipped into a degreasing agent for 16 h and followed by acetone for 16 h. After suspending in ethanoic acid solution with pepsin at 4 ◦ C, the resulting suspension was centrifuged to yield the supernatant liquor. The pH was then adjusted to 8 using NaOH solution. Collagen was then repeatedly purified by salt precipitation, centrifugation and ethanoic acid solubilization. The final collagen solution was dialyzed and stored at 4 ◦ C until use.
Table 1 Mass ratios and names of samples. Scaffold (PVP–TiO2 )1 (PVP–TiO2 )2 (PVP–TiO2 )3 (PVP–TiO2 )4
TBT with a purity of 99% was used as precursor, which was dissolved in the mixture of EtOH and HCl, and was dropped slowly into solution of HCl in distilled water under magnetic stirring for two hours. The mixture was stirred at a constant temperature for another 2 h. The molar ratio of the reactants was TBT:H2 O:EtOH:HCl = 1:200:16:0.8. The mixture gave a light blue transparent TiO2 sol. A portion of as-prepared titanium hydrosol
(Col–TiO2 )
1:0.5
(Col–PVP–TiO2 )1 (Col–PVP–TiO2 )2 (Col–PVP–TiO2 )3 (Col–PVP–TiO2 )4
1:5:0.5 1:10:0.5 1:15:0.5 1:20:0.5
was kept at 4 ◦ C for subsequent use, and a fraction was dried at 120 ◦ C for 12 h, and then grinded. 2.4. Preparation of collagen–PVP–TiO2 ternary nanocomposite scaffold Different amount of PVP was dissolved in 10 ml distilled water separately to obtain PVP solutions with various concentrations. TiO2 was added to above PVP solution in a certain stirring speed at room temperature, and resultant mixtures were named as (PVP–TiO2 )1–4 . To the above mixtures, collagen solution (2 mg/ml) was added and continuously stirred at room temperature for 2 h to give a homogeneous mixture. The gel-like solutions with the same quality were poured onto the equally-sized glass culture dishes. The samples were vacuum freeze-dried at −50 ◦ C, and then corresponding porous nanocomposite scaffold were obtained. The mass ratios and appropriate names are listed in Table 1. 2.5. Characterization SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a Mini-Protean electrophoresis apparatus. The particle size of TiO2 and micelle particles were studied by dynamic light scattering technique (DLS) using a Malvern Zetasizer Nano-ZS90 at 25 ◦ C. The attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) of the extracted collagen and ternary nanocomposites were performed using EQUINX55 in the range of 600–4000 cm−1 . The thermograms were recorded on TGA/DSC 1 synchronized thermal analyzer (METTLER TOLEDO) at the temperature range from 50 to 650 ◦ C with a heating rate 10 ◦ C/min in nitrogen atmosphere. The surface morphology of the collagen and nanocomposites was investigated using the scanning electron microscopy (SEM, FEI Quanta 200, US). 2.6. Swelling property The pre-weighed dry scaffolds were immersed in phosphate buffer saline (PBS) (pH 7.4) solution at room temperature. The weights of swollen composites were measured after the surface excess water was filtered and drained until no free water remained. The procedure was repeated every thirty minutes until the weight was stable. The degree of swelling (DS) for each sample was calculated by the following equation [23]: DS =
2.3. Preparation of TiO2 sol–gel
5:0.5 10:0.5 15: 0.5 20: 0.5
Wt − W0 W0
(1)
where Wt and W0 are the weight of sample at time t and in the dry state, respectively. Each sample was measured in quadruplicate. 2.7. Mechanical property All specimens were tensile-tested at a loading rate of 10 mm/min using an Instron Testing Machine Model 1026, after being rehydrated by immersion in neutral PBS for 0.5 or 1.5 h at
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Fig. 1. SDS-PAGE test of collagen extracted from porcine skin by pepsin. (a) Protein electrophoresis band of extract (lane 1 contains standard protein marker, lane 2 contains porcine skin type I collagen). (b) The relation between lg Mr and relative mobility (MR ) of protein molecular weight marker. (c) Parameters determination of each collagen component. Error bars represent mean ± standard deviation with n-3.
room temperature. Prior to testing, samples were cut and a polymer mesh was incorporated into the sample, for ease of handling as well as providing more accurate measure of the strength. The area between the grips was free of mesh so that only the sample was tested. Five independent measurements were performed for each sample, and the average is reported.
2.8. Hydrolytic degradation Hydrolytic degradation assay at 37.0 ± 0.5 ◦ C and pH of 7.4 was carried out in Simulated Body Fluid (SBF). SBF was prepared by dissolving 7.996 g NaCl, 0.350 g NaHCO3 , 0.224 g KCl, 0.228 g K2 HPO4 ·3H2 O, 0.305 g MgCl2 ·6H2 O, 0.278 g CaCl2 , 0.071 g Na2 SO4 , 40 ml HCl 1 N, and 6.057 g NH2 C(CH2 OH)3 (TRIS) in 1 l distilled water. A certain size of each scaffold sample was immersed in SBF until equilibrium state, then free water was removed as described in Section 2.6 and was weighed as W0 . Each sample was then immersed in SBF and degradation measurement was made every 24 h for a period of 10 days. The measured weight is Wt after eliminating the free water. Each scaffold was assayed in quadruplicate. The degradation was assessed by measuring the wet weight loss which was defined as the following expression:
Wet weight loss% =
Wt − W0 × 100% W0
(2)
3. Results and discussion 3.1. Characterization 3.1.1. Gel electrophoresis patterns and relative molecular mass of collagen Collagen has a triple helix as the basic structure motif and is a major structural protein found in the extracellular matrix [24]. Thus type I collagen is widely used in clinics for skin and tissue repairing purposes. As is the case, we have incorporated this structural protein into our TiO2 nanocomposite scaffold, with the goal of fabricating next stage skin healing scaffold. The purity of the prepared collagen was confirmed by SDS-PAGE, and the collagen concentration in the solution was determined by measuring the hydroxyproline content, according to the method reported by Woessner [25]. The two prominent bands in the electrophoretogram (Fig. 1(a)) with blurred boundary around 116 kDa is assigned to ˛1 -chain and ˛2 -chain [26], which are the major components of type I collagen [27]. The bands around and above 200 kDa belonged to ˇ-chain and -chain, respectively. So, the extracted collagen from porcine skin has its intact triple helical structure. No low molecular weight protein can be seen on the SDS-PAGE gel further indicating that there was no degradation of the pigskin collagen during the extraction process and a high purity of type I collagen has been obtained. Fig. 1(b) is the standard curve obtained by plotting relative mobility (MR ) against log of molecular weight (lg Mr ), from which the linear equation lg Mr =−1.24 MR + 5.36 was derived. The linear regression coefficient (R2 ) for the equation is 1.24, indicating a per-
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Fig. 2. Particle size analysis of TiO2 particle and micelle particle. (a) The list of average particle diameters and PDI in different solutions. (b) The bar chart of average particle diameter.
fect fit. The MR value obtained from SDS-PAGE electrophoresis was inserted into the above equation and the molecular weight of each protein components was calculated (Fig. 1(c)). 3.1.2. Particle size analysis Prior to the preparation of nanocomposite scaffold, i.e., the mixture before freeze drying comprised of water, minuscule amount of ethanol, and PVP, which is non-ionic surfactant that fulfill the basic requirement to form a microemulsion. In the binary liquid system (PVP and TiO2 ) and ternary liquid system (PVP, TiO2 and collagen), micelle particles can be formed with TiO2 as core. The hydrodynamic sizes of TiO2 particles and micelle particles in different mixtures liquid were compared and analyzed. The particle sizes in different solutions and their corresponding polydispersity indexes (PDI) are shown in Fig. 2. All samples have wide range of size distributions and differ from each other based on PDI. As shown in Fig. 2(b), the particle size of prepared TiO2 in the sol is 24.4 nm. For (PVP–TiO2 )1–4 samples, with the increase in the concentration of PVP, the particle size increased significantly and (PVP-TiO2 )4 has a final size of 916.6 nm. The adsorption of PVP onto TiO2 is achieved by the hydrogen bonding between the two reactants through acid–base mechanism, as shown in Scheme 1(a). The adsorption amount of PVP is independent of its molecular weight, but dependent on the concentration, which is in agreement with previous reports [28–30]. In this system, water and ethanol were added during the preparation of titania solution. The driving force for the adsorption of PVP on TiO2 is the interaction between carbonyl groups of pyrene ring in PVP macromolecule and hydroxyl groups on the surface of TiO2 . It can be also
observed from Fig. 2(a) that PVP can facilitate the concentrated distribution of particle size which is indicated by lower PDI. However, in the case of (Col–PVP–TiO2 )1–4 solution, compared with (PVP–TiO2 ), particle sizes are not markedly increased by increasing the amount of PVP. The incorporation of type I collagen into the solution mixture may have decreased the PVP adsorption onto TiO2 surface, which results in no significant change to particle sizes. The competing adsorption between collagen–PVP and TiO2 –PVP is another plausible explanation to the reduced particle size. In addition to the PVP–TiO2 hydrogen bond as seen with (PVP–TiO2 ) only solution, three other hydrogen bonds exist in the (Col–PVP–TiO2 ) solution. They are formed between collagen and PVP, collagen and collagen [6–8], and collagen and TiO2 [31,32]. These hydrogen-bondings have been reported and are shown in Scheme 1(b and c). Hydrogen-bonding between collagen and PVP could be studied by considering the interaction between proton-accepting carbonyl moiety in pyrrolidone rings of PVP and amino/hydroxyl side groups on collagen. Collagen–PVP shows stronger hydrogen interactions compared to both PVP–PVP and collagen–collagen, which can be proved by increased mutual miscibility. Due to the stronger hydrogen bonding formed between collagen to PVP than PVP to TiO2, the PVP adsorption amount onto the surface of TiO2 was decreased. Thus, it can be why the particle size of (Col–PVP–TiO2 ) solutions is smaller than that of (PVP–TiO2 ). It is noteworthy that the particle size obtained from (Col–TiO2 ) solution is bigger than that of TiO2 solution alone. This is a result of the enhanced adsorption of collagen onto TiO2 owing to the coordination ability of carboxylic and hydroxyl group with metal ions.
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Scheme 1. Hydrogen bonds of TiO2 –PVP (a), collagen–PVP (b), collagen–collagen (c) and collagen–TiO2 (d).
These carboxylic and hydroxyl groups probably act as the nucleation site for nanoparticle formation [33]. 3.1.3. Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) analysis Infrared spectroscopy can be widely used to monitor protein structure in films and solution. In the FTIR spectrum of the extracted pigskin collagen (Fig. 3), 3315 cm−1 and 3086 cm−1 are assigned to N H stretching vibration for amide band A and amide band B of collagen, respectively. The peak at 1652 cm−1 is due to C O stretching vibration in amide I bands, and the band in the region of 1557 cm−1 is assigned to N H bending vibration of amide II [34]. The FTIR peaks at 1402 cm−1 and 1239 cm−1 are due to COO symmetrical stretching and C N stretching of amide III, respectively [35]. The characteristic peak at 1454 cm−1 is due to pyrrolidine block for proline and hydroxyproline, which shows a complete triple helical structure of the extracted collagen [36]. The absorption intensity ratio of 1239 cm−1 to 1454 cm−1 is 1.02, which indicates that the secondary structure of collagen is undamaged [37,38]. The extent of intermolecular hydrogen bonding can be studied by determining the frequency shifts. Collagen shows a well-defined peak with maxima at 3315 cm−1 and a weak shoulder at 3423 cm−1 , due to H-bonded N H and H-bonded free N H, respectively. It can
Fig. 3. ART-FTIR spectrum of (a) extracted collagen, (b) scaffold (Col–TiO2 ) and (c) scaffold (Col–PVP–TiO2 )4 after freeze drying.
be observed that, compared to spectra of collagen, N H peak of scaffold (Col–PVP–TiO2 )4 has shifted from 3315 cm−1 to 3408 cm−1 , and peak is broadened. These indicate a very strong hydrogen bonding in this compound, which is as expected. Similarly, the scaffold (Col–TiO2 ) also shows one broad peak with peak maximum at 3316 cm−1 indicating the presence of hydrogen bonding of N H in this composite. However, the shift is much less than that of scaffold (Col–PVP–TiO2 )4 . This may be due to two different types of hydrogen bonding energies in these two scaffolds, namely N H· · ·O C for scaffold (Col–PVP–TiO2 )4 and N H· · ·O for (Col–TiO2 ). This change in hydrogen bonding energies can be understood that the interaction between collagen and PVP via hydrogen bond is stronger than that between collagen and TiO2 . For amide band B and amide band II, the peaks of (Col–TiO2 ) show somewhat shifts of 3 cm−1 and 9 cm−1 to low frequency separately, whereas these peaks of (Col–PVP–TiO2 )4 disappear and may be because of the peaks overlapping when amide band peaks shift to lower frequency caused by strong hydrogen bonding. In the FTIR spectra the shifts of the bands derived from groups taking part in the hydrogen bonds suggest that the interactions between collagen and PVP occur by hydrogen bonds [8], which is quite limited between collagen and TiO2 . It is obvious that the spectrum of (Col–PVP–TiO2 )4 is totally dominated by the spectrum of collagen. But it is also noteworthy that, probably because of the formation of hydrogen bonding between the N H and the oxygen atom on TiO2 surface, the peak at 1557 cm−1 shifted to 1548 cm−1 and the absorption intensity is weakened. 3.1.4. Thermal stability The thermal stability of nanocomposite scaffold was investigated by means of TGA and DSC. Fig. 4 shows that when more stable moiety, like PVP, is used, the thermal stability of the nanocomposite scaffold is improved. Three main temperature regions of weight loss appear in all TGA curves (Fig. 4(a)). Initial weight loss below 200 ◦ C can be attributed to the evaporation of physically absorbed water, or residual solvent in the sample. Corresponding smaller endothermic peak in DSC curve (Fig. 4(b)) represents the unfolding of triple helical structure of collagen. The significant weight loss observed between 200 and 500 ◦ C can be due to thermal decomposition of collagen and PVP [8,39], TiO2 OH [40] and the fracture of intermolecular hydrogen bonds as mentioned in Section 3.1.2. The subsequent endothermic transitions around 425 ◦ C can be observed in DSC. The pyrolysis onset temperature of (Col–PVP–TiO2 )1–4 nanocomposite slightly decreases with
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Fig. 4. (a) TGA and (b) DSC curves of (Col–TiO2 ) and (Col–PVP–TiO2 )1–4 nanocomposite scaffolds. (c) Linear plots of ln [G(ɑ)/T2 ] versus 1/T for nanocomposite scaffolds. (b) Thermal degradation apparent activation energy of nanocomposite scaffolds.
decreasing the PVP content, while its melting temperature slightly increases oppositely. It was found that the weight loss above 500 ◦ C can be neglected as only TiO2 remained. The obvious exothermic peak around 620 ◦ C in DSC curves might be due to the change of TiO2 crystal structure from anatase to rutile [41,42]. Similar TGA and DSC curves for the (Col–PVP–TiO2 ) samples suggest that increasing PVP concentration did not have a significant impact on thermal stability. However, the (Col–TiO2 ) sample has a much lower thermal stability as the scaffolds degrades at much earlier temperature range and at a faster rate. The corresponding endothermic peak around 325 ◦ C in DSC curve indicated that PVP is high in thermal stability. Thus the thermal stability of collagen-based nanocomposite scaffold was improved. The apparent activation energy is normally used to quantitatively characterize the thermal stability of scaffold. Coats-Redfern method has been widely accepted in calculating the apparent activation energy. Coats-Redfern integral formula is usually described as [43]: ln
G(˛) AR E = ln( )− RT ˇE T2
(3)
The meaning of the symbols is shown in Supporting information Table S1. This integral method lead to –Ea /R from the slope of the line as determined by plotting ln[G(˛)/T2 ] against 1/T at any certain conversion rate.
From both the TGA and DSC analyzes, temperature ranging from 200 ◦ C to 500 ◦ C has been shown as the main thermal decomposition factor of more than 70% of the polymer composite, and the decomposition conversion thereafter is less meaningful due to too high temperature and tiny sample weight-loss. Thus, the emphasis was placed on a conversion range from 0.4 to 0.9, instead of the entire process. This would offer a simplified and more meaningful way to modeling thermal decomposition behavior of nanocomposite scaffolds. Because G(˛) has thirty integral forms, the function G(˛) = −ln (1 − ˛) was most reasonable for this system and is used for comparing and evaluating the linear fitting. Fig. 4(c) shows the linear plots of ln [G(˛)/T2 ] versus 1/T˛ for both (Col–TiO2 ) and (Col–PVP–TiO2 )1–4 scaffolds. Fig. 4(d) shows the thermal degradation apparent activation energy values for certain conversion rates. As observed from the two figures, the thermal degradation apparent activation energies of scaffolds (Col–PVP–TiO2 )1–4 are higher than that of scaffold without PVP (scaffold (Col–TiO2 )). The activation energy increases with increasing the PVP content. That is suggesting that the thermo-stability for nanocomposite increases with increasing PVP content in the study, which further supports the aforementioned results. 3.1.5. Scanning electron microscopy (SEM) analysis The types of matrix used, or more precisely the exact structures of the matrix, directly affect properties of polymer products, including swelling behavior, mechanical property, cell growth and tissue formation. The surface morphologies of collagen (Fig. 5a),
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Fig. 5. SEM images of collagen (a), scaffold (Col–TiO2 ) (b) and (Col–PVP–TiO2 )4 (c), EBSD image of scaffold (Col–PVP–TiO2 )4 with EDAX.
(Col–TiO2 ) (Fig. 5b), and (Col–PVP–TiO2 )4 (Fig. 5c) were observed and compared using scanning electron microscopy (SEM). As shown in Fig. 5a (1), pure collagen after freeze drying presents with a combination of fiber and sheet morphology, with the majority of it being sheet formation. Fracture in the sheet is not due to an integrated plane, as wrinkles and grooves exist on the sheet surface. Normal collagen fibers have smooth surface but do intertwine with one another, as this phenomenon is seen in Fig. 5a (2). With the addition of nano-TiO2 particles (Fig. 5b), instead of dense sheet layer, discontinuous flakes form a tri-dimensional fluffy structure and gives a more porous nature than pure collagen. The exposed fibers become finer in structure and exhibit a more independent nature to each other. Lastly, from the SEM analysis, scaffold (Col–PVP–TiO2 )4 , stand in marked contrast to pure collagen and scaffold (Col–TiO2 ). The collagen fibers from (Col–PVP–TiO2 )4 are cemented together by PVP to achieve good cohesiveness. This leads to fewer exposed fibers on surface and affects the porous structure of original collagen. Furthermore, because of the good film-forming property of PVP, there are less flakes in the ternary system and more continuous interface than scaffold (Col–TiO2 ), along with some wrinkles and less grooves (Fig. 5c(1)). It should also be noted that, due to the enwrapping with PVP, the collagen fibers become larger in diameter and the scaffold surface become less smooth (Fig. 5c(2)). Some TiO2 particles with large diameter, which attach to the surface of the enwrapped fiber can also be detected. In a higher magnification Fig. 5c(3), irregularly-shaped TiO2 can be observed on the sheet surface of the (Col–PVP–TiO2 )4 scaffold,
which reveals that there is possible agglomerative phenomenon accompanying wrapping for nanoparticles. Fig. 5c(4) is the scanning back scattering image of the same area of Fig. 5c(3), and the EDAX spectrum recorded in the spot profile mode form one of the bright regions with the binding energy region of 0–20 keV is shown in Fig. 5c(5). The peak from the spectrum revealed the presence of Ti and O at 4.5 and 0.5 keV, respectively. The combination of Fig. 5c(4) and c(3) indicate the crystal phase uniform distribution of TiO2 in organic phase.
3.2. Swelling property Water adsorption of biomaterial is important for cell uptake, as it affects the distribution of cell suspension throughout the material and the transfer efficiency of oxygen and nutrient [44]. Thus, a faster rate can promote faster absorption of wound surface effusion. The swelling property of ternary nanocomposite scaffolds in PBS (pH 7.4) is therefore studied and is shown in Fig. 6(a) and (b). All scaffold samples reached equilibrium after being soaked in PBS for 1.5 h. The equilibrium water absorbency in PBS is in the order (Col–TiO2 ) > (Col–PVP–TiO2 )1 > (Col–PVP–TiO2 )2 > (Col–PVP–TiO2 )3 > (Col–PVP–TiO2 )4 . Some parameters may affect the swelling ratio, such as hydrophilicity, stiffness and porous structure of the matrix. The carboxyl group on collagen chain is an anionic hydrophilic group which can be ionized. Thus the space for absorbing and holding water increased due to anion–anion electrostatic repulsion [45]. Furthermore, in this ternary system, three-dimensional network of the scaffold was the
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Fig. 6. The equilibrium swelling degree (a) and time-dependent changes in DS (b) of prepared scaffolds in PBS. (c–d) Tensile properties of nanocomposite scaffolds with different PVP contents. (e) Wet weight loss of scaffolds vs. degradation time in SBF. Error bars represent mean ± standard deviation (n-4 for (a, b, e); n-5 for (c, d)).
result of interaction of collagen, PVP and TiO2 . The collagen fibers adhered to each other by the effect of PVP, and its individual pore structure was destroyed. Besides, with increasing the PVP content, the decrease in collagen relative content resulted in decreased numbers of carboxyl group with strong hydrophilicity, which is the reason why (Col–PVP–TiO2 )4 had the weakest absorbency. It is interesting to note that PVP is non-ionic polymer, and therefore electrostatic repulsion of polymeric chains is small. Hence, the DS
of scaffold (Col–TiO2 ) without PVP is the biggest, and it decreases with increasing PVP content. 3.3. Mechanical property Scaffold biomaterials are supposed to support desired cellular, process or can develop appropriate mechanical characteristics. This scaffold is designed as skin scaffold for wound healing and sup-
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posed to use under moist condition. All scaffold samples can reach equilibrium soon as shown in the swelling study. Therefore, the results of UTS and UPE depending on percentage of PVP and scaffold soaking time in PBS are graphically represented in Fig. 6c and d, respectively. As shown in Fig. 6c and d, the plasticizing action of water was obvious in all cases, independently of scaffold type. The wet strength reduced slightly when soaking time is extended from 0.5 h to 1.5 h, and scaffold with more PVP had a larger decrease. However, the ultimate elongation increased to varying degree with the increase of soaking time. Water is known to be an effective plasticizer for most biopolymers [46] and its plasticizing action is reflected in lowering of the fracture strength, elastic modulus and increasing of flexibility of the material [47]. So, the addition of more water as the plasticizer can decrease intermolecular forces that permit smaller UTS but greater UPE of the scaffold before break. Moreover, PVP could leach out more with a longer soaking time, which may cause unconsolidated structure, and as a result the strength of scaffolds reduced. For UTS, scaffolds after a soaking time of 0.5 h showed a increasing tendency from (Col–TiO2 ) to (Col–PVP–TiO2 )4 . Scaffolds with PVP exhibited higher values of ultimate tensile strength (1491 ± 20 kPa–945 ± 16 kPa), which is about 3–5 times higher than that of scaffold without PVP (304 ± 15 kPa). It suggests that (Col–TiO2 ) have lower wet strength, and the increase of UTS with PVP incorporated into the scaffolds can be due to cementation of collagen fiber with PVP increasing the joining force among collagen fibers and the H-bond between different molecules. However, the change of elongation property (Fig. 6d) with increasing PVP content did not follow the same pattern as the UTS. It showed lower values of UPE for higher concentration of PVP in the scaffold. So it is indicating that removal of the cohesive action of PVP on collagen fibers enhanced the tensile strength of scaffolds but decreased their distensibility. It should be noted that, scaffold (Col–TiO2 ) with soaking time 0.5 h showed slightly lower UPE than (Col–PVP–TiO2 )4 . Thus it can be interpreted that excess PVP can cause compact structure with poor elongation and appropriate amount of PVP can facilitate the structural continuity, which contributes to the elongation.
3.4. Hydrolytic degradation behavior The analysis of wet weight loss of scaffold in SBF (Fig. 6(e)) could reflect the degradation condition in vitro. In the early stage of analysis, it was the degradation of small molecules. It is interesting to note that during the early stage, scaffold (Col–PVP–TiO2 ) with higher PVP content has the largest wet weight loss percentage. However, after the fourth day, the degradation rates for (Col–PVP–TiO2 )4 declined and the ternary system with the lowest PVP content has the largest final overall wet weight loss. This may ascribe to the multi-sites binding of collagen, PVP and TiO2 , such as H-bond between PVP, collagen and TiO2 . This indicates indirectly the hydrogen bond can protect collagen from the quick decomposition. By the eighth day, fine cracks appeared on all four samples, but were slightly smaller and less in number in the scaffold with the higher concentration of PVP. On the tenth day, all four scaffolds fragmented. Scaffold (Col–TiO2 ) always degraded faster in SBF with cracks appearing on the fourth day of analysis, and it fragmented into pieces on the sixth day. By the eighth day, the material disintegrated completely. Therefore, the addition of PVP may increase the binding of different molecules, and then effectively maintain the integrate structure and slow the degradation of collagen-based scaffold.
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4. Conclusions A new nanocomposite scaffold composing of type I collagen, PVP and nanoparticle TiO2 in anatase form is fabricated to serve as an appropriate skin scaffold for wound healing. Based on existing experimental data, we interpret there are four main hydrogen bonds formed between collagen, PVP and titanium dioxide. It is inferred that the interaction between collagen and PVP, PVP and TiO2 are stronger than others, and this hydrogen bonding plays an important role in determining the overall properties and performance. The nanocomposites exhibit adequate mechanical properties to serve as a promising wound healing dressing. The addition of PVP can form strong interaction in this ternary system by increasing the tensile strength and improving the resistance of scaffolds in SBF, while decreasing the degree of swelling in PBS and the percentage of elongation of collagen-based scaffold. However, further studies are needed to address the nanocomposites efficacy.
Acknowledgements We would like to thank Dr. Xiaoqiang Yang, Benjamin Fook Lun Lai and Dr. Bo Deng of the Centre for Blood Research (CBR) at the University of British Columbia at Vancouver for good advices to this paper. This research was funded by the National Natural Science Foundation of China (No. 51373158).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.12. 005.
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