Research Article pubs.acs.org/journal/ascecg
Preparation and Characterization of 2,2,6,6-Tetramethylpiperidine-1oxyl (TEMPO)-Oxidized Cellulose Nanocrystal/Alginate Biodegradable Composite Dressing for Hemostasis Applications Feng Cheng,† Changyu Liu,† Xinjing Wei,† Tingsheng Yan,† Hongbin Li,‡ Jinmei He,*,† and Yudong Huang† †
MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, People’s Republic of China ‡ School of Light Industry and Textile, Qiqihar University, Qiqihar 161000, People’s Republic of China S Supporting Information *
ABSTRACT: Hemorrhage is common in surgery, and excessive bleeding is the main reason for trauma death. Effective control of bleeding is becoming more and more important in military and civilian trauma. In this work, oxidized cellulose nanocrystal/ alginate composite films and sponges were successfully prepared and their usages as the hemostatic materials were investigated. Carboxyl functionalization on the cellulose nanocrystal surface not only played a fundamental role in the structural of composites, but also contributed to absorb plasma and stimulate erythrocytes and platelets. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) spectra showed that the carboxyl groups were successfully introduced on the cellulose nanocrystal surface by TEMPO-mediated oxidization. The oxidized cellulose nanocrystals (TOCN)/alginate (SA) composites were in the presence of Ca2+ solution cross-linking. Physical properties tests results indicated that the ultrahigh porosity (sponge), surface homogeneity (film), water absorption ability, and chemical stability of TOCN-30/SA composite sponge, as well as TOCN-30/SA composite film, were all increased after ionic cross-linking, compared to the SA sponge and film, respectively. In vitro evaluation of the hemostatic effect, hemostatic time, and the blood loss in two injury models exhibited that TOCN-30/SA composite sponge had the most excellent hemostatic efficiency and could be biodegraded completely without inflammatory reaction after three weeks. In addition, the potential hemostatic mechanism of TOCN/SA composites was discussed. KEYWORDS: Cellulose nanocrystals, Oxidation, Alginate, Cross-linked sponge, Cross-linked film, Hemostatic, Biodegradability
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INTRODUCTION
As a type of natural anionic polymer, alginate obtained from brown seaweed has been widely studied and used in a variety of biomedical applications,8 such as wound dressing9 and stable gels,10 because of its good biocompatibility and low cytoxicity. Especially, the calcium-induced gels with “egg-box” structure have been proved to be formed under the cation interaction between Ca2+ and guluronate blocks in alginate.11 Alginate dressings with excellent hemostasis efficiency can absorb large volumes of wound exudate and provide a physiologically moist microenvironment for wound healing. Besides that, it can be easily removed from the wound site.12,13 However, the poor chemical stability, weak mechanical strength, and the uncontrollable structure degradation of neat alginate film or sponge limited their further application of neat alginate film or sponge.14,15 Cellulose consisted of β-1−4-linked D-anhydroglucose units as the most abundant renewable biopolymer composed and
Excessive hemorrhage is the main cause of prehospital trauma death during both military and civilian trauma, and effective hemostatic materials can quickly prevent bleeding and thus reduce the mortality.1,2 In recent years, numerous materials have been widely developed to promote rapid bleeding control,3 such as oxidized regenerated cellulose (ORC, Surgicel), 4 HemCon chitosan-based dressing,5 and the QuikClot zeolite powder.6 All of them have their own advantages and limitations. For instance, the ORC materials, which is implanted into the patient’s body, will damage the nervous system when the carboxyl content is within the range of 16%−24% and pH value is ∼3.1.7 Also, HemCon dressings prepared as gauzes are difficult to conform to deep, narrow wounds or irregularly shaped wounds.2 In addition, the study of QuikClot agents indicated that the exothermic reaction and poor biodegradability of QuikClot can even cause tissue injuries and abnormal reaction to foreign bodies.1 Thus, the challenge now is developing more alternative effective hemostatic materials to control hemorrhage. © 2017 American Chemical Society
Received: November 24, 2016 Revised: March 20, 2017 Published: March 24, 2017 3819
DOI: 10.1021/acssuschemeng.6b02849 ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
Research Article
ACS Sustainable Chemistry & Engineering
min each step (washed repeated more than three times), and then dialyzed with distilled water for ∼3−5 days until a neutral pH environment, followed by ultrasonic treatment. Finally, the CN power was obtained after freeze drying. TEMPO-Mediated Oxidation of Cellulose Nanocrystals (TOCN). TEMPO-mediated oxidation of CN was followed by using the method described in the literature.25,29−31 Briefly, ∼0.5 g CN was suspended in 50 mL of distilled water, followed by ultrasonic dispersion treatment for 15 min. TEMPO (50 mg, 0.32 mmol) and NaBr (500 mg, 4.86 mmol) were also dissolved in 50 mL of distilled water and the solution were added dropwise slowly into the CN dispersion. A certain quantity of 12 wt % NaClO (15 mL, 46.5 M) solution was then added slowly into the mixed solution for the oxidizing reaction; meanwhile, the pH value of the mixture remained at 10.8 by adding 0.5 M NaOH. The oxidation reaction was terminated by adding ethanol (1 mL). Meanwhile, the pH was adjusted to 7 with 0.5 M HCl. Finally, the aqueous dispersion of oxidized CN (TOCN) was washed thoroughly with distilled water more than three times and then freeze-dried to obtain TOCN powders. Preparation of Cellulose Nanocrystal/Alginate Cross-Linked Composite Sponges (Films). TOCN was introduced in a certain amount of alginate solution for the preparation of cross-linked sponges and films. The detailed procedure was illustrated in Figure 1. TOCN
almost inexhaustible raw material has been used in a wide variety of applications,16,17 such as food, paper, and medicine. Especially, cellulose nanocrystal (CN) with nanoscale features, high specific surface area, unique morphology, low density, mechanical strength, renewability, and biodegradability,18−21 has attracted a great deal of interest during the past decade. Furthermore, abundant active hydroxyl groups on the CN surface are suitable for chemical modification, such as oxidation and polymer grafting.22 For instance, a recent study reported that the presence of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanofibers can regulate the post-prandial blood metabolic variables and showed promising hemocompatibility and unique biological activities.23 TEMPOmediated oxidized bacterial cellulose-sodium alginate composites have also been prepared as a type of biomedical material for cell encapsulation, in which the TEMPO-mediated oxidized bacterial cellulose improved the mechanical stability, could exhibit good chemical stability of the composites, and would be a potential candidate for many biomedical applications.24 For the above-mentioned reasons, a type of cellulose nanocrystal/alginate composites was designed based on the cross-linking by external gelation in CaCl2 ethanol/water solution as a co-solvent. In addition, as the C6 primary hydroxyls made from TEMPO-oxidized materials converted to carboxyl groups on the cellulose surface, oxidized cellulose nanocrystal (TOCN) provided the possibility of participating in the construction of the cross-linking network from alginatebased composites and plays an important role in the structural, mechanical, and chemical stability of the composites.25 So far, there was no specific report referred to the hemostatic properties and biological degradable performance of TEMPOmediated oxidized cellulose nanocrystal (TOCN)/alginate composites. In this study, the morphology, chemical and physical properties, hemostatic efficiency of TOCN/SA composites both in vitro and in vivo, and the degradation in vivo were studied. It is expected that this study is useful for understanding the possible hemostatic mechanism of TOCN/ SA composites and designing an effective hemostatic material for the wound healing.
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MATERIALS
Microcrystalline cellulose (99%) was purchased from Shanghai Luan Biological Technology Co., Ltd. (China). Sodium alginate was provided by Sinopharm Chemical Reagent Co., Ltd. (China). Sodium hypochlorite (NaClO) solution was purchased from Shuang Shuang Chemical Co., Ltd. (Yantai, China). TEMPO (C9H18ON, 98%), sodium bromide (NaBr), and calcium chloride (CaCl2) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). All of the reagents were of analytical grade and used without further purification. Healthy rabbits and human blood were supplied by animal experiment center of the second affiliated hospital of Harbin medical university (Harbin, Heilongjiang Province, China). The protocol was approved by the ethics committee of the Harbin Medical University. All animals were handled according to the Chinese National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Preparation of Cellulose Nanocrystals (CNs). Procedure for the preparation of CN was the same as those described previously.25−28 Briefly, acid hydrolysis was prepared at 35 °C with 64 wt % H2SO4 (225 mL) for 2 h under vigorous stirring and then MCC powder (10 g) was slowly added into the suspension. The hydrolysis was terminated by adding a large amount of distilled water (more than 10 times the volume of the H2SO4 solution used). Subsequently, the mixture was placed overnight at 4 °C and the supernatant was discarded. After that, the system was centrifuged (10 000 rpm) for 10
Figure 1. Scheme of preparation and application for TOCN/SA composite sponge (film). solution (3 wt %) was prepared by adding the TOCN powder in distilled water under vigorous stirring for 30 min under room temperature. Similarly, sodium alginate (SA) was dissolved in distilled water and stirred for 2 h to get a 3 wt % SA solution. Then, TOCN solution was dropwise added into the prepared SA solution, followed by vigorous stirring for another 3 h. The TOCN/SA composite suspension was then cast in Petri dish plates, and a portion of them was freeze-dried and another portion was vacuum-dried. Cross-linked TOCN/SA composite sponges (films) were prepared by immersion in a CaCl2/H2O/C2H5OH (1.5 g/80 mL/20 mL) solution for 1 h.32 In addition, the TOCN/SA composites were washed with distilled water to remove residual Ca2+ ions and freeze-dried again to obtain the cross-linked sponges (dried cross-linked film at 25 °C). The obtained composite sponges and films were stored in a desiccator for more than 48 h before their use. The weight ratios and contents of TOCN/SA cross-linked samples are shown in Table S1 in the Supporting Information. Characterization. Fourier Transform Infrared (FT-IR) Analysis. Fourier transform infrared (FT-IR) spectra were used to confirm the characteristics of CN powders and TOCN/SA samples. FT-IR was 3820
DOI: 10.1021/acssuschemeng.6b02849 ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
Research Article
ACS Sustainable Chemistry & Engineering measured using the Nicolet−Nexus 670 spectrometer at room temperature. All the powder samples were recorded at the range of 4000−500 cm−1, with a resolution of 2 cm−1. X-ray Photoelectron Spectroscopy (XPS) Analysis. The elemental composition of unmodified and oxidized CNs surface was obtained from the X-ray photoelectron spectroscopy (XPS) under the ultrahigh vacuum conditions. The device was equipped with a VG Scientific ESCALAB system (220i-XLT, UK) and an X-ray source (Al Kα). X-ray Diffraction Analysis. Crystallinity of unmodified and oxidized cellulose nanocrystal was measured by X-ray diffraction (XRD) method using an XRD-6000X diffractometer with Cu Kα X-radiation. The XRD patterns of the samples were recorded in the 2θ range of 0°−40°. Morphology Analysis. Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) analysis on the TOCN/SA sponges were carried out using Quanta 200 FEG scanning electron microscope (FEI, Hong Kong) equipped with an EDS detector. The morphologies of CN and TOCN were examined via transmission electron microscopy (TEM) analysis (Model JEM-F 200, Japan) at an accelerating voltage of 80 kV. Swelling Degree of TOCN/SA Composite Sponges (Films). The degree of swelling of the sponges and films was measured gravimetrically. The sponge and film sample was cut into dimensions of 3.0 cm × 3.0 cm and immersed in the phosphate buffered saline (PBS) (pH 7.2−7.4, 37 °C) for 30 min. Then, the residual PBS was removed from the wet sample surface with filter paper and the obtained sample was immediately weighted (Wd). The swelling degree (Sd) was calculated using the following equation:33 Sd (%) =
Ww − Wd × 100 Wd
H (%) =
W2 − W1 × 100 ρV0
(3)
where Dt is the absorbance of test sample, and Dnc and Dpc are the absorbance of negative and positive control, respectively. An average value of three replicates for each test sample was determined. Cytocompatibility Assay. The sterilized materials were cut into several 1.0 mm × 1.0 mm × 1.0 mm cubes, and immersed in PBS buffer solution (5, 2.5, 0.5, and 0.1 mg mL−1) for 72 h in an air-tight glass container at 37 °C. After draining the fluid, the supernatant was sterilized for 30 min under 253.7 nm UV light. The group without any material was set as a control. Single-cell suspensions of 5000 Hela cells in 100 μL DMEM medium containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin−streptomycin (Invitrogen), were added in a 96-well plate and incubated for 48 h at 37 °C under 5% CO2. Then, 10 μL material extraction supernatant was added and incubated for another 48 h and the cell-seeded wells were washed twice with PBS to remove unattached cells. Then, 100 μL of DMEM was added and the cells were cultured with a cell counting kit-8 (CCK8) for 2 h at 37 °C. Finally, the 96-well plates were placed in a microplate reader to measure absorbance at 450 nm. The relative growth rate (RGR) of cells cultured in each group was measured to calculate the cell viability, according to the following formula:
RGR (%) =
Abs450 test × 100 Abs450 control
(4)
All results were estimated from six individual experiments and are expressed as the mean ± the standard deviation (SD). Blood Cells and Platelet Adhesion. The blood cells and platelet adhesion were conducted as described in the reported literature.34,35 In brief, for whole blood cell and platelet adhesion determination, the TOCN/SA composite sample was cut into 1 cm × 1 cm dimensions, followed by immersion into PBS (pH = 7.2−7.4) for 1 h at 37 °C. Subsequently, the whole blood was added dropwise into the sample and then incubated for 5 min at 37 °C. Platelet-rich plasma (PRP) was separated from the whole blood by centrifugation of blood at 800 rpm for 10 min. The PRP was then added dropwise into the sample and incubated for 1 h at 37 °C. All samples were then washed with PBS solution three times to remove the physical adhered blood cells and platelets, and then fixed by 2.5% glutaraldehyde for another 2 h. After that, blood cells and platelets were dehydrated with 50%, 60%, 70%, 80%, 90%, and 100% ethanol solution, with the interval for 10 min. Finally, the TOCN/SA samples were obtained after drying and SEM images were taken. Hemostatic Evaluation. Rabbit Liver Trauma Model. The hemostatic behavior of the TOCN/SA composite sponges and films was estimated by covering them on the abraded livers of male New Zealand White rabbits (4 months old and ∼3.5 kg). The composite samples were cut into pieces of required size (2.0 cm × 2.0 cm) and sterilized by the ultraviolet radiation for evaluating the hemostatic efficiency. Before undergoing an abdominal incision, the rabbits were fixed on the surgical cork board and then the ear marginal veins were injected with 3% pentobarbital sodium aqueous solution (30 mg/kg) to anaesthetize them. The composite samples were applied to the liver wound immediately when the liver was pricked with a needle (the diameter is 2 mm, and the pricked depth is 3 mm), respectively. The hemostatic evaluation was detected every 30 s, then the hemostatic time and blood loss was recorded accordingly. Rabbit Ear Artery Model. After the anesthesia of ear marginal veins through the injection of a pentobarbital sodium solution, the auricular artery of the rabbit in the middle of rabbit ear was prepared and sterilized, and the blood vessels were torn by the scalpel blade. The composite samples then were covered on the wound, and the hemostatic time and blood loss were recorded accordingly. In Vivo Degradation Behavior. The male New Zealand White rabbits (4 months old and ∼3.5 kg) were selected for the degradation test in vivo. Rabbits were randomly divided into a treatment group and a control group. All composite samples were cut into pieces of required size (1.0 cm × 1.0 cm) and sterilized by the ultraviolet radiation for testing the degradation behavior. After the anesthesia of
(1)
where Ww and Wd represents the wet and dry weight of the sample, respectively. An average value of five replicates for each sponge sample was determined. Porosity of TOCN/SA Composite Sponge. The porosity of the sponge sample was determined by the reported method.33 Briefly, the sponge samples were immersed in a certain amount of ethanol for 30 min. Subsequently, the sponge samples were weighed before and after immersion in alcohol. The porosity (P) was calculated with the following equation:
P (%) =
Dt − Dnc × 100 Dpc − Dnc
(2)
where W1 and W2 represent the weight of sponge before and after immersion in alcohol, respectively. V0 is the sponge volume and ρ is the density of ethanol (0.785 g/cm3). Five samples were measured for each type of TOCN/SA composite. Measurement of Mechanical Properties. The materials were cut into pieces with dimensions of 25 mm × 10 mm. The tensile strength was examined at a speed of 2 mm/min and recorded with an electronic universal testing machine (CMT, No. 03000227, New Think Materials Testing Co., Ltd., Shenzhen, China). At least 10 replicates were run for each sample. Hemolysis Assay In Vitro. The hemolytic study was performed on composite sponges and films extract in vitro. Sterile normal saline (NS) (20 mL) was added into the test tube which contained 25 mg sponges and films samples with incubation at 37 °C for 72 h. Hemolytic test was determined by rabbit erythrocyte suspension in vitro. In brief, 2.5 mL of sample extract solution, TOCN (1 mg/mL), TOCN (4 mg/ mL), SA (4 mg/mL) and SA (6 mg/mL) solutions were added into 2.5 mL of 2% (v/v) rabbit erythrocyte (diluted with NS) suspension incubated at 37 °C for 3h, respectively. Meanwhile, distilled water and NS was used as the positive and negative control, respectively. Whereafter, the mixture erythrocyte suspensions were separated at 1500 rpm for 10 min and determined the hemolytic rate with the UV/ vis spectrophotometer at 540 nm. The hemolytic rate (H) was calculated with the following equation:33 3821
DOI: 10.1021/acssuschemeng.6b02849 ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
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ACS Sustainable Chemistry & Engineering ear marginal veins through the injection of sodium pentobarbital, part of the operation area was sterilized, and then the sample was implanted across the ham muscle. Three rabbits from each group were examined at 7 d, 14 d, and 21 d, respectively. All the animals were carefully nurtured until the terminals were implanted. Finally, the implants and surrounding tissues were carefully removed, fixed in 10% formaldehyde solution, embedded in paraffin, and then sectioned and stained with hematoxylin and eosin (H&E). Statistical Analysis. For graphs and texts, values were expressed as mean ± standard deviation (SD). For the comparison of two groups, a Student’s t-test was used to measure the statistical significance. The probability values of p < 0.05 (using one-way analysis of variance (ANOVA) on SPSS) were considered to be statistically significant.
appeared. Moreover, as shown in the spectrum of TOCNCOOH, the peak located at 1735 cm−1 was the characteristic peak of free carboxyl groups (acidic form-COOH), indicating that the carboxylate-COONa groups in TOCN-COONa were successfully converted to free carboxyl groups (acid-COOH) via HCl treatment on oxidized CN. Therefore, the acid treatment was helpful for eliminating the interference with the absorbed water band (1640 cm−1). The XPS wide scan spectra are shown in Figure 2b; these spectra further demonstrate that the compositions on the surface of CN and TOCN also contribute to the O 1s spectrum and C 1s spectrum to some extent. Table 1 shows the XPS
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RESULTS AND DISCUSSION TEMPO-Mediated Oxidation of Cellulose. The FT-IR spectra of the unmodified MCC, CN, TEMPO-treated TOCNCOONa, and acidic form TOCN-COOH are presented in Figure 2a. The peak at 3410 cm−1 was attributed to the
Table 1. XPS Analysis of CN and TOCN Peak (eV)
Concentration (atomic %)
sample
C
O
C
O
CN TOCN
284.8 285.9
531.9 532.1
75.69 62.11
24.31 37.16
semiquantified elemental concentration for CN and TOCN. It can be found the relative amount of carboxyl groups increased after cellulose nanocrystal oxidation. With the TEMPO modification on CN, the oxygen content was increased from 24.31% to 37.16%. Therefore, XPS data indicate that TEMPO modification is a selectivity oxidation reaction that occurs on C6 with the appearance of carboxyl groups. The changes of morphology and dimension of the CN before and after oxidization were observed by TEM and using a Sepctrex laser particle counter. As shown in Figure 3a, the unmodified CN possesses a typical rodlike shape, with a length of 100−300 nm and a width of 3−15 nm, which is consistent with previous reports by other researchers.36,37 After the oxidization process, the average length is decreased from 275.5 nm (CN) to 233.8 nm (TOCN) (see Figure S1 in the Supporting Information), while the samples still maintain the rod-like shape shown in Figure 3b. These results demonstrate that CN and TOCN can maintain original morphologies and geometrical sizes of cellulose nanocrystal. The XRD patterns of CN before and after oxidation are shown in Figure 3c. It can be found that the main diffraction characteristics of TOCN from cellulose nanocrystals were quite similar (high peak at 2θ = 22.8°) and were typical for the cellulose I (2θ = 14.9° and 2θ = 16.7°), although the intensities were slightly decreased.38,39 Furthermore, the crystallinity index (Ic) values of cellulose were 88.9% (CN), 86.5% (TOCN), according to the Segal method.40 This result demonstrated that the original crystalline structure of CN can be still preserved after the chemical modification. Structure and Properties of TOCN/SA Composite. FTIR spectra of neat SA and Ca2+ cross-linking TOCN/SA composite sponges are presented in Figure 4. For neat SA, the peak at 1602 cm−1 is attributed to the stretching vibration of the carbonyl bond (−CO). And, the peaks at 1421 and 1020 cm−1 in both SA and cellulose were assigned to stretching vibration of the carboxyl (−COO−) and −C−O−C groups, respectively. However, some obvious difference can be observed in the spectrum of TOCN/SA composites (Figure 4). After the alginate was cross-linked with Ca2+, the asymmetric −CO and symmetric −COO− adsorption bands were shifted to higher wavenumbers, from 1602 cm−1 to 1607 cm−1 and from 1421 cm−1 to 1434 cm−1, and the TOCN ratio in composite sponge was also increased. These
Figure 2. (a) FTIR spectra for unmodified microcrystalline cellulose (MCC), cellulose nanocrystals (CNs), oxidized cellulose nanocrystals (TOCN-COONa), and acidic form of oxidized cellulose nanocrystals (TOCN−COOH). (b) XPS wide spectra of unmodified cellulose nanocrystals (CN), and oxidized cellulose nanocrystals (TOCN).
hydrogen bond stretching vibration of −OH groups, and the absorption at 1640 cm−1 was ascribed to the bending vibration of absorbed H2O. In addition, the peak at 2900 cm−1 was attributed to the stretching vibration of C−H. Compared to the MCC, no new peaks appeared in the FT-IR spectrum of CN, except a slight stretching vibration shoulder peak of C−H at 2900 cm−1. After TEMPO oxidization on the surface hydroxyl groups of CN, some apparent changes on the spectrum of the modified cellulose nanocrystal can be found. The new peak attributed to the stretching band of the sodium carboxylate groups (−COONa) can be observed at 1612 cm−1, which is the most important change of the carboxyl groups (CO) 3822
DOI: 10.1021/acssuschemeng.6b02849 ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
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ACS Sustainable Chemistry & Engineering
Figure 3. TEM images for (a) CN and (b) TOCN. (c) XRD spectra of CN and TOCN.
duced into SA, the rodlike nanocrystals appeared in the hole wall of sponge (Figure 5b). In addition, there was no obvious self-aggregation or microphase separation in the sponge. The results of EDS mapping showed that the Ca 2+ was homogeneously distributed on both the superficial and crosssectional surfaces of film and sponge. These results further indicated that Ca2+ not only cross-linked on the TOCN/SA composites surface (FT-IR spectra confirmed) but also that Ca2+ easily diffused into the material structure via thermodynamic driving forces, leading to the internal cross-linking reaction within the TOCN/SA composites (Figure S2 in the Supporting Information). The introduction of TOCN was built-in using the regular internal three-dimensional (3D) network of cross-linked TOCN/SA sponge. The 3D network structures were useful for nutrients metabolism of metabolic waste and were beneficial to the growth and transport of cells, and the vessels and tissues could grow successfully with materials implanted. Thus, the TOCN/SA composite sponge might accelerate the healing of the tissues within a shorter time biodegradation in vivo. Swelling Degree and Porosity. The swelling degree of the sponge and film was measured gravimetrically. The swelling degree of sponges and films is shown in Figure 6A. The TOCN-30/SA composite film and sponge had the most outstanding water absorption in all testing materials (75.2%, 1399.1%), while the alginate film or sponge was the lowest (43.35%, 825.9%). The swelling degree value of sponges was far higher than that of films. Water absorption is an important property for biomaterials and it is highly dependent on their intrinsic structure and morphology. The water molecules could smoothly pass through and penetrate the sponge, according to the sponges composed of the regular internal 3D network and high porosity. On the other hand, the introduced TOCN might disrupt the hydrogen bonding between the alginate and be conducive to increased water absorption ability with the TOCN content. Compared with the neat SA sponge (82.3%), TOCN/ SA composite sponges possess a higher density of ordered
Figure 4. FTIR spectra for neat SA, and TOCN/SA composites with different CN contents.
results could be attributed to the strong interaction between the carboxylic groups on oxidized cellulose nanocrystal, alginate, and the Ca2+ ions, according to the cross-linking. SEM Analysis. The SEM micrographs of the cross-section of sponges are shown in Figure 5. It was clear observed that the
Figure 5. SEM images of the cross-section morphology of cross-linked sponges: (a) SA and (b) TOCN-30/SA.
neat alginate sponge (SA) possesses a stratiform pore structure (Figure 5a), which is basically consistent with the previous reports.41 When cellulose nanocrystals (TOCN) were intro-
Figure 6. (A) Degree of swelling of wet sponge and film, (B) porosity evaluation of sponge, and (C) tensile strength. 3823
DOI: 10.1021/acssuschemeng.6b02849 ACS Sustainable Chem. Eng. 2017, 5, 3819−3828
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ACS Sustainable Chemistry & Engineering
Figure 7. (a) Hemolysis assay of TOCN, SA, and TOCN/SA composite extracts. (b) Hemolysis ratio in each group (n = 3). (c) Cytocompatibility of materials on Hela cells.
pores (>90%), while the porosity of TOCN-50/SA was slightly decreased (Figure 6B). It was suggested that the high porosity of TOCN/SA sponges were attributed to the internal network microstructure of the interconnection between alginate and TOCN chains (as shown in the SEM results). In addition, the highly homogeneous dispersion of nanocrystal contents may have an effect on material porosity. Furthermore, the highly porous character of the sponges would be useful to facilitate the adsorption of wound exudates. A tensile strength test was used to measure the material characteristics under an axial tensile load. Figure 6C demonstrates that the Ca2+ cross-linked composites filled with oxidized cellulose nanocrystals were generally stronger and more robust than neat alginate. The highest mechanical performance was obtained for TOCN-30/SA composites (sponge and film, respectively). The TOCN-30/SA composite film and sponge could withstand maximum stresses, which were 261.23 ± 21.33 MPa, and 35.16 ± 10.59 MPa, respectively. A possible reason might be attributed to the TOCN participating in cross-linking and have a good “egg-box” conjunction zone between TOCN and alginate during the cross-linking process. However, if superfluous CNs were added in alginate, the selfaggregation of nanoparticles may cause the reinforcing effect to be shaded-off. Hemolysis Assay In Vitro. Hemolysis tests were conducted using 2% rabbit erythrocyte suspension. As shown in Figure 7, the TOCN/SA composite extracts, TOCN solution (1 and 4 mg/mL), and SA solution (4 and 6 mg/mL) did not cause any hemolysis, while the apparent hemolysis can be observed in the positive control (distilled water). In addition, although the hemolysis ratio of the positive control (distilled water) was set to 100%, the TOCN/SA composite extracts, TOCN solution (1 and 4 mg/mL), and SA solution (4 and 6 mg/mL) were observed to exhibit
