Abstract
Lignin-carbohydrate complexes (LCC) were isolated from poplar wood after ball-milling for 48 h and 72 h. The water-insoluble LCC-48 and LCC-72 were fractionated. Porous spherical composite bio-carriers were prepared by liquid-nitrogen freezing using the water-insoluble LCCs and cellulose nanocrystals (CNC). Fourier transform infrared spectroscopy (FT-IR) showed that the bio-carriers were composed of lignin moieties and polysaccharide units. Scanning electron microscopy (SEM) revealed that the outer surface and cut surface of the spherical carrier had many pores. The specific surface areas of the LCC-72/CNC and the LCC-48/CNC composite carriers were 24.2 m2/g and 28.9 m2/g, respectively, while their pore sizes were 28.6 nm and 33.6 nm, respectively. Both carrier samples had good stability at pH 4.6 to 9.5 for about 50 days. Human hepatocytes were cultured in vitro with the bio-carriers and the cells grew well. A large number of cells adhered to the porous bio-carriers, and the cells of the experimental group to which the carriers were added exhibited higher metabolic activities. The CNC improved the biocompatibility for human hepatocytes. In summary, spherical bio-carriers prepared from LCC/CNC composite displayed high biocompatibility and have potential applications in liver tissue engineering.
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Preparation of Porous Composite Bio-carriers from Lignin-Carbohydrate Complexes and Cellulose Nanocrystals, and their Application in the Culture of Human Hepatocytes
Hongfei Wu,a Yimin Xie,a,b,* Houkuan Zhao,a Xuekuan Chen,a Chen Jiang,a Shuying Bi,a and Yanchao Liu a
Lignin-carbohydrate complexes (LCC) were isolated from poplar wood after ball-milling for 48 h and 72 h. The water-insoluble LCC-48 and LCC-72 were fractionated. Porous spherical composite bio-carriers were prepared by liquid-nitrogen freezing using the water-insoluble LCCs and cellulose nanocrystals (CNC). Fourier transform infrared spectroscopy (FT-IR) showed that the bio-carriers were composed of lignin moieties and polysaccharide units. Scanning electron microscopy (SEM) revealed that the outer surface and cut surface of the spherical carrier had many pores. The specific surface areas of the LCC-72/CNC and the LCC-48/CNC composite carriers were 24.2 m2/g and 28.9 m2/g, respectively, while their pore sizes were 28.6 nm and 33.6 nm, respectively. Both carrier samples had good stability at pH 4.6 to 9.5 for about 50 days. Human hepatocytes were cultured in vitro with the bio-carriersand the cells grew well. A large number of cells adhered to the porous bio-carriers, and the cells of the experimental group to which the carriers were added exhibited higher metabolic activities. The CNC improved the biocompatibility for human hepatocytes. In summary, spherical bio-carriers prepared from LCC/CNC composite displayed high biocompatibility and have potential applications in liver tissue engineering.
Keywords: Lignin-carbohydrate complexes; Cellulose nanocrystals; Spherical carrier; Human hepatocytes; Biocompatibility
Contact information: a: Research Institute of Pulp & Paper Engineering, Hubei University of Technology, 430068, Wuhan, China; b: Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, 430068, Wuhan, China; *Corresponding author: ppymxie@163.com
INTRODUCTION
The liver performs various essential functions for our survival, such as the control of plasma glucose levels, the synthesis of various proteins, including albumin and coagulation proteins, the synthesis of bile for the absorption of fat in the digestion process, detoxification by transaminase reactions, and the storage of several vitamins (Lee et al. 2016). However, liver disease afflicts over 10% of the world’s human population. This includes chronic hepatitis, alcoholic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Although there are treatment options for most liver diseases, such as chemotherapy, injection of blood drugs, or surgery in some cases, many types are still incurable (Zhang et al. 2013). At present, the most effective medical treatment for liver diseases, such as liver cancer and acute liver failure, is liver transplantation. The emergence of liver tissue engineering may offer a new therapy to solve the problem of insufficient liver donors. The purpose of liver tissue engineering is to construct a three-dimensional complex of hepatocytes and tissue materials to replace the diseased liver. Three important determinants to be considered in tissue engineering are the scaffold materials, seed cells, and growth regulators (Kulig and Vacanti 2004). Finding suitable scaffold materials has been one of the most important research areas of liver tissue engineering because of the basic performance of the artificial liver that mainly depends on the choice of scaffold material (Ghim et al. 2015). The available materials can be divided into two categories, namely natural scaffold materials and degradable polymer scaffold materials (Chung et al. 2002). A desirable scaffold material for liver tissue engineering must suit the following requirements: (1) have good biocompatibility and be non-toxic, bioactive, and degradable; (2) have good surface properties for cell adhesion growth; and (3) have a three-dimensional porous structure with a porosity of over 90% and retain appropriate mechanical strength (Chung and King 2011; Li et al. 2013).
Lignin-carbohydrate complexes (LCC) are a combination of hydrophobic lignin chemically bound to hydrophilic polysaccharides. Research on the connection of lignin and carbohydrates is still continuing. Within plant materials, the lignin and carbohydrates are combined by covalent bonds in LCC, and there are phenyl glycosidic bonds, ether bonds, and ester bonds between the lignin and the carbohydrates (Xie et al. 2000; Yuan et al. 2011; Nishimura et al. 2018). The polysaccharide fragment in the LCC is composed of various types of sugar units, such as glucose, arabinose, mannose, galactose, and a small amount of uronic acid (Jeffries 1990; Sakagami et al. 2005; Singh et al. 2005). Cell-wall polysaccharides containing 2% to 5% galactose moieties have the ability to recognize hepatocytes due to the presence of asialoglycoprotein receptors (ASGPR) on the hepatocytes, which binds to galactose (Cho et al. 2006; You et al. 2015; Roopan 2017). Uraki et al. (2004) prepared a temperature-sensitive gel by adding lignin and LCC. They found that the drug release temperature of the gel could be lowered because of the hydrophobic residual lignin structure. Zhao et al. (2017a) prepared a porous gel from LCC cross-linked with polyethylene glycol diglycidyl ether and successfully applied it for the culture of human hepatocytes; however, the polyethylene glycol diglycidyl ether was non-degradable.
Cellulose is the most abundant natural polymer available on the earth, and it is an important structural component of the cell wall of various plants. Apart from plants, cellulose is also present in a wide variety of living species, such as algae, fungi, bacteria, and even in some sea animals such as tunicates (Johnsy and Sabapathi 2015). When the width of a structure composed of parallel cellulose chains is below 100 nm, it could be called nanocellulose (Dufresne 2013). The nanocellulose has a large surface area and high strength. One important class of nanocellulose is obtained from renewable polymers by acid hydrolysis and has rod-like shape and revealed tunable surface chemistry (Liu et al. 2011). This kind of nanocellulose, called cellulose nanocrystals (CNC), has a large specific surface area, negative charge, and free hydroxyl groups. Therefore, as a tissue engineering material, CNC has the advantages of large specific surface area, good flexibility, and good biocompatibility (Jackson et al. 2011). In the preparation of tissue engineering scaffold materials, the addition of an appropriate amount of CNC could improve biocompatibility and mechanical strength.
In the present study, LCC/CNC porous spherical bio-carriers were prepared by mixing LCC and CNC, and freezing by liquid nitrogen, followed by freeze-drying (Fig. 1). The structure of the composite was analyzed by Fourier transform infrared (FT-IR) spectra, and the carrier morphology was observed by scanning electron microscopy (SEM). Human liver cells L-02 were cultured with the LCC/CNC porous carriers. The biocompatibility of the porous carriers was analyzed by observation of the cell-growth status and detection of metabolic activities.
Fig. 1. Mechanism of the formation of LCC/CNC composite bio-carriers
EXPERIMENTAL
Materials
Poplar (Populus euramericana) wood was obtained from the Wuhan botanical garden (Wuhan, China). Human hepatocytes were provided by the Pricells Co. (Wuhan, China). The CNC suspension prepared from acid hydrolysis from cotton was purchased from Yuhan Technology Co. Ltd. (Hangzhou, China). Bovine insulin was purchased from Aladdin (Shanghai, China). Fetal bovine serum was purchased from Shanghai Yuanmu Biotechnology Co., Ltd. (Shanghai, China). A glucose kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Phosphate buffer saline was purchased from Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, China).
Methods
Preparation of poplar LCC
Poplar wood chips were ground with a Wiley mill (FZ102, Tianjin Taishite, China) and then extracted with benzene-ethanol mixture (2/1, V/V). The powder was completely dried in vacuofor about one week. The dry wood powder was then ground for 48 h and 72 h, respectively, using a water-cooled vibratory ball-mill (MB-0, Chuo-koki.Co. Ltd, Japan). The milled sample was extracted three times using dioxane-water solution (96/4, V/V). An insoluble material was obtained after centrifugation, which was then extracted with a mixture of acetic acid and water (1/1, V/V). The material obtained after removing the solvents was then purified using the Björkman method to obtain LCC sample (Björkman 1957).
Separation of water-insoluble LCC
About 0.5 g of poplar LCC and 50 mL of distilled water were put into a 100-mL Erlenmeyer flask, heated to 50 C, and stirred for 8 h. The precipitate obtained was collected by centrifugation and then freeze-dried (-85 C, 0.006 Torr) to obtain the water-insoluble poplar LCC.
Preparation of LCC/CNC composite spherical bio-carriers
The water-insoluble LCCs (LCC-48 and LCC-72) were dissolved in 85% acetic acid solution, and the solution was poured into a 30-mL Erlenmeyer flask containing CNC suspension withstirring at room temperature. The mass fraction of LCCs and CNC used for various samples were 0%/100%, 10%/90%, 20%/80%, 30%/70%, 50%/50%, 60%/40%, 70%/30%, and 100%/0%, respectively. The mixture of LCC/CNC was then slowly added dropwise to liquid nitrogen using a sterile syringe. The spherical bio-carriers were prepared by freeze-drying (-85 C, 0.006 Torr) the mixture of LCC/CNC.
FT-IR spectroscopy
The LCC/CNC bio-carrier sample (about 1.0 mg) was mixed with 300 mg KBr via grinding. The ground powder was then pressed into a pellet. The KBr-pellet sample was analyzed using an FT-IR spectrometer (Thermofisher 6700, USA).
Observation of the surface morphology of spherical bio-carriers by SEM
The LCC/CNC composite spherical bio-carriers were put on a silicon wafer and treated by sputter coating in vacuo. The surface and cut surface morphology of the composite carriers was observed by SEM (JSM-6390LV, Tokyo, Japan).
Determination of specific surface area and pore size of the bio-carriers
The specific surface area and pore size of the spherical bio-carrier samples were determined using a BELSORP-mini II type (Ankersmid, Netherlands) high-precision surface area and pore size analyzer.
Determination of stability of the spherical carriers
The LCC/CNC composite carrier samples were allowed to soak in various buffer solutions in a 50-mL Erlenmeyer flask, i.e., acetic acid buffer (pH 4.6), phosphate buffer (pH 7.0), and NaHCO3 buffer (pH 9.5) for 50 days at room temperature. After removing the buffer, the morphology of the bio-carrier samples was observed with an optical microscope (Olympus SZX16, Japan).
Culture of human hepatocytes
Human hepatocytes (L-02) obtained from Pricells Co. (Wuhan, China) were rinsed with phosphate buffer. The hepatocytes were then inoculated into 12-well culture plates at a density of 3×105 cells/mL. The bio-carriers were sterilized in dry state at 120 C for 4 h. These were then added to the wells at a concentration of 2.5 mg/mL. The hepatocytes, together with the bio-carriers, were incubated at 37 C, 5% CO2, and 100% relative humidity. The culture medium (RPMI-1640) was supplemented with 20% FBS (fetal bovine serum), 1% penicillin, and streptomycin solution. The adhesion of human hepatocytes to the spherical bio-carriers was observed daily using a Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany). Cell-free supernatant was collected by centrifugation daily to detect the metabolic activity of the cells.
Calculating the cell number
For one week of the culture of human hepatocytes (L-02), the liquid media were collected every day in a clean bench, and the cells were washed twice with phosphate buffer followed by 0.25% trypsin digestion for 1 min to 2 min.
After the digestive medium was removed, new medium was added to terminate the digestion. The cells were dissociated into a single cell suspension. Cell suspensions (200 µL) were stained with 0.4% trypan blue solution and placed on a hemacytometer for counting on a Leica DM-6000 CS microscope (Leica Instruments Inc., Wetzlar, Germany).
SEM observation of the spherical bio-carriers
The porous carriers were taken out on the fifth day of the human liver cell culture. The cells on the porous carriers were fixed with 200 µL glutaraldehyde solution (2.5%, w/w) and then fixed with 200 µL osmic acid (10 g/L) for 5 h. After washing three times with 3 mL phosphate buffer solution (pH 7.4), the obtained cells were dehydrated with different concentrations of ethanol for 15 min.
The bio-carriers were then dried in vacuo. The dried porous bio-carriers were treated by sputter coating in vacuo. Then the cell-attached carriers were observed by SEM and photographed.
Detection of metabolic activity of the cultured human hepatocytes
Cell-free supernatant (10 µL), standard albumin (10 µL), and distilled water (10 µL) were added to a centrifuge tube. After the addition of 2.5 mL bromocresol green buffer, the samples were shaken to complete the reaction. The absorption values were monitored at 628 nm using a UV spectrophotometer (UV-2550, SHIMADZU, Japan) (Zhao et al. 2017b). The following equation (Eq. 1) was used for calculating the albumin content,
(1)
where ALB is the content (g/L) of albumin; A1 denotes the absorbance of the sample solution; A2 is the absorbance of the standard solution; C0 represents the concentration (g/L) of the standard albumin solution; and A0 is the absorbance of distilled water.
Cell-free supernatant (10 µL), 10 mmol/L blood urea nitrogen standard solution (10 µL), and distilled water (10 µL) were added to a centrifuge tube. After 15 µL oxime solution (1 g/L) and 15 µL sulfuric acid solution (0.2 g/L) were added to the centrifuge tube, the mixture was mixed, placed in boiling water for 15 min, and then cooled with cold water. The absorption values were monitored at 628 nm. Equation 2 was used for calculating the blood urea nitrogen content,
(2)
where BUN is the content (mmol/L) of blood urea nitrogen; A1 is the absorbance of the sample solution; A2 is the absorbance of the standard solution; C0 is the concentration (g/L) of the standard blood urea nitrogen solution; and A0 is the absorbance of distilled water.
About 10 µL of distilled water, 10 µL standard glucose, and 10 µL cell-free supernatant were taken in a test tube. Then, 15 µL peroxidase buffer solution (1 U/mL), glucose oxidase solution (10 U/mL), and a mixture of 1,5 dimethyl-2-phenyl-4-amino-3-pyrazolone (70 mmol), and phenol (10.6 mmol/L) were added. The mixture was mixed and allowed to react completely. The absorption values were monitored at 505 nm, and the following Eq. 3 was used for calculating the glucose content,
(3)
where C is the content (mmol/L) of glucose; A1 is the absorbance of the sample solution; A2 is the absorbance of the standard solution; and C0 is the concentration (g/L) of the glucose standard solution.
RESULTS AND DISCUSSION
FT-IR Analysis and Composition Elucidation
The FT-IR spectra of the LCC/CNC spherical carriers prepared from 48 h and 72 h ball-milling times (LCC/CNC with an LCC mass fraction of 60% is used as an example) are shown in Fig. 2.
The tentative peak assignment is shown in Table 1. From the spectra, the characteristic peaks of the LCC/CNC bio-carriers prepared by different ball-milling times were similar and displayed typical chemical structures of lignin and carbohydrates, including cellulose. From the absorption peak at 1738.1 cm-1 to 1732.2 cm-1, it can be concluded that there was a non-conjugated C=O group in the LCC. The strong absorption peak at 1045.3 to 1045.7 cm-1 in the spectra of the LCC/CNC composite is due to the C-O stretching of the polysaccharides, including cellulose of CNC and hemicellulose in LCC (Fan et al. 2006). These results indicated that the LCC/CNC bio-carriers consisted of lignin moieties and sugar units from the polysaccharide.
Fig. 2. FT-IR spectroscopy of LCC/CNC composite spherical bio-carriers. A: LCC-48/CNC; B: LCC-72/CNC
Table 1. Tentative Analysis of the Main Absorption Peaks of Composite Bio-carriers
Fig. 3. SEM images of spherical bio-carriers: A: Surface of LCC-48/CNC; B: Cut surface of LCC-48/CNC; C: Surface of LCC-72/CNC; and D: Cut surface of LCC-72/CNC
SEM Images of Spherical Bio-carriers
Figure 3 shows the surface and cut surface of the LCC/CNC bio-carriers with different ball-milling times as observed by SEM (LCC/CNC with an LCC mass fraction of 60% was used as an example). There were many holes and cracks on the surface of the LCC/CNC bio-carriers. After the carriers were cut radially, there were many holes and gullies within the carriers, which provided suitable space for cell growth.
Analysis of Specific Surface Area and Pore Diameter
The specific surface area and pore size of the composite bio-carriers were determined by BET analysis with liquid nitrogen at -196 C followed by degassing at 130 C (Wellner et al. 1998; Chirkova et al. 2011). The specific surface area of the bio-carriers was calculated using Eq. 4,
Sg = 4.36 × Vm/W (4)
where Sg is the specific surface area (m2/g) of the sample powder; Vm is the nitrogen monolayer adsorption saturation (mL); W is the sample weight (g). The average pore size of the bio-carriers was calculated by Eq. 5,
Rk = -0.414/log (P/P0) (5)
where Rk is the Kelvin radius (µm); P is liquid nitrogen’s partial pressure (Pa); and P0 is the saturated vapor pressure (Pa) of nitrogen at liquid nitrogen temperature.
The results for the average pore diameter and specific surface area are shown in Figs. 4 and 5, respectively. The specific surface areas of the LCC-72/CNC and the LCC-48/CNC bio-carriers were determined to be 24.2 m2/g and 28.9 m2/g, respectively. The average pore diameters measured were 28.6 nm and 33.6 nm, respectively. These results showed that the LCC/CNC composite bio-carriers had an appropriate pore size and enough specific surface area suitable for use in the culture of hepatocytes.
Fig. 4. BET adsorption isotherm of LCC-48/CNC bio-carriers |
Fig. 5. BET adsorption isotherm of LCC-72/CNC bio-carriers |
Stability Analysis of Spherical Bio-carriers
The stability analysis was conducted by immersing the bio-carriers in solutions of various pH values followed by measuring the change in size. The temperature of the solution was always maintained at 37 °C. As shown in Table 2, the there was no alteration in the size of the LCC/CNC bio-carriers in a wide range of pH values for 50 days in weak acid, weak alkali, and neutral medium.
Therefore, it could be concluded that the spherical carriers had excellent stability. In addition, the experiment also revealed that although CNC crystal had a certain hydrophilicity, once it became a xerogel together with water-insoluble LCC, it posed some resistance to water and maintained its original size. This may also be related to the poor water accessibility of the cellulose crystal in the CNC.
Table 2. Size Change of Spherical Bio-carriers in Various pH Conditions for 50 Days
The Growth of the Human Hepatocytes
The results of cell counting on days one to seven are shown in Figs. 6 and 7. Upon comparison with the cells cultured using the LCC/CNC spherical carriers and the cells cultured in the control solution showed an increased growth for the first 5 days, although the growth rate of the cells for the first 4 days was relatively slow. The number of hepatocytes formed was the highest on the 5th day, after which the number of cells began to decrease gradually. The cell-growth trend was as follows: LCC-72/CNC (50% LCC) > LCC-48/CNC (50% LCC)> CNC > control group. It was clear that the addition of CNC indicated some compatibility with human hepatocytes although the effect was weaker than that of LCC.