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Feng, C., Zhou, J., Xu, X., Jiang, Y., Shi, H., and Zhao, G. (2019). "Research on 3-D bio-printing molding technology of tissue engineering scaffold by nanocellulose/gelatin hydrogel composite," BioRes. 14(4), 9244-9257.

Abstract

In the biomedicine field, three-dimensional (3-D) printing of biomaterials can construct complex 3-D biological structures such as personalized implants, biodegradable tissue scaffolds, artificial organs, etc. Therefore, nanocellulose/gelatin composite hydrogels are often selected as bio-printing materials in the 3-D printing of biological scaffolds. Process parameters of 3-D printed bio-scaffolds were studied in this work because formation accuracy of scaffolds is an important part of the molding process. Firstly, the mixing proportion of nanocellulose and gelatin was explored, and the optimum proportion was selected. Then, the printing effects of different printing pressures, temperatures, speeds, and nozzle diameters were used in the 3-D printing. The filament widths were used to evaluate the molding effects. Finally, through the calculation and analysis of the grey correlation coefficient and grey correlation degree, the multi-objective optimization of the parameters was carried out. The combined effects of the process parameters and the influence degree order on the evaluation index were obtained. Using these parameters, the 3-D porous biological scaffolds were printed with high precision. Furthermore, using a microscope, the morphologies of CCK-8 cells were observed and the cell proliferation were analyzed. The results demonstrated that the printed bio-scaffolds had good biocompatibility.


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Research on 3-D Bio-printing Molding Technology of Tissue Engineering Scaffold by Nanocellulose/gelatin Hydrogel Composite

Chen Feng,a Ji-ping Zhou,b Xiao-dong Xu,a Ya-ni Jiang,b Hong-can Shi,c and Guo-qi Zhao a,

In the biomedicine field, three-dimensional (3-D) printing of biomaterials can construct complex 3-D biological structures such as personalized implants, biodegradable tissue scaffolds, artificial organs, etc. Therefore, nanocellulose/gelatin composite hydrogels are often selected as bio-printing materials in the 3-D printing of biological scaffolds. Process parameters of 3-D printed bio-scaffolds were studied in this work because formation accuracy of scaffolds is an important part of the molding process. Firstly, the mixing proportion of nanocellulose and gelatin was explored, and the optimum proportion was selected. Then, the printing effects of different printing pressures, temperatures, speeds, and nozzle diameters were used in the 3-D printing. The filament widths were used to evaluate the molding effects. Finally, through the calculation and analysis of the grey correlation coefficient and grey correlation degree, the multi-objective optimization of the parameters was carried out. The combined effects of the process parameters and the influence degree order on the evaluation index were obtained. Using these parameters, the 3-D porous biological scaffolds were printed with high precision. Furthermore, using a microscope, the morphologies of CCK-8 cells were observed and the cell proliferation were analyzed. The results demonstrated that the printed bio-scaffolds had good biocompatibility.

Keywords: 3-D printing; Biological scaffolds; Technological parameters; Grey relational degree method; Biocompatibility

Contact information: a: College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China; b: College of Mechanical Engineering, Yangzhou University, Yangzhou 225127, China; c: College of Medical, Yangzhou University, Yangzhou 225009, China; c: Yangzhou Polytechnic Institute, Yangzhou 225127, China; *Corresponding author: jpzhou@yzu.edu.cn

INTRODUCTION

It has been a challenge in the scientific and engineering communities to develop a tissue engineering scaffold and organ printing technology based on three-dimensional (3-D) printing (O’Brien et al. 2014). In the past few years, the emergence of nanomaterials has provided a new method for improving hydrogels, which requires only a few fillings that could greatly improve the toughness (Liu 2011). Previous studies report on fabricating 3-D scaffolds using a 3-D inkjet printing approach, which utilizes sodium alginate and collagen as raw materials and a calcium chloride solution as both a cross-linking agent and support material (Christensen et al. 2015; Hong et al. 2015). In addition, a 3-D scaffold was also made by a 3-D printing method using gelatin as the raw material (Lee et al. 2014; Bhattacharjee et al. 2015; Xiong 2015).

Nanocellulose, as a natural material from grass and other sources of cellulose, has a high strength, aspect ratio, biodegradability, extensive sources, and low cost (Fu et al. 2013; Lu et al. 2013; Foresti et al. 2017). Nanocellulose and its derivatives have been widely used in biomedicine and medical cosmetology (Lu et al. 2014; Pietrucha et al. 2016; Yoon et al. 2016) because it is favorable to release bioactive substances (Mariano et al. 2014; Shankar and Rhim 2016; Jiang et al. 2017).

Nowadays, CNC and its derivatives have been reported to be used as a viscosifier to improve the viscosity in the process of biological 3D printing (Shao et al. 2015). When CNC is added into hydrogel, many hydroxyl groups will be present on the surface of CNC. Thus, there will be strong interaction between CNC molecules, which will enhance the cohesion of hydrogel and increase the apparent viscosity. The increase of viscosity will be beneficial to maintaining the shape of hydrogel scaffold in the process of biological 3D printing (Wei et al. 2015).

Generally, the extrusion pressure, speed, temperature, and nozzle size may affect the freeze-casting of composite materials during the molding process of pneumatic condensation extrusion. To solve problems of over-accumulation, lap deficiency, and slobbering during the condensation extrusion, it is necessary to find a series of accurate control parameters that are suitable for a nanocellulose/gelatin composite hydrogel. In the present study, a nanocellulose/gelatin composite hydrogel was used as the bio-printing material to explore the effects of process parameters including printing pressure (Jiang 2018), temperature, speed, and nozzle diameter on the formation accuracy of bio-scaffolds. The results provided insights into the development of other materials and a relevant forming process.

EXPERIMENTAL

Materials

The nanocellulose (CNC) was extracted from the stem of humulus (HJS), a kind of grass taken from the wild (Yangzhou, China) (Jiang et al. 2015). Gelatin (GEL) was bought from Aladdin Chemistry Co. Ltd. (Shanghai, China). To test the bio-scaffold compatibility, human fibroblasts were cultured in Dulbecco’s modified eagle medium (DMEM). The CCK-8 cell viability assay kit (Shanghai Ruichu Biotech Co., Ltd., Shanghai, China) was used to test the cell proliferation and cell cytotoxicity.

Methods

Experimental equipment for 3-D printing

A platform-assisted 3-D printing system (Regenovo Biotechnology Co. Ltd., Hangzhou, China) was implemented in this study (Fig. 1a). The 3-D printing system used for the gelatin composite hydrogel was mainly equipped with a pneumatic extrusion device, a testing device, a temperature controlling device, a nozzle, and a receiving platform (Fig. 1b).

Experimental equipment for biocompatibility

To test the biocompatibility, equipment including a commingler (Shanghai Ruichu Biotech Co., Ltd., Shanghai, China), a cell culture incubator (Thermo ScientificTM, China) containing 5% CO2, a high temperature sterilizing oven (Deke Biotechnology, Shanghai, China), a laboratory centrifuge, a high temperature water bath (Deke Biotechnology, Shanghai, China), a vibrator (Sunshine, Shenzhen, China), and an enzyme-labeling instrument (Thermo ScientificTM, China) was used.

Specimen preparation

Certain amounts of gelatin (GEL) were added to a phosphate buffer solution (PBS) at 40 °C and mixed until completely dissolved. Then, different amounts of CNC suspension with a 1% concentration were added to the GEL solution and stirred for one hour at 40 °C. The mixture was then deaerated in a vacuum. Finally, the GEL concentration in the hydrogels were 5% while the CNC concentrations were 0%, 5%, 10%, and 15%. These concentrations were designated as GEL-5, 5%-CNC/GEL-5, 10%- CNC/GEL-5, and 15%-CNC/GEL-5 (Table 1), respectively.

Fig. 1. (a) Photograph of 3-D printer, (b) the establishment of 3-D printing system

Table 1. Chemical Composition of Hydrogel Samples

Three-dimensional printing method

The hydrogel materials were printed using a pneumatic extrusion method. The hydrogel stored in the cartridge entered into the irregular transition flow channel and then into the conical nozzle. It was extruded from the nozzle to form a filament. Secondly, the printer moved the nozzle according to the designed route in the platform and formed the designed pattern in the first layer. Once a layer was completed, the nozzle was raised to a higher height and the process was repeated until the pattern was complete. After printing, the filamentous hydrogel formed a specific 3-D scaffold structure according to the preset trajectory.

Fig. 2. Graphic of printing parameters in hydrogel printing

Printing setup

The rapid prototyping method of pneumatic condensation extrusion was selected according to the biological composite characteristics in this study. During the molding process, the extrusion pressure, speed, and temperature, as well as the nozzle size may affect the freeze-casting of the composite materials, as shown in Fig. 2. To evaluate the effects of the relevant parameters during the forming process, multiple experiments were designed.

The first experiment evaluated the molding process when the hydrogel concentration was changed and all other printing parameters remained consistent. The samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 were used to print a circle. The extrusion filamentous hydrogel widths and altitudes were measured and recorded at 0 s and 15 s after printing finished.

The second experiment evaluated the molding process when different nozzle diameters were used and all other printing parameters remained consistent. The samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 were printed using nozzle diameters of 0.21 mm, 0.26 mm, and 0.41 mm, respectively. The extrusion filamentous hydrogel widths were photographed and recorded at 3 min after printing finished.

The third experiment tested the molding process with different printing pressures, while all other printing parameters remained consistent. The samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 were printed under the pressures of 0.05 MPa, 0.06 MPa, 0.07 MPa, 0.08 MPa, and 0.09 MPa, respectively. The extrusion filamentous hydrogel widths were photographed and recorded at 3 min after printing finished.

The fourth experiment evaluated the molding process with different cartridge temperatures, while all other printing parameters remained consistent. The samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 were printed at the cartridge temperatures of 5 °C, 15 °C, 20 °C, and 25 °C, respectively. The extrusion filamentous hydrogels, showing the widths, were photographed and recorded at 3 min after printing finished.

The fifth experiment tested the molding process with different nozzle moving speeds while all other printing parameters remained consistent. The samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 were printed when the nozzle moved at the speeds of 10 mm/s, 15 mm/s, 20 mm/s, and 25 mm/s, respectively. The extrusion filamentous hydrogels, showing the widths, were photographed and recorded at 3 min after printing finished.

Grey relation analysis

Grey relational analysis (GRA) is an impact evaluation model to measure the degree of similarity or difference between two sequences based on the grade of relation. The GRA possesses the merit of point set topology and as such, the global comparison between two sets of data is undertaken instead of compared by measuring the distance between the two points. The process is summarized below.

The ith evaluated object can be described in Eq. 1,

X= {Xi1, Xi2,…,Xip} i=1,2,…,n (1)

where the number of alternatives is n and each alternative has p criteria.

The first step was to determine the reference sequence. According to the meaning of each criterion, the optimal value of each criterion is selected from n alternatives as the reference sequence X0, as show in Eq. 2,

 (2)

where the reference sequence X0 constitutes a relatively ideal optimal sample and is the standard of comprehensive evaluation. For each criterion, the decision maker should assign weightings and a preference index (PI). If a criterion is positive, a better alternative will occur when the criterion value is higher. If it is negative, X0 will be the minimum value. If it is a moderate scale, X0 will be the moderate value.

The second step was normalizing, as shown in Eq. 3,

 i=1, 2, …n; j=1, 2,…p (3)

where the optimal value of each index is 1 and the optimal reference sequence after normalizing could be calculated in Eq. 4:

X0 = {1, 1,…,1} (4)

The third step was to compute the distance of the maximum and minimum difference values (Δij). Equation 5 calculates the absolute difference between each alternative sequence and the reference sequence,

Δij=|Xij-1|, i=1, 2,…, n; j=1, 2, …,P (5)

where the maximum difference was designated as Δ (max), and the minimum difference as Δ(min). Equations 6 and 7 are shown below:

Δ (max) = (Δij) (6)

Δ (min) = (Δij) (7)

The fourth step was to apply the grey relational equation to compute the grey relational coefficient oi, as shown in Eq. 8,

 (8)

where ρ is the discrimination coefficient, typically between 0 and 1. In this study, ρ was 0.5.

The fifth step was to compute the grey coefficient degree (γoi). For each evaluated alternative, the mean value of the correlation coefficient between p criteria and the elements corresponding to the reference sequence was calculated. The correlation relationship between each evaluation alternative and the reference sequence was denoted as γoi as calculated in Eq. 9:

 i=1, 2 ,…n (9)

If the weights (W) of criteria are different, the grey coefficient degree (γoi) was computed through Eq. 10,

 , k=1, 2,…P (10)

where Wk is the weight of each criterion, and for decision-making processes, if any alternative had the highest γoi value, then it was the most important alternative. Thus, the alternative priorities could be ranked in accordance with γoi values.

Bio-scaffold compatibility test

First, the hydrogel was sterilized with a syringe filter (0.22 μm). Secondly, 50 μL of sterilized hydrogel was placed in each well of a 96-well plate and kept at 37 °C for 4 h. The unreacted materials were then washed 3 times with a PBS (pH 7.4). A 100 μL PBS was added to the control group. Thirdly, 100 μL cell suspensions were added to the 96-well enzyme plate. The human fibroblasts were grown in 89% DMEM supplemented with 10 vol% heat-inactivated fetal bovine serum and 1 wt.% penicillin-streptomycin under standard culture conditions at 37 °C in an incubator containing 5% CO2. Fourthly, the cells cultured on the scaffolds were observed using an inverted microscope (at 40 times and 100 times, respectively) after 1, 2, 3, and 4 days of culture. Lastly, 10 μL CCK-8 was added to each well every day. The plate was incubated in the incubator for 2 h, and the absorbance at 490 nm was measured by a microplate reader.

RESULTS AND DISCUSSION

Compatibility Test of Bio-scaffold

The hydrogel concentration determines the strength of the gel particles, and the viscosity recovery of the gel particles was the key factor affecting the molding quality of 3-D printing when the hydrogels were extruded from the nozzle. A concentration gradient of hydrogels was completed to investigate the printing effects (Fig. 3). The results showed that samples GEL-5, 5%-CNC/GEL-5, 10%-CNC/GEL-5, and 15%-CNC/GEL-5 could be extruded from the nozzle successfully. Immediately after printing was finished (0 s), the printing filament diameter of the GEL-5 sample was the largest. The diameter notably decreased when CNC was present as a filler, and 10%-CNC/GEL-5 printed the smallest filaments. Moreover, 3-D printing with GEL-5 and 5%-CNC/GEL-5 was easily dragged and molded unsuccessfully. At the moment of 0 s, the width of filaments reflects the swelling rate of hydrogel when extruded from nozzle. At the moment of 0 s, the width of 15%-CNC/GEL-5 was wider than that of 10%- CNC/GEL -5, indicating that the expansion rate of 15%-CNC/GEL-5 was higher. In addition, at the moment of 15 s, the width of 15%- CNC/GEL -5 was also wider than that of 10%- CNC/GEL -5, indicating that the shrinkage rate of 10%- CNC/GEL -5 was also greater. Thus, at the 10%-CNC/GEL-5 concentration, the hydrogels were well formed in support. However, this concentration displayed an inhomogeneous hydrogel form that led to a failed molding.

Fig. 3. Printing effects of hydrogel with different concentrations at (a) 0 s and (b) 15 s

Three-dimensional results of 10%-CNC/GEL-5 using nozzles with different diameters

The 10%-CNC/GEL-5 sample was selected to be the most suitable nozzle diameter based on the previous results. As shown in Fig. 4, the nozzle diameter influenced the molding results. During the 3-D printing process, hydrogel flows into the nozzle from the flow channel. Due to sudden changes of the flow channel and the cross-sectional area of the nozzle, fluid pressure is lost. This process is called the damping effect. When hydrogel is extruded, pressure is the power source of hydrogel outflow. Thus, under the same printing pressure, a smaller nozzle diameter caused a greater damping effect and more pressure to be lost (Zhai et al. 2010). However, the printing filaments were too thin to mold when the nozzle diameter was too small. Based on these results, the nozzle with a diameter of 0.26 mm was chosen for the following experiments.