Author’s Accepted Manuscript

3D bioprinting scaffold using alginate/polyvinyl

alcohol bioinks

Yongxiang Luo, Guilin Luo, Michael Gelinsky,

Peng Huang, Changshun Ruan

www.elsevier.com

PII: S0167-577X(16)31888-2
DOI: http://dx.doi.org/10.1016/j.matlet.2016.12.009
Reference: MLBLUE21821
To appear in: Materials Letters
Received date: 5 October 2016
Revised date: 21 November 2016
Accepted date: 3 December 2016
Cite this article as: Yongxiang Luo, Guilin Luo, Michael Gelinsky, Peng Huang
and Changshun Ruan, 3D bioprinting scaffold using alginate/polyvinyl alcohol
bioinks, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.12.009
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3D bioprinting scaffold using alginate/polyvinyl alcohol
bioinks
Yongxiang Luo1*, Guilin Luo2, Michael Gelinsky3, Peng Huang1*, Changshun Ruan2*
1
Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical
Engineering, Shenzhen University, Shenzhen, 518060, P. R. China.
2
Center for Human Tissues and Organs Degeneration, Institute of Biomedicine and Biotechnology,
Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
3
Centre for Translational Bone, Joint and Soft Tissue Research, University Hospital Carl Gustav Carus and
Faculty of Medicine, Technische Universität Dresden, Germany

luoyongxiang@szu.edu.cn

peng.huang@szu.edu.cn

cs.ruan@siat.ac.cn
*
Corresponding author: Tel.: +86 0755 86671915; Fax. +86 075586585250

Abstract

A novel 3D bioprinting of porous scaffold was developed by using highly concentrated

alginate/polyvinyl alcohol (PVA) as building block for tissue engineering. PVA sols were

partly diffused from the printed scaffolds during incubation in CaCl2 solution, creating plenty

of micropores in scaffolds. The properties of the as-prepared scaffolds such as porosity, water

adsorption and mechanical properties, can be tailored by adjusting the mass ratio of

alginate/PVA. Meanwhile, bovine serum albumin and bone morphogenetic protein 2 were

successfully co-printed into scaffolds, and their release behavior depended on the micropores.

Keywords: Biomaterials, Polymeric composites, 3D printing, Alginate, Tissue engineering

1. Introduction

Alginate with good biocompatibility and easy gelation with divalent cations under normal

physiological conditions has been extensively used for drug delivery and tissue engineering

[1-4]. Interestingly, alginate also has been widely investigated as bioink for 3D bioprinting [5,
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6]. Alginate bioinks in low concentration sols (0.5-6 %) [5, 6] are prone to diffusing after

printing (extrusion) through printing nozzles, resulting in the deformation of the whole 3D

structure. To overcome this issue, alginate sols were printed into calcium ions solution to keep

the whole 3D structure during printing scaffolds, while resulting in inhomogeneous

crosslinking [7].

Herein, a novel 3D bioprinting of porous scaffold was developed by using highly

concentrated alginate/polyvinyl alcohol (PVA) as building block. The homogeneous

alginate/PVA pastes show great potential in 3D bioprinting. The properties of the as-prepared

scaffolds such as porosity, water adsorption, mechanical property and protein delivery, can be

tailored by adjusting the mass ratio of alginate/PVA.

2. Experimental

The alginate (Manugel®; ISP Alginates Ltd. U.K.) and PVA (Mw ∼ 130 000; Sigma-Aldrich)

bioinks (pastes) were prepared in different alginate/PVA mass ratio in this study (Table 1).

Scaffolds were fabricated with a BioScaffolder 2.1 printer (GeSiM, Radeberg, Germany)

according to a previous study [8]. Afterwards, the printed scaffolds were soaked in 500 mM

CaCl2 solution for about 5 h with refreshing solution every 30 min, followed by washing with

deionized water three times and drying at room temperature.

The microstructures of printed scaffolds were analyzed by SEM (Philips XL 30/ESEM with

FEG). The water adsorption study was performed by incubating dry samples in phosphate

buffered saline (PBS) solution (pH 7.4) at 37°C. At certain time point, three samples of each

type were taken out and put on the filter paper for seconds to remove the water from the

macro pores. The water adsorption was calculated according to the following function:

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In which, W1 and W2 represent the weight of wet scaffold after soaking in PBS and the

weight of dry scaffold before soaking in PBS, respectively.

Compression test was performed by an Instron 5566 testing machine (Instron Wolpert GmbH,

Darmstadt, Germany) equipped with a 10 kN load cell. Five samples (6×6×6 mm) of each

type were tested for mean and standard deviation calculation.

Bovine serum albumin (BSA) (Sigma Aldrich) (300 mg) was dissolved in PVA or deionized

water solution (10 g) to prepare BSA loaded bioinks and scaffolds as described previously [9,

10]. The BSA loaded scaffolds were incubated in 3 mL PBS (pH 7.4, 37°C) in a shaker. At

each time point (1, 3, 6, 9, 24, 48, 72, 98 and 144 h), 1.5 mL PBS was taken and centrifuged

at 9300 g for 5.5 min. The supernatant was used for quantification of released BSA using the

Roti®-Nanoquant protein quantification assay (Roth, Germany) according to manufacturer’s

instructions. Four samples for each type were analyzed.

Recombinant human BMP-2 (rhBMP-2) (Sigma-Aldrich) was loaded in alginate

microspheres, which were prepared by oil/water emulsion according to reference [11].

Briefly, 1.5 wt% alginate sols containing 50 µg BMP-2 were added dropwise into liquid

paraffin under magnetic stirring at 9°C for 5 min. After that, 10 wt% CaCl2 solutions were

added into the emulsion under magnetic stirring. The prepared BMP-2 loaded alginate

microspheres were mixed with bioinks and printed into 3D scaffolds. The BMP-2 release

study was performed by incubating scaffolds into 2 mL PBS at 37°C in a shaker. At each time

point (1 h, 3 h, 5 h, 10 h, 1 d, 3 d, 5 d and 7 d), 500 µL PBS was taken to quantify the released

BMP-2 by BMP-2 quantikine ELISA kit (Sigma-Aldrich) according to manufacturer’s

instructions. Six samples for each type were tested.

Rat bone marrow stem cells (rBMSCs) (isolated from Sprague-Dawley rats at 5 passages)

were cultured with the addition of released BMP-2. The specific alkaline phosphatase (ALP)

activity of rBMSCs on day 7 and 14 was quantified using a kinetic colorimetric assay

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(Meridian Rd., Rochford, USA). Five samples were replicated for each type and time point.

Blank samples were cultured by adding PBS buffers without BMP-2 were performed as

control.

3. Results and discussion

In this study, bioinks (pastes) of highly concentrated alginate/PVA sols were prepared by

mixing sodium alginate with PVA solutions in different mass ratio. The concentrated

alginate/PVA pastes were homogeneous and suitable for extrusion through printing nozzles

contributing to stable strands without diffusion. The as-printed scaffolds were able to keep

their pre-designed 3D structure in air not only in calcium ions containing solutions, compared

to those fabricated by low concentration of alginate sols [5].

Alginate sols were strongly crosslinked, whlie PVA sols were partly diffused from the printed

alginate/PVA scaffolds during incubation in aqueous CaCl2 solution [12], leading to plenty of

micropores in the scaffolds (Figure 1). The total porosity of alginate/PVA scaffolds was

significantly higher than that of pure alginate scaffolds (Figure 1e). The water adsorption of

alginate/PVA scaffolds increased from 180 % to 500 % when the mass ratio of alginate/PVA

decreased from 3/1 to 1/1 during incubation in PBS buffer (Figure 1f). 3D porous scaffolds

with high porosity facilitated protein adsorption and water/medium infiltration [13].

The mechanical testing data (Figure 2) showed that the compressive strength and modulus of

pure alginate scaffolds (1/0) were significantly higher than those of all investigated

alginate/PVA scaffolds (1/1, 2/1 and 3/1) in both dry and wet state. Compressive strength and

modulus of alginate/PVA scaffolds were tailored by the porosity. In addition, the mechanical

properties of all printed scaffolds decreased sharply in wet state compared to those in dry

state, such as the compressive strength of pure alginate scaffold in wet state was 0.85 ± 0.07

MPa, which was less than 5 % of that in dry state (20.7 ± 4.1 MPa). Alginate bioinks in high

concentration were crosslinked strongly by calcium ions and the fibers were entangled

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physically after drying, which resulted in dense structures and then finally contributed to the

high mechanical properties. However, in wet state (such as in SBF or in cell culture medium),

alginate scaffolds swelled due to the efficient water adsorption. The entangling between

alginate fibers was loosened and the crosslinking calcium ions were partly exchanged, which

significantly reduced the stiffness of the alginate scaffolds.

Since the whole printing process was carried out under completely mild conditions (room

temperature, no pH shift, no organic solvent), proteins and growth factors can be loaded into

the pastes (bioinks) prior to 3D printing. The loading efficiency through this method could

achieve nearly 70 % for BSA and 90 % for growth factors encapsulation [10, 14]. In addition,

the loading amount can be easily controlled through this loading method. Due to the existence

of plenty of micropores in alginate/PVA scaffolds, BSA had a quick release from these

scaffolds. The data indicated that nearly 50 % of BSA released from alginate/PVA scaffolds

during the first 9 h of incubation (Figure 3). While BSA had a sustained release from the pure

alginate scaffolds and it showed a linear release profile during 168 h of incubation.

BMP-2 showed nearly 60 % burst release from alginate/PVA scaffolds (1/1), while about 45 %

burst release from pure alginate scaffolds (1/0) in the early stage. BMP-2 achieved a sustained

release over 7 days. The released BMP-2 significantly enhanced the ALP activity of cultured

rBMSCs, which indicated that the released BMP-2 maintained its biological activity (Figure

3).

4. Conclusions

3D printed scaffolds using highly concentrated alginate/PVA bioink were reported in this

study. The macropores of the scaffolds were controlled by 3D printing, while micropores

were related with PVA sols. The existence of micropores significantly enhanced the water

adsorption, and decreased the mechanical properties of scaffolds. BSA and BMP-2 were

5
 introduced into the bioinks thanks to the mild printing conditions, and the release profiles

were also strongly depended on the micropores.

Acknowledgement

This research was funded by the Natural Science Foundation of China (31400807), Science

and Technology Planning Project (2015A010105021) and Medical Scientific Research

Foundation (A2016253) of Guangdong Province, Basic Research Program of Shenzhen

(JCYJ20160308090821437, JCYJ20140417113430596).

References

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Fig. 1. Microscopic (a-d) and SEM (insert) images of the 3D printed scaffolds showing macro
and micro structures with different alginate/PVA mass ratios: 3/1 (a), 2/1 (b), 1/1 (c) and 1/0
(d). Scale bar = 500 µm (microscopic) and 20 µm (SEM). The porosity (e) and water
adsorption (f) of 3D printed scaffolds with different alginate/PVA ratio (*p < 0.05).

Fig. 2. The compressive strength (a, b) and Young’s modulus (c, d) of 3D printed
alginate/PVA scaffolds under dry (a, c) and wet (b, d) conditions (*p<0.05). Note the different
scales used for data obtained in dry and wet state, respectively.

Fig. 3. SEM image (a) of alginate microspheres loaded with BMP-2 (insert indicated particle
size distribution); BSA (b) and BMP-2 (c) release profile from 3D printed scaffolds in PBS
buffer (37°C, pH 7.4). Specific ALP activity of rBMSC cultured in medium with released
BMP-2 or without BMP-2 (blank) after incubation for 7 and 14 days (d) (∗p<0.05, ***
p<0.001).

Table 1. The parameters of prepared alginate/PVA bioinks

Alginate/ Alginate PVA PVA Water Printing
PVA concentration concentration solution content pressure
(mass ratio) (w/v) (g) (g) (bar)
3/1 18.2 wt% 6% 5.5 5.17 5.6
2/1 15.4 wt% 7.7 % 6.5 6.0 4.1
1/1 12.5 wt% 12.5 % 8.0 7.0 3.2
1/0 18.2 wt% - - 5.5 4.5

Highlights

 A novel concentrated alginate/PVA bioink was prepared for 3D bioprinting.

 The properties of printed alginate/PVA scaffolds were tailorable by the content of PVA.

 Proteins and growth factors were co-printed into scaffolds and they achieved a controlled release from
scaffolds.

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