Page 140 - IJB-9-1

International Journal of Bioprinting BNC-reinforced GelMa enhances property of bioprinted cartilage

applied tissue-engineered auricular cartilage based on 2.2. Animals
polyglycolic acid/polylactic acid and chondrocytes to Japanese white rabbits (female, 2 months old) and nude
clinical practice . However, it was found that the implants mice (male and female, 6 weeks old) were bought from
[9]
collapsed at varying degrees after some time. Polymer Beijing Vital River Laboratory Animal Technology
materials can easily cause aseptic inflammation , and it is Co., Ltd. (Beijing, China). All animal experiments were
[10]
challenging to achieve uniform distribution of chondrocytes approved by the Institutional Animal Care and Use
in the scaffolds using the traditional seeding cell method ; Committee of the Plastic Surgery Hospital (Approval ID:
[11]
these justify the unsatisfactory results. Due to their low 2017 – 37).
aseptic inflammation, natural hydrogel materials such as
alginate, gelatin, and hyaluronic acid have attracted attention 2.3. Preparation of hydrogel materials
recently [12-17] . The three-dimensional (3D) bioprinting BNC was produced using BC as previously described .
[30]
technology has significant advantages in the construction of In brief, BC was purified with 2% (w/v) NaOH at 80°C for
accurate anatomical contours because it can precisely define 1 h and then washed repeatedly with distilled water until
the spatial distribution of cells and materials [18,19] . Therefore, a neutral pH was obtained, was repeatedly hydrolyzed
3D bioprinting based on natural hydrogel materials is and centrifuged under acidic conditions to obtain BNC
a promising approach to overcoming the bottleneck of suspension, which was then dialyzed and freeze-dried to
cartilage tissue construction [15,20,21] . However, the insufficient produce BNC powder. GelMA lyophilized powder and
mechanical stability of hydrogel materials makes it difficult LAP were dissolved in Dulbecco phosphate-buffered saline
to ensure the shape of the scaffold in vivo. (DPBS; Gibco, Grand Island, NY, USA) to form 10% w/v

Gelatin methacryloyl (GelMA) is one of the commonly GelMA and 0.25% w/v LAP-based solutions, which
used hydrogels in 3D bioprinting, and it has been effectively were used in the control group. A certain proportion of
applied to cartilage tissue engineering [22-24] . However, due irradiated BNC was added to the GelMA solution to form
to low mechanical properties, its printing fidelity is limited, BNC/GelMA hydrogels with different concentrations
and it is difficult to produce large-scale functional tissue (Table 1).
constructs only using GelMA as the scaffold [25,26] .
2.4. Compression test on hydrogels
To improve the mechanical properties of the scaffold
TM
with natural materials, we propose adding bacterial Silicone elastomer bases (Slygard 184, Dowsil, USA)
nanocellulose (BNC) to GelMA to enhance the mechanical were heated at 70°C for 3 h to make a 10 × 4 mm disk
stability and printability of hydrogels for cartilage mold. A 300 μl hydrogels were added into the mold and
2
bioprinting. BNC is a type of naturally occurring high- irradiated with 405 nm, 30 mW/cm ultraviolet (UV)
molecular-weight polymer with good biocompatibility, light for 10 s to make it completely cross-linked to form a
high Young’s modulus, excellent water retention, and good cylindrical-shaped scaffold (10 × 4 mm) for compression
flexibility [27,28] . In addition, it has good printability and test (n = 7). A biomechanical analyzer (Instron 5967,
shear thinning characteristics, which make it suitable for USA) with a 100 N pressure sensor was used for the
[29]
3D bioprinting . In this study, we explored the appropriate compression tests. A constant compressive strain rate
was maintained at 1 mm/min until 80% of maximal
proportion of BNC in the composite hydrogel and the deformation had been reached. The hydrogels’ Young’s
feasibility of using this bio-ink to regenerate auricular modulus was measured with the slope of the stress-strain
cartilage with high mechanical performance in vivo.
curve set between 10% and 20% strain. A humidifier was
2. Materials and Methods used to maintain the surrounding air humid throughout
the tests.
2.1. Materials

Unless specified otherwise, all chemicals were obtained from Table 1. Hydrogels content concentration
Sigma-Aldrich (St. Louis, USA). GelMA lyophilized powder Groups GelMA (% BNC (% LAP
and lithium phenyl-2,4,6-trimethylbenzoylphosphinate w/v) w/v) (% w/v)
solution (LAP; 2.5% w/v) were procured from SunP 10%GelMA 10 0 0.25
Biotech (Beijing, China). Bacterial cellulose (BC) was +0.075% BNC 10 0.075 0.25
purchased from the Hainan Yide Food Co., Ltd. (Hainan,
China), while Dulbecco’s Modified Eagle Medium +0.225% BNC 10 0.225 0.25
(DMEM), penicillin-streptomycin-neomycin antibiotic +0.375% BNC 10 0.375 0.25
(PSN), and fetal bovine serum (FBS) were purchased from +0.525% BNC 10 0.525 0.25
Gibco (Grand Island, NY, USA). GelMA, Gelatin methacryloyl; BNC, Bacterial nanocellulose

Volume 9 Issue 1 (2023) 132 https://doi.org/10.18063/ijb.v9i1.631