Page 318 - IJB-9-1

International Journal of Bioprinting Sterilization effect on 3D printed inks

it is essential to address the safety issue of these printed mainly based on glycine, proline, and alanine and has
constructs from the point of view of sterilization, defined as polyampholytes nature. It is widely used in combination
a process that destroys all forms of microbial life, including with other polymers, such as alginate and methylcellulose,
the bacterial spores . If the elimination of the spores as well as with cross-linking agents, such as genipin or
[7]
cannot be ensured, the process is considered disinfection, glutaraldehyde [24,25] . On the other hand, alginate is a
instead of sterilization [7,8] . polysaccharide derived from brown algae and is a widely
Two ways are defined to ensure the sterility of final used biopolymer in biomedical science due to its ability
products: (i) final product sterilizations, which occur at to form hydrogels via chemical crosslinking with carboxyl
[24,26]
the end of the fabrication process and thus is incompatible and hydroxyl groups and divalent cations .
with works involving cell containing inks [9–11] , and (ii) While some authors have reported sterilizing
aseptic fabrication processes . acellular constructs by ultraviolet (UV) light and ethanol
[12]
baths [27,28] , little has been reported on the sterilization of
Depending on the nature of the sterilizing agent,
there are three main types of sterilization, namely inks with the potential to encapsulate cells. Therefore, the
aim of this work is to assess the effect of the commonly
physical, chemical, and physicochemical sterilizations . used sterilization techniques, such as autoclaving [29,30] ,
[13]
Additionally, physical sterilization is divided into three filtration [31,32] , and UV exposure [33,34] , on both the inks and
subtypes, which are the techniques studied in this work: the 3D-printed constructs.
autoclaving, non-ionizing radiation, and filtration .
[13]
Nevertheless, it should be noted that other techniques, In this work, different sterilization methods and
such as ethylene oxide or hydrogen peroxide plasma different media were evaluated with inks prepared with
sterilizations, were not included in this study because of gelatin and sodium alginate (SA). It is worth highlighting
their cost and security requirements [7,8,14–16] . that little has been reported on the sterilization of inks
Since cellular load inks are usually prepared with and sometimes the sterilization process used was not
cell culture media, which could modify the properties even mentioned. This work focuses on investigating
of fresh inks, the effects of those media should also be the effect of sterilization on the rheological behavior of
considered. For example, Dulbecco’s Modified Eagle’s inks, an important factor that influences the stability
Medium (DMEM), a medium used in mammalian cell and final properties of 3D-printed construct. In order to
cultures, contains a small concentration of Ca 2+[17] , which select the 3D printing parameters, rheological analysis
may interact with some biopolymers, such as alginate, was carried out, and the 3D-printed constructs were
promoting partial crosslinking and inducing a change on analyzed by Fourier transform infrared (FTIR) analysis
rheological properties [17,18] . to determine the effect of the sterilization method and
media employed.
Nowadays, there is no specific regulation with regard
to sterilization of printed biological products, although
several pharmacopoeias have begun to focus on this 2. Materials and methods
problem . To date, there are few scientific articles that 2.1. Materials
[19]
address the influence of sterilization and how this affects Gelatin (GEL; type A, bloom 300) and SA from brown
the different materials that can be printed [7,13,19–21] . Some algae were purchased from Sigma-Aldrich (Madrid,
sterilization techniques have been proposed and most of Spain). Dulbecco’s Modified Eagle’s Medium (DMEM),
them concluded that sterilization techniques should be GlutaMAX (Gibco™ F12-GlutaMax supplemented) and
TM
studied in relation to the material used [7,13,19–21] . In this phosphate-buffered saline (PBS) were purchased from
regard, recent works were focused on the assessment of Thermo Fisher (Madrid, Spain).
sterilization methods on gelatin methacryloyl (GelMA)
and alginate inks [21,22] . Furthermore, some studies 2.2. Sample preparation
have analyzed the effect of the treatment duration on Solutions with 7 wt % SA and 8 wt % GEL were prepared
rheological and physicochemical properties of alginate at 50°C under magnetic stirring for all the sterilization
inks, concluding that shorter autoclave cycles have smaller methods and culture media under study. Three sterilization
influence on the original inks . In this work, a mixture methods (autoclaving, UV radiation, and filtration) and
[23]
of two biopolymers, gelatin and alginate, was used to three media (DMEM, PBS, and water as control) were
prepare the ink for three-dimensional (3D) printing. On compared. Water was used as a control to assess the effect
the one hand, gelatin is a natural protein derived from of the electrolytes present in the culture media. Autoclaved
the hydrolysis of collagen and it is widely used because (AUTO) samples were dissolved in different media and
of its non-toxicity and biodegradability . Gelatin is then autoclaved (Selecta, ST DRY PV III 25) at 121°C and
[24]

Volume 9 Issue 1 (2023) 310 https://doi.org/10.18063/ijb.v9i1.645