Page 321 - IJB-9-3

International Journal of Bioprinting New fibrillar collagen for 3D printing and bioprinting

collagen properties (viscosity, solubility, water retention, of two native, non-soluble, fibrillar collagen inks. Each
etc.) . Usually, after the extraction, the resultant collagen formulation was specifically designed for different
[21]
collagen solution is acidic (pH 2–3) and its viscosity is 3D printing scopes: one of them (ColA) is intended as a
much lower than for fibrillar collagen . The extraction biomaterial ink (to be 3D printed and subsequently
[22]
method also determines the post-processing steps. For neutralized); the other one (ColN) is specifically designed
instance, acid-soluble collagen will require fibrillogenesis to be easily neutralized before the 3D bioprinting
induction to obtain a self-standing scaffold after printing: process, thus enabling the encapsulation of cells within
transformation of tropocollagen into collagen fibrils and, the mass and so, the creation of a collagen bioink. Both
subsequently, collagen fibers by means of a neutralization formulations have been evaluated for rheology, mechanical
process. This process implies additional optimization, thus properties, and in vitro biocompatibility. The most
being another step inducing batch-to-batch variability. For adequate printing conditions depending on the type of ink
the particular case of 3D bioprinting and TE, the use of and the concentration have also been defined. Moreover,
non-denatured collagen that keeps the fibrillar structure different ColN bioinks were prepared and loaded with
closer to the native structure present in the original tissues, two different types of cells to prove the effective and rapid
would allow a much more direct and rapid use since it neutralization and the high suitability of the formulation
involves a simpler neutralization process without further for 3D bioprinting and TE scopes. These bioinks have
crosslinking to keep a consolidated structure after printing. shown high cellular biocompatibility and proliferation for
12 days in vitro.
The importance of the optimization of extraction,
concentration, neutralization, and collagen printing 2. Materials and methods
conditions has been highlighted in a recently published
study [3,23] . In fact, bioinks with excessive collagen 2.1. Materials
concentration are known to typically compromise cell The Viscofan Fibercoll-FlexA® (ColA) and Fibercoll-
viability due to the harsher printing conditions needed FlexN® (ColN) inks (bovine, collagen type I, 5% w/w)
(high pressures for pneumatic extrusion 3D bioprinting) produced by Viscofan S.A. (Spain) were the base collagens
and the density of the resultant bioink: too dense bioinks used in this study. Each ink was subjected to a different
could jeopardize nutrients and oxygen diffusion within treatment in order to optimize their final performance.
the resultant scaffold, thus hindering cell viability and/or ColA ink is intended to be 3D printed under acidic
proliferation. Attempts to print acid collagen followed by conditions and subsequently neutralized; on the other
in situ neutralization/crosslinking have also been made, hand, ColN is also an acid collagen ink, but in this case, it
but printability issues, low cell viability, or improper is possible to neutralize the mass prior to the 3D printing
crosslinking were reported . Stepanovska et al. have (and 3D bioprinting) process, a process that is not possible
[3]
demonstrated that the optimization of the neutralization with ColA.
process is one of the key factors to be able to work with TRIS buffer, hydrochloric acid solution, and NaOH
highly concentrated collagen bioinks . Additionally, due were obtained from Sigma-Aldrich. Dulbecco’s Modified
[23]
to the low mechanical properties of pure in vitro collagen, Eagle’s medium (DMEM; ATCC, 30-2002), fetal
only few studies have reported the use of collagen as a pure bovine serum (FBS), fetal calf serum (FCS), penicillin/
substance without additives .
[24]
streptomycin and phosphate-buffered saline (PBS, 1×,
At this point, it is plainly clear that collagen is a pH 7.4) solutions were purchased from Fisher Scientific
cornerstone protein for 3D bioprinting and TE due to its (Madrid, Spain). WST-1 cell proliferation assay kit was
good biocompatibility, low immunogenicity, and natural obtained from Roche (Germany), Cell Counting Kit-8
abundance in a wide variety of tissues, making it a versatile (CCK-8) was also provided by Sigma-Aldrich, while Live/
TM
TM
ingredient for TE. However, its exploitation as a biomaterial Dead (Invitrogen ) assay was purchased from Life
is still limited due to the aforementioned drawbacks. The Technologies (Madrid, Spain).
existence of a natural, standardized, and reproducible
collagen source would be particularly useful for the progress 2.2. Ink characterization
of TE as it would minimize the batch-to-batch variability, 2.2.1. Acidic ink preparation
which usually entails the optimization of the concentration Different collagen concentrations were prepared from
and neutralization process of each batch, together with the ColA ink (Viscofan S.A., Spain). The required amounts
3D printing conditions and cellular viability. of ColA ink were mixed with water by means of Luer
lock syringes. Both syringes were connected with a Luer
The present manuscript demonstrates the female-to-female connector (Fisher, USA), and mixed for
reproducibility, reliability, and differential characteristics a total of 40 times. As an example, to prepare a 3% (w/w)

Volume 9 Issue 3 (2023) 313 https://doi.org/10.18063/ijb.712