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Xie, et al.
1.1. Limitation of the substances exchange inside to extrude microsphere-based bioink to build specific 3D
[20]
and outside centimeter-scale structure structures . Wang et al. successfully printed structures
with alginate-microspheres-based bioink fabricated by
The spontaneously formed pores by hydrogel materials microfluidic device and realized the tissue regeneration
are always in micro scale. As a result, the distribution in vivo . In the research of Hinton et al., sacrificial
[21]
of nutrients, oxygen, and cell metabolites in centimeter- gelatin microspheres were applied to supporting
scale tissues often has gradients. It is difficult for cells the extruded hydrogel filament, as widely known as
inside large tissues to survive and cell metabolites cannot suspending bioprinting . Furthermore, Jeon et al.
[22]
be smoothly discharged. Therefore, for the normal growth also designed a biodegradable and photocrosslinkable
and functionalization of cells inside large centimeter- microsphere supporting bath which could simultaneously
scale tissues, a bigger and richer network of nutrient permit smooth movement of the printing nozzle and
channels needs to be built. Traditionally, researchers tend maintain the outline of the printed structure .
[23]
to print network scaffold or microfluidic constructure As one of the most popular hydrogel materials
with designed routine program and directly form specific applied in the field of biofabrication, gelatin also has
nutrient channels network. However, the added routine its own unique thermo-sensitive properties in addition
to perform nutrient channels would largely increase the to the special characteristics obtained by modification
printing duration and difficulty, which can bring more (e.g., gelatin methacryloyl [GelMA] has irreversible
uncertainty to the printing feasibility and effectiveness. photocrosslinking characteristics) [24-28] . The response of
At present, some effective methods have been developed. non-modified gelatin precursor solution to temperature
Zhang et al. have published corresponding approach has been verified to be obvious. Unlike GelMA precursor
to form pores inside large-scale structures with phase solution which would form an irreversible and stable
separation principle . Furthermore, in our previous covalent bond after photocrosslinking, the thermo-
[4]
work, irregular gelatin fragment has also been applied to crosslinking process of non-modified gelatin precursor
form denser pores inside the large-scale tissues in vitro . solution at low temperature would be reversible and
[5]
1.2. Difficulties of 3D angiogenesis inside large- mainly depends on hydrogen bond and triple-helix
[29,30]
scale structure structure . When the temperature rises above a certain
temperature, the non-modified gelatin precursor solution
In actual organism, rich vessel network in different scales would convert to solation state. Therefore, by combining
distributes in tissue to provide enough necessary blood the special features of non-modified gelatin and GelMA
supply for the surrounding cells. Thus, to be closer to the and specific microsphere fabrication method, it is expected
tissue in organism, in addition to the tissue subject cells to fabricate the thermo-sensitive microspheres, based on
that should be loaded in centimeter-scale tissues, complex which an innovative bioink system could be designed to
network of 3D blood vessels should be also formed form richer nutrient channels within the centimeter-scale
inside [6-9] . At present, a large number of biofabrication large tissue, followed by angiogenesis and in a suitable
methods have been proposed and constructed the inducing way.
tissue structure of various kinds of loaded living cells. To address the aforementioned challenge in
However, there are only few studies of angiogenesis bioprinting of effective centimeter-scale tissue with
within bioprinted structures, and building complex 3D angiogenesis, this paper will design an innovative bioink
vessel networks within centimeter-scale tissues remains system for the extruding bioprinting scene of vascularized
challenging. centimeter-scale tissue, namely, thermo-sensitive
Microsphere has become one of the most significant sacrificial microsphere-based bioink (TSM-B). As shown
bioprinting structures due to its promising properties, such in Figure 1, the human umbilical vein endothelial cells
as tiny size, high biocompatibility, and special rheological (HUVECs)-laden gelatin microspheres with on-demand
profiles [10,11] . Various effective fabrication methods diameters were electrosprayed with high voltage electric
have been proposed by introducing external energy field and thermo-crosslinked at low temperature as the
to hydrogel precursor solution or solid bulk, namely, auxiliary component (volume ≤50%). GelMA precursor
auxiliary dripping , diphase emulsion [13,14] , lithography solution was prepared as the subject component to further
[12]
technology , and bulk crushing . It has been widely form the tissue structure by irradiation of 405 nm blue
[16]
[15]
used in the field of cell therapy , tissue models , and light. In the preparation of bioink, the electrospraying
[18]
[17]
drug releasing . In recent years, in addition to a kind process of thermo-sensitive sacrificial gelatin microsphere
[19]
of functional unit, microsphere has also been applied (TSM) was analyzed. Based on this, TSM-B containing
as bioink component or bioprinting aided tool, which TSMs with different volume-to-volume ratios and
has been widely known as “secondary printing.” For diameters were prepared and the rheological properties
example, Burdick et al. published a bioprinting method and printability were tested. In the aspect of extrusion
16 International Journal of Bioprinting (2022)–Volume 8, Issue 4