Bioprinted tissue constructs can be produced by microextrusion-based materials processing or coprinting of cells and hydrogel materials. In this paper, a gelatin–alginate hydrogel material formulation is implemented as the bio-ink toward a three-dimensional (3D) cell-laden tissue construct. However, of fundamental importance during the printing process is the interplay between the various parameters that yield the final cell distribution and cell density at different dimensional scales. To investigate these effects, this study advances a multidimensional analytical framework to determine both the spatial variations and temporal evolution of cell distribution and cell density within a bioprinted cell-laden construct. In the one-dimensional (1D) analysis, the cell distribution and single printed fiber shape in the circular cross-sectional view are observed to be dependent on the process temperature and material concentration parameters, along with the initial bio-ink cell densities. This is illustrated by reliable fabrication verified by image line profile analyses of structural fiber prints. Round fiber prints with width 809.5 ± 52.3 μm maintain dispersive cells with a degree of dispersion (Dd) at 96.8 ± 6.27% that can be achieved at high relative material viscosities under low temperature conditions (21 °C) or high material concentrations (10% w/v gelatin). On the other hand, flat fiber prints with width 1102.2 ± 63.66 μm coalesce cells toward the fiber midline with Dd = 76.3 ± 4.58% that can be fabricated at low relative material viscosities under high temperature (24 °C) or low material concentrations (7.5% w/v gelatin). A gradual decrement of Dd (from 80.34% to 52.05%) is observed to be a function of increased initial bio-ink cell densities (1.15 × 106–16.0 × 106 cells/ml). In the two-dimensional (2D) analysis, a printed grid structure yields differential cell distribution, whereby differences in localized cell densities are observed between the strut and cross regions within the printed structure. At low relative viscosities, cells aggregate at the cross regions where two overlapping filaments fuse together, yielding a cell density ratio of 2.06 ± 0.44 between the cross region and the strut region. However, at high relative viscosities, the cell density ratio decreases to 0.96 ± 0.03. In the 3D analysis, the cell density attributed to the different layers is studied as a function of printing time elapsed from the initial bio-ink formulation. Due to identifiable cell sedimentation, the dynamics of cell distribution within the original bio-ink cartridge or material reservoir yield initial quantitative increases in the cell density for the first several printed layers, followed by quantitative decreases in the subsequent printed layers. Finally, during incubation, the evolution of cell density and the emergence of material degradation effects are studied in a time course study. Variable initial cell densities (0.6 × 106 cells/mL, 1.0 × 106 cells/mL, and acellular control group) printed and cross-linked into cell-laden constructs for a 48 h time course study exhibit a time-dependent increase in cell density owing to proliferation within the constructs that are presumed to affect the rate of bio-ink material degradation.

References

References
1.
Murphy
,
S. V.
, and
Atala
,
A.
,
2014
, “
3D Bioprinting of Tissues and Organs
,”
Nat. Biotech.
,
32
(
8
), pp.
773
785
.
2.
Sun
,
R. C.
,
Emami
,
K.
,
Wu
,
H.
, and
Sun
,
W.
,
2010
, “
Biofabrication of a Three-Dimensional Liver Micro-Organ as an In Vitro Drug Metabolism Model
,”
Biofabrication
,
2
(
4
), p.
045004
.
3.
Hollister
,
S. J.
,
2005
, “
Porous Scaffold Design for Tissue Engineering
,”
Nat. Mater.
,
4
(
7
), pp.
518
524
.
4.
Ozbolat
,
I. T.
, and
Yu
,
Y.
,
2013
, “
Bioprinting Toward Organ Fabrication: Challenges and Future Trends
,”
IEEE Trans. Biomed. Eng.
,
60
(
3
), pp.
691
699
.
5.
Billiet
,
T.
,
Vandenhaute
,
M.
,
Schelfhout
,
J.
,
Van Vlierberghe
,
S.
, and
Dubruel
,
P.
,
2012
, “
A Review of Trends and Limitations in Hydrogel-Rapid Prototyping for Tissue Engineering
,”
Biomaterials
,
33
(
26
), pp.
6020
6041
.
6.
Ozbolat
,
I. T.
, and
Hospodiuk
,
M.
,
2016
, “
Current Advances and Future Perspectives in Extrusion-Based Bioprinting
,”
Biomaterials
,
76
, pp.
321
343
.
7.
Chang
,
R.
,
Nam
,
J.
, and
Sun
,
W.
,
2008
, “
Effects of Dispensing Pressure and Nozzle Diameter on Cell Survival From Solid Freeform Fabrication–Based Direct Cell Writing
,”
Tissue Eng. Part A
,
14
(
1
), pp.
41
48
.
8.
Jungst
,
T.
,
Smolan
,
W.
,
Schacht
,
K.
,
Scheibel
,
T.
, and
Groll
,
J.
,
2016
, “
Strategies and Molecular Design Criteria for 3D Printable Hydrogels
,”
Chem. Rev.
,
116
(
3
), pp.
1496
1539
.
9.
Wang
,
X.
,
Yan
,
Y.
,
Pan
,
Y.
,
Xiong
,
Z.
,
Liu
,
H.
,
Cheng
,
J.
,
Liu
,
F.
,
Lin
,
F.
,
Wu
,
R.
, and
Zhang
,
R.
,
2006
, “
Generation of Three-Dimensional Hepatocyte/Gelatin Structures With Rapid Prototyping System
,”
Tissue Eng.
,
12
(
1
), pp.
83
90
.
10.
Wu
,
Z.
,
Su
,
X.
,
Xu
,
Y.
,
Kong
,
B.
,
Sun
,
W.
, and
Mi
,
S.
,
2016
, “
Bioprinting Three-Dimensional Cell-Laden Tissue Constructs With Controllable Degradation
,”
Sci. Rep.
,
6
(
1
), p.
24474
.
11.
Kolesky
,
D. B.
,
Homan
,
K. A.
,
Skylar-Scott
,
M. A.
, and
Lewis
,
J. A.
,
2016
, “
Three-Dimensional Bioprinting of Thick Vascularized Tissues
,”
Proc. Natl. Acad. Sci.
,
113
(
12
), pp.
3179
3184
.
12.
Nichol
,
J. W.
,
Koshy
,
S. T.
,
Bae
,
H.
,
Hwang
,
C. M.
,
Yamanlar
,
S.
, and
Khademhosseini
,
A.
,
2010
, “
Cell-Laden Microengineered Gelatin Methacrylate Hydrogels
,”
Biomaterials
,
31
(
21
), pp.
5536
5544
.
13.
Rowley
,
J. A.
,
Madlambayan
,
G.
, and
Mooney
,
D. J.
,
1999
, “
Alginate Hydrogels as Synthetic Extracellular Matrix Materials
,”
Biomaterials
,
20
(
1
), pp.
45
53
.
14.
Smrdel
,
P.
,
Bogataj
,
M.
,
Podlogar
,
F.
,
Planinšek
,
O.
,
Zajc
,
N.
,
Mazaj
,
M.
,
Kaučič
,
V.
, and
Mrhar
,
A.
,
2006
, “
Characterization of Calcium Alginate Beads Containing Structurally Similar Drugs
,”
Drug Dev. Ind. Pharm.
,
32
(
5
), pp.
623
633
.
15.
Cohen
,
D. L.
,
Tsavaris
,
A. M.
,
Lo
,
W. M.
,
Bonassar
,
L. J.
, and
Lipson
,
H.
,
2008
, “
Improved Quality of 3D-Printed Tissue Constructs through Enhanced Mixing of Alginate Hydrogels
,” Nineteenth Solid Freeform Fabrication Symposium (
SFF
), Austin, TX, Aug. 4–6, pp.
676
685
.https://www.yumpu.com/en/document/view/18890599/improved-quality-of-3d-printed-tissue-constructs-through-cornell-
16.
Lee
,
K. Y.
, and
Mooney
,
D. J.
,
2012
, “
Alginate: Properties and Biomedical Applications
,”
Prog. Polym. Sci.
,
37
(
1
), pp.
106
126
.
17.
Hölzl
,
K.
,
Lin
,
S.
,
Tytgat
,
L.
,
Van Vlierberghe
,
S.
,
Gu
,
L.
, and
Ovsianikov
,
A.
,
2016
, “
Bioink Properties Before, During and After 3D Bioprinting
,”
Biofabrication
,
8
(
3
), p.
032002
.
18.
Marcotte
,
M.
,
Taherian Hoshahili
,
A. R.
, and
Ramaswamy
,
H. S.
,
2001
, “
Rheological Properties of Selected Hydrocolloids as a Function of Concentration and Temperature
,”
Food Res. Int.
,
34
(
8
), pp.
695
703
.
19.
Billiet
,
T.
,
Gevaert
,
E.
,
De Schryver
,
T.
,
Cornelissen
,
M.
, and
Dubruel
,
P.
,
2014
, “
The 3D Printing of Gelatin Methacrylamide Cell-Laden Tissue-Engineered Constructs With High Cell Viability
,”
Biomaterials
,
35
(
1
), pp.
49
62
.
20.
Chung
,
J. H. Y.
,
Naficy
,
S.
,
Yue
,
Z.
,
Kapsa
,
R.
,
Quigley
,
A.
,
Moulton
,
S. E.
, and
Wallace
,
G. G.
,
2013
, “
Bio-Ink Properties and Printability for Extrusion Printing Living Cells
,”
Biomater. Sci.
,
1
(
7
), pp.
763
773
.
21.
He
,
Y.
,
Yang
,
F.
,
Zhao
,
H.
,
Gao
,
Q.
,
Xia
,
B.
, and
Fu
,
J.
,
2016
, “
Research on the Printability of Hydrogels in 3D Bioprinting
,”
Sci. Rep.
,
6
(
1
), p.
29977
.
22.
Skardal
,
A.
,
Zhang
,
J.
, and
Prestwich
,
G. D.
,
2010
, “
Bioprinting Vessel-Like Constructs Using Hyaluronan Hydrogels Crosslinked With Tetrahedral Polyethylene Glycol Tetracrylates
,”
Biomaterials
,
31
(
24
), pp.
6173
6181
.
You do not currently have access to this content.