Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 106 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

References

1.
Woodfield
,
T. B. F.
,
Malda
,
J.
,
de Wijn
,
J.
,
Péters
,
F.
,
Riesle
,
J.
, and
van Blitterswijk
,
C. A.
,
2004
, “
Design of Porous Scaffolds for Cartilage Tissue Engineering Using a Three-Dimensional Fiber-Deposition Technique
,”
Biomaterials
,
25
(
18
), pp.
4149
4161
.
2.
Roh
,
J. D.
,
Nelson
,
G. N.
,
Udelsman
,
B. V.
,
Brennan
,
M. P.
,
Lockhart
,
B.
,
Fong
,
P. M.
,
Lopez-Soler
,
R. I.
,
Saltzman
,
W. M.
, and
Breuer
,
C. K.
,
2007
, “
Centrifugal Seeding Increases Seeding Efficiency and Cellular Distribution of Bone Marrow Stromal Cells in Porous Biodegradable Scaffolds
,”
Tissue Eng.
,
13
(
11
), pp.
2743
2749
.
3.
Bruinink
,
A.
,
Siragusano
,
D.
,
Ettel
,
G.
,
Brandsberg
,
T.
,
Brandsberg
,
F.
,
Petitmermet
,
M.
,
Müller
,
B.
,
Mayer
,
J.
, and
Wintermantel
,
E.
,
2001
, “
The Stiffness of Bone Marrow Cell-Knit Composites is Increased During Mechanical Load
,”
Biomaterials
,
22
(
23
), pp.
3169
3178
.
4.
Sin
,
L. T.
,
Rahmat
,
A. R.
, and
Rahman
,
W. A. W. A.
,
2012
,
Polylactic Acid: PLA Biopolymer Technology and Applications
,
William Andrew
,
New York
.
5.
Karst
,
D.
, and
Yang
,
Y.
,
2006
, “
Molecular Modeling Study of the Resistance of PLA to Hydrolysis Based on the Blending of PLLA and PDLA
,”
Polymer
,
47
(
13
), pp.
4845
4850
.
6.
Zhu
,
Y.
,
Gao
,
C.
,
He
,
T.
,
Liu
,
X.
, and
Shen
,
J.
,
2003
, “
Layer-by-Layer Assembly to Modify Poly(L-Lactic Acid) Surface Toward Improving Its Cytocompatibility to Human Endothelial Cells
,”
Biomacromolecules
,
4
(
2
), pp.
446
452
.
7.
Pan
,
P.
,
Zhu
,
B.
,
Kai
,
W.
,
Dong
,
T.
, and
Inoue
,
Y.
,
2008
, “
Polymorphic Transition in Disordered Poly(L-Lactide) Crystals Induced by Annealing at Elevated Temperatures
,”
Macromolecules
,
41
(
12
), pp.
4296
4304
.
8.
El-Sherbiny
,
I. M.
, and
Yacoub
,
M. H.
,
2013
, “
Hydrogel Scaffolds for Tissue Engineering: Progress and Challenges
,”
Global Cardiol. Sci. Pract.
,
2013
(
3
), pp.
316
342
.
9.
Richardson
,
S. M.
,
Curran
,
J. M.
,
Chen
,
R.
,
Vaughan-Thomas
,
A.
,
Hunt
,
J. A.
,
Freemont
,
A. J.
, and
Hoyland
,
J. A.
,
2006
, “
The Differentiation of Bone Marrow Mesenchymal Stem Cells Into Chondrocyte-Like Cells on Poly-L-Lactic Acid (PLLA) Scaffolds
,”
Biomaterials
,
27
(
22
), pp.
4069
4078
.
10.
Seppälä
,
J.
,
Korhonen
,
H.
,
Hakala
,
R.
, and
Malin
,
M.
,
2011
, “
Photocrosslinkable Polyesters and Poly(Ester Anhydride)S for Biomedical Applications
,”
Macromol. Biosci.
,
11
(
12
), pp.
1647
1652
.
11.
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
.
12.
Meng
,
Q.
,
Heuzey
,
M.-C.
, and
Carreau
,
P. J.
,
2012
, “
Control of Thermal Degradation of Polylactide/Clay Nanocomposites During Melt Processing by Chain Extension Reaction
,”
Polym. Degrad. Stab.
,
97
(
10
), pp.
2010
2020
.
13.
Melchels
,
F. P. W.
,
Feijen
,
J.
, and
Grijpma
,
D. W.
,
2009
, “
A Poly(D, L-lactide) Resin for the Preparation of Tissue Engineering Scaffolds by Stereolithography
,”
Biomaterials
,
30
(
23–24
), pp.
3801
3809
.
14.
Schagemann
,
J. C.
,
Chung
,
H. W.
,
Mrosek
,
E. H.
,
Stone
,
J. J.
,
Fitzsimmons
,
J. S.
,
O’Driscoll
,
S. W.
, and
Reinholz
,
G. G.
,
2010
, “
Poly-Epsilon-Caprolactone/Gel Hybrid Scaffolds for Cartilage Tissue Engineering
,”
J. Biomed. Mater. Res. A
,
93
(
2
), pp.
454
463
.
15.
Endres
,
M.
,
Hutmacher
,
D. W.
,
Salgado
,
A. J.
,
Kaps
,
C.
,
Ringe
,
J.
,
Reis
,
R. L.
,
Sittinger
,
M.
,
Brandwood
,
A.
, and
Schantz
,
J. T.
,
2003
, “
Osteogenic Induction of Human Bone Marrow-Derived Mesenchymal Progenitor Cells in Novel Synthetic Polymer–Hydrogel Matrices
,”
Tissue Eng.
,
9
(
4
), pp.
689
702
.
16.
Gloria
,
A.
,
Causa
,
F.
,
De Santis
,
R.
,
Netti
,
P. A.
, and
Ambrosio
,
L.
,
2007
, “
Dynamic-Mechanical Properties of a Novel Composite Intervertebral Disc Prosthesis
,”
J. Mater. Sci. Mater. Med.
,
18
(
11
), pp.
2159
2165
.
17.
Holloway
,
J. L.
,
Lowman
,
A. M.
, and
Palmese
,
G. R.
,
2010
, “
Mechanical Evaluation of Poly(Vinyl Alcohol)-Based Fibrous Composites as Biomaterials for Meniscal Tissue Replacement
,”
Acta Biomater.
,
6
(
12
), pp.
4716
4724
.
18.
Ovsianikov
,
A.
,
Deiwick
,
A.
,
Van Vlierberghe
,
S.
,
Dubruel
,
P.
,
Möller
,
L.
,
Dräger
,
G.
, and
Chichkov
,
B.
,
2011
, “
Laser Fabrication of Three-Dimensional CAD Scaffolds From Photosensitive Gelatin for Applications in Tissue Engineering
,”
Biomacromolecules
,
12
(
4
), pp.
851
858
.
19.
Van Vlierberghe
,
S.
,
Samal
,
S. K.
, and
Dubruel
,
P.
,
2011
, “
Development of Mechanically Tailored Gelatin–Chondroitin Sulphate Hydrogel Films
,”
Macromol. Symp.
,
309–310
(
1
), pp.
173
181
.
20.
Rodrigues
,
S. C.
,
Salgado
,
C. L.
,
Sahu
,
A.
,
Garcia
,
M. P.
,
Fernandes
,
M. H.
, and
Monteiro
,
F. J.
,
2013
, “
Preparation and Characterization of Collagen–Nanohydroxyapatite Biocomposite Scaffolds by Cryogelation Method for Bone Tissue Engineering Applications
,”
J. Biomed. Mater. Res. A
,
101A
(
4
), pp.
1080
1094
.
21.
Dainiak
,
M. B.
,
Allan
,
I. U.
,
Savina
,
I. N.
,
Cornelio
,
L.
,
James
,
E. S.
,
James
,
S. L.
,
Mikhalovsky
,
S. V.
,
Jungvid
,
H.
, and
Galaev
,
I. Y.
,
2010
, “
Gelatin–Fibrinogen Cryogel Dermal Matrices for Wound Repair: Preparation, Optimisation and In Vitro Study
,”
Biomaterials
,
31
(
1
), pp.
67
76
.
22.
Vishnoi
,
T.
, and
Kumar
,
A.
,
2012
, “
Conducting Cryogel Scaffold as a Potential Biomaterial for Cell Stimulation and Proliferation
,”
J. Mater. Sci. Mater. Med.
,
24
(
2
), pp.
447
459
.
23.
Chang
,
K.-H.
,
Liao
,
H.-T.
, and
Chen
,
J.-P.
,
2013
, “
Preparation and Characterization of Gelatin/Hyaluronic Acid Cryogels for Adipose Tissue Engineering: In Vitro and In Vivo Studies
,”
Acta Biomater.
,
9
(
11
), pp.
9012
9026
.
24.
Schuurman
,
W.
,
Khristov
,
V.
,
Pot
,
M. W.
,
van Weeren
,
P. R.
,
Dhert
,
W. J. A.
, and
Malda
,
J.
,
2011
, “
Bioprinting of Hybrid Tissue Constructs With Tailorable Mechanical Properties
,”
Biofabrication
,
3
(
2
), p.
021001
.
25.
Noe
,
R.
,
Henne
,
A.
, and
Maase
,
M.
,
2003
, “
Acyl- und Bisacylphosphinderivate Acyl and Bisacylphosphine
,” Patent Application, Germany.
26.
Abramoff
,
M. D.
,
Magalhães
,
P. J.
, and
Ram
,
S. J.
,
2004
, “
Image Processing With ImageJ
,”
Biophotonics Int.
,
11
(
7
), pp.
36
42
.
27.
Fedorovich
,
N. E.
,
Oudshoorn
,
M. H.
,
van Geemen
,
D.
,
Hennink
,
W. E.
,
Alblas
,
J.
, and
Dhert
,
W. J. A.
,
2009
, “
The Effect of Photopolymerization on Stem Cells Embedded in Hydrogels
,”
Biomaterials
,
30
(
3
), pp.
344
353
.
28.
Williams
,
C. G.
,
Malik
,
A. N.
,
Kim
,
T. K.
,
Manson
,
P. N.
, and
Elisseeff
,
J. H.
,
2005
, “
Variable Cytocompatibility of Six Cell Lines With Photoinitiators Used for Polymerizing Hydrogels and Cell Encapsulation
,”
Biomaterials
,
26
(
11
), pp.
1211
1218
.
29.
Lee
,
B.-H.
,
Li
,
B.
, and
Guelcher
,
S. A.
,
2012
, “
Gel Microstructure Regulates Proliferation and Differentiation of MC3T3-E1 Cells Encapsulated in Alginate Beads
,”
Acta Biomater.
,
8
(
5
), pp.
1693
1702
.
30.
Nicodemus
,
G. D.
, and
Bryant
,
S. J.
,
2008
, “
Cell Encapsulation in Biodegradable Hydrogels for Tissue Engineering Applications
,”
Tissue Eng. Part B Rev.
,
14
(
2
), pp.
149
165
.
31.
Rehmann
,
M. S.
, and
Kloxin
,
A. M.
,
2013
, “
Tunable and Dynamic Soft Materials for Three-Dimensional Cell Culture
,”
Soft Matter
,
9
(
29
), pp.
6737
6746
.
32.
Kehrer
,
J. P.
,
1993
, “
Free Radicals as Mediators of Tissue Injury and Disease
,”
Crit. Rev. Toxicol.
,
23
(
1
), pp.
21
48
.
33.
Cadet
,
J.
,
Sage
,
E.
, and
Douki
,
T.
,
2005
, “
Ultraviolet Radiation-Mediated Damage to Cellular DNA
,”
Mutat. Res.
,
571
(
1–2
), pp.
3
17
.
34.
Kappes
,
U. P.
,
Luo
,
D.
,
Potter
,
M.
,
Schulmeister
,
K.
, and
Rünger
,
T. M.
,
2006
, “
Short- and Long-Wave UV Light (UVB and UVA) Induce Similar Mutations in Human Skin Cells
,”
J. Invest. Dermatol.
,
126
(
3
), pp.
667
675
.
35.
Fairbanks
,
B. D.
,
Schwartz
,
M. P.
,
Bowman
,
C. N.
, and
Anseth
,
K. S.
,
2009
, “
Photoinitiated Polymerization of PEG-Diacrylate With Lithium Phenyl-2,4,6-Trimethylbenzoylphosphinate: Polymerization Rate and Cytocompatibility
,”
Biomaterials
,
30
(
35
), pp.
6702
6707
.
36.
Cheng
,
J.
,
Jiang
,
S.
,
Gao
,
Y.
,
Wang
,
J.
, and
Sun
,
F.
,
2014
, “
Tuning Gradient Property and Initiating Gradient Photopolymerization of Acrylamide Aqueous Solution of a Hydrosoluble Photocleavage Polysiloxane-Based Photoinitiator
,”
Polym. Adv. Technol.
,
25
(12), pp.
1412
1418
.
37.
Liu
,
M.
,
Li
,
M.-D.
,
Xue
,
J.
, and
Phillips
,
D. L.
,
2014
, “
Time-Resolved Spectroscopic and Density Functional Theory Study of the Photochemistry of Irgacure-2959 in an Aqueous Solution
,”
J. Phys. Chem. A
,
118
(
38
), pp.
8701
8707
.
38.
Bahney
,
C. S.
,
Lujan
,
T. J.
,
Hsu
,
C. W.
,
Bottlang
,
M.
,
West
,
J. L.
, and
Johnstone
,
B.
,
2011
, “
Visible Light Photoinitiation of Mesenchymal Stem Cell-Laden Bioresponsive Hydrogels
,”
Eur. Cell. Mater.
,
22
, pp.
43
55
; Discussion 55.
39.
Hammer
,
J.
,
Han
,
L.-H.
,
Tong
,
X.
, and
Yang
,
F.
,
2014
, “
A Facile Method to Fabricate Hydrogels With Microchannel-Like Porosity for Tissue Engineering
,”
Tissue Eng. Part C Methods
,
20
(
2
), pp.
169
176
.
40.
Gandavarapu
,
N. R.
,
Alge
,
D. L.
, and
Anseth
,
K. S.
,
2014
, “
Osteogenic Differentiation of Human Mesenchymal Stem Cells on α5 Integrin Binding Peptide Hydrogels is Dependent on Substrate Elasticity
,”
Biomater. Sci.
,
2
(
3
), pp.
352
361
.
41.
Chen
,
Y.-C.
,
Su
,
W.-Y.
,
Yang
,
S.-H.
,
Gefen
,
A.
, and
Lin
,
F.-H.
,
2013
, “
In Situ Forming Hydrogels Composed of Oxidized High Molecular Weight Hyaluronic Acid and Gelatin for Nucleus Pulposus Regeneration
,”
Acta Biomater.
,
9
(
2
), pp.
5181
5193
.
42.
Solchaga
,
L. A.
,
Tognana
,
E.
,
Penick
,
K.
,
Baskaran
,
H.
,
Goldberg
,
V. M.
,
Caplan
,
A. I.
, and
Welter
,
J. F.
,
2006
, “
A Rapid Seeding Technique for the Assembly of Large Cell/Scaffold Composite Constructs
,”
Tissue Eng.
,
12
(
7
), pp.
1851
1863
.
43.
Pei
,
M.
,
Solchaga
,
L. A.
,
Seidel
,
J.
,
Zeng
,
L.
,
Vunjak-Novakovic
,
G.
,
Caplan
,
A. I.
, and
Freed
,
L. E.
,
2002
, “
Bioreactors Mediate the Effectiveness of Tissue Engineering Scaffolds
,”
J. Off. Publ. Fed. Am. Soc. Exp. Biol.
,
16
(
12
), pp.
1691
1694
.
44.
Griffon
,
D. J.
,
Sedighi
,
M. R.
,
Schaeffer
,
D. V.
,
Eurell
,
J. A.
, and
Johnson
,
A. L.
,
2006
, “
Chitosan Scaffolds: Interconnective Pore Size and Cartilage Engineering
,”
Acta Biomater.
,
2
(
3
), pp.
313
320
.
45.
Lu
,
L.
,
Peter
,
S. J.
,
Lyman
,
M. D.
,
Lai
,
H. L.
,
Leite
,
S. M.
,
Tamada
,
J. A.
,
Uyama
,
S.
,
Vacanti
,
J. P.
,
Langer
,
R.
, and
Mikos
,
A. G.
,
2000
, “
In Vitro and In Vivo Degradation of Porous Poly(DL-Lactic-Co-Glycolic Acid) Foams
,”
Biomaterials
,
21
(
18
), pp.
1837
1845
.
46.
Yang
,
J.
,
Shi
,
G.
,
Bei
,
J.
,
Wang
,
S.
,
Cao
,
Y.
,
Shang
,
Q.
,
Yang
,
G.
, and
Wang
,
W.
,
2002
, “
Fabrication and Surface Modification of Macroporous Poly(L-Lactic Acid) and Poly(L-Lactic-Co-Glycolic Acid) (70/30) Cell Scaffolds for Human Skin Fibroblast Cell Culture
,”
J. Biomed. Mater. Res.
,
62
(
3
), pp.
438
446
.
47.
Huang
,
H.
,
Ding
,
Y.
,
Sun
,
X. S.
, and
Nguyen
,
T. A.
,
2013
, “
Peptide Hydrogelation and Cell Encapsulation for 3D Culture of MCF-7 Breast Cancer Cells
,”
PLoS ONE
,
8
(
3
), p.
e59482
.
48.
Georges
,
P. C.
, and
Janmey
,
P. A.
,
2005
, “
Cell Type-Specific Response to Growth on Soft Materials
,”
J. Appl. Physiol.
,
98
(
4
), pp.
1547
1553
.
49.
Sunyer
,
R.
,
Jin
,
A. J.
,
Nossal
,
R.
, and
Sackett
,
D. L.
,
2012
, “
Fabrication of Hydrogels with Steep Stiffness Gradients for Studying Cell Mechanical Response
,”
PLoS ONE
,
7
(
10
), p.
e46107
.
50.
Yeung
,
T.
,
Georges
,
P. C.
,
Flanagan
,
L. A.
,
Marg
,
B.
,
Ortiz
,
M.
,
Funaki
,
M.
,
Zahir
,
N.
,
Ming
,
W.
,
Weaver
,
V.
, and
Janmey
,
P. A.
,
2005
, “
Effects of Substrate Stiffness on Cell Morphology, Cytoskeletal Structure, and Adhesion
,”
Cell Motil. Cytoskeleton
,
60
(
1
), pp.
24
34
.
51.
Trappmann
,
B.
, and
Chen
,
C. S.
,
2013
, “
How Cells Sense Extracellular Matrix Stiffness: A Material’s Perspective
,”
Curr. Opin. Biotechnol.
,
24
(
5
), pp.
948
953
.
52.
Marklein
,
R. A.
, and
Burdick
,
J. A.
,
2009
, “
Spatially Controlled Hydrogel Mechanics to Modulate Stem Cell Interactions
,”
Soft Matter
,
6
(
1
), pp.
136
143
.
53.
Chou
,
Y.-F.
,
Dunn
,
J. C. Y.
, and
Wu
,
B. M.
,
2005
, “
In Vitro Response of MC3T3-E1 Preosteoblasts Within Three-Dimensional Apatite-Coated PLGA Scaffolds
,”
J. Biomed. Mater. Res. B Appl. Biomater.
,
75B
(
1
), pp.
81
90
.
54.
St-Pierre
,
J.-P.
,
Gauthier
,
M.
,
Lefebvre
,
L.-P.
, and
Tabrizian
,
M.
,
2005
, “
Three-Dimensional Growth of Differentiating MC3T3-E1 Pre-Osteoblasts on Porous Titanium Scaffolds
,”
Biomaterials
,
26
(
35
), pp.
7319
7328
.
55.
Qiao
,
P.
,
Li
,
F.
,
Dong
,
L.
,
Xu
,
T.
, and
Xie
,
Q.
,
2014
, “
Delivering MC3T3-E1 Cells Into Injectable Calcium Phosphate Cement Through Alginate-Chitosan Microcapsules for Bone Tissue Engineering
,”
J. Zhejiang Univ. Sci. B
,
15
(
4
), pp.
382
392
.
You do not currently have access to this content.