Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI
Article
Laminin-111 Peptides Conjugated to Fibrin Hydrogels Promote Formation of Lumen Containing Parotid Gland Cell Clusters Kihoon Nam, Joshua P Jones, Pedro Lei, Stelios T. Andreadis, and Olga Juliana Baker Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00588 • Publication Date (Web): 05 May 2016 Downloaded from http://pubs.acs.org on May 11, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
Laminin-111 Peptides Conjugated to Fibrin
2
Hydrogels Promote Formation of Lumen Containing
3
Parotid Gland Cell Clusters
4
Kihoon Nam1, Joshua P. Jones2, Pedro Lei3, Stelios T. Andreadis3, 4, 5 and Olga J. Baker1*
5
1
School of Dentistry, 2Department of Bioengineering, The University of Utah, Salt Lake City,
6
UT 84108, USA, 3Department of Chemical and Biological Engineering, 4Department of
7
Biomedical Engineering, School of Engineering and Applied Sciences, 5Center of Bioinformatics
8
and Life Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260,
9
USA.
10
11
ABSTRACT
12
Previous studies showed that mouse submandibular gland cells form three-dimensional
13
structures when grown on Laminin-111 gels. The use of Laminin-111 for tissue bioengineering is
14
complicated due to its lack of purity. In contrast, the use of synthetic peptides derived from
15
Laminin-111 is beneficial due to their high purity and easy manipulation. Two Laminin-111
16
peptides have been identified for salivary cells: the A99 peptide corresponding to the α1 chain
17
from Laminin-111 and the YIGSR peptide corresponding to the β1 chain from Laminin-111,
ACS Paragon Plus Environment
1
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 30
1
which are important for cell adhesion and migration. We created three-dimensional salivary cell
2
clusters using a modified fibrin hydrogel matrix containing immobilized Laminin-111 peptides.
3
Results indicate that the YIGSR peptide improved morphology and lumen formation in rat
4
parotid Par-C10 cells as compared to cells grown on unmodified fibrin hydrogel. Moreover, a
5
combination of both peptides not only allowed the formation of functional three-dimensional
6
salivary cell clusters but also increased attachment and number of cell clusters. In summary, we
7
demonstrated that fibrin hydrogel decorated with Laminin-111 peptides supports attachment and
8
differentiation of salivary gland cell clusters with mature lumens.
9
KEYWORDS: Salivary Gland, Laminin-111 Peptides, Fibrin Hydrogel, Rheology
10 11
INTRODUCTION
12
Proper salivary gland function is critical for oral health. Autoimmune disorders (such as
13
Sjögren’s syndrome), genetic diseases (such as ectodermal dysplasia), and γ-irradiation therapies
14
(for head and neck cancers) cause salivary secretory dysfunction and lead to severe dryness of
15
the oral cavity.1-3 Dry mouth can lead to oral infections, sleep disturbances, oral pain and
16
difficulty in chewing or swallowing food. 4-6 However, the current treatments for dry mouth only
17
provide temporary relief and no tissue engineering approaches are currently available for patients
18
suffering with dry mouth.7 Therefore, it is important to design a clinically safe matrix capable of
19
supporting the growth of a functional salivary gland structure first in vitro and then in vivo
20
applications.
21
Previous studies showed that rat parotid gland (Par-C10) cells and mouse parotid cells form
22
three-dimensional (3D) cell clusters displaying tight junctions (TJ) and agonist-induced secretory
ACS Paragon Plus Environment
2
Page 3 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
responses when grown on Growth Factor-Reduced Matrigel (GFR-MG).8, 9 Matrigel is rich in
2
extracellular matrix (ECM) proteins, with the major components being Laminin-111 (L1) (60%)
3
and collagen IV (30%). Although Matrigel allows the formation of 3D salivary constructs from
4
single acinar cells, clinical application may be hindered by reports of basement membrane
5
matrices supporting tumor growth. Also, it is derived from mouse cancer cells, which could
6
trigger possible immune reactions in humans.10, 11
7
Hydrogels are widely used as a tissue engineering scaffold.12 Among the hydrogels, fibrin
8
hydrogel (FH) is the most extensively studied hydrogel. FHs are water-swollen, polymeric
9
structures that form scaffolds capable of supporting cell viability and differentiation for long
10
periods of time by interaction of cells with fibrin. Fibrin forms a hydrogel at physiological
11
temperatures and contains native arginine-glycine-aspartic acid (RGD) sites that promote cell
12
attachment.13 In addition, several studies demonstrated engineering of FH with conjugated
13
growth factors, genes and recombinant viruses for multiple applications ranging from wound
14
healing, vascular tissue engineering and lentiviral arrays. These include delivery of keratinocyte
15
growth factor (KGF) to promote wound healing14, a peptide-TGF-β1 fusion protein to improve
16
the contractile function, extracellular matrix synthesis and mechanical properties of vascular
17
grafts15,
18
microarray platforms 17-19.
16
, and plasmid DNA and recombinant lentivirus for engineering gene delivery
19
Previous studies also showed that a combination of GFR-MG and FH allowed Par-C10 cells to
20
form 3D structures with a hollow lumen resembling salivary glands. Additionally, critical
21
components within GFR-MG (i.e., a combination of EGF and IGF-1) enhanced salivary gland
22
differentiation when polymerized within FH.8 However, EGF and IGF-1 were found to be
23
incapable of independently producing organized cell clusters.8 More recently, another critical
ACS Paragon Plus Environment
3
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 30
1
component of GFR-MG, L1 was able to produce organized salivary cell clusters using primary
2
mouse submandibular (mSMG) cells.20 Nonetheless, the full L1 sequence may not be suitable for
3
clinical applications, as some protein domains are known to promote tumorigenesis or
4
immunogenic response that may outweigh the potential benefits provided by the whole
5
molecule.21, 22 Conversely, the use of synthetic peptides is relatively simple and inexpensive as
6
compared to animal derived proteins. In addition, controlled densities of peptides can be
7
conjugated to the target material surfaces. Moreover, it is possible to minimize the immune
8
reactivity or pathogen transfer.23 In 1999, Yoshida et al. reported that the YIGSR peptide has an
9
inhibitory effect on tumor growth and an anti-proliferative effect.24 Moreover, the cell
10
attachment and proliferation of mouse fibroblasts were improved on RGD-modified films in
11
2012.25 Therefore, the use of a combination of peptides (RGD and YIGSR) within the L1
12
sequence may provide the synergistic effects while minimizing the risks: a) the A99 (RGD)
13
peptide corresponding to the α1 chain from L1 (important for cell adhesion),
14
YIGSR peptide corresponding to the β1 chain from L1 (also important for cell adhesion,
15
inhibitory effect on tumor growth and migration). 24, 26, 28
25-27
and b) the
16
The goal of this study was to test whether L1 peptides (corresponding to regions that promote
17
intact salivary gland formation, see Table 1) were able to induce lumen formation in Par-C10
18
salivary cell clusters. Our results indicate that cell clusters were formed on the FH modified with
19
YIGSR peptide. Specifically, it improved morphology and lumen formation in rat parotid Par-
20
C10 cells as compared to cells grown on unmodified FH. Moreover, a combination of YIGSR
21
and A99 peptides not only allowed the formation of functional 3D salivary cell clusters, but also
22
increased attachment and cell cluster numbers. In summary, FH decorated with Laminin-111
ACS Paragon Plus Environment
4
Page 5 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
peptides supports attachment and differentiation of salivary gland cell clusters with mature
2
lumens.
3 4
MATERIALS AND METHODS
5
Materials
6
Lyophilized fibrinogen from human plasma was purchased from EMD Millipore (Billerica,
7
MA). Spectra/Por® 7 dialysis membrane (MWCO = 3.5 kDa) was purchased from Spectrum
8
Laboratories (Rancho Dominguez, CA). Whatman™ syringe filter (0.8 µm) was purchased from
9
GE Healthcare Life Sciences (Pittsburgh, PA). Millex syringe filter (0.22 µm) was purchased
10
from Merck Millipore (Billerica, MA, USA). Paraformaldehyde (PFA) was purchased from
11
Baker (Phillipsburg, NJ). Insulin-transferrin-sodium selenite media supplement, retinoic acid,
12
hydrocortisone, gentamicin, epidermal growth factor (EGF) from murine submaxillary gland,
13
DL-dithiothreitol (DTT) and ε-aminocaproic acid (εACA) were purchased from Sigma-Aldrich
14
(St. Louis, MO). Rabbit anti-zonula occludens-1 (ZO-1) antibody was purchased from Invitrogen
15
(Carlsband, CA). Dulbecco's modified eagle medium/nutrient mixture F-12 (1:1) (DMEM/F12
16
(1:1)), fetal bovine serum (FBS), glutamine, Lab-Tek® chambered coverglass (8-well),
17
sulfosuccinimidyl
18
Fluor® 488 conjugated goat anti-rabbit secondary antibody, Alexa Fluor® 568 conjugated
19
phalloidin, TO-PRO®-3 iodide nuclear stain and Fura-2-acetoxymethylester were purchased from
20
Thermo Fisher Scientific (Newington, NH). Peptides were synthesized by University of Utah
21
DNA/Peptide synthesis core facility.
6-(3'-(2-pyridyldithio)propionamido)hexanoate
(Sulfo-LC-SPDP),
Alexa
22 23
Peptide Synthesis
ACS Paragon Plus Environment
5
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 30
1
Two biologically active peptides derived from L1 were synthesized on an ABI431 or ABI433
2
peptide synthesizer using a standard Fmoc solid-phase peptide synthesis as follows: Amino acids
3
were protected at their amino terminus by the Fmoc (9-fluorenylmethoxycarbonyl) group and
4
coupled to the growing chain after activation of the carboxylic acid terminus. Then, the Fmoc
5
group was removed by piperidine treatment and the process repeated. After the peptide was
6
assembled it was removed from the resin by treatment with trifluoroacetic acid (TFA). At the
7
same time, protecting groups on amino acid side chains were removed yielding the crude linear
8
peptide. Finally, one-step purification by reverse-phase HPLC yielded peptides with >95%
9
purity. Two scrambled peptides were synthesized as a control. The overall synthesis scheme of
10
scrambled peptides was the same as described above. All peptides were synthesized with a
11
cysteine and two glycine residues (Cys-Gly-Gly, CGG) at the N-terminus. A cysteine free thiol
12
group was used for coupling with thiol-reactive fibrinogen and the two glycine residues used as a
13
spacer, respectively. A list of these peptides is shown in Table 1. Peptide
Sequence
Molecular mass
CGGALRGDN-amide
860.9
A99
L1 sequence Laminin α1 chain
(RGD)
(1145-1150) Laminin β1 chain
YIGSR
CGGADPGYIGSRGAA-amide
1350.5 (925-936) Scrambled peptide
RAD
CGGALRADN-amide
875.0 for A99 Scrambled peptide
SGIYR
CGGADPGSGIYRGAA-amide
1350.5 for YIGSR
14
Table 1. Sequence of synthetic peptides
ACS Paragon Plus Environment
6
Page 7 of 30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
Peptide Conjugated Fibrinogen
2
Lyophilized fibrinogen was dissolved in 0.1 M Phosphate-Buffered Saline (PBS, pH 7.2, 0.15
3
M NaCl, 1 mM EDTA) and dialyzed using a disposable cellulose membrane (MWCO = 3.5 kDa)
4
overnight. Then, the fibrinogen solution was purified using a 0.8 µm filter. In order to produce a
5
thiol-reactive fibrinogen, 7.2 equivalents of Sulfo-LC-SPDP was added to the purified fibrinogen
6
solution and incubated for 1 h at room temperature. Subsequently, the excess Sulfo-LC-SPDP
7
and its hydrolysis products (N-hydroxysulfosuccinimide, Sulfo-NHS) were removed by dialysis.
8
The level of LC-SPDP-modification was determined by measuring the absorbance of pyridine-
9
2-thione at 343 nm. Briefly, 10 µL of DTT (15 mg/mL) was added to 1 mL of modified
10
fibrinogen. After 15 min of incubation, absorbance at 343 nm was measured and calculated the
11
change in absorbance: ∆A343 = (A343 after DTT) – (A343 before DTT). The level of SPDP
12
modification was calculated using the following equation: =
13 14
∆ 341 × (1) 8080 /
Where 341 kDa reflects the molecular weight of fibrinogen and the value 8080 reflects the extinction coefficient for pyridine-2-thione at 343 nm: 8.08 x 103 M-1 cm-1.29, 30
15
For peptide conjugation, LC-SPDP activated fibrinogen was dissolved in 50 mM PBS (pH 7.2,
16
0.15 M NaCl, 10 mM EDTA). Two equivalents of peptide per 2-pyridyldithiol groups of LC-
17
SPDP-fibrinogen were added to the solution, and the mixture was reacted for 18 h at room
18
temperature. The reaction was monitored by Tin-Layer Chromatography (TLC). Finally, the
19
product was dialyzed against ultrapure water using a dialysis membrane (MWCO = 3.5 kDa) as
20
described above and products were filtered using a 0.22 µm syringe filter from Merck Millipore.
21
Then, peptide conjugated fibrinogen was lyophilized and stored at - 80 ºC until use.
22
The concentration of fibrinogen was calculated using the following equation:
ACS Paragon Plus Environment
7
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
" (/ ) = 1 2
Where ℇ*+,
,
the
extinction
Page 8 of 30
#$% ×
&' " (' (2) ℇ*+,
coefficient
at
280
nm
for
human
fibrinogen,
is
1.51 01 (01.31
3 4
Molecular Weight Determination
5
Static light scattering has been used to determine the size and molecular weight of
6
macromolecules since the 20th century.32 When the light hits a macromolecule (e.g., a polymer or
7
protein), some of the light is absorbed and re-emitted in all directions. The Rayleigh equation (3)
8
describes the relationship between molecular weight and scattered light. By using this equation,
9
the molecular weight of the modified fibrinogen can be determined. 2 3 ℎ 56& ' ∶
10
Where:
11
K is an optical constant
12
C is the sample concentration,
13
θ is the measurement angle,
14
Rθ is the Rayleigh ratio,
15
M is the molecular weight,
16
A2 is the second virial coefficient.
89 1 = ; + 2# 9> (3) 2:
