Manipulation of Extracellular Matrix Remodeling ... - ACS Publications

Subscriber access provided by GUILFORD COLLEGE

Article

Manipulation of Extracellular Matrix Remodeling and Neurite Extension by Mouse Embryonic Stem Cells using IKVAV and LRE Peptide Tethering in Hyaluronic Acid Matrices. T. Hiran Perera, Skyler M Howell, and Laura A. Smith Callahan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00578 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 22, 2019

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 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 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.

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 55 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

Manipulation of Extracellular Matrix Remodeling

2

and Neurite Extension by Mouse Embryonic Stem

3

Cells using IKVAV and LRE Peptide Tethering in

4

Hyaluronic Acid Matrices.

5

T. Hiran Perera, †, ‡ Skyler M. Howell†, ‡ and Laura A Smith Callahan*,†,‡,,┴

6

†Vivian

7

University of Texas Health Science Center at Houston McGovern Medical School,

8

Houston, TX 77030, United States

9

‡Center

L. Smith Department of Neurosurgery, McGovern Medical School at the

for Stem Cell and Regenerative Medicine, Brown Foundation Institute of

10

Molecular Medicine, McGovern Medical School at the University of Texas Health

11

Science Center at Houston, Houston, TX 77030, United States

12

┴Graduate School of Biomedical Sciences, MD Anderson Cancer Center UTHealth,

13

Houston, TX 77030, United States

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 55

1

2

CORRESPONDING AUTHOR

3

* 1825 Pressler Suite 630F, Houston TX 77030 E-mail:

4

[email protected]. Phone: 713-500-3431. Fax: 713-500-2424

5

6

7

8 9

ABSTRACT

Cellular remodeling of the matrix has recently emerged as a key factor in promoting neural differentiation.

Most strategies to manipulate matrix remodeling focus on

10

proteolytically cleavable crosslinkers, leading to changes in tethered biochemical

11

signaling and matrix properties. Using peptides that are not the direct target of enzymatic

12

degradation will likely reduce changes in the matrix and improve control of biological

13

behavior. In this study, laminin derived peptides, IKVAV and LRE, tethered to

14

independent sites in hyaluronic acid matrices using Michael addition and strain-promoted


2

Page 3 of 55 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

azide–alkyne cycloaddition are sufficient to manipulate hyaluronic acid degradation,

2

gelatinase expression and protease expression, while promoting neurite extension

3

through matrix metalloprotease dependent mechanisms in mouse embryonic stem cells

4

encapsulated in hyaluronic acid matrices using an oxidation-reduction reaction initiated

5

gelation. This study provides the foundation for a new strategy to stimulate matrix

6

remodeling that is not dependent on enzymatic cleavage targets.

7 8

KEYWORDS. Hydrogel, Neural Tissue Engineering, Extracellular Matrix Remodeling, Strain-

9

Promoted Azide–Alkyne Cycloaddition, Oxidation-Reduction Reaction, Thiol-ene Chemistry.

10 11


3


1

2

Page 4 of 55

INTRODUCTION

In the central nervous system (CNS), numerous studies indicate the axons of the grafted cells

3

do not express mature markers1-5 and minimal ingrowth of host axons into the graft area has

4

been observed6, which limit graft-host relay formation and functional recovery. The addition of

5

support matrices promote neural maturation and neurological function recovery7-14. However,

6

recent evidence indicates that matrix remodeling is a key component to neural differentiation and

7

axon extension15-20, but matrix development has not traditionally focused on formulation

8

development that modulates matrix remodeling beyond the use of degradable crosslinkers to

9

improve migration through increased porosity15, 21-26. The development of matrices that modulate

10

this remodeling to alter differentiation and integration represents a new strategy to manipulate

11

the behavior of transplanted cells in cell therapy treatments in the CNS.

12

Matrix metalloproteases (MMP) are principally responsible for CNS extracellular matrix

13

(ECM) remodeling27-29, making MMP a promising molecular target to promote neuronal

14

maturation and axon extension in therapeutic applications30-34. Previous studies have shown that

15

blocking MMP activity alters cellular proliferation, plasticity and axon extension35-38, and the

16

structural organization of the tissue39. Little is known about the role of ECM remodeling during

17

axon guidance40, but inhibiting MMP activity leads to defects in axon extension toward and

18

guidance to the target35-38, 41, 42. ECM composition43, 44 and stiffness45, and cytoskeletal

19

tension45, 46 alter MMP expression and activity. Binding of latent MMP to the ECM can lead to

20

cleavage of MMP to their active form44. These established interactions of MMP with the

21

biochemical and mechano-transduction pathways makes utilization of biomaterial matrices to


4

Page 5 of 55 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

manipulated MMP expression and activity a viable approach to modulate cellular ECM

2

remodeling and thus differentiation and axon extension.

3

Hyaluronic acid (HA), a major component ECM in the CNS47, 48, has a long history of use in

4

medical products in the clinic49, 50. Its repeating disaccharide structure provides multiple

5

modification sites that has led to its common use in tissue engineering hydrogels51-57. HA

6

hydrogels have been found to improve angiogenesis, cellular migration and implant-host

7

integration in the CNS58. In vitro studies demonstrate that HA supports neural stem cell growth

8

and differentiation54, 59. The addition of laminin has been found to further enhance these

9

effects60, however laminin is degraded by MMP, which exposes bioactive cryptic sites within the

10

protein61-63. Utilization of laminin derived bioactive peptides allows for greater control of

11

bioactive signal concentration and cellular exposure time compared to laminin molecules.

12

Isoleucine-lysine-valine-alanine-valine (IKVAV), a peptide derived from a cryptic site in

13

laminin61, 64, has a number of biological affects in the CNS65, 66, but does not support the cellular

14

attachment of all neural cells types in all species67-69. Use of additional bioactive signaling

15

peptides with IKVAV to further modulate cellular behavior is becoming common70-73, but it is

16

necessary to test of a wider array of bioactive peptide sequences to achieve the desired effects74.

17

Leucine-arginine-glutamic acid (LRE) is one such potentially complementary bioactive signaling

18

peptide to IKVAV that has not been widely studied, but is being included with IKVAV in

19

complex peptide signaling combinations to emulate cellular niches in the body75, 76. Originally

20

identified in laminin77, LRE occurs in a wide range of CNS proteins77-79 and promotes adhesion,

21

axon guidance, and other cell behaviors not stimulated by IKVAV77, 78, 80-82. Both IKVAV and

22

LRE modulate MMP expression and activation83-85, which can increase neurogenesis, neuronal

23

differentiation, and axon extension16, 86, 87. In the present study, strain-promoted azide–alkyne


5


1

cycloaddition reaction and Michael addition were used to covalently bind IKVAV and LRE at

2

independent concentrations to orthogonally functionalized reactive moieties on the HA

3

backbone54. Both reactions have high efficiency, mild reaction conditions and orthogonality of

4

the reactants88-90. IKVAV and LRE peptide functionalized HA (PEP-HA) was then mixed with

5

methacrylated HA (mHA) and gelled using radical initiated polymerization, which has a rapid

6

gelation rate while overcoming the disadvantages of photopolymerization91, 92. The effects of

7

IKVAV and LRE tethered peptide signaling were then examined on the proliferation,

8

differentiation, neurite extension, and ECM remodeling of encapsulated mouse embryonic stem

9

cells (mESC) encapsulated in the hyaluronic acid hydrogels.

Page 6 of 55

10

MATERIALS and METHODS

11

Materials and analytical instrumentation: HA was purchased from Lifecore (Chaska, MN).

12

1H

13

were referenced to the residual proton. The degree of functionalization was probed by NMR

14

analysis upon normalization to the integrals of the HA backbone methyl peak. Chemicals were

15

purchased from Thermo Fisher Scientific (Waltham, MA) or VWR International (Radnor, PA)

16

unless specified otherwise.

17

Peptide Synthesis: LRE with a C-terminus three glycine linker to a cysteine (LREGGGC, MW

18

= 691.2 gmol-1) and IKVAV with a N-terminus glycine linker to a acryloyl group

19

(Ac(GIKVAV), MW = 640.3 gmol-1) were synthesized with an fluorenylmethyloxycarbonyl

20

(Fmoc)/tert-butyl strategy using a Biotage Initiator+Alstra automated microwave peptide

21

synthesizer (Charlottesville, VA). Briefly, for LREGGGC synthesis Fmoc-S-trityl-L-cysteine 4-

22

alkoxybenzyl alcohol resin (0.5 mmol/g, Chem-Impex, 0.5 mmol scale) was used. The coupling

and 13C NMR spectra (Bruker, Billerica, MA) were recorded at 600, or 150 MHz in D2O and


6

Page 7 of 55 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

of amino acids was carried out with 2.5 mmol of each amino acid, 2.25 mmol of

2

hydroxybenzotriazole activator, and 2.5 mmol of diisopropyl carbodiimide. Final deprotection

3

and cleavage of the peptide from the resin were performed with a mixture of trifluoroacetic acid

4

(TFA) /triisopropylsilane/Water: 95/2.5/2.5 by volume. The resin beads were then filtered and

5

the filtrate was precipitated in cold diethyl ether. The crude peptide was further purified with a

6

Biotage Isolera/Dalton2000 flash purification system in reverse phase with acetonitrile and water

7

containing 0.1% TFA and then lyophilized (Labconco, Kanas City, MO). The peptide was

8

obtained in the pure form as a white powder with a yield of 84% (116 mg). Ultraviolet (254 nm)

9

tracing from the Biotage Isolera/Dalton2000 flash purification system after mass based sorting

10

and mass spectra recorded on an Advion Mass express ESI-MS spectrometer (Ithaca, NY) were

11

used to confirm purity of the peptide. The peptide Ac(GIKVAV) was synthesized and purified in

12

a similar fashion.

13

Synthesis of Dibenzocyclooctyne (DBCO)-Maleimide-LREGGGC complex: A 50mL round

14

bottom flask was charged with an amount of 5mg of Dibenzocyclooctyne (DBCO)-Maleimide,

15

5mg of LREGGGC 10.00mL of PBS and a stir bar and mechanically mixed overnight at room

16

temperature. The final solution was dialyzed (MWCO = 500 Da) with double distilled water

17

(DDW) for 3 days (3 L). DDW was changed every day. The dialyzed product was frozen

18

overnight at 80 C, lyophilized (Labconco, Kansas City, MO) to obtain a white powder, which

19

was analyzed by mass spectrometry (supplemental Figure S1).

20

Dual functionalization of HA with thiol and azide (DIFF-HA): HA was functionalized in a

21

manner similar to that previously described54. The functionalization strategy is shown in Figure

22

1A. Briefly, the pH of a 0.5 percent weight/volume (w/v) solution of sodium HA (average Mw =

23

75 kDa) in DDW was raised to 10 with 1 M sodium hydroxide (NaOH). A two-fold molar excess


7


1

of ethylene sulfide was added, and the HA solution mechanically mixed overnight at room

2

temperature. A 3 cm bed of Celite® 545 (Sigma-Aldrich, St. Louis, MO) was used to filter the

3

solution. Next, a five-fold molar excess of dithiothreitol (DTT) was added and 1 M NaOH was

4

used to adjust the pH to 8.5. The reaction was mechanically stirred overnight at room

5

temperature then 2-(N-morpholino)-ethanesulfonic acid buffer (50mM, pH=4, Sigma-Aldrich,

6

St. Louis, MO), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10 mM, EDC-

7

HCl, Advanced Chemtech, Louisville, KY), N-hydroxysuccinimide (62 mM, NHS, and 11-

8

azido-3,6,9-trioxaundecan-1-amine (88 mM, AA, Sigma-Aldrich, St. Louis, MO) were added to

9

the solution, and the solution was mechanically stirred for 24 hours at room temperature. The

Page 8 of 55

10

solution (total volume = 80 mL) was dialyzed (MWCO = 12 – 14 kDa) against 1 M sodium

11

chloride (1 L) in DDW for 1 day, and then dialyzed against additional DDW for 5 days (3 L)

12

with DDW changed every day. The dialyzed product was frozen at -80C and lyophilized

13

(Labconco, Kansas City, MO) to obtain di-functional HA powder, which was analyzed by 1H-

14

NMR spectroscopy (supplemental Figure S2). The degree of functionalization for thiol groups

15

was determined by calculating the integrated intensity of the corresponding signals with respect

16

to the acetyl-methyl signal at 2.041ppm in the 1H-NMR spectra.

17

Functionalization of DIFF-HA with peptides (PEP-HA): The functionalization strategy is

18

shown in Figure 1A. Briefly, a 50mL round bottom flask was charged with 100mg of HA

19

functionalized with thiol and azide groups (DIFF-HA), 20.00mL of PBS and a stir bar. The pH

20

of the solution was raised to 8.5 using 1 M NaOH. Next, 1.024mg of Ac(GIKVAV) was added

21

and mechanically mixed overnight at room temperature to allow for base catalyzed Michael

22

Addition90, 93, 94. The final solution was dialyzed (MWCO = 10 kDa) DDW for 3 days (3 L) with

23

DDW changed every day. This solution was transferred back to a 50mL round bottom flask and


8

Page 9 of 55 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

an amount of 3mg of DBCO-Maleimide-LREGGGC complex was added and mechanically

2

mixed overnight at room temperature. The final solution was dialyzed (MWCO = 10 kDa) DDW

3

for 3 days (3 L) with DDW changed every day. The dialyzed product was frozen overnight at 80

4

C, lyophilized (Labconco, Kansas City, MO) to obtain a white powder, which was analyzed by

5

1H-NMR

6

peptides were determined by calculating the integrated intensity of the corresponding signals

7

with respect to the acetyl-methyl signal at 2.041ppm in the 1H-NMR spectra (supplementary

8

figure S4).

spectroscopy (supplementary figure S3). The degree of functionalization of thiol and

9

10 11

Figure 1. Schematic of hyaluronic acid functionalization strategies A) dual functionalization

12

with thiol and azide and subsequent tethering of IKVAV and LRE peptides. B) methacrylation.

13

Functionalization of HA with methacrylate (mHA) HA was functionalized with methacrylate

14

groups in a manner similar to previously described54 (Figure 1B). Briefly, a 10-fold molar excess


9


Page 10 of 55

1

of methacrylic anhydride (Sigma-Aldrich, St. Louis, MO) was slowly mixed into a 1.0 % (w/v)

2

solution of sodium hyaluronic acid in DDW at 4°C overnight. The pH of the solution was

3

maintained between 8 and 9 with 10 M NaOH. A 10-fold volume of chilled 95% ethanol was

4

used to precipitate the mHA. The precipitate was then pelleted by centrifugation at 2,000 rpm for

5

10 min. The supernatant was discarded, the pelleted mHA dissolved in DDW, and then dialyzed

6

(MWCO = 8 – 10 kDa) against DDW for 2 days. The DDW was changed several times per day.

7

The solution was then frozen at -20°C overnight and lyophilized to obtain mHA powder. The

8

product was analyzed by 1H-NMR spectroscopy (supplementary figure S5). Based on the

9

acetyl-methyl signal in 1H-NMR spectrum, approximately 31% of the HA backbone was

10

functionalized.

11

Hydrogel Fabrication and characterization: A 1% solution of HA containing 33% DIFF-HA

12

or PEP-HA and 66% mHA was used to from polymer disks (diameter 6.27 mm and height 2.86

13

mm) in molds with 10mM ammonium persulfate (APS)/ tetramethylethylenediamine (TEMED)

14

and allowed to form gels for 25 minutes at room temperature (RT). Samples were characterized

15

for swelling ratio (n=5), water content (n=5), mechanical properties (n=3) and degradation in

16

hyaluronidase (n=3) as previously described54, 92, 95, 96. Briefly, the initial mass of the polymer

17

disks was measured immediately after fabrication, while the polymer disk’s wet mass were

18

obtained after swelling the disk overnight in neural differentiation media at 37°C in 5% CO2.

19

The samples were then lyophilized until completely dry and measured again to obtain the dry

20

mass. The swelling ratio is obtained by normalizing the wet mass to the dry mass and the water

21

content is the difference of between the wet mass and the dry mass97, 98. The Young’s Moduli of

22

the polymer disks were quantified using a TA.XTplus Texture Analyzer (Stable Micro Systems,

23

Surrey, UK) with a ¼ inch spherical probe, which compressed the samples at a rate of 0.01mms-1


10

Page 11 of 55 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

until 10% strain was reached. Young’s modulus (E) was calculated using the following

2

equations99-101:

3

a = R1/2δ1/2

4

σ=

F πa2 3π(1 ― ν2)σ

5

E=

6

Force (F), depth of indentation () and strain () data were recorded to calculate the contact

7

radius (a), indentation stress (), and Young’s modulus (E). R is the radius of the ¼ inch

8

spherical probe.  is the Poisson’s ratio102 (0.5). For degradation studies, the polymer disks were

9

degraded in solutions of 100 U hyaluronidase (Sigma Aldrich) per ml of phosphate buffered

10

saline (PBS, replaced at specified intervals of 1, 3, 6, 12, 24, 48, 72, 96, and 120 hours, and

11

stored at -20 C until analysis) at 37°C on an orbital shaker at 50 rpm. The time for complete

12

degradation of the hydrogel disks is reported. The amount of uronic acid (a degradation product

13

of HA) released during degradation was measured using a previously established carbazole

14

reaction technique103. Briefly, 100 ul of the degradation solution was added to a concentrated

15

sulfuric acid/sodium tetraborate decahydrate (Sigma Aldrich) solution and heated to 100⁰C for

16

10 minutes. A 100 ul of 0.125% carbazole (Sigma Aldrich) in absolute ethanol was added to the

17

solution and heated to 100°C for 15 minutes. The absorbance of the solution at 530 nm was

18

measured with a microplate reader (Tecan Infinite M1000, Maennedorf, Switzerland). The

19

amount of uronic acid was determined using solutions of known concentrations of the 60 kDa

20

HA as a standard. The percent of total uronic acid released at each time point is reported.

20ε


11


Page 12 of 55

1

Determination of hydrogel gelation time: Hydrogel gelation time was determined in a manner

2

similar to that previously described92. Briefly, 1% HA solutions were charged into molds with 5

3

or 10 mM APS/TEMED and incubated at either room temperature or 37°C. Gelation was

4

determined by attempting to lift the hydrogel from the mold with a spatula to see if the solution

5

was liquid (flowing around the spatula) or solid (rigid and lifted by the spatula). The solutions

6

were tested every minute from 1 to 30 min (n=6, supplemental Table S1).

7

Murine embryonic stem cell culture (mESC) and 3D hydrogel composites: D3 mESC (P68)

8

were cultured as previously described54, 92, 104, 105. Briefly, maintenance culture occurred on 0.1%

9

gelatin-coated tissue culture plastic in mESC expansion media (DMEM media supplemented

10

with 10% FBS, 10-4 M b-mercaptoethanol, 1.33 µg/mL HEPES, 0.224 µg/mL L-glutamine, and

11

1000 units/mL human recombinant LIF (Millipore Sigma, Burlington, MA)). For experiments,

12

160 µL of 1% HA solutions containing DIFF-HA or PEP-HA with mHA were mixed with 106

13

mESC per mL and 10mM APS/TEMED were placed in molds and allowed to gel for 20 min.

14

Each sample was placed individually in a well of a 48 well-plates. The 3D hydrogel composites

15

were then cultured in neural differentiation media (80% F-12, 20% Neurobasal medium, 1%

16

penicillin/streptomycin, 0.8X N2, 0.2X B27, 10 mM sodium pyruvate and 2 µM retinoic acid)

17

for 9 days in a humidified incubator (37 ⁰C, 5% CO2). Fresh neural differentiation media (300

18

µL) was then added to each well every other day for the duration of the experiments.

19

Cytotoxicity: mESC were encapsulated in the hydrogels with either 10mM APS/TEMED

20

initiated gelation as described above or a photoinitiated gelation similar to our previous study54.

21

For photoinitiated gelation, APS/TEMED were removed from the matrix solution and replaced

22

with 0.1% Irgacure 2959 (Ciba Specialty Chemicals, Basel, Switzerland). Once charged in the

23

mold, gelation was initiated with 5 min. of exposure to 2.3 mJ/cm ultraviolet A (UVA) light.


12

Page 13 of 55 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

This photoinitated gelation is a more commonly used gelation initiator than the oxidation-

2

reduction reaction by APS/TEMED106-108. Similar to our previous study92, the effects of the

3

radical initiators on cellular viability were assessed.

4

cultures were transferred to a 48 well plate and cultured in mESC expansion media in a

5

humidified incubator (37 ⁰C, 5% CO2). Twenty four hours after cellular encapsulation a

6

LIVE/DEAD Viability/Cytotoxicity Assay (Thermo Fisher) was performed according to

7

manufacturer protocol to determine the cytotoxicity of the gelation catalysts and the hydrogel.

8

Briefly, the 2 mM calcein AM (live stain) and 4 mM ethidium homodimer-1 (dead stain) in 10

9

mM PBS was added to the cell culture media for 30 min. The hydrogels were then immediately

Briefly, after polymerization the 3D

10

imaged using a confocal microscope (Leica, Surrey, Canada) for live cells (494 nm

11

excitation/517 nm emission) and dead cells (528/617 nm). The confocal images were condensed

12

along the z-axis to form a 2D projection image. The number of live cells was calculated as the

13

percentage of the total number of cells (sum of live and dead cells). Over 1000 cells were

14

analyzed per group (n=3).

15

Immunofluorescence: At designated time points, 3D cultures were fixed with 4%

16

paraformaldehyde for 20 minutes. Then, a solution of 0.1% Tween X in PBS was added for 30

17

minutes. The samples were then blocked with 5% donkey serum in PBS for 1 hr to minimize

18

nonspecific antibody binding, incubated with purified rabbit anti-mouse neuron-specific class III

19

β-tubulin (TUJ1, 1:500) antibodies at 4 C overnight and treated with fluorescently conjugated

20

donkey anti-rabbit IgG (1:1000) antibodies 4 C overnight. Finally, cell nuclei were stained with

21

DAPI (1:1000). All the steps were followed by several washes of PBS. All the images were

22

taken using an inverted fluorescence microscope (Nikon TE2000-E, Tokyo, Japan) and (1) the

23

percentage of cells stained positive for TUJ1 and expressing neurites (n=5 with over 200 cells


13


Page 14 of 55

1

analyzed per formulation for day 9) and (2) the length of neurite extension defined as the

2

distance from the termination of TUJ1 staining in projections of a cell body to the closest edge of

3

the nucleus were measured (n=5 samples with >80 axons measured per formulation at day 9).

4

Proliferation and alkaline phosphatase (ALP) assays: Cellular proliferation and ALP

5

expression (a marker of pluripotency) were measured over 9 days of culture in the hydrogels in

6

both mESC expansion media and neural differentiation media as previously described104, 105.

7

DNA and ALP content were determined according to manufacture protocols using Quant-iT Pico

8

Green dsDNA Fluorescence Kit and a p-nitrophenyl phosphate (pNPP) method kit (AnaSpec,

9

Fremont, CA). ALP concentrations were normalized to the DNA content from the same sample

10

(n=3).

11

Cellular degradation of HA and protease and gelatinase expression and activity assays:

12

Samples placed in 500 µL of 0.1% tween X for 15 min and then mechanically broken using a

13

spatula. The solution was centrifuged for 5 min at 237 rpm. The solution was then vortexed,

14

mechanically broken using a spatula, incubated for 15 minutes and centrifuged again for 5 min.

15

at 237 rpm. For each assay, 100 µL of sample was used. Uronic acid release (a degradation

16

produce of HA) was determined using the carbazole reaction technique103 similar to described

17

above. To assess the activity of protease and gelatinase in the cellular lysate, the ability of

18

enzymes to digest casein and gelatin was assessed using self-quenched Fluorescein

19

isothiocyanate (FITC)-Casein and boron-dipyrromethene (BODIPY)-gelatin. Enzymatic

20

digestion of the proteins leads to fluorescence through reduced quenching. To determine total

21

expression of the enzymes, latent MMP in the pro-form was activated using 1 µL of 1mM 4-

22

aminophenylmercuric acetate (AMPA) in the cellular lysate. For determination of protease

23

activity, 250 µL PBS was added to the sample with 0.02 ng/ µL of FITC-casein protease


14

Page 15 of 55 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

substrate and incubated at 37 °C, 50 rpm for 3 hours. The solution was then excited at 485 nm

2

and emission at 530 nm measured. To determine gelatinase expression, 100 µL of 0.5M

3

tris(hydroxymethyl)aminomethane in PBS with 50mM CaCl2, 100 µL DDW water and 0.5 µL of

4

a 1:2 dilution of BODIPY gelatin in PBS was added to the cellular lysate. The samples were

5

incubated at 37°C for 3 hr in the dark and vortexed briefly every hr. The solution was then

6

excited at 490 nm and emission at 520 nm measured with a microplate reader (Tecan Infinite

7

M1000, Maennedorf, Switzerland). Fluorescent intensity was normalized to the DNA content of

8

each sample (n=9).

9

Matrix metalloprotease (MMP) blocking studies: After 3 days of neural differentiation

10

encapsulated in the matrices, cells were found to be TUJ1+ and beginning to polarize in both

11

matrices, but not yet extending significant axons (DIFF-HA=8.2 µm ± 1.0 µm and PEP-HA: 11.2

12

± 1.3 µm, n=3 with >25 axons measured per condition). To assess the effect of matrix

13

metalloproteases on axon extension without affecting initial neuronal commitment, 1mM of

14

broad spectrum MMP blocking agent, GM6001, or Negative control substance for GM6001, N-t-

15

butoxycarbonyl-L-leucyl-L-tryptophan methylamide, were added to the culture media after 3

16

days of encapsulated neural differentiation. Media with blocking agent or control substance

17

were changed every other day. After a total of 9 days of neural differentiation, samples were

18

processed for immunofluorescent analysis as described above (n=5 samples with >90 axons

19

measured per formulation and media condition).

20

Statistics: Quantitative data is presented as mean ± standard deviation of the mean. N-way

21

ANOVA followed by Bonferroni’s multiple comparison post hoc tests was conducted where

22

appropriate using GraphPad Prism version 5.0 for Mac (GraphPad Software, La Jolla, CA). For


15


1

comparisons between two groups, an unpaired two tailed student T-test was used to determine

2

significance. In all analysis, a p-value of less than 0.05 was determined as significant.

3

RESULTS

4

Page 16 of 55

IKVAV functionalization consumed roughly half of the available thiols on the HA backbone

5

(Figure 2) and led to ~0.1 ppm downfield shifts in Peak a in PEP-HA from HA functionalized

6

with thiol and azide groups (DIFF-HA). The degree of IKVAV functionalization and remaining

7

thiolation was determined to be 7 % ± 2 % and 6 % ± 1 %, respectively, in PEP-HA, while

8

thiolation on DIFF-HA was determined to be 15% ± 1 % over three independent

9

functionalization reactions. The degree of LRE functionalization was determined to be 4 % ± 1

10

% (Figure 3) in PEP-HA.

11 12 13

Figure 2. 1H-NMR spectra of IKVAV tethering to hyaluronic acid (HA) through dual functionalization (DIFF-HA) to peptide functionalization (PEP-HA).


16

Page 17 of 55 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 2 3 4

Figure 3. 1H-NMR spectra of LRE tethering to hyaluronic acid (HA) through dual functionalization (DIFF-HA) to peptide functionalization (PEP-HA). The mechanical properties of hydrogels made with DIFF-HA and PEP-HA were found to not

5

vary significantly (Table 1). Both DIFF-HA and PEP-HA hydrogels degraded at a similar rate

6

when exposed to similar amounts of hyaluronidase (Figure 4). The gelation time of the

7

hydrogels could be tailored by altering the concentration of the free radical initiators or the

8

temperature at which the reaction occurred (Supplemental Table S1). The use of APS/TEMED

9

as the free radical initiator lead to a cellular viability similar to that of Irgacure 2959 with UVA,

10

a more commonly used free radical initiator system that is often considered the gold standard, 24

11

hrs after mESC encapsulation (Supplemental Figure S6, Irgacure 2929/UVA: DIFF-HA=

12

94.8% ± 2.8% (average ± standard deviation) & PEP-HA = 92.4% ± 7.4%, APS/TEMED: DIFF-

13

HA= 93.3% ± 5.8% & PEP-HA = 93.6% ± 2.9%).

14 15


17


MATERIAL

DIFF-HA

PEP-HA

1.193 ± 0.024

1.238 ± 0.034

kPa

kPa

Swelling ratio

18.73 ± 0.73

17.47 ± 0.44

Water content

94.65 ± 0.21 %

94.27 ± 0.14 %

PROPERTY

Young’s Modulus

1

Page 18 of 55

Table 1. Summary of Matrix Characterization

2 3 4 5

Figure 4. Degradation of hydrogels with (PEP-HA) and without (DIFF-HA) IKVAV and LRE peptides in 100 U hyaluronidase per mL PBS at 37°C over a 120 hrs. After 9 days of encapsulated culture, the media formulation significantly impacted the

6

cellular content encapsulated in DIFF-HA matrices, but not PEP-HA matrices (Figure 5A).

7

Significantly more cells were found in PEP-HA matrices cultured in neural differentiation media

8

compared to DIFF-HA matrices cultured in the same media. Over the time course, all matrix


18

Page 19 of 55 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

formulations in both media formulations significantly reduced expression of pluripotency marker

2

ALP compared to the seeded cellular population (Figure 5B). Use of differentiation media lead

3

to a significant reduction in ALP expression in cells encapsulated in both DIFF-HA and PEP-HA

4

matrices compared to day 1 levels by day 6 of encapsulation.

5

6

Figure 5. Cellular proliferation (A) and loss of pluripotency marker alkaline phosphatase (B)

7

over 9 days of culture in either pluripotency maintenance or neural differentiation media. A) *

8

indicates p

Manipulation of Extracellular Matrix Remodeling ... - ACS Publications

Recommend Documents