Protease-Sensitive Hydrogel Biomaterials with Tunable Modulus and

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Article

Protease-Sensitive Hydrogel Biomaterials with Decoupled Modulus and Adhesion Ligand Gradients for 3D Vascular Sprouting Yusheng J. He, Daniel A. Young, Merjem Mededovic, Kevin Li, Chengyue Li, Kenneth Tichauer, David Venerus, and Georgia Papavasiliou Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00519 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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Biomacromolecules

Protease-Sensitive Hydrogel Biomaterials with Tunable Modulus and Adhesion Ligand Gradients for 3D Vascular Sprouting Yusheng J. He,† Daniel A. Young,† Merjem Mededovic,† Kevin Li,† Chengyue Li,† 1

Kenneth Tichauer,† David Venerus,‡ and Georgia Papavasiliou∗,† †Illinois Institute of Technology, Biomedical Engineering Department, Chicago, IL, USA ‡Illinois Institute of Technology, Chemical and Biological Engineering Department, Chicago, IL, USA E-mail: [email protected] Phone: 1-312-567-5959

2

Abstract

3

Biomaterial strategies focused on designing scaffolds with physiologically relevant

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gradients provide a promising means for elucidating 3D vascular cell responses to spatial

5

and temporal variations in matrix properties. In this study we present a photopoly-

6

merization approach, ascending photo-frontal free-radical polymerization, to generate

7

proteolytically degradable hydrogel scaffolds of poly(ethylene) glycol (PEG) with tun-

8

able continuous gradients of (1) elastic modulus (slope of 80 Pa/mm) and uniform

9

immobilized RGD concentration (2.06±0.12 mM) and (2) immobilized concentration of

10

the RGD cell-adhesion peptide ligand (slope of 58.8 µM/mm) and uniform elastic mod-

11

ulus (597± 22Pa). Using a co-culture model of vascular sprouting, scaffolds embedded

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with gradients of elastic modulus induced increases in the number of vascular sprouts

13

in the opposing gradient direction, while RGD gradient scaffolds promoted increases in

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ACS Paragon Plus Environment

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the length of vascular sprouts towards the gradient. Furthermore, increases in vascular

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sprout length were found to be prominent in regions containing higher immobilized

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RGD concentration.

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Introduction

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The inability to promote a stable microvascular network within implantable scaffolds is cur-

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rently a major hurdle to the clinical translation of biomaterial-based strategies in tissue

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engineering. 1 The engineering of large volume, metabolically demanding tissue requires the

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formation of rapid, stable, and functional neovascularization (new blood vessel formation)

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for oxygen and nutrient transport and removal of waste products to support and maintain

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viability, function, and restoration of newly formed tissue. Neovascularization is dependent

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on vascular cell response to multiple spatiotemporal signals including, soluble and immobi-

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lized biochemical factors, as well as gradients of mechanical properties and physical structure

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provided by the 3D extracellular matrix (ECM). 2 Direct measurement of these gradients in

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native ECM is challenging; thus, a precise understanding of the role of different types of gra-

15

dients on vascularized tissue formation is difficult to achieve using animal models. To this

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end, a variety of biomaterial approaches have been developed to create gradient scaffolds to

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elucidate the role of spatiotemporally presented signals on neovascularization of engineered

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tissues in vitro and in vivo.

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Extensive research has focused on creating tissue engineered scaffolds with persistent gra-

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dients of diffusible angiogenic growth factors to promote rapid and stable neovascularization

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within implantable scaffolds. 3–6 Other types of gradients have proven critical to this process,

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including gradients of ECM structural and adhesive proteins, 7–9 peptide composition 10–12 as

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well as gradients in mechanical and structural properties including matrix modulus 13–15 and

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proteolytic degradation rate. 14 While these studies have shown promising results for stimu-

25

lating guided vascular cell response within 3D scaffolds, most have been limited to exploring

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cellular responses to gradients of a single factor (i.e. ECM components or growth factor 2


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Biomacromolecules

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composition). For example, 3D culture models utilizing collagen scaffolds containing concen-

2

tration gradients of hyaluronic acid (HA) have demonstrated directional vascular sprouting

3

from cell spheroids downstream of the HA gradient. 8 Depth-wise gradients of the cell ad-

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hesive RGD peptide ligand have been shown to enhanced cellular infiltration within fibrous

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HA scaffolds towards increasing RGD concentration as compared to the reverse gradient

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orientation. 12 Finally, immobilized gradients of VEGF in porous collagen scaffolds promote

7

chemotactic endothelial cell migration in 3D culture. 4,16 Gradients in mechanical properties

8

and physical structure are present in a variety of native tissues and play a critical role in

9

biological repair processes, such as nerve regeneration and osteochondral tissue repair. 17,18

10

Limited studies, however, have investigated the influence of multiple types of scaffold em-

11

bedded gradients (i.e. mechanical, degradative and biochemical) on 3D cell responses, and

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more specifically on 3D vessel assembly.

13

Various polymerization methods have been developed to create biochemical and mechan-

14

ical continuous gradients within crosslinked hydrogel scaffolds for tissue engineering applica-

15

tions. Among these, two common fabrication approaches have been extensively exploited. In

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one approach, gradient scaffolds are formed using a two-step process. The first step involves

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establishment of a monomer concentration gradient using a fluidic device, such as a source-

18

sink diffusion device, 19 a tree-like microfluidic device, 20,21 or a device involving mixing of

19

prepolymer input streams using multiple syringe pumps. 10,22 This is followed by a second

20

step where the gradient is stabilized upon crosslinking. Since diffusion acts to equilibrate

21

the established concentration gradient during the stabilization step, the final gradient profile

22

may be different from that imposed prior to crosslinking. The second approach involves

23

variation in the degree of crosslinking which results in a gradient of mechanical or structural

24

properties. This has been achieved by exposing prepolymer solutions to variable amount

25

of light during photocrosslinking using sliding 15 or gradient greyscale photomasks. 22 In this

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method, the established crosslinking gradient may simultaneously induce concentration gra-

27

dients of immobilized bifunctional cues (cell adhesion ligand or growth factor composition) 3



1

in addition to spatial variations in mechanical properties.

2

As an alternative to the above-mentioned techniques, frontal polymerization has been

3

utilized to created polymer scaffolds with precise and complex gradient profiles. Frontal

4

polymerization occurs directionally from a localized reaction zone within which an increase

5

in the rate of polymerization occus due an input of energy (i.e. heat, light, or increase in

6

viscosity through the addition of a polymer seed) leading to the establishment of a polymer

7

reaction front. 23–25 The ability to create polymeric fronts using an external flux of long-

8

wavelength light irradiation, photofrontal polymerization, provides a rapid, one-step process

9

for generating scaffolds with precise control in continuous gradient properties for tissue en-

10

gineering applications.

11

In previously published work we developed a frontal photopolymerization approach to

12

create proteolytically degradable poly(ethylene) glycol (PEG) hydrogel scaffolds with tun-

13

able and continuous gradients of mechanical properties and embedded bifunctionality to

14

explore their role on guiding vascular cell response in 3D culture. 14 Localized and controlled

15

perfusion of a photoinitiator within a monomeric mixture resulted in the estabilishment of a

16

localized reaction zone and in the formation of a photopolymerizable front that propoagated

17

through the polymer mixture to form crosslinked gradient hydrogel scaffolds upon exposure

18

to visible light. Using this approach, scaffolds with simultaneous gradients in elastic modu-

19

lus, immobilized RGD concentration, and matrix metalloproteinase (MMP)-sensitivity were

20

created. 14,26,27 Scaffolds presenting simultaneous gradients of these factors induced 3D bi-

21

directional vascular sprout alignment and invasion; however, the coupled gradients made it

22

difficult to clearly elucidate the effects of these factors on 3D neovascularization responses.

23

This motivated the development of an alternative, distinct and robust method to create

24

proteolytically degradable scaffolds with decoupled gradients of initial (i.e. prior material

25

degradation) mechanical properties and immobilized RGD concentration to elucidate their

26

role on vascular sprouting in 3D culture.

27

Here we present a distinct free-radical frontal photopolymerization technique that en4


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Biomacromolecules

1

ables modulation of continuous gradients in crosslink density and/or elastic modulus and

2

immobilized peptide concentration in biomimetic hydrogel scaffolds. This is achieved using

3

a dual programmable syringe pump system to control the composition and flow rates of

4

two distinct prepolymer solutions in the feed stream entering a propagating reaction front

5

in a polymerization reaction chamber exposed to visible light. This allows for rapid sta-

6

bilization of concentration gradients in the precursor solutions enabling precise control of

7

the established gradients within the scaffolds. Using this approach, we created two types

8

of gradient scaffolds: (1) scaffolds with an elastic modulus gradient (slope = 80 Pa/mm)

9

and uniform immobilized concentration of RGD ( 2.06±0.12 mM) and (2) scaffolds with

10

gradients of immobilized RGD concentration (slope = 58.8 µM/mm) and uniform elastic

11

modulus ( 597± 22Pa). In each case we fully characterized the gradient scaffolds in terms of

12

spatial variations in mechanical properties, immobilized RGD concentration, and proteolytic

13

degradation. Finally, we evaluated the role of gradients of elastic modulus and immobilized

14

RGD concentration on 3D vascular sprouting using an in vitro co-culture assay of sprouting

15

angiogenesis. We hypothesized that scaffolds with individual gradients in elastic modulus or

16

Immobilized RGD would stimulate uni-directional vascular sprouting in response to spatial

17

variations of these factors.

18

Experimental

19

Materials

20

Dimethylformamide (DMF), O-benzotriazole-N,N,N’,N’-tetra- methyl-uronium-hexafluoro-

21

phosphate (HBTU), trifluoroacetic acid (TFA), Fmoc-amino acids and the Wang resin were

22

obtained from AAPPTec (Louisville, KY). N,N-Diisopropylethylamine (DIEA), thioanisole,

23

triisopropylsilane (TIS) and diethyl ether were obtained from Fisher Scientific (Hanover

24

Park, IL). Piperidine, phenol, N-vinylpyrrolidone (NVP), triethanolamine (TEA) and eosin

25

Y were obtained from Sigma Aldrich (St. Louis, MO). 5



1

Peptide Synthesis, Design, and Purification

2

To render the scaffolds susceptible to cell mediated proteolysis, an MMP-sensitive peptide

3

sequence (GGVPMS↓ MRGGK, ↓ denotes cleavage point) was synthesized and incorpo-

4

rated within network crosslinks as described below. This peptide sequence is susceptible

5

to cleavage by numerous MMPs including collagenase-1 (MMP-1), gelatinase A (MMP-2)

6

and gelatinase B (MMP-9), which each play an important role in ECM remodeling. 28 The

7

peptide sequence YRGDS was also synthesized and immobilized into the hydrogel matrix

8

to enable cell adhesion. Both peptides were synthesized using solid-phase peptide synthesis

9

based on standard Fmoc chemistry on a Focus Xi automated peptide synthesizer (AAPPTec,

10

Louisville, KY). Amino acid coupling was carried out on a Wang resin in the presence of

11

DIEA and HBTU. The Fmoc group was deprotected with 20% piperidine in DMF. Peptides

12

were cleaved from the resin and deprotected in a TFA cleavage cocktail (90% TFA, 2.5% TIS,

13

2.5% thioanisole, 2.5% (w/v) phenol and 2.5% deionized water) for 2.5 hr, precipitated in cold

14

diethyl ether and purified by reverse-phase high-perfomance liquid chromatography (HPLC).

15

Peptide purity was confirmed by ion trap time-of-flight (IT-TOF) mass spectroscopy. The

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HPLC chromatograms and IT-TOF mass spectra for both peptides are included in the Sup-

17

porting Information (Figures S3 and S4.) Purified peptides were lyophilized and stored at

18

-20 ◦ C until use.

19

Synthesis of Difunctional and Monofunctional Photopolymerizable

20

PEG Macromers

21

Three types of photopolymerizable macromers were synthesized and used to create cell adhe-

22

sive and proteolytically degradable hydrogel scaffolds with tunable homogeneous and gradi-

23

ent properties. The synthesized macromers included: (1) an MMP-sensitive PEG diacrylate

24

crosslinker (MMP-sensitive PEGDA) to render network crosslinks susceptible to proteolytic

25

degradation, (2) an RGD containing PEG monoacrylate macromer (RGD-PEGMA) to in-

6


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Biomacromolecules

1

corporate tethered cell-adhesive peptide ligands in the hydrogel and (3) a non-biofunctional

2

PEG monoacrylate macromer (PEGMA) produced with molecular weight similar to RGD-

3

PEGMA used as a surrogate during polymerization. The PEGMA macromer was used to

4

control hydrogel properties (elastic modulus, degradation and immobilized RGD concentra-

5

tion) without inducing variations in polymerization kinetics during the gradient fabrication

6

process as described below.

7

The MMP-sensitive peptide (GGVPMS↓ MRGGK) was conjugated to Acryl-PEG5000-

8

SVA (Laysan Bio, Arab, AL, MW = 5kDa) in a 2:1 PEG to peptide molar ratio. This resulted

9

in the formation of an MMP-sensitive PEGDA difunctional crosslinker Acryl-PEG5000-

10

GGVPMS↓MRGGK- PEG5000-Acryl (MW ∼11kDa). The cell adhesive peptide YRGDS

11

was conjugated to Acrylate-PEG3400-SVA in a 1:1 PEG to peptide molar ratio yielding

12

a photopolymerizable RGD-PEGMA monofunctional macromer, Acryl-PEG3400-YRGDS

13

(MW ∼ 4kDa). A tyrosine residue (Y) was added to the peptide sequence to track its

14

incorporated concentration in the scaffolds via radiolabeling with

15

Peptides conjugation reactions were carried out in 50mM NaHCO3 solution (pH 8.0) for 4

16

hours while protected from light. The conjugated solution was then dialyzed for 24 hours to

17

removed unreacted reagents, lyophilized, and stored at -20 ◦ C until use.

125

I as described below.

18

The unmodified PEG monoacrylate macromer, PEGMA (MW =5kDa), was synthe-

19

sized through acrylation of monomethoxy-PEG (MW 5kDa) with a 2-fold molar excess of

20

acryloyl chloride relative to free hydroxyls in the presence of trimethylamine in anhydrous

21

dichloromethane under argon overnight in the dark. The resulting product was separated

22

from aqueous byproducts by the addition of 2M K2 CO3 and collection of the organic phase

23

in a separatory funnel. The final PEGMA solution was precipitated in ice cold ether, fil-

24

tered, dried under vacuum overnight, and stored at -20 ◦ C until use. Acrylation efficiency of

25

PEGMA was confirmed to be above 95% using 1 H-NMR.

7



1

Fabrication of Uniform Hydrogel Scaffolds

2

To create scaffolds with tunable gradients of embedded biochemical composition and me-

3

chanical properties, polymerization experiments involving the synthesis of hydrogel scaffolds

4

with uniform properties were first executed to screen formulations that decoupled variation

5

in immobilized RGD concentration and elastic modulus. Uniform hydrogel scaffolds were

6

formed using visible light free-radical photopolymerization as described in our previously

7

published studies. 29 Precursor solutions consisted of 37mM N-vinyl pyrrolidone (an acceler-

8

ator and comonomer), 225mM TEA (co-initiator), 0.05mM eosin Y (photo-sensitizer) and

9

varying concentrations of the photopolymerizable macromers (RGD-PEGMA, PEGMA and

10

MMP-sensitive PEGDA) dissolved in 10mM HEPES buffered saline (HBS; 10 mM HEPES

11

sodium salt and 137 mM NaCl) with pH=7.4 adjusted by addition of HCl. Modulation in

12

modulus were achieved through adjustments in the concentration of MMP-sensitive PEGDA

13

(1.5, 2 and 2.5 mM) with fixed concentration of RGD-PEGMA (2mM). To induce variations

14

in immobilized RGD concentration without imposing variations in scaffold modulus, the

15

molar ratio of RGD-PEGMA to PEGMA (25:75, 50:50 and 75:25) was adjusted while the

16

total concertation of PEG monoacrylate species (RGD-PEGMA and PEGMA) and MMP-

17

sensitive PEGDA macromers was fixed at 2mM and 1.5mM, respectively. Hydrogels were

18

formed by placing 100 µl of the precursor solution into a well of a 96-well plate followed

19

by photopolymerization with visible light ( λ=514nM) for 5 min using an Argon Ion Laser

20

(Coherent, Inc., Santa Clara, CA) at a laser flux of 100 mW/cm2 . After polymerization,

21

hydrogels were rinsed with HBS and swollen to equilibrium prior to quantifying their me-

22

chanical and physical properties and embedded biochemical composition.

23

Fabrication of Gradient Hydrogel Scaffolds

24

A schematic of the gradient hydrogel scaffold frontal photopolymerization fabrication process

25

is shown in Figure 1. Hydrogel scaffolds containing gradinets in modulus or immobilized RGD

26

were created by mixing two distinct precursor solutions (precursor A and precursor B defined 8


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Biomacromolecules

1

in Table 1), consisting of 37mM of NVP (accelerator and comonomer), 225mM TEA (co-

2

initiator), 0.05mM eosin Y (photo-initiator) with varying concentrations of RGD-PEGMA,

3

PEGMA and MMP-sensitive PEGDA dissolved in HBS(pH=7.4). The total macromer com-

4

position was varied in a manner that allowed us to decouple gradients of immobilized RGD

5

concentration from elastic modulus as well as modulate gradient characteristics (i.e. magni-

6

tude and slope of the immobilized RGD gradient). Four gradient scaffolds were created using

7

this approach: (1)scaffolds with an elastic modulus gradient and a uniform immobilized RGD

8

concentration, and scaffolds with uniform elastic modulus containing (2) steep, (3) interme-

9

diate, and (4) shallow RGD gradient slopes. To create scaffolds with an elastic modulus

10

gradient, two distinct precursor concentrations of the MMP-sensitive PEGDA crosslinker

11

were used (2mM for precursor A and 1.5mM for precursor B) containing an equivalent con-

12

centration of RGD-PEGMA in precursor A and precursor B. Sscaffolds containing an RGD

13

gradient and uniform elastic modulus were created by polymerizing precursor A and B with

14

a the concentration of the MMP sensitive PEGDA crosslinker while the concentrations of

15

RGD-PEGMA and PEGMA were varied in each precursor (A and B) to induce variations

16

in gradient slope. Specifically, the RGD-PEGMA precursor concentration was adjusted to

17

2mM, 1mM and 0.5mM in Precursor A and kept at 0mM in Precursor B, while the con-

18

centration of PEGMA was varied from 0mM, 1mM and 1.5mM in Precursor A and kept at

19

2mM in Precursor B targeting steep, intermediate, and shallow RGD gradients, respectively.

20

Table1 summarizes the precursor composition used to create the different types of gradient

21

scaffolds.

22

The targeted gradients were formed as a result of continuous feeding of an ascending pho-

23

topolymerizable reaction front with imposed changes in macromer composition over time.

24

This was achieved by perfusion of distinct precursor solutions whose delivery was controlled

25

using two programmable syringe pumps (NE-1000, New Era Pump System Inc., Farming-

26

dale, NY) shown in Figure1A. The flow rates of each precursor solution were programmed

27

to be linearly increasing form 0 µl/min to 120 µl/min (Precursor A) or decreasing from 120 9



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Table 1: Precursor compositions for gradient hydrogel fabrication Gradient Scaffold Group modulus gradient (uniform RGD)

MMP-sensitive PEGDA (µM)

PEGMA-RGD (µM)

PEGMA (µM)

A

2

2

0

steep RGD Gradient (uniform modulus)

B

1.5

2

0

A

1.5

2

0

intermediate RGD gradient (uniform modulus)

B

1.5

0

2

A

1.5

1

1

shallow RGD gradient (uniform modulus)

B

1.5

0

2

A

1.5

0.5

1.5

B

1.5

2

0

Pump

1

µl/min to 0 µl/min (Precursor B) over a period of 5 min (Figure1B). Subsequently precur-

2

sor solutions were mixed and dripped into a transparent reaction chamber (5mm x 10mm x

3

20mm) and immediately photopolymerized by exposure to visible light ( λ=514 nm) using an

4

Argon Ion Laser (Coherent, Inc., Santa Clara, CA) at a laser flux of 100mW/cm2 . Hydrogel

5

crosslinking occured continuously with the ascending reaction front as precursor solutions

6

were fed to the reaction vessel. While the syringe pumps were programmed to deliver precur-

7

sor for 5 min, the photopolymerization was carried out for a total of 10 min to ensure that

8

every region of the precursor was photopolymerized for at least 5 min. Following photopoly-

9

merization, gradient hydrogels were removed from the reaction vessel, soaked in HBS for 48

10

hours to allow for equilibrium swelling, and then cut into seven 2mm thick sections using an

11

array of razor blades. The first and last sections were discarded (to avoid chracterization of

12

resulting edge effects,) and the remaining five sections were were used to quantify variation

13

in elastic modulus, degradation kinetics and immobilized RGD concentration as described

14

below (Figure1C).

10


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Biomacromolecules

Figure 1: Gradient Hydrogel Fabrication, Charaterization, and Quantification of 3D Vascular Sprouting in Response to Imposed Gradients (A) Schematic of gradient hydrogel fabrication process of photo-frontal polymerization. (B) Flow rate profiles of precursor solutions using two programmable syringe pumps to control their delivery during crosslinking. (C) Schematic representation of hydrogel sectioning and spheroid placement in gradient scaffolds for quantifying spatial variations of physical and mechanical properties, biochemical composition and vascular sprouting. (D) Representation of methodology used to quantify directional vascular sprouting parameters in responses to the embedded gradients. Vascular sprout response towards high RGD or modulus (90◦ ), low RGD or modulus (270◦ ) or in directions perpendicular to the imposed RGD or modulus gradients (0◦ , 180◦ ).

11



1

Fabrication of Fluorecent Gradient Hydrogel Scaffolds

2

A near-infrared fluorophore, indocyanine green (ICG, MW=774.96 g/mol), was physically

3

entrapped in the scaffolds during crosslinking to visualize and confirm the presence of a

4

gradient through spatial variations in fluorescence. ICG was selected because its fluorescence

5

spectrum (Ex 780nm/Em830nm) does not overlap with that of the photo-initiator eosin

6

Y (Ex 524nm/Em544nm) used during the photopolymerization. To create scaffolds with

7

gradients of fluorescence, 0.1 mM ICG was included in precursor A. Concentration gradients

8

of soluble ICG in the scaffolds were imaged immediately after hydrogel formation using a

9

Pearl near-infrared fluorescent and bioluminescent small animal imaging system (LI-COR,

10

Biosciences). Fluorescence intensity gradients were quantified by analyzing the images with

11

MATLAB (R2017a).

12

Quantification of Elastic Modulus Gradients

13

Spatial variations in elastic modulus were quantified using compression experiments. Prior

14

to mechanical testing, hydrogel sections were swollen in HBS for 48 hours with three buffer

15

changes. Scaffolds sections were loaded onto a TA RSA3 mechanical tester (TA Instruments)

16

controlled by TA Orchestrator software. Samples were compressed at a rate of 0.5 mm/min

17

and the elastic modulus of each section was quantified from the slope of the linear region

18

(0.95) of the stress-strain curve (provided in the supporting information)

19

as previously reported. 26 ). The elastic modulus of scaffolds with uniform properties and

20

biochemical composition was similarly quantified.

21

Quantification of Immobilized RGD concentration Gradients

22

Immobilized RGD concentration in scaffolds was quantified by radiolabeling tyrosine residues

23

(Y) with

24

five mg of YRGDS was dissolved in reaction buffer (1x PBS, pH=6.5), combined with 1mCi

125

I prior to gel formation according to previously reported techniques. 26 Briefly,

12


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Biomacromolecules

1

sodium iodine (Perkin-Elmer Health Sciences) in the presence of iodination beads (Pierce)

2

and allowed to react for 15 min. Radiolabeled RGD (125 I-YRGDS) was conjugated to Acryl-

3

PEG3400-SVA as described above to generate a radiolabeled version of the RGD-PEGMA

4

macromer. To track the spatial incorporation of the YRGDS adhesion ligand in gradient

5

scaffolds and in uniform hydrogel controls, radiolabeled Acryl-PEG3400-YRGDS was added

6

to the precursor in an amount not exceeding 20 wt % of the total peptide. In the case of

7

gradient scaffolds, hydrogels were sectioned as described above, and sections were swollen in

8

HBS for 48 hours. Following washing, the radioactivity of each section was measured using

9

a Packard Cobra II gamma counter (Perkin-Elmer Health Sciences). The immobilized RGD

10

concentration in the scaffold [RGD]gel was calculated according to following the equation: R × SA [RGD]gel = m mdried swollen − mdried + ρwater ρP EG

(1)

11

where R is the measured radioactivity, SA is the specific activity (mole/Ci) that was

12

measured and used to convert the measured radioactivity to moles of peptide, mswollen is

13

the equilibrium the swollen weight of the hydrogel, mdried is the weight of the hydrogel in

14

the dried state, ρwater is density of water at room temperature (0.997 g/ml) and ρP EG is the

15

density of PEG (MW=10 kDa) at room temperature (1.3 g/ml).

16

Quantification of Gradients in Proteolytic Scaffold Degradation

17

Spatial variations in scaffold degradation were quantified from gravimetric measurements in

18

the wet weight of hydrogel sections during incubations in interstitial collagnease (Collagenase-

19

1A, MMP-1,Sigma Aldrich) in 10mM HBS with 1mM CaCl2 (pH =7.4) at a fixed concen-

20

tration of 1 µg/mL. Gradient hydrogel sections were incubated in collagenase degradation

21

buffer solutions at 37◦ C over a desired time period, the enzyme solution was removed, and the

22

swollen weight of each section was recorded every hour until complete hydrogel degradation

23

was achieved. 13



1

Evaluation of 3D Vascular Sprout Formation in Response to Scaffold

2

Embedded Gradients

3

The effects of the scaffold embedded gradients on neovascularization was evaluated using a

4

3D co-culture spheroid sprouting assay of sprouting angiogenesis. 14 This assay was chosen

5

because it recapitulates the multicellular and coordinated process of neovascularization in

6

vivo. Cell spheroids composed of 50% human umbilical vascular endothelial cells (HUVECs,

7

Lonza) and 50% human umbilical arterial smooth muscle cells (HUASMCs, Lonza) were

8

formed by suspending both cells types (5000 total cells/well) in culture media containing

9

0.24% (w/v) methyl cellulose in round-bottom, low binding, 96 well-plates and incubated

10

overnight at 37◦ C and 5% CO2 . Following scaffold fabrication, and swelling for 48 hours in

11

EGM-2, cell spheroids were gently placed inside the scaffolds using a micropipette tip. In

12

the case of gradient scaffolds, aggregates were positioned in three distinct regions along the

13

gradient with a maximum of two spheroids per regions spaced at least 8mm apart. Each

14

region presented distinct scaffold properties based on the type of gradient. In the case of

15

scaffolds containing gradients in elastic modulus, spheroids were seeded in high, intermediate

16

,or low modulus regions. In the case of RGD gradient scaffolds, spheroids were seeded in

17

regions containing high, intermediate ,and low immobilized concentrations of the cell adhe-

18

sion ligand (Figure 1). Spheroids were imaged using an Axiovert 200 inverted microscope

19

(Carl Zeiss Inc.) at days 1, 3, 5, and 7. Culture media (EGM-2) was changed every other

20

day. Phase contrast images were acquired from days 1 to 7 and used to quantify spheroid

21

sprout invasion over time in terms of projected spheroid area as well as the directionality of

22

vascular sprouting by measuring the length of individual sprouts at day 7 using Axiovision

23

4.2 image analysis software (Carl Zeiss Inc., GÃűttingen, Germany). The projected area and

24

individual sprout was traced manually for each image with acquired measurements blinded

25

to the samples. Examples of analyzed images are provided in the Supporting Information

26

(Figure S1). Specifically, the image field centered on the spheroid was divided into four

27

directional sectors as shown in Figure 1, where the 90◦ and 270◦ angles represent the direc14


Page 14 of 42

Page 15 of 42 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

tions of increasing and decreasing elastic modulus and RGD concentration along the gradient

2

direction, respectively, while the 0◦ and 180◦ angles correspond to directions perpendicular

3

to the imposed gradients. The analysis of directionality was evaluated by measuring the

4

average length and number of sprouts at each of these angles (Figure 1D).

5

Results

6

Characterization of Uniform Hydrogel Scaffolds with Tunable Elastic

7

Modulus and Immobilized Concentration of RGD Peptide Ligands

8

Prior to creating scaffolds with tunable gradients of mechanical properties and immobilized

9

RGD, studies involving the formation of scaffolds that presented these factors uniformly

10

(isotropic scaffolds) were performed to determine the polymerization conditions for decou-

11

pling these matrix properties. The elastic modulus and immobilized RGD concentration in

12

isotropic hydrogel scaffolds were individually tuned by adjusting the precursor concentration

13

of MMP-sensitive PEGDA, RGD-PEGMA, and PEGMA. Adjustments in the precursor con-

14

centration of MMP-sensitive PEGDA from 1.5, 2, 2.5 mM resulted in variations in elastic

15

modulus of 1.2±0.05 kPa, 3.2±0.02 kPa, and 6.0 ±0.07kPa (Figure 2A) respectively, while

16

the immobilized concentration of RGD remained constant (1.39±0.05 mM, Figure 2B). Ad-

17

justments in the molar ratio of RGD-PEGMA to PEGMA (25:75, 50:50,75:25) while keeping

18

the total concentration of PEG monoacrylates (RGD-PEGMA and PEGMA) fixed (2mM)

19

and the concentration of the MMP-sensitive PEGDA (1.5 mM) constant led to variations in

20

immobilized RGD concentration of 0.33±.02 mM, 0.61±0.04 mM, and 0.87±0.08mM, respec-

21

tively, (Figure 2D) while the scaffold elastic modulus was fixed at 1.2±0.08 Pa (Figure 2C).

22

It is worth noting that the immobilized RGD concentration is lower than the concentration

23

of RGD-PEGMA in the precursor solution because the RGD concentration in the scaffold is

24

measured in the fully swollen state and the hydrogels typically result in a 10% increase in

25

gel volume from the relaxed state. The immobilized RGD concentration in the fully swollen 15


Biomacromolecules

gel is calculated using equation 1, which considers the ratio of moles of RGD incorporated

2

and the volume of the swollen gel which contributes to an observed decrease of incorporated

3

RGD. Furthermore, the presence of multiple monomers (PEGDA, RGD-PEGMA and NVP)

4

in this copolymerization system influence the cross-propagation kinetics which impact the

5

amount of monomers incorporated into the crosslinked network. Regardless, these results

6

indicate that variation in hydrogel modulus and immobilized RGD concentration may be de-

7

coupled by controlling crosslinker concentration and the RGD-PEGMA and PEGMA molar

8

ratio in the precursor.

B

Elastic Modulus (Pa)

8000

*

*

6000

*

4000 2000 0 1.5 mM

2 mM

2.5 mM

PEGDA Concentration

C

D 2000 1500 1000 500 0 25:75

50:50

75:25

PEGMA-RGD to PEGMA Ratio

Immobilized RGD Concentration (mM)

A


1


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 16 of 42

2.0 1.5 1.0 0.5 0.0 1.5 mM

2 mM

2.5 mM

PEGDA Concentration

* 1.0 0.8

*

*

0.6 0.4 0.2 0.0 25:75

50:50

75:25

PEGMA-RGD to PEGMA Ratio

Figure 2: Characterization of isotropic hydrogel scaffolds with decoupled variations in immobilized RGD concentration and elastic modulus. Variations in crosslinker (PEGDA) concentration results in changes in (A) elastic modulus without inducing variations in (B) immobilized RGD concentration. Variations in the ratio of PEGMARGD to PEGMA at fixed PEG monoacrylate concentration does not induce alterations in (C) elastic modulus but results in variation in (D) immobilized RGD concentration in the scaffolds. Asterisk (*) indicates statistical significance with p < 0.05 (n = 4). 16


Page 17 of 42 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

Characterization of Hydrogel Formation via Photo-frontal Polymer-

2

ization

3

To fabricate gradient scaffolds using photo-frontal polymerization, the flow rate of the feed

4

stream was carefully selected to match the propagation speed of the polymerization front

5

while maintaining a constant thin liquid layer of precursor solution on the upper surface

6

of the propagating polymerization front (Supplementary Video 1). When higher flow rates

7

were selected, convection in the liquid precursor resulted in lack of control of desired gra-

8

dients while lower flow rates led to discrete spatial variations in scaffold properties rather

9

than formation of continuous gradients (data not shown). The process of hydrogel formation

10

was characterized by terminating the polymerization at various light exposure times ranging

11

from 1 to 10 min after which the thickness of the formed gel as well as the liquid precursor

12

layer were quantified. Figure 3A displays a series of hydrogels produced by photo-frontal

13

polymerization at various exposure times. The thickness of the hydrogels is observed to

14

increase with time and to eventually reached a plateau after 6 min (Figure 3B). The rate of

15

increase in gel thickness, related to the propagation speed of the polymerization front, follow-

16

ing an exposure time of 6 min was determined to be 2.36±0.08 mm/min, which corresponds

17

to the programmed rate of the rising precursor liquid level (2.4mm/min). The thickness of

18

the liquid precursor layer was calculated by subtracting the gel thickness from the overall

19

thickness. Figure 3C shows that the precursor layer thickness is maintained at 0.65± 0.23

20

mm for first five minutes and is completely depleted after 6 min of light exposure. Overall

21

these results confirm that the selected flow rate in the feed stream (120 µl/min) sustains

22

the propagation of the polymerization front and maintains a thin precursor layer to ensure

23

proper gradient generation.

17


Biomacromolecules

B

C

15

+"#$% ,"#$% -"#$% !".#$% )"#$% *"#$% ("#$% &"#$% '"#$% !"#

%$10

5

0 0

1

2

3

4

5

6

7

8

9

10

Time (min)

Precursor Layer Thickness (mm)

A

Gel Thickness (mm)

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 18 of 42

1.5

1.0

0.5

0.0 0

1

2

3

4

5

6

7

8

9

10

Time (min)

Figure 3: Characterization of photo-frontal polymerization used to create gradient hydrogel scaffolds. (A) Hydrogels formed at various exposure time. (B) Changes in scaffold height as a function of visible light exposure times. (C) Variations in precursor thickness over light exposure time. 1

Visualization of Continuous Gradient Formation during Ascending

2

Photo-frontal Polymerization

3

To verify that continuous gradients could be created using our proposed methodology, the

4

near-infrared fluorophore, ICG, was included in the precursor solution fed by pump A at

5

a concentration of 0.1 mM to generate an ICG gradient ranging from 0 to 0.1 mM along

6

the gradient direction (y-axis). A continuous gradient of fluorescence intensity was visual-

7

ized along the y-axis with minimal variation along the x-axis within the scaffold following

8

photopolymerization as demonstrated in Figure 4A. Acquired images were analyzed using

9

MATLAB to quantify spatial variations in fluorescence intensity within scaffolds. The data

10

in Figure 4B indicate gradual decreases in fluorescence intensity with increasing distance

11

from the region at which precursor was continuously fed to the polymerization front. These

12

results indicate that controlled perfusion of polymeric precursors during free-radical frontal

13

photopolymerization can be used to create continuous and specific gradients within hydrogel

14

scaffolds along the vertical (y-axis) direction.

18


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

A

B

y

Fluorescence Intensity (a.u.)

Page 19 of 42

x

0.6

0.4

0.2

0.0 0.0

0.4

0.8

1.2

Length (mm)

Figure 4: Visualization of gradient formation. (A) Fluorescent image of a hydrogel scaffold embedded with concentration gradient of ICG. (B) Fluorescence intensity change along the gradient direction. 1

Characterization of Hydrogel Scaffolds with Elastic Modulus Gradi-

2

ents

3

Scaffolds with gradients of elastic modulus were created by gradually changing the concen-

4

tration of the MMP-sensitive PEGDA crosslinker in the precursor feed stream while keeping

5

the feed concentration of RGD-PEGMA constant during photopolymerization. As shown

6

in Figure 5A, this resulted in the formation of hydrogel scaffolds with linearly increasing

7

gradients of elastic modulus from 660 Pa to 1460 Pa over a span of 10mm, with a gradient

8

slope estimated to be 80 ± 5 Pa/mm. Quantification of spatial variaiton in the immobilized

9

RGD revealed that the concentration of cell adhesive peptide remained uniform (at 2.06 ±

10

0.12mM) in modulus gradient scaffolds (p=0.996).

11

Gradient hydrogel scaffolds were also rendered proteolytically degradable by including

12

MMP-sensitive peptide sequences within network crosslinks. Since gradients in elastic mod-

13

ulus were induced as a result of gradients in crosslink density due to adjustments in the com-

14

position of the MMP-sensitive PEGDA crosslinker in the precursor, the resultant scaffolds

15

also presented spatial variations in MMP-sensitive crosslinked sites available for enzymatic 19


Biomacromolecules

1

cleavage. This was confirmed by quantifying spatial variations in proteolytic degradation ki-

2

netics obtained by gravimetric measurements in the wet weight of hydrogel sections (2mm)

3

over time in collagenase incubation. As shown in Figure 5B, scaffolds containing gradients

4

of elastic modulus exhibited spatial variations in degradation kinetic profiles. The time

5

required for complete degradation progressively increased with increasing elastic modulus

6

and/or crosslink density. Hydrogel sections with the lowest modulus (0-2 mm) exhibited the

7

shortest time for complete degradation at 4 hours, while sections with intermediate elastic

8

modulus (4-6 mm) required 6 hours for complete degradation, and sections possessing the

9

highest elastic modulus (8-10 mm) degraded the slowest with complete degradation occurring at 8 hours.

B 140

6

Elastic Modulus 1500 4 1000 2 500 0 0

2

4

6

0-2 mm

2000

RGD Concentration

8

120

% Reamining

A


10


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 20 of 42

4-6 mm

80

6-8 mm

60

8-10 mm

40 20

0 12

10

2-4 mm

100

0 0

Length (mm)

2

4

6

8

Time (hrs)

Figure 5: Characterization of hydrogel scaffolds with gradients of elastic modulus and proteolytic degradation. (A) Scaffolds with spatial variations of elastic modulus and uniform immobilized RGD concentration. (B) Spatial variations in hydrogel degradation kinetics in collagenase enzyme solution (1 µg/mL).

11

Characterization of of Hydrogel Scaffolds with Immobilized RGD

12

Concentration Gradients

13

RGD gradient hydrogel scaffolds were created by gradually changing the molar ratio of RGD-

14

PEGMA and PEGMA while keeping the total concentration of the monoacrylate macromers

15

and the crosslinker concentration constant in the precursor solution fed into the reaction 20


Page 21 of 42 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

vessel over time. The immobilized RGD concentration gradient linearly increased 205%

2

from 0.48mM to 0.98 mM over a span of 10 mm, resulting in a gradient slope of 58.8±4.8

3

µM/mm while the elastic modulus remained relatively uniform at 597±22Pa (Figure 6A).

4

Quantification in proteolytic degradation kinetics along the RGD gradient scaffolds revealed

5

similar degradation profiles and time for complete degradation (4 hours, Figure 6B). This

6

was expected since scaffolds of uniform crosslink density possess crosslinks susceptible to

7

proteolytic degradation thereby resulting in uniform degradation kinetics. Based on our

8

observed results with uniform hydrogel scaffolds, changes in the molar ratio of RGD-PEGMA

9

and PEGMA induced RGD concentration gradients without inducing spatial variations in

10

crosslink density and/or elastic modulus and consequently proteolytic degradation kinetics.

11

Characteristics in gradients of biochemical composition, including magnitude and slope,

12

have also been found to be important in controlling cell responses. 11 Therefore, we created

13

hydrogel scaffolds where the slope of the immobilized RGD concentration gradient was var-

14

ied while the modulus remained uniform. This was achieved by adjusting the concentration

15

range of RGD-PEGMA in the precursor feed stream during gradient hydrogel formation, as

16

indicated in Table 1. The RGD-PEGMA concentration ranges were varied to create shallow,

17

intermediate, and steep gradients while the total concentration of PEGMA species was kept

18

constant at 2mM by supplementing with PEGMA surrogate in the precursor to maintain

19

the same crosslink density (and elastic modulus) in each case. As shown in Figure 7, all

20

three RGD gradient hydrogels exhibited a linearly increasing immobilized RGD concentra-

21

tion with distinct slopes at 9.0±2.1 µM/mm (shallow), 32.7±5.6 µM/mm (intermediate) and

22

58.8±4.8 µM/mm (steep). These results demonstrate that the slope of the RGD gradient

23

can be modulated without inducing variations in scaffold elastic modulus and degradation

24

kinetics by varying the RGD-PEGMA precursor concentration while keeping total concentra-

25

tion of total PEG monoacrylate macromers (RGD-PEGMA and PEGMA) constant during

26

polymerization.

21


Page 22 of 42

B 1.5

Elastic Modulus 1500 1.0 1000 0.5 500 0.0 0

2

4

6

140

0-2 mm

120

2-4 mm

100

4-6 mm

2000

RGD Concentration

8

% Reamining

A


8-10 mm

60 40 20

0 12

10

6-8 mm

80

0 0

2

Length (mm)

4

6

8

Time (hrs)

Figure 6: Characterization of hydrogel scaffolds with gradients in immobilized RGD concentration (A) Scaffolds with spatial variations in immobilized RGD concentration and uniform elastic modulus and (B) Uniform degradation kinetics in collagenase incubation (1µg/mL).

Immobilized RGD Concentration (µM)

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

1400

Shallow (9 µM/mm)

1200

Intermediate (34 µM/mm)

1000

Steep (59 µM/mm)

800 600 400 200 0 0

2

4

6

8

10

12

Length (mm)

Figure 7: Characterization of scaffolds with varying slope and magnitude of immobilized RGD concentration.

22


Page 23 of 42 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

Quantification of 3D Vascular Sprout Formation in Gradient Hydro-

2

gel Scaffolds

3

A co-culture model of sprouting angiogenesis was used to investigate the effect of the imposed

4

modulus or immobilized RGD gradients on 3D vascular sprout formation. Cell spheroids

5

were placed into gradient scaffolds at three distinct regions along the imposed gradients. In

6

these specific regions, the cells were exposed to different gradient magnitudes but the same

7

gradient slope. In scaffolds containing a modulus gradient of 80 ±5 Pa/mm and a uniform

8

immobilized RGD concentration ( ∼2mM) spheroids were placed in three regions along the

9

gradient, with each region composed of a distinct modulus; (1) a high elastic modulus region

10

(∼1300Pa), an intermediate modulus region( ∼1000 Pa), and a low modulus region(∼700

11

Pa). Among the three types of RGD gradient scaffolds, vascular cell response was evaluated

12

only in the case of a steep RGD gradient (58.8 ±4.8µM/mm with spheroids placed in regions

13

containing high (∼900 µM), intermediate (∼700 µM), and low (∼500 µM), immobilized RGD

14

concentrations.

15

To evaluate the effects of specific gradients and gradient characteristics on 3D neovascu-

16

larization, we quantified in vitro vascular sprout invasion over time from acquired brightfield

17

images of cell spheroids within the different scaffold regions at days 1, 3, 5, 7 (Figure 8) .

18

As shown in Figure 9, the normalized invasion area in both elastic modulus and RGD gra-

19

dient increased progressively over time with up to a 5-fold increase at day 7. Cell spheroids

20

seeded in the low elastic modulus region exhibited significantly higher sprout invasion area

21

as compared to the high modulus region by days 5 and 7 (Figure 9A). However, no statistical

22

significant differences in invasion areas among the three modulus regions were observed at

23

earlier time points. Differences in invasion area were not found to be statistically signifi-

24

cant when spheroids were placed in different regions (different distances from the imposed

25

gradient) within the RGD gradient scaffolds (Figure 9B).

26

Immobilized or soluble gradients of biochemical factors have been previously reported to

27

result in sprout polarization in the direction towards the imposed gradients. 8,30 To determine 23


Biomacromolecules

RGD Gradient

A Modulus Gradient

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

500µm

Page 24 of 42

D

B

E

C

F

Figure 8: Vascular sprout invasion in gradient hydrogel scaffolds. Day 7 representative brightfield images of vascular sprout formation of spheroids embedded in different regions of scaffolds containing an elastic modulus gradient (A) high, (B) intermediate and (C) low modulus regions, and in scaffolds containing an immobilized RGD gradient (D) high RGD, (E) intermediate RGD, and (F) low RGD concentration regions.

24


Page 25 of 42

B 8

*

High Modulus Intermediate Modulus

*

6

Normalized Invasion Area

A Normalized Invasion Area

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

Low Modulus

4 2 0 Day1

Day3

Day5

8

High RGD Intermediate RGD

6

Low RGD 4 2 0 Day1

Day7

Day3

Day5

Day7

Figure 9: Kinetics in 3D Vascular Sprout Invasion Area. Projected area of vascular sprouts in (A) modulus gradient hydrogels and (B) RGD gradient hydrogels. * indicates statistical significance with p < 0.05 (n = 8). 1

whether the imposed gradients result in guided vascular sprout responses, the average sprout

2

length and sprout number at day 7 were quantified along 4 directions: parallel and towards

3

gradient ( θ= 90◦ ), parallel and opposing gradient ( θ= 270◦ ) and in direction perpendicular

4

to the gradients ( θ= 0◦ and 180◦ ). As shown in Figure 10A, in scaffolds containing gradi-

5

ents of elastic modulus, the length of vascular sprouts was found to be significantly higher

6

in regions of low modulus (691±167 µm) towards the opposing gradient direction (towards

7

low crosslink density, θ= 270 ◦ ), as compared to the sprout length at 90

8

However, in gradient scaffold regions of high and intermediate modulus, no statistical sig-

9

nificant differences in directional vascular sprout length was found to occur. In terms of the

10

number of vascular sprouts, however, significant increases in the opposing gradient direction

11

( θ= 270 ◦ ) were noted as compared to all other directions. This result was found to be

12

consistent in all regions of the scaffold (high, intermediate, and low modulus, Figure 10B).

13

Furthermore, the number of sprouts towards the gradient ( θ = 90 ◦ ) was also found to be

14

significantly higher as compared to the perpendicular directions ( θ=0 ◦ , 180 ◦ ). In summary,

15

these results indicate that gradients in mechanical properties of the matrix influence vascular

16

sprout formation with increased vascular sprout polarization and sprout length towards the

17

opposing gradient direction. These results are consistent with previous studies that indicate

18

that sprout formation and migration decrease with increase in matrix density as additional

25


◦

(494±76 µm).

Biomacromolecules

1

proteolytic activity is required for 3D matrix remodeling. 29–31

A

B

1000

*

800

90° 180°

600

*

20

0°

Number of Sprouts

Mean Sprout Length (µm)

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 26 of 42

270° 400 200 0

15

*

* *

*

*

* *

*

*

0° 90°

*

*

*

180° 270°

10 5 0

High Modulus Region

Intermediate Modulus Region

High Modulus Region

Low Modulus Region

Intermediate Modulus Region

Low Modulus Region

Figure 10: Directional Analysis of Vascular Sprout Parameters in Response to Gradients of Elastic Modulus. Quantification of directional (A) mean sprout length and (B) number of sprouts in gradient scaffold regions containing high, intermediate and low modulus at day 7. * indicates statistical significance with p < 0.05 (n = 8).

2

In contrast to the results of vascular sprouting in scaffolds embedded with gradients of

3

elastic modulus, opposite trends are observed in response to immobilized gradients of RGD

4

concentration. As shown in Figure 11A, when vascular cell spheroids were placed in high

5

RGD containing regions, significant increases in sprout length were found to occur towards

6

the gradient (689±77 µm, θ = 90 ◦ ) as compared to the opposing gradient direction (330±58

7

µm, θ= 270 ◦ ) and the perpendicular directions (448±101 µm , θ =0

8

= 180 ◦ ). Similar finding were observed to occur in scaffold regions of intermediate RGD

9

concentration with significant increases in sprout length towards the imposed gradient (600

10

± 55µm, θ = 90◦ ) as compared to opposing gradient direction (432±50 µm, θ= 270◦ ). In

11

scaffold regions of low RGD concentration, the average sprout length along the different di-

12

rections was not found to be statistically significant. In scaffold regions of intermediate RGD

13

concentration, the number of sprouts was significantly higher in the direction of the gradi-

14

ent (θ= 90◦ ) as compared to the perpendicular directions (θ= 0◦ , and 180◦ ), (Figure 11B).

15

Interestingly, in regions of low immobilized RGD concentration, significant increases in the

16

number of sprouts were observed opposing (θ= 270◦ ) and towards (θ= 90◦ )the gradient as

17

compared to the perpendicular directions (θ=0 ◦ , 180 ◦ ). In summary, these results demon26


◦

, 415±111 µm, θ

Page 27 of 42

1

strate that the imposed RGD gradient results in increases in sprout length and number when

2

spheroids are localized to regions of high and intermediate immobilized RGD concentration.

A

B

1000 800

*

*

*

0°

*

90° 180°

600

* *

20

Number of Sprouts

Mean Sprout Length (µm)

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

270° 400 200

15

*

*

*

*

0° 90° 180° 270°

10 5

0 High RGD Region

Intermediate RGD Region

Low RGD Region

0 High RGD Region

Intermediate RGD Region

Low RGD Region

Figure 11: Directional Analysis of Vascular Sprout Parameters in Response to Gradients of Immobilized RGD Concentration. Quantification of (A) mean sprout length and (B) number of sprouts in gradient scaffold regions containing high, intermediate and low immobilized RGD concentration at day 7. (* indicates statistical significance with p < 0.05 (n = 8).

3

Discussion

4

A primary factor limiting the volume of tissue that can be engineered is the extent to which

5

stable blood vessels can be stimulated to form within the implantable scaffold. An ex-

6

tensive, rapid and stable blood supply is required to the meet mass transport demands

7

in newly formed engineered tissues. A potential solution is to induce rapid directional

8

ingrowth of a vascular network guided by spatiotemporal cues embedded within an im-

9

plantable 3D biodegradable scaffold. Significant progress has been made to elucidate the

10

role of growth factor gradients on neovascularization using microfluidics and biomaterials-

11

based platforms. 6,16,31–33 In addition to growth factors, ECM composition, cell adhesion

12

ligands, as well as mechanical properties of the matrix play an important role in blood ves-

13

sel assembly. 34,35 Gradient hydrogel scaffolds fabricated using natural, synthetic, or hybrid

14

polymer combinations embedded with spatial variations in cell adhesion ligand concentra27



1

tion or mechanical properties have been shown to lead to guided vascular cell migration on

2

2D surfaces. 10,11,13,36 Limited studies, however, have attempted to elucidate the role of these

3

gradients in 3D culture or on vascularized tissue remodeling in vivo. Biomaterial platforms

4

that enable selective and precise control of matrix gradients and gradient characteristics

5

would provide significant insight on individual and combinatorial effects of multiple types of

6

scaffold gradients which are critical for engineering vascularized tissue formation for a variety

7

of damaged tissues.

8

In this study, we present a novel gradient hydrogel scaffold fabrication technique to create

9

proteolytically degradable PEG hydrogel scaffolds with tunable spatial variations in elastic

10

modulus and immobilized RGD concentration. The key feature of this technique is that it

11

allows for precise control of gradient generation due the inherently high rate of polymer-

12

ization of a photopolymerizable ascending reaction front. Unlike conventional crosslinking

13

methods, free-radical frontal photopolymerization enables immediate stabilization of concen-

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tration gradients before diffusion acts to equilibrate the gradients, resulting in precise control

15

of gradient profiles. In our presented gradient scaffold fabrication approach, polymerization

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is localized to the reaction front; a thin layer (

Protease-Sensitive Hydrogel Biomaterials with Tunable Modulus and

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