Supervisors: J. Chang (University of California San Diego)

S. Varghese (University of California San Diego)

D. van der Schaft (Eindhoven University of Technology)

Improving the properties of PEGDA

hydrogels by adding clay particles, without

reducing the biocompatibility

October 2009

A.C.C. van Spreeuwel

BMTE 09.36

2

Contents

Abstract 3

1. Introduction 4

2. Materials and Methods 6 2.1 Hydrogel preparation 6

2.2 Swelling properties 6

2.3 Mechanical properties 6

2.4 Cell encapsulation 6

2.5 Live/dead assay 6

2.6 Biochemical assays 7

3. Results 8 3.1 Hydrogel preparation 8

3.2 Swelling properties 8

3.3 Mechanical properties 9

3.4 Cell encapsulation 10

3.5 Live/dead assay 10

3.6 Biochemical assays 11

4 Discussion 12

5 Conclusion 14

References 15

Supplements 16 A. Protocol: Making a nanocomposite hydrogel 16

B. Protocol: Live/dead assay of cells encapsulated in a hydrogel construct 17

C. Protocol: Papain digestion 18

D. Protocol: DNA assay 19

E. Protocol: GAG assay 21

3

Abstract This study focuses on improving the properties of PEGDA hydrogels by adding clay particles to these

hydrogels, without reducing the biocompatibility. PEGDA hydrogels with different molecular weight and an

increasing amount of clay were made by using photo polymerization. The gels were tested for their swelling

properties, mechanical properties and biocompatibility. Swelling measurements were performed to investigate

the effects of molecular weight, clay concentration and osmotic pressure on the swelling properties of the

gels. Compressive and tensile tests were done to asses the mechanical properties of the hydrogels. Bovine

chondrocytes were encapsulated in these gels to test the cell viability and the production of extracellular

matrix proteins, which was done with a live/dead assay and biochemical assays. The addition of clay particles

did not interfere with the photo polymerization. The results demonstrated that adding up to 5% clay to the

PEGDA hydrogels resulted in better or similar swelling behavior. The overall mechanical properties were

improved by adding up to 10% clay. Furthermore, results showed that adding clay to the PEGDA hydrogel

did not decrease the biocompatibility.

4

1. Introduction

Hydrogels consist of insoluble polymer networks and large quantities of water thus mimicking the soft tissues

in the body. They are suitable for various biomedical applications, such as drug delivery [1], wound healing

[2] and tissue engineering [3]. But the main disadvantage of hydrogels is that they are usually very soft and

fragile due to the high amount of water in a swollen hydrogel.

Hydrogels made of poly(ethylene glycol) (PEG) have been studied extensively for biomedical applications

because of their hydrophilicity and biocompatibility [1]. PEG hydrogels contain a lot of water which

promotes the diffusion of nutrients inside the gel. The biological tissue mimicking properties make these

suitable for cell encapsulation and cell culture. High cell viability is observed for various types of cells

encapsulated in PEG hydrogels [4].

This material has a lot of properties that mimic the natural environment of cartilage. Cartilage consists of at

least 65% water and the chondrocytes receive their nutrients through diffusion because there are no blood

vessels in the tissue, so these hydrogels should be very suitable to culture chondrocytes. If the hydrogels are

strong enough, they might be very useful to make tissue engineered cartilage that can be used to repair or

replace damaged cartilage.

Functional groups, such as the RGD peptide can be introduced to improve the functionality of the PEG

hydrogel [1]. To fabricate PEG hydrogels, acrylated PEG derivates are often used because they can be easily

polymerized using photopolymerization. Photopolymerization uses UV-light to polymerize the monomers in

the hydrogel solution. The exposure to UV-light causes photo initiators to generate free radicals that initiate

the polymerization to form the PEG network. A PEG hydrogel can be formed by exposure to UV-light for as

short as 3 minutes [5]. Due to the short-term UV exposure, photopolymerization is generally considered as a

safe method to encapsulate cells [4]. Another advantage of in situ polymerization is that specific shapes can

be tailor-made to fit exactly into the tissue defects needed to be repaired.

The soft and fragile hydrogels have limited wide-spread biomedical applications. It would be useful to

improve the mechanical properties of these hydrogels. Recently, a new class of nanocomposite (NC)

hydrogels has been developed by in-situ polymerization of acrylate or acrylamide monomers with clay

nanoparticles. These new NC hydrogels exhibit robust mechanical properties compared to hydrogels prepared

by conventional polymerization in which chemical crosslinkers are usually employed [6, 7, 8]. Although the

interactions between polymer and clay particles are not fully elucidated yet, it is believed that the clay

particles can act as multi-functional cross-linkers and can form a physical gel within the polymer network [9].

This study focuses on NC hydrogels that are made of PEG diacrylate (PEGDA) and clay. A nanocomposite

hydrogel made of PEGDA and clay is expected to have improved properties compared to the conventional

pure PEGDA hydrogel. The mechanical properties should improve because of the interactions between PEG

and clay. This NC hydrogel should have good cell viability because of the PEG and should be easy to

polymerize by using photopolymerization. To investigate the effects of the addition of clay nanoparticles to

PEGDA hydrogels, hydrogels with different molecular weights PEGDA and increasing amounts of clay were

tested for their swelling and mechanical properties as well as their biocompatibility.

To investigate the effect of clay particles on the swelling properties, pure PEGDA and NC hydrogels were

swollen in phosphate buffered saline (PBS) and in solutions with an increasing amount of NaCl. To

investigate the mechanical properties of these new hydrogels, compressive and tensile tests were performed

on both pure PEGDA and NC hydrogels.

5

Biocompatibility is crucial for the use of NC hydrogels in tissue engineering applications [1,4]. The cells

should be able to proliferate and live in this new material. To test the biocompatibility, chondrocytes isolated

from bovine cartilage were encapsulated in the NC and pure PEGDA hydrogels. Live/dead assays were used

to determine the cell viability at different time intervals after the encapsulation. Biochemical assays including

DNA assay and glycosaminoglycans (GAGs) assay were conducted to assess the ability of encapsulated

chondrocytes to proliferate and produce cartilage specific extracellular matrix.

6

2. Materials and Methods

2.1 Hydrogel preparation Pure PEGDA hydrogels were prepared by dissolving PEGDA 3.4k, 10k or 20k (10% w/v) in water with

Irgacure 2959 (0.05% w/v) as a photoinitiator. Nanocomposite hydrogels were prepared by mixing an

aqueous solution of Laponite XLS (Southern Clay Products, Texas) with a stock solution of PEGDA,

followed by adding the photoinitiator. The solutions were poured into the molds followed by subjecting them

to UV exposure for five minutes to form the hydrogels. Caps of 1.5 ml tubes were used as a mold for the

cylindrical hydrogels prepared for compression and swelling tests as well as for the cell encapsulation. The

sides of the caps were precut before pouring the solution into the molds, to make sure the gel can be removed

easily after photopolymerization. Dog-bone shaped molds with a total length of 62 mm were used to prepare

hydrogels for the tensile tests. The thin middle part of the dog-bone shaped mold had a length of 18 mm, a

width of 1.3 mm and a thickness of 1.1 mm.

2.2 Swelling properties To examine the swelling properties of the hydrogels, two different tests were performed. In the first test, the

effect of PEG molecular weight (3.4, 10 and 20k) and clay concentration (0%, 2.5%, 5% and 10%) on

hydrogel swelling were studied. After photopolymerization, hydrogels were washed in deionized water for

two days to remove unreacted monomers and photoinitiator while frequently changing the water. Next, the

hydrogels were dried until a constant dry weight was reached. The gels were swollen in PBS and their weight

was measured at various time intervals until the gels reached a constant weight. In the second study, the effect

of osmotic pressure on the swelling of hydrogels was studied by incubating hydrogels in different

concentrations of NaCl solutions (0M, 0.05M, 0.15M, 0.3M, 0.5M and 2M). The hydrogels were allowed to

swell for two days to reach equilibrium after which their weight was measured. All tests were done for three

constructs per group. Swelling ratio was determined by dividing the swollen weight by the dry weight.

2.3 Mechanical properties To study the effects of clay particles on the mechanical properties of the nanocomposite hydrogels, PEGDA

10k hydrogels with 0%, 2.5%, 5% and 10% clay were used for compression tests and gels with 0% and 10%

clay were used for tensile tests. Gels were swollen in PBS for a few days after preparation before doing the

tensile test. Compression tests were done at a rate of 10mm/min and for the tensile tests a rate of 20 mm/min

was used. At least three samples per group were used for these tests. Maximum strain and stress at the

moment of fracture was recorded and compression or elastic modulus was calculated from the first 10%

compression or elongation. Toughness was calculated by taking the area under the stress-strain curve.

2.4 Cell encapsulation Chondrocytes were isolated from bovine cartilage and used for cell encapsulation in PEGDA 10k hydrogels

with 0%, 2.5% and 5% clay. For the cell encapsulation, PEGDA was dissolved in PBS instead of water. The

chondrocytes were centrifuged and then resuspended in the PEGDA-Laponite solution with photoinitiator

(0.05%). The solution was poured into the molds and then polymerized by exposure to UV light for five

minutes. Constructs were cultured in chondrogenic medium for up to three weeks. Constructs to be used for

biochemical assays were frozen at 1, 7 and 21 days of culture.

2.5 Live/dead assay To compare the viability of cells encapsulated in pure PEGDA and NC hydrogels, a live/dead assay was done

using the live/dead viability cytotoxicity kit (Molecular Probes). This kit contains Calcein-AM to stain the

living cells and ethidium homodimer-1 to stain the dead cells. Fluorescence microscopy was used to visualize

the green living and the red dead cells. Before staining, the hydrogels were cut into several thin slices to

promote a good staining. Live/dead assay was done right after 0 and 7 days of culture.

7

2.6 Biochemical assays Cell constructs were lyophilized for two days and then crushed in papain buffer by using a pellet pestle.

Samples were incubated in papain buffer for 16 hours at 60°C. After vortexing and centrifuging, the

supernatant can be used for different biochemical assays. DNA content was determined using the Quant-iT

PicoGreen dsDNA Kit (Invitrogen). Fluorescence of the samples was measured using a plate reader and

compared to lambda DNA standard, provided by the kit. GAGs content of the digested samples was

determined by using 1,9-Dimethylmethylene blue (DMMB) dye. Samples were measured at a wavelength of

525nm using a spectrophotometer and compared to a standard of chondroitin sulfate (CS). Only one construct

for each group was used to carry out the assay for day 1 and a minimum of three constructs for each group

was used for the biochemical assays of day 7 and 21.

8

3. Results

3.1 Hydrogel preparation Hydrogels with different concentrations of clay were made by using photopolymerization. It was noticed that

a small amount of the solution formed physical gels right after mixing clay with PEGDA (10k and 20k). The

formation of physical gels could be attributed to the physical interactions between clay particles and PEGDA

polymer. These physical gels can be re-dissolved into solution by vortexing vigorously for at least 20 minutes.

Furthermore, it was observed that the presence of clay particles did not interfere with the UV-mediated

photopolymerization of the acrylate groups on PEG. Hydrogels with clay particles (up to 10%) could be

polymerized easily by exposing them to UV-light for five minutes, just like the pure PEGDA hydrogels.

3.2 Swelling properties Hydrogels with increasing amounts of clay were swollen in PBS and their weight was measured at different

time intervals until they reached equilibrium. It was observed that gels with 2.5% clay have higher swelling

ratio than gels without clay particles (fig 1). However, adding more clay decreases the swelling ratio. Gels

with 10% clay have similar or even lower swelling ratio than pure PEGDA hydrogels. This effect is more

obvious for hydrogels with a higher molecular weight.

To see if the clay particles affect the osmotic pressure, hydrogels with 0% and 10% clay were also swollen in

solutions with increasing molarity. Figure 2 shows that increasing the NaCl concentration decreases the

swelling for both the pure PEGDA hydrogels and the gels with 10% clay, but the effect is more significant for

the latter.

0.0 2.5 5.0 7.5 10.00

10

20

30 PEG3400

PEG10000

PEG20000

Clay Content (%)

Sw

ell

ing

Ra

tio

(%

)

Figure 1: Swelling ratio as a function of clay content Figure 2: Percentage of weight change as a function of

for different molecular weights. NaCl concentration for PEGDA 10k hydrogels with

0% and 10% clay.

0.0 0.5 1.0 1.5 2.0 40

60

80

100

120 Clay/PEG

PEG

NaCl concentration (M)

We

igh

t c

ha

ng

e (

%)

9

0.0

2.5

5.0

10.0

0

1000

2000

3000

4000

Clay Content (%)

Fra

ctu

re S

tress (

kP

a)

0.0

2.5

5.0

10.0

0.0

0.1

0.2

0.3

Clay Content (%)

To

ug

hn

ess (

MJ/m

3)

A

B

0.0

2.5

5.0

10.0

0

1000

2000

3000

4000

Clay Content (%)

Fra

ctu

re S

tress (

kP

a)

0.0

2.5

5.0

10.0

0.0

0.1

0.2

0.3

Clay Content (%)

To

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ess (

MJ/m

3)

A

B

PEG

Cla

y/PEG

0

50

100

150

200

250

Fra

ctu

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tress (

kP

a)

PEG

Cla

y/PEG

0

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300

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Ela

sti

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ou

gh

ness

(kJ/m

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tress (

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0

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Ela

sti

c T

ou

gh

ness

(kJ/m

3)

A

B

Figure 3: Fracture stress (A) and toughness (B) from Figure 4: Fracture strain (A) and toughness (B) from

compression tests for PEGDA 10k hydrogels with tensile tests for PEGDA 10k hydrogels with 0 and

increasing clay content. 10% clay.

3.3 Mechanical properties PEGDA 10k hydrogels with 0%, 2.5%, 5% and 10% clay were used for compression tests. Fracture stress (fig

3A) increased dramatically with increasing clay content. Fracture stress for the NC hydrogel with 10% clay

was more than ten times higher than the fracture stress for the pure PEGDA hydrogel. The same trend was

observed for the toughness (fig 3B). Fracture strain and compression modulus both showed the same

increasing trend but it was less obvious.

PEGDA 10k hydrogels with 0% and 10% clay were used for tensile tests. Figure 4A shows that the fracture

stress was more than three times higher for the gel with 10% clay compared to the pure PEGDA hydrogel.

Toughness (fig 4B) was also strongly increased for the hydrogel with 10% clay compared to the pure PEG

hydrogel. The same trend was observed for both the fracture strain and the elastic modulus.

10

3.4 Cell encapsulation Bovine chondrocytes were encapsulated in PEGDA 10k hydrogels with 0%, 2.5% and 5% clay. Cell

encapsulation was not performed in PEGDA 10k hydrogels with 10% clay due to the difficulty of mixing the

cells with this highly viscous polymer/10% clay solution.

3.5 Live/dead assay To check the viability of the chondrocytes, a live/dead assay was done after 0 and 7 days of encapsulation.

Figure 5 seems to show slightly more dead cells for the pure PEGDA hydrogels compared to the hydrogels

with 5% clay. From day 0 to day 7 the cell viability did not change a lot for the hydrogels with 0% clay as

well as for the hydrogels with 5% clay.

Figure 5: Live/dead assay for pure PEGDA 10k hydrogels on day 0 (A) and day 7 (B) and PEGDA 10k hydrogels with

5% clay on day 0 (C) and day 7 (D).

11

3.6 Biochemical assays DNA and GAGs assays were performed for cell constructs of day 1, 7 and 21 after encapsulation. Average

DNA content increases from 1.5 µg per construct on day 0 to 2.5 µg on day 21 and this was similar for all

three groups. Figure 6 shows an increase in GAGs compared to the DNA content of the constructs, and this

trend was the same for all three groups. There was practically no GAGs secretion on day 1 and it increased to

about 100 µg of GAGS per µg of DNA on day 21.

Figure 6: GAGs/DNA for PEGDA 10k hydrogels with 0%, 2.5% and 5% clay on day 1, 7 and 21.

1 7 21 1 7 21 1 7 21

0

50

100

150

5% clay

2.5% clay

0% clay

Time (days)

GA

Gs/D

NA

12

4. Discussion

To investigate the effects of the addition of clay nanoparticles to PEGDA hydrogels, gels with increasing

amounts of clay were tested for their swelling properties, mechanical properties and on their biocompatibility.

During preparation of these NC hydrogels, the strong interactions between PEG and clay nanoparticles were

observed. For example, physical gels were formed right after mixing clay and PEG. This phenomenon might

be attributed to the various interactions between clay and PEG such as hydrogen bondings and hydrophobic

interactions [9]. Furthermore, it was demonstrated that the clay particles do not interfere with the UV-

mediated photo polymerization of the acrylate groups on PEG. The same photo polymerization time could be

used for the pure PEG and the NC hydrogels, which is considered as safe for the encapsulated cells [4].

The swelling ratios of pure PEG and PEG/clay NC hydrogels (2.5, 5 and 10% clay) were studied. Among

them, NC gels with 2.5% clay showed the highest swelling ratio. The increase in swelling ratio could be

explained by the osmotic pressure from the clay particles. Clay particles are negatively charged and they

absorb counter ions in aqueous solution [9]. The accumulation of counter ions results in an increase of

osmotic pressure. Because of that, the gel absorbs more water than it would do without clay. But adding more

clay decreased the swelling ratio instead. Hydrogels with 10% clay had similar or even lower swelling ratio

than pure PEGDA hydrogels. This could be a result of the interactions between clay and polymer, which

increases the apparent crosslinking density [9]. Nevertheless, in the presence of clay particles, the NC

hydrogels maintained a highly aqueous environment (>85% water content) which is essential for cell growth.

To demonstrate the effects of osmotic pressure by the clay particles, PEGDA hydrogels with a clay content of

0% and 10% were swollen in solutions with a series of NaCl solutions. Both the pure PEGDA and the NC

hydrogels showed a decrease in swelling with an increase in NaCl concentrations, although the effect was

more significant for the NC hydrogels. The decrease in swelling for the pure PEGDA hydrogels can be

explained by the higher concentrations of ions outside the hydrogel, which compensate the osmotic effect

from the PEG polymer network. The fact that the effect is more obvious for the gels with 10% clay supports

the hypothesis that clay particles contribute to the osmotic pressure. With increasing molarity, the osmotic

pressure caused by the clay particles decreases and this resulted in less swelling. Therefore, deswelling

properties of the pure PEGDA hydrogels were caused by only one effect while deswelling of the NC

hydrogels were caused by two different effects.

To investigate the effect of clay particles on the mechanical properties of the hydrogels, compressive and

tensile tests were studied for PEGDA hydrogels with clay content up to 10%. The results of compression tests

demonstrate that the addition of clay particles resulted in stronger and tougher hydrogels. An explanation for

this phenomenon could be that clay particles are able to absorb the force applied to the hydrogels, which

results in a higher compressive modulus. If clay particles and polymer are cross-linked via physical

interactions, which are non-covalent, both components can move with respect to each other during the

deformation without causing the hydrogel to break [9]. This property could also explain the higher tensile

strength of the nanocomposite hydrogels.

Biocompatibility is crucial for NC hydrogels if they are going to be used for tissue engineering applications

[1, 4]. Chondrocytes were encapsulated in PEGDA hydrogels with different amounts of clay to see the effect

of clay particles on the encapsulated cells. During the preparation of these gels, it was observed that the

resultant solution of 10% PEGDA10k/ 10% clay was too viscous for cell encapsulation. PBS was used to

dissolve the PEGDA, while water was used for the other experiment so this higher viscosity could be due to

the higher amount of ions in PBS. In order to maintain high solubility of clay particles in aqueous solution,

clay particles are fully negatively charged. The repulsion force between clay particles prevents their

aggregation [9]. In the presence of salt (0.15 M NaCl for PBS), the surface of clay particles was neutralized

13

and this increases the chance of aggregation. These formed clay particle aggregates may increase the viscosity

of the solution.

The results of the live/dead assay suggest that slightly more cells stay alive when encapsulated in PEGDA

hydrogels with clay compared to the pure PEGDA hydrogels, although this effect was very small and the

live/dead assay was done for only one construct. But at least these results demonstrate that cell viability for

chondrocytes did not decrease by adding clay to the hydrogel. DNA assay confirmed this, since DNA content

from the pure PEG and NC hydrogels was similar.

GAGs assay showed that the GAGs production per weight of DNA from PEG/clay NC hydrogels was

comparable to that of pure PEGDA hydrogels. As expected, there was almost no GAG production after one

day, since there was insufficient time to produce GAGs yet. The increase in GAGs after one and three weeks

demonstrates that the cells are able to live and produce extracellular matrix in this new clay containing

hydrogel system. These results demonstrate that adding clay to the hydrogel did not decrease the production

of GAGs by the encapsulated chondrocytes.

In future studies, tensile tests for PEGDA hydrogels with 2.5% and 5% clay should be performed. The

live/dead assay should be repeated for more constructs. Also, other biochemical assays, such as a collagen

assay, could be used to provide more information about the extra cellular matrix proteins produced by the

encapsulated cells. Furthermore, it could be interesting to investigate the ability of this material to support the

differentiation of stem cells into chondrocytes.

14

5. Conclusion

The results demonstrated that the same photo polymerization technique could be used for pure PEG and NC

hydrogels. Also, adding up to 5% clay to the PEGDA hydrogels resulted in better or similar swelling

behavior. The swelling of NC hydrogels were governed by a balance between the osmotic effect of clay

particles and the apparent crosslinking density from a clay-mediated second network. The overall mechanical

properties of PEGDA hydrogels were improved by adding up to 10% of clay. Live/dead and biochemical

assays confirmed that adding clay to the PEGDA hydrogel did not decrease the cell viability or the production

of GAGs by the encapsulated chondrocytes.

15

References [1] Lin, C.C., Anseth, K.S., PEG Hydrogels for the Controlled Release of Biomolecules in Regenerative Medicine,

Pharmaceutical Research, 26(3): 631-43, 2009

[2] Kokabi, M., Sirousazar, M., Hassan, Z.M., PVA–clay nanocomposite hydrogels for wound dressing, European

Polymer Journal, 43: 773-781, 2007

[3] Haraguchi, K., Takehisa, T., Ebato, M., Control of Cell Cultivation and Cell Sheet Detachment on the Surface

of Polymer/Clay Nanocomposite Hydrogels, Biomacromolecules, 7(11): 3267-75, 2006

[4] Varghese, S., Elisseeff, J.H., Hydrogels for Musculoskeletal Tissue Engineering, Advances in Polymer Science,

203: 95-144, 2006

[5] Nguyen, K.T., West, J.L., Photopolymerizable hydrogels for tissue engineering applications, Biomaterials, 23:

4307-4314, 2002

[6] Liu, Y., Zhu, M., Liu, X., Zhan, W., Sun, B., Chen, Y., Adler, H.P., High clay content nanocomposite hydrogels

with surprising mechanical strength and interesting deswelling kinetics, Polymer, 47: 1-5, 2006

[7] Lin, J., Xu, S., Shi, X., Feng, S., Wang, J., Synthesis and properties of a novel double network nanocomposite

hydrogel, Polymers Advanced Technologies, 2008

[8] Okay, O., Opperman, W., Polyacrylamide-Clay Nanocomposite Hydrogels: Rheological and

Light Scattering Characterization, Macromolecules, 40: 3378-3387, 2007

[9] Schexnailder, P., Schmidt, G., Nanocomposite polymer hydrogels, Colloid and Polymer Science, 287: 1-11,

2009

[10] Strehin, I.A., Elisseeff, J.H., Characterizing ECM production by cells encapsulated in hydrogels, Methods in

Molecular Biology, Extracellular Matrix Protocols, 552(23): 349-362, 2009

16

Supplements

Supplement A

Making a nanocomposite hydrogel This protocol is to make a 10% clay, 10% PEGDA hydrogel. The same protocol can be used for

different percentages of clay and polymer.

Materials:

- Clay: Laponite XLS

- PEGDA

- Irgacure 2959

- Ethanol

- dd H2O

Protocol:

1. Weigh 100 mg Laponite XLS and put it into a 1.5ml tube.

2. Add 560 µl of H2O and vortex until the solution is clear.

3. Make a 30% PEGDA solution in H2O and vortex a couple of times.

4. Make a 10% Irgacure 2959 solution in ethanol.

5. Add 334 µl of PEGDA solution to the 1.5ml tube and vortex a couple of times. Make

sure the solution is completely mixed. The clay and polymer can form a physical gel.

6. Add 5 µl of 10% Irgacure 2959 to the eppendorf tube and vortex.

7. Put the solution into the mold.

8. Expose the samples to UV for 5 minutes.

9. Take the hydrogels out of the molds carefully, put the hydrogels in H2O and refresh the

water a few times before putting the samples in PBS.

17

Supplement B

Live/dead assay of cells encapsulated in a hydrogel construct

Materials:

- Hydrogel constructs

- live/dead viability cytotoxicity kit (Molecular probes)

- Pen/strep

- Sterile PBS

- DMEM

- 2 razor blades

Prepare live/dead assay solution:

Calcein is susceptible to hydrolysis when exposed to moisture.

- Add 1 ml of DMEM into a 1.5 ml tube, add 0.5 µl of calcein AM and 2 µl of EthD-1 to

the DMEM to make the live/dead assay solution.

Protocol:

1. Take one construct.

2. Use two razor blades to slice the construct into several thin slices in a petri dish.

3. Rinse the slices with 1 ml of PBS for 3 times.

4. Make the live/dead assay solution.

5. Add 0.5 ml of the solution to the construct slices.

6. Incubate for 30 minutes at 37°C.

7. Remove the solution.

8. Rinse the slices with PBS.

9. Place the petri dish with slices under the microscope to visualize with fluorescent light.

Red cells � Dead, nucleus staining

Green cells � Live, cytoplasm staining

18

Supplement C

Papain digestion protocol Based on protocol from “Characterizing ECM Production by Cells Encapsulated in Hydrogels”

[10]

Materials needed:

- PBE Buffer: 0.1 M Na2HPO4, 10mM EDTA Na2.2H2O, pH 6.5, filter sterilize. Store at

4°C for up to 3 months.

- Papain buffer: 9.3 units papain type III per 1 ml of PBE buffer.

- Pellet pestles

Protocol:

1. Weigh the wet and dry weights of the hydrogel constructs. To obtain the wet

weight, remove the constructs from media, lightly dry them with a delicate task

wipe (i.e., Kimwipe TM ) and weigh them. Then lyophilize the samples for 2

days and weigh them again to obtain the dry weights.

2. To the dry constructs, add 0.5 mL papain buffer and homogenize the constructs

using a pellet pestle. Add an additional 0.5 mL papain buffer and vortex. Place in

60°C water bath for 16 h.

3. Vortex the construct and then centrifuge (14,000 rpm, 10 s) to get the undigested

scaffold to pellet on the bottom of the centrifuge tube. Use the supernatant to

complete the different assays. (samples can be stored at -20°C)

19

Supplement D

DNA assay protocol Based on protocol written by Allison Finger, February 26, 2009

See package insert for Quant-iT PicoGreen dsDNA kit for more information.

Materials needed

- Samples digested with papain (see papain digestion assay for biochemical analysis)

- Quant-iT PicoGreen dsDNA Kit (Invitrogen) (stored at -20°C) which includes:

o Quant-iT PicoGreen dsDNA reagent

o 20X TE (may be stored at room temperature)

o Lambda DNA standard (may be stored at 4°C)

- DEPC-treated water

- Plate reader with nucleic acid wavelength filter

Note: In most cases, it is best to dilute samples with DEPC treated water for this assay to save

the sample in case the assay must be repeated.

Note: Allow the PicoGreen reagent to warm to room temperature before using. Make sure to keep

the PicoGreen in a dark place while thawing to avoid photo bleaching.

Procedure

1. Determine plate setup. Use the clear 96-well plates for this assay. Leave 15 wells for the

standards, and 30 wells if you intend to run both the high and low range standards (high

range standard can detect 1 µg/ml -1 ng/ml DNA and low range standards detect 25

ng/ml – 25 pg/ml DNA). Run all samples and standards in triplicate (3 wells per sample

or standard).

Note: It is best to run no more than 16 samples and 5 standards per plate. The reaction is time

sensitive, and the longer it takes to pipet PicoGreen into all the wells at the end of the assay the

more likely it is that there will be variation in the fluorescence readings from one side of the plate

to the other.

2. Dilute samples if necessary with 1x TE buffer. Each well will have 100 µl of

sample/diluted sample. If diluting the samples, make sure to make extra for pipetting

error.

3. Calculate how much PicoGreen reagent you will need. You will pipet 100 µl per well,

and make extra for pipetting error.

[(# of samples x 3) + (# of standards x 3) + 2] x 0.1 ml = total volume of PicoGreen.

The supplied PicoGreen reagent is diluted 200-fold before using (e.g. if you need 20mL

diluted PicoGreen, mix 20/200 =100 µl PicoGreen +19.9 ml 1X TE)

4. Mix up 1X TE buffer from the 20X stock provided in the kit. Calculate how much 1X TE

buffer you will need (include the volume needed for diluting samples, preparing DNA

Stock solutions- see example below, and preparing diluted PicoGreen. 20X TE should be

diluted 20-fold with DEPC-treated water (e.g. if you need 40 ml 1X TE, mix 40/20 =2 ml

20X TE + 38 ml DEPC-treated water).

5. Mix up standards using lambda DNA provided in the kit. Stock DNA is supplied at 100

µg/ml and must be diluted with 1X TE buffer to 2 µg/ml (high range standard) or 50

20

ng/ml (low range standard).

6. Make sure plate reader is turned on at this point to allow the machine to warm up for 10

to 15 minutes before using. You may want to go ahead and enter the plate setup in a new

PicoGreen protocol document. Make sure the correct files are in place.

7. Pipet samples and standards onto plate in triplicate according to plate setup.

8. Mix up PicoGreen reagent. Turn down the lights in the area where you are working to

prevent photo bleaching. Wrap aluminum foil around the tube to protect the reagent from

light. Mix the stock PicoGreen with 1X TE to dilute 200-fold.

9. Pipet 100 µl diluted PicoGreen into each sample and standard well. Cover plate with

aluminum foil as soon as you are finished pipetting the PicoGreen. Incubate plate for 2-5

minutes at room temperature before measuring fluorescence reading in plate reader.

21

Supplement E

GAG assay protocol Based on protocol from “Characterizing ECM Production by Cells Encapsulated in Hydrogels”

[10]

Materials needed

- PBE Buffer: 0.1 M Na2HPO4, 10mM EDTA Na2.2H2O, pH 6.5, filter sterilize. Store at

4°C for up to 3 months.

- PBE/Cys solution: 0.0875 g Cysteine in 50 ml PBE buffer.

- CS stock solution: 50 mg/ml CS in PBE/Cys solution. Store at -20°C.

- CS working solution: 25 µl CS stock solution, 12.5 ml PBE/Cys solution.

- DMMB dye stock solution: 40mM Glycine, 40mM naCl, pH3. Dissolve 16 mg DMMB

and check that OD525 is between 0.31 and 0.34. Store in dark at 25°C for up to 3 months.

- Papain digests

- UV Spectrophotometer

- Cuvettes

Protocol

1. Dilute the CS working solution with PBE to get a final volume of 100 µl such that you

have appropriate solutions for the standard curve (minimum absorbance should be 20

time dilution).

2. Add 2.5 ml of DMMB dye to 100 µl blank sample (no CS working solution) and use a

disposable transfer pipette to mix the solution, then quickly place the blank sample in the

spectrophotometer and read at 525 nm.

3. Measure the other standards in the same way.

4. Dilute samples if necessary with PBE buffer and measure in the same way.