A Collagen Peptide-Based Physical Hydrogel For

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1426A Collagen Peptide-Based Physical Hydrogel forCell EncapsulationaCharles M. Rubert Perez, Alyssa Panitch, Jean Chmielewski*Collagen peptide-based hydrogels are prepared and characterized for application in 3D cellgrowth. Physical hydrogels are formed by covalently linking a collagen-based peptide to an8-arm poly(ethylene glycol) star polymer. The resulting viscoelastic hydrogels have the abilityto melt into a liquid-like state near the melting temperature of the collagen triple helix andreform back into an elastic-state at roomtemperature, adding a thermorespon-sive feature to the material. In addition,the hydrogels possess desirable stiffness,as well as a highly cross-linked networkof pores where cells are found to reside,making the hydrogels promising scaf-folds for the culture of hMSCs.Introduction

Collagen is the most abundant protein in mammals, found

in connective tissue, ligaments, skin, bone, and cartilage as

well as being one of the main components of the

extracellular matrix (ECM).[1] The most common primary

structure of collagen consist of repeating units of an Xaa-

Yaa-Gly tripeptide sequence, where Xaa and Yaa are

occupied by L-proline (Pro) and 4(R)-hydroxy-L-proline

(Hyp) residues, respectively.[2] Single collagen strands

adopt a polyproline type II (PPII) helical conformation that

assemble into super-coiled right handed triple helices the

major structural motif exhibited by collagen.[3] This triple-C. M. Rubert Perez, J. ChmielewskiDepartment of Chemistry, Purdue University, 560 Oval Drive,West Lafayette, IN 47907, USAE-mail: [email protected]. PanitchWeldon School of Biomedical Engineering, Purdue University, 206S. Martin Jischke Drive, West Lafayette, IN 47907, USA

a Supporting Information for this article is available from the WileyOnline Library or from the author.

Macromol. Biosci. 2011, 11, 14261431

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinehelical structure forms the basis for higher order assemblies

of collagen into fibers and fibrous networks.[4]

Natural collagen derived from animal sources has been

used to construct collagen-based scaffolds for applications

such as drug delivery,[5] bone repair,[6] cell growth,[7] and

tissue engineering.[8] However, a number of disadvantages

of using naturally derived collagen has limited potential

use, including the heterogeneity of the materials, the

possibility of immunogenic response due to the transfer of

toxic agents,[9] and difficulties in further modifying the

collagen sequence and structure to alter its physical

properties. In order to overcome these disadvantages,

chemically synthesized collagen peptides have provided a

new alternative for the formation of improved biomater-

ials. When using synthetic collagen peptides, one is able to

chemically control the amino acid sequence by rational

design and alter the overall self-assembly of collagen

peptide triple helices into high order structures to provide

more controllable materials for matrix engineering.[10]

In this study, we focused on the conjugation of a

synthetic collagen peptide sequence to a multiarm star

polymer in an effort to form a collagen-polymer hydrogel

with tunable properties for cell encapsulation and growth.

Hydrogels are highly hydrated materials that are able tolibrary.com DOI: 10.1002/mabi.201100230

Figure 1. Schematic representation of hydrogel formation with a collagen triple helical peptide, CGG-POG8, and an 8-arm PEG-MAL starpolymer.

A Collagen Peptide-Based Physical Hydrogel for Cell Encapsulation

www.mbs-journal.deform fibrous 3D networks, therefore hydrogel materials are

some of the most used scaffolds for mimicking the ECM.[11]

Collagen peptide hydrogels have been used for thermo-

responsive drug delivery,[12] cell encapsulation,[13] and

particle tracking analysis.[14] Our hydrogel design consisted

of an 8-arm 40 kDa poly(ethylene glycol) star polymer with

terminal maleimide functionality (8-arm PEG-MAL), and a

collagen peptide consisting of eight repeating units of the

tripeptide sequence Pro-Hyp-Gly, with an N-terminal Gly-

Gly-Cys triad [CGG-(POG)8]. The collagen peptide was

designed to maintain a stable triple helical structure under

physiologically relevant conditions,[10b,15] whereas the

cysteine residue provides the point of reactivity with the

maleimide group of the star PEG polymer. This strategy

would lead to arms of the polymer that are functionalized

with the collagen peptide (Figure 1). Hydrogel formation is

proposed to form, therefore, through physical crosslinks

between the collagen peptide triple helices that are

attached to the star polymer arms. This architecture would

allow for multiple sites of attachment for each PEG star unit

to potentially form a highly ordered network.Experimental Section

Materials

H-Rink Amide-ChemMatrix1

resin was purchased from BioMatrix

Inc. (Quebec, Canada). Fmoc-Gly-OH and Fmoc-Pro-OH amino acids

were purchased from Anaspec Inc. (Fremont, CA). Fmoc-Hyp(t-

butyl)-OH amino acid and O-(benzotriazol-1-yl)-N,N,N,0N0-tetra-

methyluroniumhexafluorophosphate (HBTU) were purchased

from Aapptec Inc. (Louisville, KY). Fmoc-Cys(Trt)-OH amino acid

was purchased from SynPep Corp. (Dublin, CA). N-hydroxybenzo-

triazole hydrate (HOBT) was purchased from Oakwood Products,

Inc. (West Columbia, SC). 2,4,6-Trimethylpyridine or 2,4,6-collidine

(TMP) was purchased from Sigma-Aldrich Chemical Co. (St. Louis,

MI) CellTiter 96 AQeous One Solution Cell Proliferation Assay waswww.MaterialsViews.com

Macromol. Biosci. 2011

2011 WILEY-VCH Verlag Gmbpurchased from Promega (Madison, WI). 8-arm PEG-MAL (MW

40 kDa) was purchased from Nektar Inc. (Huntsville, AL). Tris(2-

carboxyethyl)phosphine (TCEP) was purchased from TCI America

Inc. (Portland, OR). Live/dead cell viability/toxicity kit was

purchased from Invitrogen (Carlsbad, CA). BD Falcon cell culture

inserts (0.3 cm2 growth area) were purchased from BD Biosciences

(West Chester, PA). All other chemicals were purchased from Sigma

Chemical Co. (St. Louis, MI).

Peptide Synthesis and Purification

Peptides were synthesized using a solid-phase fluorenylmethox-

ycarbonyl (Fmoc)-based approach on the ChemMatrix resin with

HBTU as the coupling reagent. For cysteine coupling, a combination

of HBTU and HOBT (1 equiv. each) were used for couplings.[16] After

the final Fmoc deprotection the resin was treated with acetic

anhydride to acetylate the amino-terminus. The peptide was

cleaved from the resin using a trifluoroacetic acid (TFA) cocktail

solution [90% TFA, 1% triisopropylsilane (TIPS), 1% thioanisole, 2.5%

ethanedithiol, 2.5% anisole]. The resulting mixture was filtered, the

resin was washed with additional TFA and concentrated in vacuo.

The residue was triturated with cold diethyl ether, and the

precipitate was collected by centrifugation. For purification via

high-performance liquid chromatography (HPLC), the crude pep-

tide was dissolved in containing 0.01 M TCEP and purged with N2 for

2 h for disulfide bond reduction. Peptide solution was then purified

by reverse phase HPLC using a Phenomenex Luna C18

(50 21.20 mm, 100 A, 5mm) with an eluent consisting of solventA (CH3CN/0.1% TFA) and solvent B (H2O/0.1% TFA) with a 230%

solvent-A gradient over 60 min and a flow rate of 10.00 mL min1

(l214nm andl260nm). Purity of the peptides was verified by analyticalreverse-phase HPLC (see Supporting Information) using a Phenom-

enex Luna C18 column (2504.6 mm, 100 A, 5mm) with an eluentconsisting of solvent A (CH3CN/0.05% TFA) and solvent B (H2O/

0.05% TFA) with a 250% solvent-A gradient over 30 min and a flow

rate of 1.20 mL min1 (l214nm). The structure of the peptide wasconfirmed by mass spectrometry {matrix-assisted laser deso-

rption/ionization time-of-flight (MALDI-TOF) analysis of CGG-

(POG)8 [MH]: calcd. 2 412.06; found 2 411.94}., 11, 14261431

H & Co. KGaA, Weinheim1427

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C. M. Rubert Perez, A. Panitch, J. ChmielewskiCircular Dichroism (CD)

CD wavelength spectra scans were performed on a JASCO Model

J810 CD spectropolarimeter (Easton, MD) equipped with a PFD-425S

Peltier temperature control unit at 4 8C using a 0.1-cm path-lengthquartz cell. The spectra was averaged over three scans taken from

300 to 210 nm with a data pitch of 0.1 nm with a bandwidth of

1 nm. The scan rate of was 100 nm min1 with a response time 1 s.The CD data obtained was processed into from degrees of rotation

to mean residue ellipticity. CD melting curves were determined by

measuring the mean residue ellipticity at 225 nm, while running a

temperature slope between 4 and 90 8C at 6 8C h1 with a datapitch of 0.2 8C, bandwidth of 4 nm and response time of 4 s. Thepeptide sample was prepared by making a 2104 M CGG-(POG)8solution in 0.01 M phosphate buffer pH 7.4 and 5103 M TCEP.The polymer/peptide sample was prepared by heating a 4% PSP-

POG8 hydrogel and aliquoting the required volume to make a

2104 M PSP-POG8 solution in 0.01 M phosphate buffer pH 7.4.Rheology

All rheological analyses were performed on a TA instrument ARG2

rheometer (New Castle, DE) using a 20-mm cone and plate

geometry with a 18 angle and a sample gap of 200mm. Forrheological experiments, an 8-arm PEG-MAL solution in phosphate

buffer at pH7.4 (5% w/v, 50mL) was mixed with a CGG-(POG)8peptide solution also in phosphate buffer at pH7.4 (0.01 M, 50mL)on the rheometer plate to produce the 4% PSP-POG8 hydrogel

(100mL). The 8% PSP-POG8 hydrogel was prepared in the same way,

but with double the concentration of the star polymer and peptide.

To avoid evaporation, a solvent trap was placed around the sample.

For each sample, three different measurements were performed.

First the storage and loss moduli was monitored while applying an

oscillation stress between 0 and 500 Pa with a constant frequency

of 5 Hz. Then, a frequency sweep was performed between 1 and

30 Hz with an constant oscillation stress of 5 Pa. Lastly, the

temperature sweep was monitored between 2560 8C and 6025 8Cwith a constant oscillation stress of 5 Pa and a frequency of 5 Hz

with a gradient of 5 8C min1.Encapsulation Studies

For cell encapsulation studies, an 8-arm PEG-MAL solution (5 or

10 wt%, 35mL) in PBS buffer pH 7.4 was mixed in a Falcon cellculture insert with a CGG-(POG)8 peptide solution made by

dissolving the solid peptide (0.6 or 1.2 mg) in 35mL of cell

suspension (1.5 106 hMSCs mL1 in PBS buffer pH 7.4). Afterhydrogel formation occurred, inserts were placed in a 24-well plate

with 700mL of mesenchymal stem cell basal medium (MSCBM)

supplemented with the correspondent growth supplements

(MCGS, Wakersville, MD). For the 3-(4,5-dimethylthiazol-2-yl)-5-

(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS) cell viability assay, cells were incubated for 24 h and cells

growing in 2D and cells encapsulated within the 4% PSP-POG8 and

8% PSP-POG8 hydrogels were treated with 20mL of the CellTiter 96

Aqeous One solution and incubated at 37 8C for 4 h. Followingincubation, each of the wells containing the corresponding sample

was removed from the insert and dissolved with 700mL of media.Macromol. Biosci. 2011

2011 WILEY-VCH Verlag GmbAbsorbance at 490 nm was measured using the TECAN Spectra-

Fluorplus microplate reader (Durham, NC). Percent cell viability

was calculated by comparing the absorbance of cells growing in 2D

versus the cells growing inside of the hydrogel. For the Calcein-AM

cell viability assay, 2D cells and cells encapsulated within the 4%

PSP-POG8 hydrogel were incubated with Calcein AM (106 M) for

30 min after 24 h of cell culture. Cells were then visualized for green

fluorescence using a Optical Microscope Olympus BX51 equipped

with a CCD camera (Center Valley, PA). Calcein-AM was excited

using a U-MWB2 filter with excitation of 460495 nm and the

fluorescence emission was collected using a 520 nm filter.

Cryo-Scanning Electron Microscopy (Cryo-SEM)

Imaging

Hydrogel samples were prepared in situ on the Cryo-SEM slit

sample holder as described in the previous section. Samples were

frozen with liquid nitrogen and transferred to the Gatan Alto 2500

pre-chamber (cooled to 170 8C). The surface of the sample wasfractured in various locations using a scalpel to produce free-break

surfaces before being sublimated for 20 min at 85 8C. Pt sputtercoating followed for 120 min and then sample was transferred to

the microscopes cryo stage (130 8C) for imaging. Samples wereimaged with a FEI NOVA nanoSEM field emission (FEI Company,

Hillsboro, Oregon) using the through-the-lens (TLD) or Everhart-

Thornley (ET) detector at 5 kV accelerating voltage and a working

distance (WD) of 5 mm at different magnifications.Results and Discussion

Synthesis of CGG-(POG)8 and Hydrogel Formation

The CGG-(POG)8 peptide was synthesized using solid phase

methods on the Rink amide ChemMatrix resin, purified to

homogeneity by reverse phase HPLC and analyzed by

MALDI mass spectrometry. The purified CGG-(POG)8 pep-

tide was tested for its ability to induce hydrogel formation

when conjugated to the 8-arm PEG-MAL. To this end, star

polymer solutions (5 and 10 wt%) were added to a solution

of peptide both in phosphate-buffered saline (PBS) at

pH 7.4 to achieve a ratio of 1:12, respectively. Gelation ofthe samples was observed within 1 min in each case,

yielding hydrogels that were 4 and 8% in the peptide/PEG

star polymer (PSP-POG8).

Physical Characterization of the PSP-POG8 Hydrogels

CD was used to evaluate if the peptides within the hydrogel

maintained their ability to adopt a collagen triple helical

conformation, as compared to the peptide in free solution.

Both the peptide in solution and peptide in the 4% hydrogel

displayed a maximum in the CD spectrum at approximately

225 nm, a feature that is characteristic of a collagen triple

helix (See Supporting Information).[10b,17] To determine

what role the hydrogel architecture plays on triple helix

stability, we performed thermal denaturation studies by CD, 11, 14261431

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Table 1. Thermal denaturation (Tm) and storage modulus (G0) datafor PSP-POG8 hydrogels.

Sample Tm[-C]

G( [Pa]

Before

thermal

annealing

After

thermal

annealing

CGG-(POG)8a) 53 ND ND

4% PSP-POG8 hydrogelb) 56 685 798

8% PSP-POG8 hydrogel n.d.c) 1 337 1 714

a)2 104 M peptide solution in 0.01 M phosphate buffer atpH 7.4 with 5 103 M TCEP; b)An aliquot was taken from thehydrogel to make a solution that was 2 104 M in peptide in0.01 M phosphate buffer at pH7.4; c)Not determined.


www.mbs-journal.de(Table 1). The collagen peptide alone, CGG-(POG)8, exhibited

a Tm of 53 8C (Table 1), a value that is close to that reportedfor the (POG)8 peptide.

[18] The triple helix of the polymer-

conjugated peptide was found to melt at a slightly higher

temperature, most likely due to intramolecular scaffold

stabilization of the helical structure, as has been observed

for collagen sequences tethered to dendrimer scaffolds.[19]

These data demonstrate that the multiarm polymer allows

for the formation of a stable triple helix within the context

of the hydrogel matrix.

Rheology experiments were performed to probe the

viscoelastic properties of the peptide/PEG star polymer

hydrogels (PSP-POG8). Oscillation stress and frequency

sweep experiments were carried out to determine the linear

dynamic range of the two hydrogels by measuring the

storage (G0) and loss (G00) moduli. For instance, at a constant

oscillation stress of 5 Pa, average G0 values of 685 and

1 337 Pa were obtained for the 4 and 8% PSP-POG8Figure 2. Temperature sweep experiments were performed by the rhrepresent the first analysis of the hydrogels and open symbols represtorage modulus (^), G00 loss modulus (D).

www.MaterialsViews.com


2011 WILEY-VCH Verlag Gmbhydrogels, respectively, with a frequency sweep of

130 Hz (See Table 1 and Supporting Information). The G0

values for both hydrogels dominated over the G00 values

over the course of the analysis. These data demonstrate that

CGG-(POG)8 crosslinks the polymer to form hydrogel whose

stiffness can be modulated through changes in the

concentration of the starting materials, with the 8%

hydrogel showing on approximately a twofold increase

in storage modulus over the 4% hydrogel.

Studies above investigated the thermal denaturation of

the collagen triple helix within the hydrogel by CD. Since

the collagen peptide is the major physical crosslinking

component of the hydrogel, we performed rheometry

temperature sweep experiments to determine the effect of

increasing temperature on the properties of the hydrogels.

Using a constant oscillation stress of 5 Pa and a frequency of

5 Hz, the hydrogels were submitted to increasing tempera-

ture. In these experiments, the storage modulus was found

to decrease as the 4 and 8% hydrogels were heated, with the

materials completely changing into a liquid-like state at

approximately 60 8C (Figure 2), a value that is in the samerange as the collagen triple helix melting temperatures

determined by CD. These data confirm that as the collagen

triple helix melts, the physical properties of the hydrogels

change significantly from a polymer network to a liquid-

like state. These data also provide supporting evidence that

the collagen peptide triple helix is a major factor in hydrogel

formation within the PEG star polymer.

These melted materials were found to reform a

gelatinous material when cooled to room temperature,

and the cooled hydrogels were found to have higher storage

modulus values as compared to their starting, unheated

states (Table 1). The increase in the storage modulus was

somewhat larger for the 8% PSP-POG8 hydrogel, perhaps

indicating a change in the morphology of the hydrogel

through the thermal annealing cycle. The annealedeometry for (a) 4% PSP-POG8 and (b) 8% PSP-POG8. Filled symbolssent analysis after recooling the samples from the first analysis. G0

, 11, 14261431


Figure 3. Internal hydrogel morphology was investigated by cryo-SEM. (a) and (b) 4%PSP-POG8 at two different magnifications, (c) and (d) 8% PSP-POG8 at same magni-fication before and after thermal annealing, respectively.

Figure 4. (a) Fluorescence images of viable hMSCs stained with Calcein AM encapsulatedwithin the 4% PSP-POG8 hydrogel. (b) Cryo-SEM images of hMSCs occupying the porousstructure of the 4% PSP-POG8 hydrogel.

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C. M. Rubert Perez, A. Panitch, J. Chmielewskihydrogels were also submitted to a

second temperature sweep analysis,

and, although the starting G0 values were

higher this time; liquid-like states were

obtained again at a temperature of

60 8C (Figure 2).Cryo-SEM was used to visualize the

internal morphology of the hydrogels

and any changes due to thermal anneal-

ing. Both the 4 and 8% PSP-POG8 hydro-

gels were analyzed before thermal

annealing, and the morphology of the

hydrogels was found to consist of a

fibrous, honeycomb-like structure, with

round compartments or pores that were

525mm in size (Figure 3ac). Withinsome pores, the fibrous sheets seemed to

be broken or incomplete. Upon thermal

annealing the cryo-SEM of the 4% PSP-

POG8 hydrogel remained essentially

unchanged, whereas the 8% hydrogel

had the same overall morphology, but

now with significantly smaller pore sizes

of 2mm and with more complete pores(Figure 3d). Interestingly, this more con-

centrated material was found to respond

to the annealing and a more crosslinked

structure resulted.

Cell Encapsulation Experimentswith the PSP-POG8 Hydrogels

For the PSP-POG8 hydrogels to have

applications in tissue engineering and

regenerative medicine, it was essential to

determine if cells could be encapsulated

within the hydrogel and remain viable.

To test this, a suspension of human

mesenchymal stem cells (hMSCs) in a

CGG-(POG)8 solution in PBS buffer

pH 7.4 was treated with 5 or 10% ofthe PEG-MAL star polymer to successfully

provide the 4 or 8% PSP-POG8 hydrogels.A colorimetric MTS analysis was used to determine cell

viability of encapsulated hMSCs within the hydrogel. It was

determined that the cells were95% viable after 1 d in both4 and 8% hydrogels, favorably comparable to the viability

found for hMSCs that were grown in 2D cell culture plates.

Fluorescence microscopy was also used to provide visual

evidence for cell viability (Figure 4a). hMSCs that were

encapsulated within the 4% hydrogel for 24 h were treated

with the viability stain Calcein AM, and green fluorescence

was observed for live cells throughout the hydrogel.

Encapsulated cells remained viable for more than 5 d andMacromol. Biosci. 2011

2011 WILEY-VCH Verlag Gmbwere able to be transferred to a 2D plates to continue their

growth (Data not shown). Cumulatively these data provide

evidence that the hydrogel has low toxicity on the

encapsulated cells.

The internal morphology of the above 4% hydrogel with

the encapsulated hMSCs was also visualized using cryo-

SEM (Figure 4b). The cells were observed to occupy the pores

of the hydrogel, thereby providing structural support for the

cells within the matrix. At a higher magnification the

boundary between the hydrogel network and the cell

membrane could be distinguished, providing evidence of, 11, 14261431

H & Co. KGaA, Weinheim www.MaterialsViews.com


www.mbs-journal.dethe physical contacts between cell and collagen-based

scaffold (see Supporting information).Conclusion

In summary, we have designed a collagen peptide-based

hydrogel using an 8-arm-PEG star polymer to generate a 3D

cell culture matrix with appropriate viscoelastic properties.

This material has the ability to melt into a liquid-like state

at increased temperatures and reverts back into a gel upon

cooling, presumably due to the denaturation and refolding

of the collagen peptide triple helix. The internal morphol-

ogy of the hydrogel exhibited a pore size that is reasonable

for cell encapsulation as shown by cryo-SEM, and the

hydrogel demonstrates low cell toxicity. Since the collagen

triple helix plays a significant role in the properties of the

hydrogel, a range of materials for cell culture can be

envisioned through judicious changes to the collagen

peptides used to form the hydrogels, including peptides

containing cell adhesion signals or collagenase sequences.Acknowledgements: We are grateful to the NSF (0848325-CHE) forsupport of this research, to D. Sherman for assistance with SEM,and to J. Paderi for assistance with rheology.

Received: June 8, 2011; Published online: August 9, 2011; DOI:10.1002/mabi.201100230

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A Collagen Peptide-Based Physical Hydrogel For