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A Collagen Peptide-Based Physical Hydrogel For
A Collagen Peptide-Based Physical Hydrogel For
Collagen peptide-based hydrogels are prepared and characterized for application in 3D cell
growth. Physical hydrogels are formed by covalently linking a collagen-based peptide to an
8-arm poly(ethylene glycol) star polymer. The resulting viscoelastic hydrogels have the ability
to melt into a liquid-like state near the melting temperature of the collagen triple helix and
reform back into an elastic-state at room
temperature, adding a thermoresponsive feature to the material. In addition,
the hydrogels possess desirable stiffness,
as well as a highly cross-linked network
of pores where cells are found to reside,
making the hydrogels promising scaffolds 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 XaaYaa-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-
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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 star
polymer.
Experimental Section
Materials
1
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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 200 mm. For
rheological experiments, an 8-arm PEG-MAL solution in phosphate
buffer at pH 7.4 (5% w/v, 50 mL) was mixed with a CGG-(POG)8
peptide solution also in phosphate buffer at pH 7.4 (0.01 M, 50 mL)
on the rheometer plate to produce the 4% PSP-POG8 hydrogel
(100 mL). 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 8C
with 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%, 35 mL) in PBS buffer pH 7.4 was mixed in a Falcon cell
culture insert with a CGG-(POG)8 peptide solution made by
dissolving the solid peptide (0.6 or 1.2 mg) in 35 mL of cell
suspension (1.5 106 hMSCs mL1 in PBS buffer pH 7.4). After
hydrogel formation occurred, inserts were placed in a 24-well plate
with 700 mL 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 20 mL of the CellTiter 96
Aqeous One solution and incubated at 37 8C for 4 h. Following
incubation, each of the wells containing the corresponding sample
was removed from the insert and dissolved with 700 mL of media.
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Absorbance at 490 nm was measured using the TECAN SpectraFluorplus 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.
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Sample
Tm
[-C]
CGG-(POG)8a)
4% PSP-POG8 hydrogel
8% PSP-POG8 hydrogel
53
b)
G( [Pa]
After
Before
thermal
thermal
annealing annealing
ND
ND
56
685
798
n.d.c)
1 337
1 714
Figure 2. Temperature sweep experiments were performed by the rheometry for (a) 4% PSP-POG8 and (b) 8% PSP-POG8. Filled symbols
represent the first analysis of the hydrogels and open symbols represent analysis after recooling the samples from the first analysis. G0
storage modulus (^), G00 loss modulus (D).
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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 magnification before and after thermal annealing, respectively.
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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 morphology 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.
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