Harnessing Traction-Mediated Manipulation of the Cell-Matrix … · 2010-06-15 · Harnessing Traction-Mediated Manipulation of the ... To form alginate hydrogels, a calcium-sulfate

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nmat2732

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Harnessing Traction-Mediated Manipulation of the Cell-Matrix Interface to Control Stem Cell Fate

Supplemental Materials

Methods

Hydrogel Synthetic ECM Analogs Sodium alginate with high molecular weight and high

guluronic acid content (MVG) or high mannuronic acid content (MVM) was purchased from FMC

Biopolymer (Princeton, NJ). High molecular weight, GA rich alginate was irradiated by a 5 Mrad

Cobalt source to produce a low molecular weight alginate with high GA content. When this low Mw

material is mixed with a critical amount of high Mw MVG, the binary mixture (binary MVG) crosslinks

to a high degree in the presence of divalent cations, but has a pre-hydrogel viscosity similar to that of

pure MVG at low concentrations1. The lower viscosity of binary MVG pre-polymer solution (compared

to pre-polymer solution formed by the same weight percent of unary MVG or MVM) facilitates cell

encapsulation without loss of cell viability2. The weight-averaged molecular weight Mw and gyration

radius rg were previously calculated using gel permeation chromatography2.

Following physical chain modifications, the adhesion peptide sequence G4RGDASSKY-OH was

coupled to alginate. For FRET experiments, either G4RGDASSK(5,6-carboxyfluorescein)Y-OH or

G4RGDASSK(5,6-tetramethylrhodamine)Y-OH, were used in place of the baseline peptide sequence,

whereas for integrin-binding ELISA, the sequence G4RGDASSK(biotin)Y-OH was used. All peptides

were purchased from Peptides International (Louisville, KY) and characterized at > 95% purity by the

manufacturer. Peptides were coupled to alginate polymers using published carbodiimide chemistry3.

The concentration of peptides and polymer was adjusted to yield between 1 and 20 peptides per polymer

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SUPPLEMENTARY INFORMATION doi: 10.1038/nmat2732

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chain; the efficacy of this labeling reaction for multiple alginate types was previously characterized

using 125I labeled RGD peptides3. The biotin and fluorescent labels used to identify RGD peptides in

various assays are not expected to affect this coupling efficiency. Moreover, the fluorescence intensity

of fluorophore-RGD-coupled alginate did not vary significantly from batch-to-batch or from one type of

alginate polymer to another.

Following peptide modification, alginate was dialyzed, treated with activated charcoal, filter

sterilized (0.22μm) and freeze-dried. Lyophilized alginate was reconstituted to 2-8% wt in media

without serum or phenol red. To form alginate hydrogels, a calcium-sulfate slurry (1.22M in deionized

water; Sigma) was mixed with 1% wt solutions of MVM, or either unary or binary MVG using luer-

lock syringes, and crosslinked between two glass plates separated by a spacer (750μm – 2mm). The

various alginate hydrogel pre-polymer formulations and crosslinking agents are summarized in Table

S.1. Alginate hydrogels were crosslinked for 45 minutes before discs were punched out and transferred

to media.

Standard agarose (“Agarose for Routine Use”; Sigma; Table S.2) was modified with RGD as

previously described4. Briefly, four-fold excess G4RGDASSKY-OH peptide was reacted with 10-fold

excess of the crosslinker N-Sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (sulfo-

SANPAH; Thermo Scientific) in Dulbecco’s PBS (dPBS) over 4 hr in the dark at 25°C. Next, the

solution of peptide-coupled crosslinker was combined with melted agarose in dPBS to a final

concentration of 2.5mg/mL, and maintained at 45°C. The polymer solutions combined with peptide and

sulfo-SANPAH were then exposed to 354nm UV light for 15 minutes while incubating in a 45°C water

bath. Agarose was gelled at 4°C, and then disinfected in 70% ethanol. Following disinfection, matrices

were washed extensively in dPBS. The reaction efficiency for RGD-agarose coupling was previously

characterized4.



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Poly(ethylene glycol) dimethacrylate (PEGDM) prepolymers and acryloyl-PEG-GRGDS were

prepared as described previously5,6. Briefly, PEG (Mw 10,000 or 35,000 Da; Table S.2), methacrylic

anhydride (MA), diethyl ether, dichloromethane and triethylamine (TEA) were purchased from Sigma-

Aldrich and used as received. Photoinitiator Irgacure 2959 (I2959) was obtained from Ciba Specialty

Chemicals and used as received. Acryloyl-PEG-N-hydroxysuccinimide (ACRL-PEG-NHS, 3400 g/mol)

was purchased from Laysan Bio, Inc. GRGDS-OH peptide was purchased from Bachem Bioscience Inc.

PEGDM10k and PEGDM35k were prepared from the reaction of various PEGs and MA in

dichloromethane for 48 hr at room temperature. Next, the solution was precipitated into diethyl ether.

The product was filtered, dried in a vacuum oven overnight at room temperature, and then dialyzed for

3 days against de-ionized water. GRGDS-OH peptide was dissolved in anhydrous dimethyl formamide

(DMF) containing 4 M excess of TEA. ACRL-PEG-NHS was also dissolved in anhydrous DMF and,

immediately after, mixed with 1.1 M excess of peptide. After incubating for 3 h at room temperature,

ACRL-PEG-GRGDS was precipitated twice in cold anhydrous diethyl ether and dried in a vacuum oven

overnight at room temperature.

To prepare hydrogels, PEGDM (5-20% wt) and aqueous I2959 solution (0.05% wt) were mixed

in dPBS. Cylindrical samples were prepared by pouring the polymer solutions into a Teflon mold and

then curing with a long wavelength UV source (365 nm, 300 µW/cm2) for 20 min at room temperature.

Mechanical Characterization of Hydrogels Immediately after being cast, alginate hydrogels (12.2mm

diameter and 2mm thickness) were subjected to shear rheology using a Bohlin controlled strain

rheometer (Malvern, MA). The frequency response of hydrogels was determined between 0.01 and 10

Hz (Fig. S2); alginate hydrogels tested in this study behaved in a nearly linear-elastic manner over a

broad frequency range that includes 1 Hz. This characteristic behavior was independent of crosslinking



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chemistry, except at the lowest crosslinking densities or in MVM. Even in those matrices, the departure

from linear-elastic behavior was only at extreme frequencies. For elastic modulus measurements,

alginate, PEGDM and agarose hydrogels were subjected to an unconfined compression test

(1mm/minute) in an Instron 3342 mechanical apparatus (Norwick, MA), immediately after being cast

(alginate, agarose) or after swelling to equilibrium in dPBS for 2 hr (PEGDM). The elastic modulus E

was calculated as the slope of the linear portion (first 10% of strain) of the stress vs. stain curves (Fig.

S2). As in previous studies with ionically crosslinked alginate hydrogels, E for hydrogels with 1%

weight alginate dropped to approximately half its initial value after 24 hr in DMEM (Fig. S2), and

thereafter remained constant (data not shown).

Routine Cell Culture and Cell Characterization Human mesenchymal stem cells (hMSC7)

were purchased from Lonza (Basel, Switzerland). hMSC were maintained in low glucose DMEM

supplemented with 20% FBS and 1% penicillin/streptomycin (Invitrogen), and used between passages

4-5. Clonally derived murine bone marrow stromal mesenchymal stem cells (mMSC), originally

obtained from Balb/c mice (D18-10) were purchased from American Type Cell Culture (ATCC) and

maintained in standard DMEM supplemented with 10% Fetal Bovine Serum and 1%

penicillin/streptomycin (Invitrogen), and used between passages 20-24.

Cells were maintained at lower than 80% confluency in culture. Baseline expression of

Alkaline Phosphatase (ALP), activity (a biomarker for matrix synthesis concurrent with osteogenic

lineage specification) was assessed by plating naïve MSC (5,000 cells/cm2) onto Labtek chamber slides

(Thermo Scientific) in standard culture media, then fixing in 4% parformaldehyde and staining for ALP

24 hr after plating cells. ALP activity was visualized by Fast Blue staining (500μg/mL naphthol-AS-

MSC phosphate, NAMP and 500μg/mL Fast Blue BB, Sigma) in alkaline buffer (100mM Tris-HCl,



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100mM NaCl, 0.1% Tween-20, 50mM MgCl2, pH 8.2). Nuclei were counterstained with 1μg/mL

Hoescht 33342 (Invitrogen). After mounting (Vectashield), cells were visualized on a Nikon E800

upright microscope equipped with a 20x (0.45 NA) magnification lens (Nikon; Tokyo, Japan) and an

Olympus DP-70 (Tokyo, Japan) color camera. The density of ALP-expressing cells in the naïve

populations after tissue culture expansion was calculated by counting the number of cells in five

randomly selected fields (20x magnification), and normalizing to the total number of cells detected by

Hoescht staining (Fig. S1).

Stem Cell Differentiation in 3D Matrix Culture mMSC and hMSC in flasks were

trypsinized (0.05% trypsin/EDTA, Invitrogen), washed twice with Dulbecco’s PBS (dPBS), filtered

through a 70μm-mesh cell strainer (BD Pharmigen) and resuspended into serum free media at a

concentration of 80 million viable cells/mL (mMSC) or 60 million viable cells/mL (hMSC). The

concentration and viability of cells were determined using a Coulter Vicell (Beckman Coulter,

Fullerton, CA). Cell suspensions were transferred to an orbital shaker for 15 minutes to allow adhesion-

mediated signaling to decay to basal levels. For alginate-based 3D culture studies, cell suspensions were

mixed with unary or binary MVG pre-polymer solutions to a final density of 2 x 107 cells/mL (mMSC)

or 1.5 x 107 cells/mL (hMSC) in 1-5% wt/wt alginate. The calcium concentration used to crosslink

hydrogels was varied from 6.25-50mM to yield hydrogels with elastic moduli from 2.5-110 kPa after

the first day of culture (Fig. S2). Alginates were modified to present a final density of either 189 or

754μmol/L RGD peptides within the cell-encapsulating matrices. The RGD density was manipulated by

controlling the number of RGD peptides grafted to a single alginate polymer chain.

In some studies, RGD-modified agarose or PEGDM were used as alternative cell-encapsulating

matrices. For agarose studies, RGD-modified agarose (0.25% wt) was diluted with unmodified agarose



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(0-1.25% wt) and cells at 45°C, then cell-polymer solutions were allowed to gel at 25°C for 20 minutes.

The rigidity of agarose hydrogels was manipulated by altering the concentration of unmodified agarose

(Fig. S2) but the final concentration of RGD was always maintained constant at 754μmol/L. For

PEGDM studies, ACRL-PEG-GRGDS was mixed with PEGDM (5-20% mass fraction depending on

the desired hydrogel elastic modulus, Fig. S2) and cells suspended into dPBS, and hydrogels were

formed by UV crosslinking as described above. The final concentration of GRGDS was maintained

constant at 754μmol/L after mixing with cells. PEGDM hydrogels were washed once and media was

replaced at 2 hr to prevent any affects on cells by excess crosslinker. For both agarose and PEGDM

based 3D culture studies, the final concentration of mMSC within hydrogels was 2 x 107 cells/mL.

After hydrogel matrices were formed, discs 1mm thickness and 20mm in diameter were punched

out with a metal dye. Matrices were maintained in the same medium used for cell expansion. To induce

differentiation, 3D mMSC cultures were also supplemented with 10mM β-glycerol phosphate (Sigma),

50μg/mL ascorbic acid (Sigma) and 0.1μM dexamethasone (Sigma), as dexamethasone alone has

demonstrated ability to induce adipogenesis of D1 in-vitro10. 3D hMSC cultures were supplemented

with the same concentrations of β-glycerol-phosphate and ascorbic acid, and, additionally, were cycled

between an adipogenic cocktail consisting of 1μM dexamethasone, 50μM indomethacin (Sigma),

0.5μM 3-isobutyl-1-methylxanthine (IBMX; Sigma) and 10μg/mL human recombinant insulin

(Invitrogen), and an adipogenic maintenance supplement containing only insulin and 0.1μM

dexamethasone11. The hMSC adipogenic supplement was cycled every time the cells were fed.

In a subset of studies, alginate hydrogels containing encapsulated mMSC were incubated in the

same medium as alginate hydrogels containing no cells. By varying the crosslinking concentration of

the cell-free hydrogels, the total amount of bio-available soluble calcium in the induction medium was

decoupled from the rigidity of the cell-encapsulating matrix. (Fig. S3) The concentration of free calcium



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in the medium after 2 or 7 days in culture was measured using the colorimetric, calcium-sensitive dye

Arsenazo III (Sigma) and comparing the results to a standard curve prepared from calcium chloride

dissolved into 0.3M saline.

Histological Analysis of MSC Lineage Specification After 1 week of culture, cell-encapsulating

matrices were fixed (4% parformaldehyde, PFA in serum free, phenol red free DMEM with 0.1%

sodium azide, 0.1% Triton-X-100 and 0.1% Tween-20) for 30 minutes at 25°C and washed in serum

free media with 0.1% Tween-20. ALP activity (osteogenic biomarker) and neutral lipid accumulation (a

functional marker for adipogenesis) were visualized by Fast Blue and Oil Red O (ORO) staining11,

respectively, on whole mounted alginate matrices. After fixing, the matrices were cut to nearly identical

dimensions with a razor blade, then equilibrated into alkaline staining buffer (100mM CaCl2, 100mM

Tris-HCl, 100mM NaCl, 0.1% Tween-20, 50mM MgCl2, pH 8.2), and then incubated in the same buffer

with the addition of 500μg/mL NAMP and 500μg/mL Fast Blue BB. After washing in alkaline staining

buffer, the samples were equilibrated back to neutral pH using serum free media with 100mM CaCl2

and 0.1% Tween-20 then stained with a solution of ORO (600μg/mL in isopropyl alcohol for 2 hr at

25°C). Nuclei were counterstained with 1μg/mL Hoescht 33342, and the constructs were washed, fixed

a second time in 4% PFA with 100mM CaCl2 and 0.1% Tween-20, washed, and mounted in

Vectashield. Color micrographs were acquired using a Nikon E800 upright microscope and an Olympus

DP-70 color camera. Because the softest agarose hydrogels displayed significant heterogeneity in

micro-architecture, only cells that appeared to be completely encapsulated into the material were

analyzed histologically.

For indirect immunofluorescence analysis of cell proliferation via expression of Ki-67 and

osteogenesis via expression of osteocalcin (OCN), alginate matrices were fixed (4% PFA in PBS with



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divalent cations, cPBS), washed in cPBS containing 100mM BaCl2 and then in cPBS, then equilibrated

to 30% sucrose in cPBS overnight at 4°C. Subsequently, cell-matrix constructs were equilibrated into

optimal cutting temperature medium (OCT) before being frozen in liquid nitrogen and sectioned on a

cryotome to 5μm thickness. For OCN staining, sections were blocked (5% FBS in a solution of 0.1%

BSA and 0.1% Triton-X-100 in dPBS), then probed with goat-anti-mouse OCN antibodies (Table S.3)

diluted into antibody-dilution buffer (0.1% BSA, 0.1% Triton-X-100 in PBS) overnight at 4°C,

followed by washes and staining with Alexa Fluor 488 conjugated donkey anti-goat IgG (5μg/mL,

Invitrogen). For Ki-67 staining, sections were blocked (5% goat serum in antibody-dilution buffer), then

probed with rat anti-mouse Ki-67 primary antibodies (Table S.3) overnight at 4°C, followed by washes

and staining with Alexa Fluor 488 conjugated goat-anti-rat IgG (5 μg/mL, Invitrogen). After secondary

antibody incubations (2 hr at 25°C), sections were washed and mounted in ProLong Gold with DAPI

(Invitrogen). Fluorescence images were acquired using an Olympus IX81 inverted microscope equipped

with a Coolsnap HQ2 camera (Prior Scientific, Rockland, MA) and a Carv II Nipkow-type Spinning

Disc Confocal Attachment (BD Biosciences, San Jose, CA). Digital micrographs were acquired with

2x2 CDC binning via either 63x, 1.4 N.A. oil immersion or 20x, 0.3 N.A. lenses (Olympus) and a

Coolsnap HQ2 camera. Monochrome micrographs were processed and pseudocolored using Adobe

Photoshop.

Western Analysis of MSC Lineage Specification in Alginate Matrices After 1 week of 3D culture,

alginate matrices were washed with dPBS, then immersed in 8mL of 50mM ethylenediaminetetraacetic

acid (EDTA; Sigma) in dPBS (pH = 7.4) for 25 minutes at 37°C. Cells were pelleted and lysed into

Radio Immunoprecipitation Assay (RIPA) buffer (Sigma) with Minitab Protease Inhibitors (Roche).



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Protein content of lysates was determined with the bicinchoninic acid (BCA) protein assay

(Thermo Scientific). 30 μg of protein were separated by SDS-PAGE electrophoresis on 16% Tris-

Glycine Gels, and transferred to PVDF membranes (BioRad, Hercules, CA). Membranes were blocked

(3% nonfat milk/TBST, 1 hr), and then incubated with primary antibodies (Table S.3) overnight at 4°C,

washed in TBST, and probed with HRP-tagged secondary antibodies (goat anti-rabbit for Cbfa-1 and

fibronectin, rabbit anti-mouse for Actin) from Cell Signaling (Danvers, MA), or biotinylated secondary

antibodies (mouse-anti rabbit for PPAR-γ, Adn and Collagen I, rabbit anti-rat for OPN) followed by

streptavidin-HRP (Jackson Immunolabs; West Grove, PA). Blots were developed using

Bioluminescence X-ray film (Kodak) and the Enhanced Chemiluminescence substrate system (Thermo

Scientific). Actin was used as an internal loading control.

Digital images of Western Blots were quantified using Image-J software. Band intensity of

Cbfa-1 and PPAR-γ for each sample was normalized to the intensity of the sample from the 2.5 kPa

matrix (PPAR-γ) or 20 kPa matrix (Cbfa-1) from the same film. Actin bands were scanned to normalize

for loading differences between samples. Multiple X-ray film exposures were performed to ensure that

sample exposure times fell within the linear range of the detection system (Fig. S1).

Analysis of Macromolecular Transport in Alginate Matrices of Varying Rigidity To determine

whether changing the mechanical properties of alginate hydrogels within the range of E used to

investigate MSC fate significantly affects macromolecular transport12, we directly measured diffusion

coefficients for a representative protein, bovine serum albumin (BSA, 67 kDa; Sigma), within alginate

matrices. Protein was labeled with rhodamine (1:4 ratio of BSA to rhodamine) via reaction of BSA with

5,6-tetramethylrhodamine succinimidyl ester (Invitrogen) in bicarbonate buffer (0.1M NaHCO3, pH 8.5)



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over 24 hr at 25°C. The products were purified by dialysis, and the degree of coupling was confirmed

by UV-vis spectrophotometry.

Next, BSA diffusion out of alginate hydrogels was measured. For these studies, alginate

hydrogels of varying mechanical properties encapsulating 1mg/mL TAMRA-labeled BSA were cast and

equilibrated over 24 hr into medium (without serum or phenol red) containing the same concentration of

BSA-TAMRA. Hydrogel discs (9.33mm diameter, 2mm thickness discs) were transferred to serum and

phenol red free medium (10mL), and aliquots of this bathing media were taken periodically to measure

molecular diffusion out of the hydrogels. The experiments were performed on an orbital shaker to

prevent boundary-layer effects from complicating diffusion measurements. BSA-TAMRA

concentration was measured using a Fluorescence plate reader (Biotek; Winooski, VT). At short times,

the concentration of BSA inside the hydrogel was assumed to be unperturbed, leading to the semi-

infinite slab approximation13, where the molecular flux out of the hydrogel matrix, j, at a given time t is

given by Equation 113:

( )t

Dccj matrixmatrixmedia π

−= Equation 1

where cmedia was assumed to be zero, cmatrix was the initial concentration of encapsulated BSA-TAMRA,

and Dmatrix is the diffusion coefficient within hydrogel matrices. Assuming that cmedia and cmatrix did not

change significantly over the course of the experiment because of the semi-infinite slab approximation,

Equation 1 was integrated to yield Equation 213:

πmatrix

bath

matrixmatrixmedia

DtVAcc 2= Equation 2

where Amatrix is the surface area of the alginate hydrogel through which flux occurs. Dmatrix was

calculated based on the slope of linear curves of cmedia versus the square-root of t; the assumption that



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cmedia and cmatrix did not change sufficiently to invalidate Equation 1 was confirmed by the fit of these

curves to a linear curve (R2 > 0.8 in all cases) over the range of t1/2 tested. The data suggest changes in

macromolecular diffusion did not appear to underlie the sensitivity of MSC populations to E (Fig. S3).

These findings are consistent with previous studies, in which the average pore-mesh size of alginate

hydrogels was determined by differential scanning calorimetry to be approximately 5nm, and that this

radius did not change significantly even when the mechanical properties of the hydrogels changed due

to changes in initial formulation or degradation14. Other studies have shown directly that diffusion of

small metabolites (e.g. oxygen, glucose) within alginate matrices does not change significantly with

respect to either polymer concentration or the extent of crosslinking15.

Cell, Cytoskeletal and Nuclear Morphology in 3D Alginate Matrices We assessed gross,

cytoskeletal and nuclear morphology of encapsulated D1 mMSC either 2 or 24 hr after cell

encapsulation, with the earlier time-point used to determine possible effects on morphology before

significant changes in protein synthesis could occur, and the latter time-point used to determine longer-

term reorganization. We observed no apparent differences between the different time points, and

therefore performed the measurements at 2 hr. All representative images shown in the text are taken

from the axial plane in which cross-sectional area was highest.

Morphology was assessed after incubating hydrogels in phenol red free DMEM (Invitrogen)

with 10% FBS. In some cases, a broad spectrum Myosin II inhibitor, 2,3-Butanedione monoxime

(BDM; 20mM) was added to cells while they were incubating on the orbital shaker before

encapsulation, and also to the media bathing the 3D culture. Gross cell morphology was assessed via

differential-interference contrast (DIC) imaging. Photo-micrographs were obtained using the same

microscope and software used to acquire immunofluorescence images, via a 63x, 1.4 N.A. oil-



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immersion lens. To assess F-actin morphology, alginate-encapsulated cells were fixed in 4%

paraformaldehyde diluted into serum free media with 0.1% Triton-X-100, then washed and stained with

Alexa Fluor 568-phalloidin in-situ (Invitrogen; 4 U/mL in serum-free media with 0.1% Triton-X-100)

for 30 minutes at 25°C, washed in PBS containing divalent cations (cPBS; Invitrogen) and mounted in

Vectashield. Confocal images of F-actin were obtained with a Carv II Nipkow-type Spinning Disc

Confocal Attachment (BD Biosciences, San Jose, CA) to the Olympus IX81. Nuclear morphology was

assessed by acquiring confocal scans of nuclei labeled with Ethidium Homodimer (EtD-1; Invitrogen),

using a Zeiss LSM 510 Meta Laser Scanning Confocal Microscope. For all metrics of cytoskeletal and

nuclear morphology, a Z-sampling rate of 0.25μm was used for image acquisition.

To quantify gross cell shape and F-actin morphology, cells were segmented manually using

Scanalytics IPLab software (BD Biosciences; Rockvlle, MD), and the projected cross-sectional area was

measured using the Matlab Image Processing Toolbox (Mathworks, Natick, MA). The cross-section of a

given cell with the largest projected area was used for quantitative analyses. Cortical (F-actin)

protrusions into the matrix were manually counted and measured using Scanalytics IPLab. These

protrusions were counted if they projected out perpendicular to the prominently stained membrane (Fig

S4). The protrusion length was defined by the shortest straight line segment that could connect the tip of

the protrusion to the point at which it touched the rest of the membrane (Fig. S4). Nuclear morphology

was quantified using Matlab-based image processing of confocal stacks. Briefly, nuclei were identified

against background via a global threshold (the most intense pixels in a given nucleus) and median and

Gaussian smoothing filters were applied to reduce noise. After segmenting nuclei, the surface area A

and volume V of each nucleus was measured via numerical integration of the perimeter (to measure A)

and the area (to measure V) of each confocal slice. Nuclear morphology was assessed quantitatively

using nuclear sphericity, a non-dimensional parameter related to A and V of nuclei by Equation 3:



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( )AV 3

231

6πψ = Equation 3

where ψ = 1 for a perfect sphere. Sphericity was used in lieu of nuclear shape index (NSI16), a

parameter identified in previous studies as correlating with cell fate in 2D culture becauseψ does not

require a-priori knowledge of the relative orientation of the nucleus, which would be difficult to assess

in 3D where there is no preferential axis of polarization. Representative images in Figure 2.C are iso-

surfaces fit to segmented nuclei.

Image analyses of morphology were performed on a minimum of 10 cells per condition, and

box-plots were generated in Matlab.

Indirect Immunofluorescence (IF) Analysis of 2D Integrin-ECM Binding IF analysis of specific

integrins used by mMSC to adhere to surfaces coated with natural ECM proteins was performed using

the method described by Keselowsky and Garcia17. Either human plasma vitronectin or fibronectin

(Sigma) dissolved into 0.1M carbonate buffer (pH 9.2), and coated at saturating densities (1μg/cm2)

onto the surfaces of Labtek chamber slides for 24 hr at 4°C. Surfaces were subsequently washed in

dPBS. Next, mMSC were suspended into dPBS with 2mM dextrose and seeded at a surface density of

5,000 cells/cm2. Integrins were crosslinked to ECM proteins 2 hr after seeding using a cell-

impermeable, hydrophilic amine crosslinker, 3,3´-Dithiobis(sulfosuccinimidylpropionate) (DTSSP,

Thermo Scientific). The crosslinker was quenched with 50mM Tris, and non-crosslinked cell

components were extracted with 0.1% SDS. After several dPBS washes, integrins were visualized using

indirect immunofluorescence of either α5 or αV integrin subunits (Table S.3). Because this procedure is

designed to extract all non-crosslinked components of the cell including the nucleus, Hoescht 33342

was used as a nuclear counterstain to ensure complete extraction17.



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Imaging α5-Integrin Localization in 3D Matrices Plasmid DNA encoding EGFP-tagged human α5-

integrins18 was kindly provided by Rick Horwitz (University of Virginia). DNA encoding EGFP was

purchased from Clonetech (Mountain View, CA). Plasmid DNA was amplified in E. coli and isolated

using a MaxiPrep Kit (Qiagen; Valencia, CA). DNA yield and purity were determined with a DU530

UV/Vis Spectrophotometer (Beckman Coulter). Cells were transfected with plasmids for EGFP-α5-

integrin or naked EGFP using Lipofectamine 2000 in OptiMEM (1 μg/cm2 plasmid DNA, 0.2% v/v

Lipfectamine). A population of D1 stably expressing EGFP-α5-integrin was generated based on

resistance to G418-Sulfate (Invitrogen; 300μg/mL), and FACs to sort for EGFP-positive cells at the

Harvard University Center for Systems Biology on a MoFlo Cell Sorter (Dako Cytomation, Denmark).

D1 expressing EGFP or EGFP-α5-integrin were encapsulated into alginate matrices (either unmodified

or modified with 15μM RGD) and at 2 hr matrices were fixed with 4% paraformaldehyde in serum free

media. Confocal micrographs were obtained at 63x magnification with a 1.4 N.A. oil immersion lens

using the previously described Olympus system.

ELISA to Analyze Specific Integrins Binding to RGD Presented by in 2D and 3D Matrices A

novel method, inspired by the peptide-ELISA used routinely for antibody epitope mapping, was

developed to probe for specific integrin subunits clonally derived mMSC use to bind RGD presented by

2D and 3D matrices (Fig. S5). Biotinylated RGD peptides were used to facilitate isolation via

Neutravidin coated stripwells (Thermo Scientific) of RGD(biotin)-bound integrins. Next, integrins

bound to the RGD-biotin were probed with a standard ELISA, using polyclonal antibodies against either

α5 or αV integrin subunits (Table S.3), HRP-tagged anti-rabbit secondary antibodies (Jackson

Immunolabs), and QuantaBlue fluorogenic HRP substrate (Thermo Scientific). We note that an

analogous approach, which does not utilize protein crosslinking, has been used previously to study



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fibronectin-β1-integrin interactions via co-immunoprecipitation and Western Blot19. The present

technique was first validated in studies of integrin binding to RGD peptides mixed with mMSC

suspended into 2mM dextrose in dPBS (2 x 107/mL) and incubated on an orbital shaker. After 15

minutes, cells were pelleted (1200 rpm, 4°C) and lysed into RIPA. The protein content of clarified cell

lysates was determined via BCA, and an equal quantity of protein (5μg) was added to each of the

neutravidin coated stripwells; the protein was incubated on the plates for 2 hr at 25°C on a plate shaker.

Before coating cell lysates, the plates were washed twice with Dulbecco’s PBS and blocked for 15

minutes with StartingBlockTM (Pierce) at 25°C. After probing lysates for 2 hr (25°C), the plates were

washed in dPBS and probed for 2 hr at 37°C with primary antibodies (Table S.3), then washed and

probed for 1 hr at 37°C with HRP-tagged secondary antibody. After washing, plates were exposed to

QuantaBlue for 45 minutes at 37°C. The developed substrate was transferred to a black bottomed-plate

for fluorescence analysis in a Biotek Plate Reader with 325nm excitation and 460nm emission.

For studies of 3D-matrix based integrin-RGD binding, biotinylated RGD peptides were coupled

to alginate polymers used to form mMSC-encapsulating hydrogels as described for Nb analysis (cell

density of 2 x 107 cells/mL). At the same 2 hr time point where other 3D cell adhesion assays were

performed, alginate matrices were washed in dPBS and digested with alginate lysase (250μg/mL). Cell

lysates were prepared and probed as described above. The relative number of bonds between integrins

and RGD was calculated according to Equation 4:

blankcontrol

blanksampleb RFIRFI

RFIRFIN

X −−

=α, Equation 4

where the blank corresponds to lysate of cells not exposed to RGD-biotin, and the control was an

arbitrarily selected sample. The control sample for all 3D assays and for the direct 2D to 3D comparison

was the condition in which mMSC were encapsulated into 22 kPa matrices with a 1:4 ratio of 2 RGD-



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biotin / polymer to unmodified polymer (15μM RGD-biotin). In control studies, binding of RGD to both

integrins was inhibited when integrins were saturated with unlabelled RGD, when RGD-biotin was

replaced with RGE-biotin, or when cell traction forces blocked with BDM (Fig. S5). 2D studies of

specific integrin-RGD bonds were performed with the same RGD-biotin-coupled RGD. For 2D studies,

alginate substrates were prepared (37μM RGD-biotin) and incubated in serum free media for 3 days.

Next, mMSC were seeded at a surface density of 200,000 cells/cm2. Integrin-RGD binding analyses

were performed 2 hr after seeding cells. The number of bonds was calculated relative to αV-RGD bonds

formed for cells cultured on a 22 kPa substrate. The relationship between E and the relative number of

αV-RGD bonds was fit to a hyperbolic curve of the form:

EEEN

N bb V +

⋅=

21

max,,α Equation 5

where E1/2 is the elastic modulus of the substrate at which half the maximum number of bonds was

formed. Because bond formation was normalized to unity, Nb,max was arbitrarily set to 1. Curve fitting

was performed in Matlab using non-linear regression.

FRET Measurements of RGD-Cell Bond Number in RGD-modified Alginate Hydrogels

The FRET system used in our studies is analogous to FRET systems used to monitor protein-

protein interaction dynamics in living cells20,21; however, instead of labeling integrins, the entire cell

membrane is labeled non-specifically with the lipid-intercalating dye 5-hexadecanoylaminofluorescein,

and RGD is labeled with tetramethylrhodamine22. For FRET measurements, mMSC were labeled with

5-hexadecanoylaminofluorescein (4nmol/cm2; Invitrogen) in tissue culture flasks for 24 hr, before being

prepared for encapsulation as per differentiation studies. In some cases, BDM (20mM), or



17

cyclohexamide (20 μg/mL, to inhibit protein synthesis) were added to cells while they were incubating

on the orbital shaker (37°C, 5% CO2) before encapsulation. Cell suspensions were mixed with RGD-

TAMRA-modified alginates to a final concentration of 2 x 107 cells/mL and 1-4 weight percent alginate

solution. The density of RGD-TAMRA peptides was controlled by varying the number of peptides

coupled to one polymer chain. After mixing cells with alginate and crosslinking, hydrogel discs (750μm

thick, 9.33mm diameter) were incubated for 2 hr on an orbital shaker in phenol red free medium (10%

FBS) at 37°C, 5% CO2 before FRET measurements. The 2 hr time-point was chosen to minimize any

effects of new protein synthesis on integrin-RGD bond formation. RGE-TAMRA peptides were used as

negative controls.

To obtain micrographs depicting FRET between RGD-TAMRA and 5-

hexadecanoylaminofluorescein labeled cell membranes, mMSC were encapsulated into hydrogels at a

density of 106 cells/mL, and 2 hr after cell encapsulation, cells in hydrogels were imaged with a 63x 1.0

N.A. water-immersion lens (Zeiss) on the same Zeiss Confocal Microscope used for nuclear

morphology analysis. Fluorescein (green; 500-530nm) and TAMRA (red; 565-615nm) emission were

detected simultaneously in two independent emission channels. Images were then prepared by

overlaying the thresholded green and red emission channels in Adobe Photoshop.

To obtain quantitative data on integrin-RGD bond formation, emission spectra from hydrogel

discs were collected after excitation at 488nm using a Fluoromax 3 Spectroscope (Jobin Horiba, Edison,

NJ). Spectra were normalized to the cell number in each hydrogel, by dissolving the hydrogel in 50mM

EDTA in dPBS (Sigma) and then counting cells in a Z2 Coulter Counter (Beckman Coulter, Fullerton,

CA). The degree of energy transfer was calculated according to Equation 6:

0

1IIDFRET −= Equation 6



18

where I refers to emission intensity at 520nm, the peak fluorescein emission, and I0 to the 520nm

emission intensity in a donor control sample with the same peptide density, elastic modulus and drug

treatment but with RGD peptides rather than RGD-TAMRA peptides. Because the concentration of

RGD-TAMRA peptides was varied in some studies, DFRET was calculated based on diminished

fluorescein emission rather than sensitized rhodamine emission. DFRET values were calculated based on

the averaged I and I0 values (n = 4-5). The number of integrin-RGD bonds was used calculated based on

a linear calibration curve generated by performing parallel FRET-binding and 125I-RGD binding studies

in solution22.

To estimate the minimum depth into the alginate hydrogel an integrin must reach to access

RGD, we made the following assumptions: 1) RGD is distributed homogenously within the alginate

matrix, 2) mMSC are spherical, with radius r of 10μm, and can bind any RGD peptide in a spherical

shell surrounding them (schematic in Figure 5.A). The minimum depth, h, was calculated based on the

minimum volume of the spherical shell, which in turn was estimated by comparing the number of bound

RGD peptides per cell to the concentration of RGD in the hydrogel, CRGD (Equation 7).

24 rNcNh

ARGD

b

π⋅⋅= Equation 7

where NA is Avagadro’s number.

RGD-Cell Bond Formation Measurements in Covalently Crosslinked Alginate Hydrogels

MVG alginates were oxidized to form aldehyde groups with sodium periodate (15% theoretical

degree of oxidation23), then dialyzed and freeze-dried. The lyophilized products were coupled to either

RGD or RGD-TAMRA peptides using the same carbodiimide chemistry described above. To form

covalently-crosslinked alginate dialdehyde hydrogels, oxidized high Mw, high GA alginate (2-8% wt)



19

was mixed with poly(acrylic-co-hydrazide) (PAH24) to a final concentration of 25-300 mM hydrazide

groups, using luer-lock syringes. The elastic moduli of covalently crosslinked hydrogels were measured

using the same Intron apparatus used for calcium-alginate matrices (Fig. S7). Although it is established

that calcium-alginate is a highly compatible material for cell encapsulation, cell viability within the

covalently crosslinked alginates described here has not been assessed. Thus, we assessed short-term

(e.g. over 1-2 days) viability of mMSC encapsulated into covalently crosslinked hydrogels. D1 mMSC

were encapsulated at 107 cells/mL. Cell viability was assessed qualitatively with a Live/Dead Stain kit

(Invitrogen) and Alamar Blue (Ab Serotec, Raleigh, NC) reduction. (Fig. S7).

FRET measurements of cell-RGD bond formation in covalently crosslinked hydrogels were

performed as described for calcium-alginate matrices, except that hydrogels were digested with

250μg/mL alginate lyase (Sigma) in dPBS instead of 50mM EDTA to analyze encapsulated cell

number.

FRET Measurements of Cell Traction-Mediated Nanoscale RGD Clustering were performed using

a FRET technique originally described by Kong et al.25, with modifications to allow population-level

measurements in 3D hydrogels. Briefly, suspensions of unlabelled mMSC were prepared as described

for Nb measurements, and mixed with MVG containing an equal proportion of RGD-TAMRA-modified

polymer : RGD-carboxyfluorescein-modified polymer, to a final concentration of 2x107 cells/mL and

1% wt polymer. The density of RGD peptides was kept constant (1 RGD / polymer; overall density of

37μmol/L). After mixing cells with alginate, hydrogels were crosslinked, and incubated for 2 hr in

phenol red free media with serum as with Nb measurements. Emission spectra were normalized to cell

number and the degree of energy transfer was estimated by Equation 6. These FRET measurements are



20

specific to clustering of RGD by cells, as shown by control experiments performed with MC3T3-E1

pre-osteoblasts encapsulated into matrices with 2 RGD / polymer (Fig. S8).

Analysis of Extracellular Matrix Synthesis and by mMSC in 3D Alginate Matrices The time-

course of cellular ECM synthesis was analyzed via Western analyses. D1 mMSC were cultured for 3, 5

or 7 days in differentiation media within alginate matrices with varied rigidity and 754μM RGD. ECM

proteins were recovered along with cells by chelating calcium-alginate with 50mM EDTA/dPBS, then

lysing the recovered cell pellet in RIPA. For Western analysis, 25μg/protein were loaded in 4-12%

gradient Tris-glycine gels, separated by SDS-PAGE, and transferred to PVDF. Membranes were then

blocked with 5% BSA in TBST, probed with primary antibodies (Table S.3), then with HRP-tagged

secondary antibodies and visualized with X-ray film as in lineage commitment studies. Purified ECM

protein standards (mouse Type I Collagen and mouse Fibronectin) were purchased from RayBiotech

(Norcross, GA) and Innovative Research, Inc. (Novi, MI).

Analysis of mMSC Fate in Extended 3D Matrix Culture Clonally derived mMSC were

cultured in 3D alginate matrices in differentiation media for 21 days. Medium was collected on day 21

for analysis, and matrices were treated with a solution of 2.5mg/mL Collagenase P (Pierce) in 0.025%

Trypsin/EDTA to digest ECM proteins, followed by immersion in 50mM EDTA/dPBS to isolate cells

from alginate. Cell pellets were lysed into RIPA and clarified. To measure total DNA, the resulting

pellets were air-dried and subjected to 3 freeze-thaw cycles at -80°C, then thawed into CyQuant DNA

Lysis Buffer (Invitrogen). DNA content was measured using Hoescht 33342 (0.1μg/mL) based on a

standard curve prepared from calf thymus DNA samples (Invitrogen). Clarified cell lysates were used

for Western Analysis. Secreted Osteocalcin was quantified using a Mouse Osteocalcin ELISA kit



21

(Biomedical Technologies, Stoughton, MA) according to the manufacturer’s protocol, and was

normalized to total cellular DNA content.

Antibody Blocking Studies Clonally derived mMSC were prepared as described for 3D

differentiation studies, and during the 15 minute incubation on the orbital shaker, cells were mixed with

function blocking antibodies against α5-integrins (H10-27), αV-integrins (RMV-7), or Isotype Controls

(Table S.3). The same antibodies were added to the media used for feeding cells. The viability of

encapsulated cells exposed to function blocking antibodies was assessed with a Live/Dead Staining Kit

(Invitrogen), and proliferation was assessed via Ki-67 immunofluorescence (Fig. S9).



22

Supplemental Tables

Table S.1. Alginate Formulations

Table S.2. Agarose and PEGDM Hydrogel Formulations

Alginate Type Ratio of M/G blocks

Alginate backbone modification

Weight % High Mwpolymer

Weight % 5 Mrad MVG

Crosslinking Molecules

Unary MVG 0.62 None 1 0 Calcium Sulfate (CaSO4)

Binary MVG 0.62 None 1 1-4 Calcium Sulfate (CaSO4) MVM 0.36 None 1 0 Calcium Sulfate (CaSO4)Alginate Dialdehyde

0.62 15% theoretical degree of oxidation

1 0 poly(acrylamide-co-hydrazide) (PAH)

Polymer Type Mw (kDa) Polymer backbone modification

Weight % High Mwpolymer

Crosslinking Molecules

Agarose Unknown None 0.25-1.5 Physical Crosslinking of Agarose

PEGDM 35 None 7-10 Free radical polymerization PEGDM 10 None 5-20 Free radical polymerization



23

Table S.3. Primary AntibodiesProtein Antigen Clone, Catalog #

and Vendor Host species and reactivity

Concentration or Fold Dilution of Polyclonal Antibody Stock Solution used and Application

α5-integrin (CD49e) H10-27, 553318, BD Pharmigen

Rat anti-mouse integrin α5

5-50 μg/mL (Blocking)

α5-integrin (CD49e) Polyclonal AB1921, Chemicon

Polyclonal rabbit anti-mouse IgG

1:500 (Indirect immunofluorescence) 1:1000 (Integrin-binding ELISA)

αV-integrin (CD51) Polyclonal AB1923, Chemicon

Rabbit anti-human integrin-αV

11:500 (Indirect immunofluorescence) 1:1000 (Integrin-binding ELISA)

αV-integrin (CD51) RMV-7, 552299, BD Pharmigen

Rat anti-mouse integrin αV

50μg/mL (Blocking)

Actin Clone C4; MAB1501, Chemicon

Mouse anti-actin 1:1000 (Western Blot)

Adiponectin (Adn) C45B10, 2789, Cell Signaling

Rabbit anti-mouse Adiponectin

1:500 (Western Blot)

Collagen I Polyclonal AB765P, Chemicon

Rabbit anti-mouse collagen Type I

2μg/mL (Western Blot)

Core Binding Factor α1 (Cbfa-1)

ab23981, Abcam Rabbit anti-Cbfa-12 2μg/mL (Western Blot)

Fibronectin Polyclonal, Ab1954, Chemicon

Rabbit anti-Fibronectin 0.1μg/mL (Western Blot)

Ki67 Antigen TEC-3, Dako Rat anti-mouse Ki67 Antigen

74μg/mL (Indirect Immunofluorescence)

Mouse IgG 107.3, 554721, BD Pharmigen

Rat anti-mouse TNP-keyhole limpet hemocyanin

50 μg/mL (Blocking)

Osteocalcin (OCN) BT-592, Biomedical Technologies

Goat anti-mouse Osteocalcin

1:200 (Indirect immunofluorescence)

Osteopontin (OPN) MPIIIB10, Developmental Studies Hybridoma Bank

Rat anti-osteopontin 1:250 of Ascites Fluid (Western Blot)

Peroxisome proliferator-activated receptor gamma (PPAR-γ)

C26H12, 2435, Cell Signaling

Rabbit anti-PPARγ2 1:500 (Western Blot)

1. The epitope recognized by antibody AB1923 is conserved between human and mouse, according to the antibody manufacturer

2. The epitope recognized by ab23981 and C26H12 are synthetic peptides derived from the human proteins, and the antibodies react with human, mouse and rat proteins



24

Figure S1

0

4

8

12

naïve primaryhMSC

naïve clonallyderived mMSC

line

% A

LP P

ositi

ve

Heterogeneity in Human Mesenchymal Stem Cells and their Response to 3D Matrix Mechanics(A-B). Representative images of cells from (A) the clonally derived murine mesenchymal stem cell line (D1; mMSC) and (B) human mesenchymal stem cell (hMSC) populations after in-vitro expansion, stained with the alkaline phosphatase (ALP) substrate Fast Blue. (C). Quantification of the number of the percentage of cells in randomly selected fields that expressed significant ALP activity (* p < 0.01, t-test). (D-E). Quantification of Cbfa-1 ( ) and PPAR-γ ( ) expression levels in (D) mMSC (* p < 0.05, t-test comparing Cbfa-1 expression; ** p < 0.01, t-test comparing PPAR-γ expression) and (E) hMSC (* p < 0.05, t-testcomparing expression of either protein) encapsulated into matrices presenting 754μM RGD. Note the greater heterogeneity in hMSC gene expression. (F). In-situ staining for ALP activity (blue) and Oil Red O for Neutral Lipids (red) in hMSC cultured for 1 week in matrices presenting 754μM RGD but varying stiffness. Error bars are SD, n = 3-5 . Scale bars (A,B,F): 100μm.

A B C *

D E

F



25

Figure S2

Mechanical Properties of Cell-Encapsulating Syntheic Extracellular Matrix Analogs for Physiological Cell Encapsulation. (A-B). Elastic moduli of 1 weight percent alginate solutions of high Mw, high GA, (♦) and high Mw, high MA, ( ) alginate crosslinked with calcium sulfate, tested (A) immediately after being cast or (B)24 hr after incubation in DMEM. (C). Elastic moduli of binary alginate comprised of 1 weight percent high Mw,high GA alginate combined with 0-4 weight percent of low Mw, high GA alginate for a total polymer concentration of 1-5 weight percent, which were crosslinked with 50mM CaSO4 (♦). (D-E). Rheological analysis of hydrogel storage moduli over a broad frequency range indicate minimal frequency response, and that G’ is significantly larger than G”, suggesting the hydrogels behave in a nearly linear-elastic manner at the 1Hz compression rate at which E measurements were performed. (F). Elastic moduli of crosslinked solutions of varying concentrations of agarose. (G). Elastic moduli of solutions of PEGDM polymers of MW either 35 kDa (♦)or 10 kDa ( ) crosslinked with an excess of photo-initiator via free-radical polymerization. Error bars are SD, n = 3-4. All data on elastic modulus versus either crosslinker concentration or polymer weight percent were fit by linear regression (R2 > 0.9) and differences in elastic modulus as a function of either crosslinker concentration or polymer concentration were statistically significant based on 1-way ANOVA (p < 0.05).

10

100

1000

10000

100000

0.01 0.1 1 10Frequency (Hz)

Mod

ulus

(Pa)

MVG, 50mMCaSO4 (G')

MVG, 50mMCaSO4 (G")

MVG, 12.5mMCaSO4 (G')

MVG, 12.5mMCaSO4 (G")

0.1

1

10

100

1 10 100Crosslinker Concentration (mM)

Elas

tic M

odul

us (k

Pa)

0

50

100

150

0 1 2 3 4 5Polymer Weight Percent

Elas

tic M

odul

us (k

Pa)

A B

D E

0.1

1

10

100


Elas

tic M

odul

us (k

Pa)

C

0.1

1

10

100

1000

0 5 10 15 20 25Polymer Weight Percent

Elas

tic M

odul

us (k

Pa)

F

0

20

40

60

80

100

120

140

0 0.25 0.5 0.75 1 1.25 1.5

agarose concentration (g/100mL)

E (k

Pa)

G0.01

0.1

1

10

100

0.001 0.01 0.1 1 10 100Frequency (Hz)

G' (

kPa)

MVM, 50mM Ca

MVG, 25mM Ca

MVG, 50mM Ca

MVG, 12.5mM Ca



26

Figure S3

Stem Cell Fate is Correlated to Matrix Rigidity but not Macromolecular Transport or Soluble Calcium (A). Relative diffusion coefficient (Deff) for bovine serum albumin within gels of varying stiffness. Deff values are normalized to Deff measured for the softest (2.5 kPa) matrices. No statistically-significant differences between any pair of data points (p > 0.05) were found. (B). Schematic depicting a standard 3D MSC differentiation study (left), and the study in which cell-encapsulating hydrogels were combined in the same medium with cell-free hydrogels in order to equalize free (soluble) Ca2+ in all gel conditions. (C). Table indicating the concentrations of calcium used to crosslink hydrogels in a standard experiment and in the study to decouple soluble Ca2+ from matrix rigidity, and the concentration of soluble calcium in the media at day 2 measured via Arsenazo III. Note that in either experiment, by day 7, levels of Ca2+ in the media were ~ 1.8mM (the Ca2+ concentration of standard DMEM) for all conditions (n = 4 for soluble Ca2+ measurements; data not shown). (D). Representative Western analysis of Cbfa-1 and PPAR-γ expression in clonally derived mMSC cultured within matrices of varying rigidity cultured in the presence of Ca2+ releasing cell-free hydrogels were comparable to results obtained without the addition of cell-free hydrogels to the culture media, indicating minimal affects of the concentration of dissolved calcium on mMSC fate. E values represent the values for hydrogels after 1 day in culture.

Estimated Ca2+

concentration in cell-

encapsulating hydrogel

(mM)

Estimated Ca2+

concentration in cell-free hydrogel

(mM)

MeasuredCa2+

concentration in media on

day 2, standard

experiment(mM)

MeasuredCa2+

concentration in media on

day 2, multiplehydrogel

experiment(mM)

6.25 50 2.6 ± 0.1 4.5 ± 0.1

12.5 37.5 3.6 ± 0.1 3.5 ± 0.1

25 25 4.1 ± 0.1 3.3 ± 0.3

50 6.25 6.8 ± 0.2 3.1 ± 0.3

BA

C D0

0.4

0.8

1.2

1.6

2

0 20 40 60 80 100 120Elastic Modulus (kPa)

Def

f



27

Figure S4

Quantitative Analysis of Cell Morphology in 3D Matrices. (A). Maximum projected cross-sectional area for mMSC in matrices with 754μM RGD obtained from Differential Interference Contrast images. (B).Example of an encapsulated D1 mMSC in which cortical actin protrusions (grayscale micrograph) into the surrounding matrix were manually identified (red segments) and measured. (C-F). F-actin morphology for mMSC encapsulated into matrices with 15μM RGD (C,E) or 754μM RGD (D,F). Quantifying the number of protrusions per cell (C,E) revealed that protrusions were absent in BDM treated cells or cells in which integrins were saturated with soluble RGD before encapsulation (p < 10-4 compared to 22 kPa, Holm-Berferonni test). However, the difference in the numbers of protrusions per cell as a function of matrix rigidity was only significant when directly comparing 110 kPa to 5, 10 or 22 kPa matrices for 754μM RGD and only when comparing 22 kPa to 110 kPa matrices for 15μM RGD (pair-wise, 2-tailed t-test, p < 0.05). Measuring individual protrusion length (D,F) revealed that protrusion length correlated negatively with matrix stiffness; however, this correlation was not significant (p > 0.1, 1-way ANOVA) at either RGD density. (G-H). Nuclear morphology for mMSC in matrices with 754μM RGD: (G) nuclear volume and (H) nuclear sphericity as a function of matrix modulus. A minimum of 10 cells per condition were used for cell-morphology analyses; whiskers on box-plots display the interquartile range, and outliers are noted as red crosses. E valuesshown are for hydrogels at the time of cell encapsulation. Cell morphology was assessed 2 hr after cell encapsulation, and E values represent the values for hydrogels immediately after being cast. Scale bars (A): 10μm.

DB

E F G H

CA



28

Figure S5

Quantitative Analysis of Specific Integrins used to Bind RGD in 2D and 3D Matrices (A). Schematic of ELISA to quantify specific integrins bound to biotinylated RGD presented by alginate matrices. (B). Sample of raw fluorescence data demonstrating dose-dependent binding of α5 and αV integrins to RGD-biotin peptides mixed with mMSC cell suspensions as assessed with the integrin-binding ELISA. (C).Negative controls for α5-integrin-RGD binding ELISA: relative bond numbers (normalized to value for control, a 22 kPa matrix presenting 15μM RGD-biotin) between RGD-biotin and α5-integrin or RGE-biotin and α5-integrin for clonally derived mMSC encapsulated into 3D matrices (* p < 0.05 versus all other conditions, Holm-Bonferroni test). (D). αV-integrin binding to RGD presented by 2D substrates presenting 37 μM RGD. Integrin binding analyses were performed 2 hr after encapsulating cells, and E values shown are for hydrogels at the time of cell encapsulation. Data (♦) were fit by a hyperbolic curve (-- ; E1/2 = 2.31 kPa, R2 = 0.88). Error bars are SD, n = 3-6 (B,C) or SEM, n = 4-5 (D).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control 20mM BDM 1mM SolubleRGD

15uM RGE 754uM RGE

BA

*

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

1 10 100 1000 10000 100000N RGD/ cell (x105)

Rel

ativ

e Fl

uore

scen

ce In

tens

ity

alpha-V

alpha-5

C

0

0.2

0.4

0.6

0.8

1

1.2

0 25 50 75 100 125 150 175 200

Elastic Modulus (kPa)

Rel

ativ

e α

V-R

GD

Bon

ds

1. Alginate Lyase 2. Cell Lysis

ELISA for particularintegrin

*D



29

Figure S6

FRET Technique to Measure Integrin-RGD Bond Formation in 3D Matrices (A). Schematic of FRET technique to quantify cell-RGD bond formation. When the cell membrane is labeled with 5-hexadecanoylaminofluorescein, 488nm excitation yields 520nm (green) emission, unless rhodamine (Rho) labeled RGD-peptides are presented by the alginate matrix and bound to integrins, in which case 520nm emission is diminished and 580 nm (red) emission is enhanced. (B). Emission spectra of clonal mMSC encapsulated into RGD-modified hydrogels with a constant density of RGD (37 μM) but different E, or 37μM RGE at 22 kPa. Spectra from FRET samples were normalized by spectra from matched donor controls with the same mechanical properties.

A B

0

0.25

0.5

0.75

1

500 525 550 575 600 625 650Emission Wavelength (nm)

Emis

sion

Inte

nsity

Rel

ativ

e to

M

atch

ed D

onor

Con

trol

1.6 kPa

6 kPa

22 kPa

45 kPa

22 kPa RGE



30

Figure S7

Integrin-RGD Bond Formation Depends on Cell Traction and ECM Rigidity but not Matrix Associated Calcium (A). Elastic moduli of 4 weight percent alginate dialdehyde crosslinked with varying concentrations of PAH. Concentrations denote the density of hyrazide functional groups presented by PAH. (B).Elastic moduli of hydrogels formed by crosslinking a pre-polymer of various weight percent of alginate dialdehyde with 300mM hydrazide groups presented by PAH. (C). Live-Dead Stain performed 8 hr after encapsulating cells, and (D). Alamar Blue reduction performed 8 hr ( ) or 48 hr ( ) after encapsulating D1 mMSC into alginate hydrogels crosslinked with calcium (calcium-MVG) or alginate-dialdehyde hydrogels crosslinked with PAH (PAH-alginate-dialdehyde), indicating that mMSC viability is not significantly affected by encapsulation into covalently crosslinked PAH-alginate dialdehyde hydrogels. (E). mMSC-RGD bond numbers obtained using different alginate polymers and crosslinking agents are plotted against the calcium concentration used to crosslink the hydrogel. Data show poor correlation between calcium concentration and Nbond / cell. Error bars are SD for Alamar Blue reduction and elastic modulus measurements (n = 3) and SEM for FRET analysis (n= 3-5). Scale bars: (C): 100μm. Cell-RGD bond formation was analyzed 2 hr after cell encapsulation, and Evalues represent the values for hydrogels immediately after being cast.

D E

0.1

1

10

100


Elas

tic M

odul

us (k

Pa)

0

50

100

150

200

0 2 4 6 8Polymer Weight Percent

Elas

tic M

odul

us (k

Pa)

0

5000

10000

15000

20000

Calcium-Alginate PAH-Alginate Dialdehyde

Ala

mar

Blu

e R

educ

tion

(RFU

) Day 0Day 2

A

0

5

10

15

20

0 10 20 30 40 50 60Calcium Concentration (mM)

Nbo

nd /

cell

(x10

4 )

B

C



31

Figure S8

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Cells (-) Cells (+) Cell Free

DFR

ET

Verifying the ability of FRET to measure RGD clustering by cells encapsulated in 3D RGD-alginate matrices. The degree of energy transfer was calculated according to Equation 1 for alginate-RGD hydrogels, either with or without (Cell Free) encapsulated cells. Energy transfer increased in the presence of cells (MC3T3-E1 pre-osteoblasts; Cells(-)) but less so when cell traction forces were inhibited with Cytochalasin D (1μM; Cells (+)). Hence, FRET measurements of RGD clustering reflect traction-mediated reorganization of the matrix on the nanometer scale and not aggregation of peptides attached to different alginate polymer chains. Error bars are SD (* p < 0.01 versus all other conditions, Holm-Bonferroni test). Nanoscale RGD reorganization was analyzed 2 hr after cell encapsulation, and E values represent the values for hydrogels immediately after being cast.

*

*

*



32

Figure S9

0

5

10

15

20

25

30

IgG Control H10-27 RMV-7

% K

i-67

Posi

tive

Cel

ls

Affect of Integrin Function Blocking Antibodies on Stem Cell Fate in 3D Matrix Culture (A). Representative images of in-situ Live-Dead Staining (Calcein-AM: Green, Ethidium Homodimer: Red) of mMSC after 1 week of culture in matrices of various rigidity and in the presence of either integrin blocking antibodies H10-27, RMV-7 or control IgG. (B). Representative images of indirect immunofluorescence staining for Ki-67 (green) with DAPI counterstain (blue), of cryosectioned 5 kPa matrices after 1 week in culture. Results were similar in 22 and 110 kPa matrices (data not shown). Scale bars: (A), 100μm, (B), 20μm. E values are for hydrogels after 1 day in culture. (C). Quantification of Ki-67 expression in randomly selected fields. No statistically significant differences between any two conditions were found (p > 0.5, 2-tailed t-test). (D). Western analysis of Cbfa-1 as a function of α5-integrin blocking antibody dose in 20 kPa matrices. (E). Western analysis of Cbfa-1 and PPAR-γ in mMSC cultured for 1 week in the presence of 50μg/mL dose of function blocking antibodies against α5 or αV-integrins in either 2.5 or 20 kPa matrices. Error bars are SD, n = 3.

Aaa

B

C

2.5 kPa 20 kPa

α5

Cbfa-1

αv IgG

PPAR-γ

Actin

α5 αv IgG

D

E



34

Supplemental References

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2. Kong HJ, Smith MK, Mooney DJ. Designing alginate hydrogels to maintain viability of immobilized cells. Biomaterials 24, 4023-9 (2003).

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6. Bencherif SA et al. Influence of the degree of methacrylation on hyaluronic acid hydrogels properties. Biomaterials29, 1739-49 (2008).

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Harnessing Traction-Mediated Manipulation of the Cell-Matrix … · 2010-06-15 · Harnessing Traction-Mediated Manipulation of the ... To form alginate hydrogels, a calcium-sulfate