Hydrogels with Dual Gradients of Mechanical and Biochemical Cues

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Hydrogels with Dual Gradients of Mechanical and Biochemical Cues for Deciphering Cell-Niche Interactions Xinming Tong, James Jiang, Danqing Zhu, and Fan Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00074 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016

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ACS Biomaterials Science & Engineering

Hydrogels with Dual Gradients of Mechanical and Biochemical Cues for Deciphering Cell-Niche Interactions Xinming Tong†, James Jiang‡, Danqing Zhu§, Fan Yang*,†,§ †

Department of Orthopaedic Surgery, Stanford University, CA, 94305 (USA).

‡

Department of Human Biology, Stanford University, CA, 94305 (USA).

§

Department of Orthopaedic Surgery and Bioengineering, Stanford University School of Medicine, 300 Pasteur Dr., Edwards R105, CA, 94305 (USA).

KEYWORDS: Hydrogels, gradient, cell niche, biochemical, mechanical.

ABSTRACT. Cell niche is a multifactorial environment containing complex interactions between biochemical and physical cues. While extensive studies have examined the effects of biochemical or physical cues alone on cell fate, how biochemical and mechanical signals interact to influence cell fates remains largely unknown. To address this challenge, here we report a polyethylene glycol-based gradient hydrogel platform as biomimetic cell niche containing independently tunable matrix stiffness and biochemical ligand density. The versatility of this platform is demonstrated by fabricating and characterizing single gradient or orthogonally aligned dual gradient hydrogels. These gradients result in differential elongation and spreading of human fibroblasts. Both 1 ACS Paragon Plus Environment


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hydrogel stiffness and biochemical ligand density are independently tunable by sequential photopolymerization. By controlling light exposure, a broad range of hydrogel stiffness and different types/doses of biochemical ligands can be incorporated. Such tunability facilitates customization of this platform for investigating complex cell-niche interactions associated with various cell types, such as stem cells and cancer cells. The outcomes of such studies may help identify optimal niche cues to promote desiralbe stem fates and tissue regeneration or inhibit diseases progression.

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INTRODUCTION Cells reside in a multifactorial environment in which they respond to various niche inputs to change their fate and functions.1-3 Various biological molecules, such as cytokines and growth factors, have been studied to elucidate how cells interact with these regulators.4 In addition, recent studies have demonstrated that insoluble factors, including mechanical properties and immobilized biochemical ligands, play essential roles in regulating cellular responses.5-8 For example, varying the elastic modulus of a substrate over a range from soft to stiff shifts stem-cell differentiation toward neurogenic, myogenic, and osteogenic fates, respectively.5-8 Changing the distribution of Arg-Gly-Asp (RGD) peptide, a cell adhesion ligand, alters stem-cell fate from osteogenic to adipogenic,9 while changing the concentration of RGD affects the extent of osteogenic differentiation of stem cells.10 Despite our awareness that cells respond to an intertwined regulatory signaling network, most studies to date have investigated individual types of microenvironmental cues. How the complex interplay among niche cues collectively influences cell fate and functions remains an unaddressed challenge. High-throughput screening of cell-niche interactions using combinatorial hydrogels has emerged as a promising approach to help elucidate complex cell-niche interactions, and to identify optimal niche properties that lead to desired cellular responses.11-13 Previous studies have shown that cells often respond to interactive niche signals in a non-linear manner,11-13 highlighting the importance of performing such systematic screening. Despite the progress made so far using combinatorial screening approaches, variations in niche properties can only be applied discretely, which is likely to miss important information due to discontinuities in variation. This strategy also requires large number of samples, time and complex assays for screening and data analyses14. Furthermore, native tissues are often characterized with gradual 3 ACS Paragon Plus Environment


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transition of niche cues in a gradient manner, which cannot be mimicked using discrete hydrogel compositions.15-18 For example, articular cartilage is organized with gradually varying moduli and concentrations of extracellular-matrix molecules such as glycosaminoglycans and collagen, from the superficial layer to the subchondral layer.15-20 Many essential biological processes are mediated by gradients in biophysical and biochemical properties instead of by constant and homogenous distributions of forces or molecules.21-24 Hence, it would be highly desirable to develop artificial cell niche with gradient properties to increase screening efficiency and better mimic native tissue zonal organization. Recent work has explored the development of gradient materials for the purpose of mimicking the zonal organization of tissues.25-30 However, previous efforts have focused largely on developing biomaterials with single gradients, such as a mechanical or biochemical gradient alone, which cannot capture the multifactorial nature of cell niche. Thus, there remains a critical need to develop biomaterials with spatiotemporal gradients in multiple niche properties to help better decipher cell-niche interactions in a high-throughput and physiologically relevant manner. Here, we report the development of a hydrogel platform with dual gradients in mechanical and biochemical cues to help decipher cell-niche interactions. We first crosslinked the mechanical supporting matrix (Figure 1a-c), then incorporated a biochemical ligand (Figure 1d-f); both steps relied on a photocontrolled thiol-ene radical reaction. The hydrogel stiffness and biochemical ligand density can be controlled separately using a photomask to control the dose of UV light (365 nm) in each step (Figure 1b, e). We characterized the single mechanical and biochemical gradients achieved in each step as well as the dual gradient obtained by sequentially applying the two gradients to one substrate (Figure 1f). To demonstrate the versatility of this method, we aligned the two gradients in an orthogonal manner by simply changing the sliding 4 ACS Paragon Plus Environment

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direction of the photomask during the two steps. We then further explored the influence of stiffness and adhesion-ligand density of the hydrogel substrate on the adhesion and morphology of human fibroblasts, a model cell type commonly used for studying wound healing and tissue regeneration-applications.

Figure 1. Scheme for fabricating mechanical and biochemical dual-gradient hydrogel substrates. MATERIALS AND METHODS Materials. Eight-arm PEG (molecular weight ~10 kDa) was purchased from JenKem Technology USA. Linear PEG (~1.5 kDa) was purchased from Sigma-Aldrich USA. Eight-arm PEG-norbornene (PEG8NB) and linear PEG-dithiol (PEG2SH) were then synthesized in accordance with a previous study.31-32 Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) was synthesized as reported previously.33 Peptides CRGDS and FITC-CRGDS were purchased from Bio Basic Inc. All other reagents and solvents were obtained from Fisher unless otherwise noted.



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Hydrogel Preparation. PEG precursor solutions of 8-arm PEG-norbornene and PEG-dithiol in phosphate-buffered saline (PBS) (10% (w/v), with 0.05 wt% LAP) were mixed at a volume ratio of 20:9. To make hydrogels of lower concentration, this solution was diluted with PBS plus 0.05 wt% LAP. To crosslink the hydrogel, the mixed precursor solution was loaded into a chamber sandwiched between a glass slide and a coverslip, with one coverslip as the spacer (one coverslip provides 0.2 mm of thickness). The crosslinked hydrogel sheet had dimensions of 2.5 x 2.5 x 0.02 cm. UV light (365 nm) at 1.5 mW/cm2 was used for crosslinking. To fabricate hydrogels with homogenous mechanical stiffness, the entire chambered solution was exposed to UV light for a predetermined period. To introduce a mechanical gradient, a gradient of UV exposure was achieved by sliding a photomask over the precursor solution, using a syringe pump. The exposure time window, which determines the stiffness range of the gradient, was tuned by adjusting the sliding speed and total exposure time. Crosslinked hydrogels with mechanical gradients were soaked in PBS overnight to wash out uncrosslinked, free precursors. All groups were made with 3 replicates. Biochemical Ligand Incorporation. The CRGDS peptide was dissolved in PBS plus 0.05 wt% LAP to a final concentration of 1 mM. Washed hydrogels with mechanical gradients were placed in a 50 mL conical tube and briefly centrifuged to remove surface water. Then, 0.2 mL of CRGDS solution were dropped on top of the hydrogel and a coverslip was placed over the solution. The thickness of the solution covering the hydrogel was ~0.4 mm, achieved by using extra coverslips as spacers. Similar to hydrogel crosslinking, homogenous ligand incorporation was achieved by exposing the entire chamber to UV light for a predetermined period. Sliding a photomask over the system yielded ligand incorporation in a gradient, while adjusting the sliding


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speed and total exposure time affected the range of ligand density. All groups were made with 3 replicates. Mechanical Testing. For mechanical testing, hydrogels were constructed with a thickness of 3 mm and surface dimensions of 2.5 x 2.5 cm. Cylinder-shaped samples (6 mm in diameter) were extracted to fit the loading cell of the equipment. To measure hydrogel stiffness, unconfined compression testing was performed with the Instron 5944 testing system fitted with a 10-N load cell (Interface Inc.). The mechanical testing set up is consisted of custom made aluminum compression platens lined with PTFE to minimize friction. The diameter and thickness of all hydrogels were measured using digital calipers. Before each test, a preload of approximately 1 mN was applied. The upper platen was then lowered at a rate of 1% strain/sec to a maximum strain of 30%. Load and displacement data were recorded at 100 Hz. All tests were conducted in PBS at room temperature. All stress measurements were based on the the initial cross sectional area of the hydrogels. Compressive moduli were calculated using the linear curve fits of the stress versus strain curve for strain ranges of 10-20%. Three samples per each group were tested. Characterization of Peptide Incorporation. To quantify the density of incorporated peptide, the fluorescence intensity of FITC-CRGDS was measured. To avoid saturation, FITC-CRGDS and non-tagged CRGDS were mixed at molar ratio of 1:2. Hydrogels were made with dimensions of 2.5 x 2.5 x 0.02 cm, which can fit into a standard 6-well plate. Fluorescence intensity was measured with a SpectraMax M2 microplate reader using well-scanning mode. A standard curve was constructed by measuring the fluorescence intensities of hydrogels with known peptide concentrations. To capture the full distribution of incorporated peptide, hydrogels were also imaged with a Molecular Imager Gel Doc XR System (Bio-Rad) using the fluorescence-scanning mode. 7 ACS Paragon Plus Environment


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Cell Adhesion and Morphology. Human fibroblasts (line BJ) were obtained from American Type Culture Collection (ATCC line CRL-2522) and expanded in culture medium (Dulbecco’s modified Eagle medium (DMEM, Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/ mL penicillin and 0.1 mg/mL streptomycin). To examine cellular responses to hydrogel stiffness and to the incorporation of biochemical ligand, hydrogels were thoroughly washed and soaked in PBS for one day before they were seeded with human fibroblasts at 2x103 /cm2. Ultra-low attachment 6-well plates (Corning) were used to ensure that cells attached only to hydrogels. Cells were maintained at 37 °C and 5% CO2 in culture medium and allow adhere to the hydrogels and to spread for 24 h before morphology assays were carried out. Cytoskeleton Staining and Analysis. After removing the culture medium, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and thoroughly washed with PBS. Factin was stained with phalloidin-tetramethylrhodamine B (Sigma) and nuclei were stained with Hoechst 33342 (Invitrogen). Images were taken with a Zeiss fluorescence microscope. Cell spreading area and elongation were analyzed using ImageJ (NIH). Data was represented as means ± tandard deviation of at least three replicates. RESULTS Varying mechanical stiffness. Previous research using hydrogel substrates with discrete mechanical stiffness has shown that matrix stiffness plays an important role in modulating cell fates such as proliferation and differentiation34-35. Here we aim to develop hydrogel substrates with continuous gradient of mechanical stiffness, which better mimics the in vivo niche and will facilitate high-throughout screening studies using significantly reduced number of cells and


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materials. To construct a gradient in mechanical stiffness in the hydrogel substrate, the time for which the polyethylene glycol (PEG) precursor solution was exposed to UV light was changed to vary the degree of photo-activated crosslinking. With a concentration of 5% (w/v) PEG precursor, increasing the exposure time from 5 s to 120 s increased the hydrogel stiffness from 0.5 kPa to 3.0 kPa (Figure 2a). Similarly, increases in stiffness from 4.2 kPa to 12.0 kPa and from 24.6 kPa to 37.4 kPa were obtained by increasing the UV exposure time when the precursor concentration was 7.5% (w/v) and 10% (w/v), respectively (Figure 2a). We next examined the responses of living cells to the stiffness of hydrogel substrates. After cell culture for 24 h, human fibroblasts exhibited enhanced spreading on hydrogels with higher stiffness. Specifically, increasing the hydrogel stiffness from 4.2 kPa to 12.0 kPa increased cell elongation by 0.7 fold and spreading area by 0.8 fold (Figure 2b). Higher cell attachment density was observed on hydrogels with higher stiffness (Figure 2c).



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Figure 2. Varying hydrogel stiffness affects fibroblast morphology. (a) Mechanical stiffness of hydrogels increased as a function of increasing PEG concentration and increasing UV exposure time. (b) Quantification of elongation and area of fibroblasts on hydrogel substrates with the indicated mechanical stiffness. (c) Representative immunofluorescence images of cell morphology as a function of the mechanical stiffness of the hydrogel substrate. Red, F-actin; blue, nuclei. Scale bars, 100 µm. Data was represented as means ± standard deviation of at least three replicates. Varying and characterizing the biochemical ligand. To create the desired biochemical gradient, a cell adhesion-supporting peptide, Cys-Arg-Gly-Asp-Ser (CRGDS),36 was used as model ligand. This peptide was labeled with fluorescein isothiocyanate (FITC), a fluorescent marker, to facilitate the characterization of peptide incorporation in the hydrogels. Fluorescein such as FITC is subject to photobleaching when exposed to high intensity UV for long time, which can affect the accuracy of measured signals. To avoid this problem, we have chosen thiolene chemistry, which is known to have high efficiency for crosslinking. This allows us to use very low UV intensity (1.5 mW/cm2) for very short time (2 mins maximum). A bandpass filter was also used to avoid photo-toxic low wavelegnth UV. Our results confirmed no significant change of FITC intensity was observed after 2 mins of exposure at this condition (data not shown). A standard curve relating peptide density with fluorescence intensity over a wide range (0 to 1.75 mM) was obtained without obvious saturation of the fluorescence signal (Figure 3a). UV exposure time was changed to vary the degree of photoactivated incorporation of the peptide; when we increased the exposure time from 15 s to 120 s, the peptide density increased from 0.225 mM to 0.75 mM (Figure 3b).


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Cellular responses to peptide density were also examined. Increasing peptide (FITC-CRGDS) density led to substantial higher fibroblast adhesion and spreading. Increasing the peptide density from 0.25 mM to 0.75 mM resulted in an approximately 0.6-fold increase in cell elongation and a 1.4-fold increase in cell spreading area (Figure 3c). Higher cell density was observed with hydrogels with 0.5 - 0.75 mM peptide than with 0.25 mM peptide (Figure 3d).

Figure 3. Effects of cell adhesive ligand (CRGDS) density on fibroblast morphology. (a) Standard curve correlating fluorescence intensity with FITC-CRGDS density. (b) CRGDS density increases as a function UV exposure time for incorporation into the hydrogel. (c) Quantification of elongation and area of fibroblasts on hydrogel substrates versus CRGDS density. (d) Representative images of cell morphology as function of CRGDS density on the



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hydrogel substrate (units are in mM). Red, F-actin; blue, nuclei. Scale bars, 100 µm. Data was represented as means ± standard deviation of at least three replicates. Single gradients of mechanical stiffness and biochemical ligand. We successfully obtained a gradient of mechanical stiffness via our photomask-sliding strategy (Figure 1b). Specifically, with a concentration of 7.5% (w/v) PEG, a gradient of mechanical stiffness was obtained with a 1.7-fold change from 7.9 kPa to 13.4 kPa by allowing the exposure to UV light to proceed for 530 s across the precursor solution (Figure 4a). An exposure time of 5-120 s led to a gradient with a 1.3-fold change from 12.8 kPa to 16.7 kPa (Figure 4a). With a concentration of 10% (w/v) and 5-30 s UV exposure, a slight variation in stiffness from 42.6 kPa to 46.6 kPa across the hydrogel was obtained (Figure 4a). Similar to the mechanical gradient, to achieve a gradient of biochemical ligand in our hydrogels, the degree of photoactivated incorporation was varied by changing the duration of UV exposure (Figure 1e). A full view of the ligand distribution cross hydrogel was obtained via fluorescence imaging of the hydrogels. Incorporating peptide (CRGDS) with constant, uniform UV exposure resulted in a homogenous fluorescence distribution; increasing exposure time led to increased fluorescence intensity (Figure 4b). Sliding the photomask to allow varied UV exposure across the hydrogel (covered with peptide solution) yielded smoothly changing fluorescence intensity (Figure 4b). A window of 5-30 s of UV exposure generated a smooth increase in fluorescence intensity across the hydrogel, while extending the time window to 60 s or 120 s seemed to result in earlier signal saturation on fluorescence imaging (Figure 4b, S1). In addition to imaging, quantitative measurement by plate reading showed a smooth increase in fluorescence intensity across all UV exposure time windows (Figure 4c). Specifically, 0-30 s of UV exposure resulted in a 5.2-fold change in peptide density from 0.075 mM to 0.34 mM, while


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extending the exposure to 60 s and 120 s led to 6.8-fold and 10.3-fold changes, respectively (Figure 4c). We then examined the cellular responses to the peptide density at a homogenous hydrogel stiffness of 10 kPa. Enhanced fibroblast adhesion density and spreading was observed at regions with higher peptide density (Figure 4d).

Figure 4. Hydrogels with stiffness gradient or biochemical gradient only.

(a) Mechanical

stiffness of hydrogels from three regions of single stiffness gradient hydrogels confirms increasing hydrogel stiffness. (b) Representative fluorescence images of a biochemical gradient hydrogel with FITC-CRGDS incorporated in a uniform distribution or in a gradient. (c) Quantified RGD density of regions of single biochemical gradient hydrogels. (d) Representative images of cell spreading on biochemical gradient hydrogels with an increasing gradient of RGD density. Red, F-actin; blue, nuclei. Scale bars, 100 µm. Data was represented as means ± standard deviation of at least three replicates.



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Dual gradients. We next constructed hydrogels that contained a mechanical gradient, and then incorporated a peptide gradient. To demonstrate flexibility in the directions of the two gradients, we applied the biochemical gradient orthogonal to the mechanical gradient. The gradient of mechanical stiffness was obtained over range of 5~15 kPa; peptide density varied over a range of 0 to 0.7 mM. When seeded on hydrogels with this dual gradient, fibroblasts spread according to both mechanical stiffness and peptide density (Figure 5). Specifically, keeping the matrix stiffness constant while increasing the peptide density promoted cell spreading, while increasing the stiffness with constant peptide density increased cell spreading (Figure 5).

Figure 5. Representative micrographs and quantification of elongation/area of human fibroblasts cultured on dual-gradient hydrogels (day 1). Keeping matrix stiffness constant while increasing RGD density promoted cell spreading. Similarly, increasing stiffness while maintaining a constant RGD density led to increased cell spreading. Red, F-actin; blue, nuclei. Scale bars, 100 µm.


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DISCUSSION Here we have demonstrated a strategy to construct hydrogels with dual gradients in mechanical and biochemical properties as substrates for probing cell-niche interactions. We utilized photoactivated thiol-ene radical addition for both crosslinking and biochemical ligand incorporation. Thus, mechanical stiffness and ligand density were tuned by varying the UV exposure time, which determines the degree of crosslinking and ligand incorporation. We anticipate that the strategies reported here would be broadly applicable to both basic-science investigations of cell-nice interactions as well as tissue-engineering applications. Although photoa-ctivated polymerization has already been widely used to create hydrogel substrates with gradients, most reported strategies employ acrylate- and acrylamide-based monomers.37-38 These monomers are usually made into hydrogels following a chain-growth radical polymerization mechanism, which allows only one-step fabrication because the post-polymerized chains are deactivated ‘dead’ chains.39 This intrinsic limitation prohibits flexibility, often yielding a single gradient.30, 38 Instead, here we employed the photo-controlled thiol-ene radical addition for both crosslinking and ligand incorporation, allowing sequential fabrication of substrates with multiple gradients. Specifically, active norbornene groups that are not consumed in crosslinking can be exploited in subsequent ligand incorporation. Sequential crosslinking and incorporation can then allow independent control over mechanical and biochemical properties to apply dual gradients (Figure 1). We used 8-arm PEG-norbornene to maximize the immobilization of norbornene groups while using linear PEG-dithiol to minimize the existence of residual, unreacted free thiols in order to avoid interference by extra thiols with ligand incorporation. In the crosslinking step, we mixed 8-arm PEG-norbornene with PEG-dithiol in a ratio that yielded excess norbornene groups to ensure that sufficient (no less than 7 mM of residual norbornene groups even at



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concentration of 5 % (w/v) PEG) were available after incorporation, even at high degrees of crosslinking. In this study, gradients of mechanical stiffness were synthesized by sliding a photomask over the substrate in order to control UV exposure time. Varying this time changed the degree of crosslinking and achieved a wide range of mechanical stiffness (Figure 2a, 4a). In the present study, we reported a stiffness gradient ranging from 8 to 47 kPa. For example, with an initial concentration of 7.5% (w/v) PEG, increasing UV exposure from 5 s to 120 s resulted in a 3-fold increase in hydrogel stiffness (from 4.2 kPa to 12.0 kPa), covering a range that exhibited in tissues spanning fat to muscle.2 The stiffness range may be further customized by changing polymer structures and crosslinking mechanism. For example, changing PEG to hyaluronic acid will yield a stiffness range from 3.6 to 53.6 kPa with UV exposure from 5 to 15 mins.

48

Maintaining the precursor concentration but varying the degree of crosslinking basically changes the crosslinking functionality (the number of arms that have been crosslinked). This variation in functionality may avoid significant changes in network framework and hydrogel swelling behavior, which usually occur when the precursor concentration is changed.40 Thus, varying the exposure to UV light to control mechanical stiffness is advantageous over changing the precursor concentration, as the latter results in significant variations in network structure that subsequently influence cell-niche interactions.41 Further, the creation of gradient via changes in precursor concentration usually requires a large volume of solution

28-29

or tedious manipulation,25,

42

neither of which is desirable for large-scale screens of complex niche interactions. To achieve a biochemical gradient in our hydrogels, we specifically left extra norbornene groups available after crosslinking in order to promote photo-controlled biochemical ligand incorporation. Again, sliding a photomask over the substrate to control UV exposure enabled 16 ACS Paragon Plus Environment

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ligand density to be varied over a wide range. For example, CRGDS, a model ligand, was incorporated with a density gradient of 0.065 to 0.35 mM using a UV exposure time of 5-30 s (Figure 4c), yielding a smooth gradient that was confirmed via fluorescence imaging (Figure 4b). As a tuning mechanism independent of crosslinking, this ligand-incorporation strategy could minimize interference by hydrogel network structure and mechanical properties. In contrast, conventional methods usually utilize ligand-functionalized monomers during copolymerization while crosslinking the hydrogel to achieve a biochemical gradient.26, 37 This strategy results in changes in hydrogel network structure when varying the ligand density by changing the concentration of the functionalized monomer,43 hence affecting related mechanical properties. Alternative strategy utilizes diffusion of biochemical molecules in precursor solution before crosslinking44, but the drawback is that it replies on the tedious microfluidic methods and the diffusion is highly depended on the choice of biochemical molecules and may need optimization for each biochemical cue of interest. We successfully constructed dual gradient hydrogels by sequential crosslinking to control hydrogel stiffness and biochemical ligand density (Figure 1f, 5). Unlike conventional one-step fabrication, this sequential strategy enables independent control over gradients in terms of range and direction. This strategy can maximize the screening of cell responses to both types of niche cues, thus facilitating the elucidation of cell-niche interactions. For example, we fabricated hydrogels with mechanical and biochemical gradients that orthogonally crossed, which allowed us to study cell responses to both mechanical stiffness and adhesion-ligand density in a single hydrogel (Figure 5). Our hydrogels also empowered us to robustly probe fibroblast responses to hydrogel niche properties, including mechanical stiffness and adhesion-ligand density. Increasing both 17 ACS Paragon Plus Environment


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mechanical stiffness and adhesion-ligand density facilitated cell spreading (Figure 2c, 3d), which is consistent with previous reports.45-47 Changes in the density of the cell-adhesion ligand CRGDS led to more dramatic differences in cell adhesion and morphology (Figure 3c), elongation, and cell-spreading area (Figure 3d). However, here we did not observe a response of obvious cell alignment to the gradient of CRGDS density (Figure 4d, 5), as previously reported.26, 37 This discrepancy may be due to differences in gradient slope and the relatively smaller size of our hydrogels. The dual-gradient hydrogels that we developed here also allowed us to explore the synergy between these two cues, which to date has not been well studied. Within the tested ranges of 5-15 kPa stiffness and 0- 0.7 mM CRGDS density, although both cues played important roles in fibroblast response, there were overwhelming effects at the higher end of each gradient. For example, at high ligand densities of 0.7 mM, decreasing the hydrogel stiffness from 15 kPa to 5 kPa only led to limited decreases in cell spreading (Figure 5). On the other hand, at high stiffness, changes in CRGDS density did not results in strong differences in cell morphology (Figure 5). This observation highlights the importance of screening biophysical and biochemical cues at the same time when evaluating cellular behaviors, which also emphasizes the advantages of using hydrogels with dual gradients, as fabricated in this study. CONCLUSIONS Here, we report a facile method to fabricate hydrogels with dual gradients in mechanical and biochemical properties. Both hydrogel stiffness and biochemical ligand density are independently tunable by sequential photopolymerization. By controlling light exposure, a broad range of hydrogel stiffness and different types/doses of biochemical ligands can be incorporated. Our platform allows aligning two niche gradients with variable arrangements such as orthogonal (as shown in this study), same direction, or opposite direction. Such tunability facilitates 18 ACS Paragon Plus Environment

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customization of this platform for investigating complex cell-niche interactions associated with various cell types (i.e. stem cells, cancer cells) or niche cues to match tissue of interest. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Figure of the plot of fluorescent intensity using ImageJ. (PDF) AUTHOR INFORMATION Corresponding Author * Prof. Fan Yang, Assistant Professor of Orthopaedic Surgery and Bioengineering, Director of Stem Cells and Biomaterials Engineering Laboratory, Stanford University School of Medicine, 300 Pasteur Dr., Edwards R105, Stanford, CA, 94305. E-mail: [email protected]. ACKNOWLEDGMENT The authors would like to thank the following funding sources for support including Stanford Chem-H Institute New Materials for Applications in Biology and Medicine Seed Grant, NIH R01DE024772-01, NIH R01AR063717-01, NIH R01AR055650-05A1, National Science Foundation CAREER award program (CBET-1351289), California Institute for Regenerative Medicine Tools and Technologies award ( RT3-07804), Stanford Child Health Research Institute, Stanford Bio-X Interdisciplinary program, and Alliance for Cancer Gene Therapy. The authors would like to thank Anthony Behn for his technical assistance and helpful discussion in the mechanical testing. REFERENCES 19 ACS Paragon Plus Environment


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For Table of Contents Use Only. Hydrogels with Dual Gradients of Mechanical and Biochemical Cues for Deciphering CellNiche Interactions Xinming Tong, James Jiang, Danqing Zhu and Fan Yang


Hydrogels with Dual Gradients of Mechanical and Biochemical Cues

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