Poly(ethylene glycol) Hydrogels with Tailorable ... - ACS Publications

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Poly(ethylene glycol) Hydrogels with Tailorable Surface and Mechanical Properties for Tissue Engineering Applications Nehal R Patel, Anna K Whitehead, Jamie J Newman, and Mary Caldorera-Moore ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00233 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 13, 2016

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ACS Biomaterials Science & Engineering

Poly(ethylene glycol) Hydrogels with Tailorable Surface and Mechanical Properties for Tissue Engineering Applications Nehal R. Patel◊, Anna K. Whiteheadǂ, Jamie J. Newmanǂ*, Mary E. Caldorera-Moore◊* ◊ Department of Biomedical Engineering, Louisiana Tech University, Ruston, Louisiana 71272, United States ǂ School of Biological Sciences, Louisiana Tech University, Ruston, Louisiana 71272, United States *Corresponding Authors

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Abstract: Advanced cellular biomanufacturing requires the large-scale production of biocompatible materials that can be utilized in the study of cell-matrix interactions and directed stem cell differentiation as well as the generation of physiologically-relevant tissues for therapeutic applications. Herein we describe the development of a hydrogel based platform with tailorable mechanical properties that supports the attachment and proliferation of both pluripotent and multipotent stem cells. The biomimetic hydrogel scaffold generated provides biocompatible compositions for generating various tissue-like elasticities for regenerative medicine applications and advanced biomanufacturing.

Keywords: Regenerative Medicine; stem cells; matrix elasticity; biomimetic hydrogel


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The restoration and direct replacement of diseased cell and tissues is becoming a clinical possibility due to advances in biologically mimicking (biomimetic) materials constructs and advances in stem cell technologies. To make this a clinical reality requires large-scale production of fully functional cells and tissues which has lead to the formation of the field coined advanced biomanufacturing.1 Advanced biomanufacturing relies on the reproducible and efficient generation of biocompatible materials for the study of cellular properties and directed differentiation as well as the creation of clinically-relevant tissues for therapeutic applications.1,2 Current techniques in biomanufacturing use a variety of processes and materials to optimize platforms for these two goals. These techniques include electrospinning,3 freeze-drying,4 melt molding,5 membrane lamination,6 gas foaming,7 and 3-D printing,8 which can be expensive and complex processes that are difficult for large scale production of physiologically relevant materials. Thus, there is a need for research to develop more accessible high throughput biomanufacturing of these materials. Hydrogels are three-dimensional polymeric networks that are attractive material platforms for biomanufacturing applications because of their high biocompatibility, hydrophilicity, tissue like architecture, and innate biomimetic properties.9 Hydrogels offer researchers the ability to control scaffold chemical and mechanical material properties by adjusting the monomer and/or polymer chemical composition and the polymerization conditions (curing time and energy intensity). Hydrogelbased materials give researchers the ability to incorporate physical properties into scaffolds to obtain the necessary physiological responses. These include encapsulating growth factors into the hydrogel to promote cell growth and proliferation or factors that encourage the degradation of the hydrogel materials.10 This is why a significant portion of materials used in the biomedical field for biomanufacturing of cells and tissues involves crosslinked polymeric hydrogel networks. Specifically, poly(ethylene glycol) (PEG) based hydrogels are biocompatible and bio-inert in nature. PEG based hydrogels have been extensively investigated for their application as coatings,11 adhesives,12 and for cartilage repair.13 However, despite the large number of studies utilizing PEGs and hydrogel based materials as tissue scaffolds there is still a lack of clear understanding of the correlation between material properties and cell response; which is paramount for stem cell research where environmental cues (chemical, physical, and stimulus) control cell fate. The process of poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel synthesis in this study does not require the use of specialized equipment and can be performed in most laboratory settings. The biocompatibility of our hydrogels was tested using stem cells as a model because the continuously growing field of regenerative medicine requires the generation of biomimetic platforms that convey persistent elasticity and direct stem cell differentiation. Stem cells have the unique properties of self-renewal and differentiation potential that make them ideal for use in the large scale production of other functional cells and tissues. Embryonic stem cells have long been used as a model for the study of early development and pluripotency; that is the ability to differentiate down any of the over 200 lineages of an adult human.14 Today, there is greater interest in the study of more clinically relevant cell types including: human induced pluripotent stem cells (hiPSCs)15 and human adult stem cells, primarily mesenchymal16 and adipose stem cells17, as they are easy to access and, in all three cases, patient specific. This project uses murine embryonic stem cells (mESCs), human adipose stem cells (hASCs), and human bone marrow-derived mesenchymal stem cells (hMSCs) to demonstrate the biocompatible nature of the tailorable scaffolds developed. These cells are common laboratory models that offer therapeutic potential in the areas of regenerative medicine and tissue engineering. Murine ESCs are a pluripotent cell type that serves as a model for the use of other



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Figure 1: A) A schematic representing synthesis of PEGDMA-MAA hydrogel network using free radical photopolymerization method. B) The effects of varying the total PEGDMA 1000 percentage on hydrogel elasticity. A decrease in the total polymer percentage resulted in a decreased elastic modulus, indicating more elastic hydrogels. Each experiment represents a compilation of over 10,000 discrete data points. C) An increase in the percent wt. of high molecular weight PEGDMA into the PEGDMA 1000 hydrogels resulted in a decreased elastic modulus. The composition of each hydrogel includes the given percentage of higher molecular weight (Mw) polymer with the remainder of the hydrogel composed of PEGDMA 1000. All hydrogels include 2% (wt.) MAA for surface functionalization.

pluripotent stem cells, including iPSCs. Induced PSCs are patient specific, but the method for reprogramming remains inefficient. Human ASCs and hMSCs are multipotent cells, with more limited differentiation and replicative potential. However, they can be derived from the individual, therefore evading any immune rejection, and are currently being used in nearly 800 clinical trials.18 Current approaches in tissue engineering and regenerative medicine use a variety of cell types to determine the most efficient and effective therapy option. In all cases, the success depends not only on the cell, but the chemical and physical cues that direct the cells towards a particular cell fate.19-21 There is a need in the field to continue to identify reliable and reproducible methods for directed differentiation and clinical application. The motivation for this work was to develop biomimetic materials that support cell attachment and viability and that can ultimately be tailored to direct the differentiation of stems cells toward specific lineages. The hydrogel scaffolds generated in this study can be tailored to mimic the natural elasticity of desired tissue types, making them a promising biomaterial for therapeutic tissue generation. Hydrogel films containing 20-30% wt. total poly(ethylene glycol) dimethacrylate (PEGDMA) were prepared (Figure 1A) using standard photopolymerization methods. Different molecular weights (Mw) (1000, 8000, and 20,000) PEGDMA were used to adjust the overall hydrogel network mesh size. All hydrogel blends were prepared with 2% wt. methacrylic acid (MAA) and will be referred to as PEGDMA-


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MAA hydrogels for the remainder of the paper (see supplemental information). During the photopolymerization process, MAA becomes tethered to the PEGDMA hydrogel network leading to carboxyl groups on the surface of the synthesized hydrogels. As shown in Figure 1A, these carboxyl groups can be activated post synthesis, to become amine reactive for specific functionalization of the hydrogel surface with peptides and proteins. Elasticity of each hydrogel sample was determined using the material tester system, ADMET expert 2600. Surface functionalization of the synthesized PEGDMAMAA hydrogels with fibronectin (FN) was completed using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and Sulfo- N-hydroxysulfosuccinimide (Sulfo-NHS) chemistry. Mouse ESCs, hMSCs, and hASCs were maintained in culture at 37°C with 5% CO2. The number of cells seeded onto functionalized hydrogels and tissue culture treated wells in 24 well tissue culture plates were: mESCs (40,000 cells), hMSCs (4,000 cells), and hASCs (4,000 cells). Cell viability assays were performed at 72 hours in mESCs and 120 hours in hMSCs and hASCs. Quantitative RT-PCR was performed to determine the expression of a pluripotency marker in mESCs and multipotency markers in hMSCs and hASCs. Initially, PEGDMA-MAA hydrogels of Mw 1000 with varying polymer percentages (% wt.) were synthesized to evaluate the effect of polymer percentages on the elastic modulus of the hydrogel. The graph of stress vs. strain displayed a decrease in slope with a decrease in the polymer percentage (Figure 1B). The elastic modulus decreased as the polymer percentage decreased in the hydrogel composition. This trend is due to less PEGDMA being available to be crosslinked into the hydrogel network which results in a larger network mesh size and subsequently a more elastic network. Following the trend observed, 20% wt. PEGDMA-MAA hydrogel blends containing PEGDMA 1000 and a higher molecular weight polymer (Mw 20,000 or Mw 8,000) were synthesized. The higher the Mw of the PEGDMA the larger the resulting network mesh size. Both blends of PEGDMA-MAA containing PEGDMA Mw 1000 and 8000 and PEGDMA Mw 1000 and 20,000 displayed a decrease in the elastic modulus with an increase in the percentage of higher molecular weight polymers (%wt.) (Figure 1C). The overall elastic modulus of the hydrogel was easily modulated by varying the percentage of higher molecular weight polymers within the composition. 20% blends of PEGDMA-MAA with PEGDMA Mw 1000 and 20,000 displayed a wide range of elasticity mimicking different tissues in vivo. The PEGDMA-MAA hydrogel blends of 10% PEGDMA Mw 1000 and 10% PEGDMA Mw 20,000, 5% PEGDMA Mw 1000 and 15% PEGDMA Mw 20,000, and 3% PEGDMA Mw 1000 and 17% PEGDMA Mw 20,000 each yielded an elastic modulus in the following ranges: 50-60 kPa (bone), 25-30 kPa (cartilage), and 8-10 kPa (muscle) respectively. These results demonstrate that without altering the chemistry of the hydrogel composition, this protocol provides a simple method for studying the role of elasticity on stem cell differentiation. 20% PEGDMA-MAA hydrogels of Mw 1000 were functionalized through 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC)/ Sulfo- N-hydroxysulfosuccinimide (Sulfo-NHS)mediated coupling of fibronectin to the methacrylic acid groups on the hydrogel surface. Murine ESCs, hMSCs, and hASCs displayed attachment within 24 hours post-seeding on functionalized hydrogels. All three cell types cultured on the hydrogels demonstrated attachment and growth patterns similar to the control cells cultured on tissue culture plates (Figure 2A). Murine ESCs on hydrogels had a high nucleusto-cytoplasm ratio and were localized in colonies, consistent with the characteristics of embryonic stem



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Figure 2: A) Microscopic images of mESCs, hMSCs, and hASCs on tissue culture plates (control) and 20% PEGDMA1000MAA hydrogels at 72 hours in culture (10X, scale 400 μm). Phalloidn (red) and DAPI (blue) were used to provide better resolution of the attachment of hMSCs and hASCs to the hydrogels. B) Live/dead analysis of mESCs, hMSCs, and hASCs cultured on 20% PEGDMA-MAA hydrogels of Mw 1000. Live cells were shown by blue nuclear stain and dead cells were shown by red nuclear stain. C: Quantitative RT-PCR of the pluripotency marker murine pou5f1 and nanog in mESCs and the multipotency markers human tfrsf10d and cd44 in hMSCs, and human pvlr3 and pou5f1 in hASCs. Each bar represents the average of triplicate qRT-PCR reactions for n=3. Relative expression levels between the control and experimental groups were analyzed using the two tailed t-test (Confidence Interval 95%, p-value < 0.05 is considered significant).

cells. Both hMSCs and hASCs on hydrogels displayed fibroblast-like phenotypes, consistent with mesenchymal stem cells in culture. Cell attachment is shown through phase-contrast imaging of the mESCs which are easily visualized because of their colony formation. However, because hMSCs and hASCs grow as flat, individual cells, they are more difficult to see with phase-contrast imaging and so phalloidin, an F-Actin cell stain, was used to better demonstrate cell attachment and allow for comparison between tissue culture treated plastic and hydrogels. A viability assay performed on mESCs (72 hours post-seeding), hMSCs (120 hours post-seeding), and hASCs (120 hours post-seeding) on


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hydrogels demonstrated a majority of viable cells (Figure 2B). The biocompatibility of PEGDMA-MAA hydrogels was demonstrated by the attachment and growth of the three cell types. Quantitative reverse transcriptase PCR (qRT-PCR) was performed after 72 hours for mESCS and 120 hours for both hMSCs and hASCS to determine the expression of pluripotency markers in mESCs and multipotency markers in hMSCs and hASCs. There was no significant difference in the expression of pluripotency and multipotency markers in cells seeded on hydrogels in comparison with cells seeded on tissue culture plates, demonstrating that 20% PEGDMA-MAA hydrogels of Mw 1000 allowed attachment and proliferation without promoting cell differentiation (Figure 2C). In conclusion, hydrogels were synthesized by varying the percentages of higher molecular weight polymers in the overall hydrogel composition. This allowed the development of materials of varying elasticities while maintaining the same chemical composition. The elasticity of the different hydrogel blends ranged from 8 – 400 kPa, mimicking tissues ranging in stiffness from muscle to bone. Surface functionalization was accomplished using EDC/Sulfo-NHS chemistry, a technique that facilities the attachment of a number of peptides or proteins to the surface of the scaffold for a variety of applications in cell studies and biomanufacturing. Here we functionalized the hydrogels with fibronectin to support attachment of cells. The mESCs, hMSCs, and hASCs seeded on hydrogels displayed similar morphologies as those cells seeded on tissue culture treated plastic. In addition, cell viability assays performed on cells seeded on hydrogels revealed a majority of viable cells, confirming the biocompatibility of the hydrogels synthesized. Lastly, the cells cultured on the hydrogels maintained their pluripotency and multipotency markers displaying a similar gene expression profile as observed in the control cells. In summary, the method implemented in this study is efficient and reproducible, which allows the possibility of synthesizing a wide range of biocompatible scaffolds on a bulk scale. We have successfully synthesized a biocompatible and highly tunable PEGDMA-MAA based hydrogel scaffold that can be utilized for studying cell response to matrices of varying stiffness, while holding the chemistry of the hydrogels consistent. A complete understanding of stem cell responses to substrate stiffness is important for advancing stem cell-based clinical applications and optimization of physiologically relevant materials for tissue engineering. Initially, scaffold-based tissue engineering was limited to synthesis of scaffolds on a small scale to determine the optimal scaffold properties for tissue engineering applications. As the demand for transplants increases every second, there is a huge need for bulk scale manufacturing of scaffolds for generating functional tissues in vitro. Acknowledgements: This research is supported by the Malcom Feist Partners across campuses (PAC) grant by the Center for Cardiovascular Diseases and Sciences at LSU Health Shreveport, Sigma Xi Grants in Aid of Research, and the LaSPACE Undergraduate Research Assistantship Program and Minority Research Scholars Program. We would like to thank Dr. Yuri Lvov for allowing us to use the ADMET expert 2600 material tester system and Dr. Bruce Bunnell for providing us with hMSCs used in the cell culture studies. We would also like to acknowledge College of Applied & Natural Sciences and College of Engineering & Sciences at Louisiana Tech University. Artwork was generated by Nick Bustamante, Chair and Associate Professor of Studio Art at Louisiana Tech University.



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Supporting Information Available: The following files are available free of charge: detailed materials and methods sections, table of hydrogel compositions and curing times for varying elasticity, table of RNA concentrations used for cDNA synthesis for each cell type, and table listing primer pairs or pluripotency and multipotency markers.


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0.7424

Control (Tissue culture plate)

htfrsf10d

hpou5f1 0.3056

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20% PEGDMA-MAA Mw 1000 Hydrogel


0.0596

hcd44 0.2656

Poly(ethylene glycol) Hydrogels with Tailorable ... - ACS Publications

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