RGD Modified Hydrogel

Hydrogels are increasingly being utilized in cell culture studies to better mimic the extracellular milieu that cells must navigate through during development, immune response, and disease1. To enhance hydrogel versatility, researchers have incorporated covalently bound bioactive peptides into hydrogels. As such, hydrogels can be modified to support cell functionality. For example, the addition of cell adhesion peptides, derived from the extracellular matrix (ECM), can improve adhesion2, spreading3, viability4, proliferation5, migration6, invasion7, and neurite extension8. The most commonly incorporated cell adhesion peptide is the tri-amino acid arginine-glycine-aspartate (RGD). RGD, the primary integrin-binding domain of ECM proteins, is extensively used to promote cell attachment to different biomaterials, including VitroGel. A plethora of ECM proteins contain this amino acid sequence, including fibronectin9, vitronectin10, collagen11, and laminin2. RGD offers several advantages over native proteins, including resistance against denaturation and exquisite control of density and patterning within hydrogels13.

The use of 3D hydrogels allows scientist to study disease progression in an environment more physiologically relevant than 2D in vitro system while allowing for more control than in vivo models. For instance, investigating tumor growth and metastasis in 3D hydrogels offers a more accurate experimental system because, in vivo, cancer cells adhere, invade, and disseminate into tissues in 3D. Furthermore, understanding the mechanisms of cancer cell adhesion in 3D environments is critical to finding therapeutics to combat tumor growth and metastasis. By adding an adhesive peptide, such as RGD, to hydrogels, researchers have expanded our understanding of cancer progression. RGD has been shown to increase the adhesion of cancer cell lines to various biomaterials 2,14,15. For example, VitroGel hydrogels have been developed with covalently bound RGD (VitroGel RGD) and shown to have effects on cancer progression 1618. Previous studies have demonstrated that RGD is a robust adhesive substratum for cancer cells leading to increased spreading 2,15, which is shown to be dependent on peptide density 9. Balanced stabilization and turnover of adhesion complexes control cancer cell migration20. Therefore, RGD has been used to examine the formation of adhesion complexes and stress fibers in motile cancer cells. In one study, RGD was shown to accelerate migration, due to greater adhesion stabilization, of glioma cells2. This effect was shown to be dependent on substratum elasticity. Adhesion is also critical for cancer cell proliferation and tumor growth. Therefore, using 3D hydrogel systems, experimenters can study the mechanisms underlying proliferation and growth of cancer cells. For instance, prostate cancer cells cultured in VitroGel RGD hydrogels were found to be highly proliferative and invasive in response to nerve growth factor18. RGD modified hydrogels were also found to induce the formation of spheroids, analogous of tumors, in two different ovarian cancer cell lines (OV-MZ-6 and SKOV-3)21. Spheroid formation was additionally observed using VitroGel RGD17. In this study, the authors evaluated tumor progression by utilizing three osteosarcoma cell lines, OSA 1777, MNNG-HOS, and U2OS. VitroGel RGD was shown to have differential effects on spheroid growth over time, with OSA 1777 clusters developing more slowly. OSA 1777 spheroids were also shown to be more susceptible to chemotherapy mediated cell death. Therefore, VitroGel RGD offers researchers a powerful opportunity to not only study the mechanisms of cancer development but to differentiate between individual cancer cell lines. This tool will allow scientists to further tailor the development of therapeutics to individuals with varying genetic mutations within the same cancer type. Finally, hydrogels provide researchers the ability to study the molecular mechanisms of cancer progression. Using VitroGel RGD, Shamloo and colleagues investigated the role of adenosine kinase (ADK) in tumorigenesis, as dysregulated adenosine signaling is implicated in breast cancer pathogenesis16. Breast cancer cell proliferation and migration were found to be dependent on the expression of specific ADK isoforms. Overall, because cancer growth and metastasis is such a complex process, RGD modified hydrogels allow researchers to independently parse out specific processes, as well as collectively study these processes.

The ECM plays a principal role in providing the structural support needed for cell proliferation and differentiation during tissue development. Consequently, adhesive peptides are frequently employed in 3D hydrogels to elucidate the mechanisms of cell adhesion, proliferation, and differentiation22,23. Hydrogels with RGD as an adhesive substratum have been shown to promote adhesion of cells from diverse tissues, including bone24, muscle25, cartilage26, vascular27, cardiac28,29, lung30, and connective tissue31. Furthermore, upon attachment, sufficient adhesion, and ECM remodeling is needed for cells to asymmetrically spread and receive cues from the environment. While this has been studied in 2D, in vivo stem cells and precursor cells receive information from a 3D environment. Therefore, studies focusing on these cellular processes using 2D platforms do not properly recapitulate the range of cues involved. VitroGel RGD provides an excellent opportunity to focus on these processes in a more relevant manner. RGD has been shown to promote cell spreading in osteoblasts24, fibroblasts32, and stem cells33. Cell adhesion and spreading initiates activation of intracellular signaling pathways that are essential for proliferation and differentiation, and 3D environments provide the most accurate platform for studying the signals underlying these processes. For example, VitroGel RGD was utilized to examine 3D ovarian follicle formation and oocyte maturation34. In comparison to 2D cultures, VitroGel RGD accelerated the formation of follicles and oocyte complexes, further demonstrating the value of 3D hydrogels relative to 2D cultures. In addition, multiple studies have used this 3D platform to study osteogenesis, the development of bone24,35,36. RGD has been shown to induce attachment and spreading of osteoblasts, cells that play a critical role in bone development via secretion of the requisite matrix material24. Furthermore, incorporation of RGD in 3D hydrogels has been shown to induce differentiation of both goat35 and rat36 bone marrow stromal cells into osteogenic cells. RGD modified hydrogels have additionally been shown to induce proliferation of myoblasts5,25, chondrocytes 26,37,38, human umbilical vein endothelial cells27,39, and dermal fibroblasts31. Moreover, differentiation of precursor cells into defined target cell types has been shown with bone marrow cells35,36, myoblasts5, and stem cells40,41. These studies demonstrate the utility of RGD modified hydrogels in studying a variety of developmental processes.

RGD mediated cell adhesion has also been shown to be useful in studying migration, neurite formation and outgrowth, and regenerative processes. For example, cancer cells show increased migrate rates in the presence of RGD2. These effects on migration have also been observed in human umbilical vein endothelial cells27, fibroblasts42, and mesenchymal stem cells43. This is likely due to the RGD modulation of adhesion dynamics, as RGD has been shown to increase adhesion duration2 and support the formation of stress fibers26. The mechanisms for neurite outgrowth are similar to cell migration. By using chick dorsal root ganglion neurons, Lampe and colleagues measured the effect of RGD on neurite outgrowth8. They found that RGD increased neurite lengths, rate of outgrowth, and neurite number. Finally, by injecting VitroGel RGD into lesions or degenerative sites, researchers have been able to show the regenerative abilities of this platform44,45. Specifically, an injectable RGD containing hydrogel was used to study regenerative processes in lesioned rat cortices44. The adhesive hydrogel was able to support new vascularization and inhibit glial scarring, indicating successful regeneration. In another study, researchers injected hydrogels with RGD into injured rat spinal cords and observed angiogenesis and axon regeneration45. Furthermore, scientists were able to show cell differentiation and partial restoration of the ECM in vivo after injecting VitroGel RGD loaded with mesenchymal stem cells derived from the nucleus pulposus into degenerating rat intervertebral disc. Overall, RGD has been extensively studied and been shown to be an extremely useful peptide for studying a variety of cellular processes in different tissue types.

Cell Type Behavior Reference Table for VitroGel RGD

Bone
Cell TypeBehavior
Goat bone marrow stromal cellsPromoted osteogenic differentiation
Rat bone marrow stromal cellsPromoted osteogenic differentiation
Rat osteoblastsIncreased cell attachment and spreading
Cancer/Tumor
Cell TypeBehavior
Biphasic synovial sarcoma SYO-1Cell proliferation and cell matrix interactions
Bone OSA 1777Cell proliferation and cell matrix interactions
Breast 4T1Cell proliferation, division, migration, and invasion
Breast AU-565Cell proliferation and cell matrix interactions
Breast Cancer MCF-7Cell proliferation, intercellular connections
Breast E0771Cell proliferation and cell matrix interactions
Breast MDA-MB-231Cell proliferation, division, migration, and invasion
Breast T47DCell proliferation, division, migration, and invasion
Colorectal adenocarcinoma DLD-1 cellsCell proliferation and cell matrix interactions
Epithelial ovarian OV-MZ-6Promoted spheroid formation and proliferation
Epithelial ovarian SKOV-3Promoted spheroid formation and proliferation
Fuji CellsCell proliferation and cell matrix interactions
Glioblastoma SF 268Cell proliferation and cell matirx interaction
Glioblastoma SF 295Cell proliferation and cell matirx interaction
Glioblastoma SNB75Cell proliferation and cell matirx interaction
Glioblastoma U-251 MGCell proliferation and cell matirx interaction
Glioma U87-MGIncreased cell spreading and actin stress fiber assembly
Glioma U87-MGCell proliferation and cell matirx interaction
Glioma U373-MGIncreased cell adhesion duration and migration (on higher stiffness)
HEK 293Cell proliferation and cell matrix interactions
Huaman colon carcinoma HCT-8Cell proliferation and cell matirx interaction
Human colorectal carcinoma HCT 116cell proliferation, cell survival, and intercelluar networking
Human pancreatic cancer PANC-1cell proliferation and cellular interactions
Insulinoma ins-1 (Rat)Cell proliferation and cell matrix interactions
Liver carcinoma HepG2Cell proliferation and cell matirx interaction
Melanoma CellsCell proliferation and cell matrix interactions
Ovarian carcinoma OVCAR-3Cell proliferation and invasion
Primary glioblastom U87cell proliferation and cellular interactions
Prostate adenocarcinoma LNCaPIncreased cell attachment
Prostate CRPCCell proliferatin and invasion
Prostate DU145Cell proliferation and invasion
Prostate PC3Cell proliferation and invasion
Cartilage
Cell TypeBehavior
Bovine chondrocytesIncreased cell viability and proliferation
Bovine chondrocytesPromoted cell attachment, viability, and stress fiber formation
Human chondrocytesPromoted cell viability and proliferation
Connect Tissues
Cell TypeBehavior
Fibroblast NIH3T3Promoted cell spreading
Fibroblasts NIH3T3Increased directional cell migration toward gradient
Human dermal fibroblastsPromoted cell survival and spreading
Human dermal fibroblastsIncreased cell adhesion and proliferation
Human foreskin fibroblastsPromoted cell spreading
Epithelial Cells
Cell TypeBehavior
A549 cellsCell proliferation and invasion
MCF-12ACell proliferation and invasion
Mouse ovarian follicle cellsCell proliferation and invasion
Immune Cells
Cell TypeBehavior
Beta TC3 CellsCell proliferation and cellular interactions
Kidney
Cell TypeBehavior
Human embryonic kidney HEK293Promoted spheroid formation
Madin-Darby Canine KidneyPromoted formation of structured epithelial cysts
Liver
Cell TypeBehavior
Human hepatocytesIncreased number of filopodia and synthesis of albumin
Mouse hepatocytesPromoted cell viability
Lung
Cell TypeBehavior
Alveolar epithelial A549Inhibited cell detachment
Alveolar epithelial RLE-6TNIncreased cell attachment and mesenchymal differentiation
Muscle
Cell TypeBehavior
Mouse skeletal myoblastsPromoted cell attachment, proliferation, and myofibril formation
Myoblasts C2C12Promoted cell proliferation and differentiation
Neural
Cell TypeBehavior
Chick dorsal root ganglion cellsIncreased neurite length, neurite outgrowth, and neurite number
In vivo lesioned rat cortexSupported angiogenesis and inhibited glial scars
In vivo lesioned rat spinal cordSupported angiogenesis and axon regeneration
Stem Cells
Cell TypeBehavior
Human embryonic stem cellsIncreased retinal pigmented epithelium and optic vesicle development
Human iPSCCell proliferation, and cell matrix interactions
Human mesenchymal stem cellsIncreased cell viability
Mouse embryonic stem cellsPromoted endothelial cell differentiation
Mouse mesenchymal stem cellsPromoted cell spreading and migration
Rat mesenchymal stem cellsIncreased cell adhesion and spreading
Rat mesenchymal stem cellsPromoted cell attachment and differentiation
Vascular/Cardiac
Cell TypeBehavior
Human aortic smooth muscle cellsPromoted cell attachment
Human umbilical vein endothelial cellsIncreased cell adhesion, proliferation, migration, and angiogenesis
Human umbilical vein endothelial cellsIncreased cell adhesion and proliferation
Rat neonatal cardiacPromoted cell attachment and tissue regeneration and prevented apoptosis

Reference

Reference

[1]      S. R. Caliari and J. A. Burdick, “A practical guide to hydrogels for cell culture,” Nature Methods. 2016, doi: 10.1038/nmeth.3839.
[2]      Y. Kim and S. Kumar, “CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility,” Mol. Cancer Res., 2014, doi: 10.1158/1541-7786.MCR-13-0629.
[3]      S. P. Massia, S. S. Rao, and J. A. Hubbell, “Covalently immobilized laminin peptide Tyr-Ile-Gly-Ser-Arg (YIGSR) supports cell spreading and co-localization of the 67-kilodalton laminin receptor with α-actinin and vinculin,” J. Biol. Chem., 1993.
[4]      L. Jongpaiboonkit, W. J. King, and W. L. Murphy, “Screening for 3D environments that support human mesenchymal stem cell viability using hydrogel arrays,” Tissue Eng. – Part A, 2009, doi: 10.1089/ten.tea.2008.0096.
[5]      Y. Yeo, W. Geng, T. Ito, D. S. Kohane, J. A. Burdick, and M. Radisic, “Photocrosslinkable hydrogel for myocyte cell culture and injection,” J. Biomed. Mater. Res. – Part B Appl. Biomater., 2007, doi: 10.1002/jbm.b.30667.
[6]      C. M. M. Motta, K. J. Endres, C. Wesdemiotis, R. K. Willits, and M. L. Becker, “Enhancing Schwann cell migration using concentration gradients of laminin-derived peptides,” Biomaterials, 2019, doi: 10.1016/j.biomaterials.2019.119335.
[7]      A. V. Taubenberger et al., “3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments,” Acta Biomater., 2016, doi: 10.1016/j.actbio.2016.03.017.
[8]      K. J. Lampe, A. L. Antaris, and S. C. Heilshorn, “Design of three-dimensional engineered protein hydrogels for tailored control of neurite growth,” Acta Biomater., 2013, doi: 10.1016/j.actbio.2012.10.033.
[9]      M. D. Pierschbacher and E. Ruoslahti, “Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule,” Nature, 1984, doi: 10.1038/309030a0.
[10]    M. A. Arnaout, B. Mahalingam, and J.-P. Xiong, “INTEGRIN STRUCTURE, ALLOSTERY, AND BIDIRECTIONAL SIGNALING,” Annu. Rev. Cell Dev. Biol., 2005, doi: 10.1146/annurev.cellbio.21.090704.151217.
[11]    J. J. Ames et al., “Identification of an endogenously generated cryptic collagen epitope (XL313) that may selectively regulate angiogenesis by an integrin yes-associated protein (YAP) mechano-transduction pathway,” J. Biol. Chem., 2016, doi: 10.1074/jbc.M115.669614.
[12]    K. ‐I Tashiro et al., “The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth,” J. Cell. Physiol., 1991, doi: 10.1002/jcp.1041460316.
[13]    S. L. Bellis, “Advantages of RGD peptides for directing cell association with biomaterials,” Biomaterials, 2011, doi: 10.1016/j.biomaterials.2011.02.029.
[14]    M. Abdul Kafi, W. A. El-Said, T. H. Kim, and J. W. Choi, “Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays,” Biomaterials, 2012, doi: 10.1016/j.biomaterials.2011.10.003.
[15]    B. Ananthanarayanan, Y. Kim, and S. Kumar, “Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform,” Biomaterials, 2011, doi: 10.1016/j.biomaterials.2011.07.005.
[16]    B. Shamloo et al., “Dysregulation of adenosine kinase isoforms in breast cancer,” Oncotarget, 2019, doi: 10.18632/oncotarget.27364.
[17]    P. Thanindratarn, X. Li, D. C. Dean, S. D. Nelson, F. J. Hornicek, and Z. Duan, “Establishment and Characterization of a Recurrent Osteosarcoma Cell Line: OSA 1777,” J. Orthop. Res., 2019, doi: 10.1002/jor.24528.
[18]    M. di Donato, G. Cernera, A. Migliaccio, and G. Castoria, “Nerve growth factor induces proliferation and aggressiveness in prostate cancer cells,” Cancers (Basel)., 2019, doi: 10.3390/cancers11060784.
[19]    N. Orgovan, B. Peter, S. Bosze, J. J. Ramsden, B. Szabó, and R. Horvath, “Dependence of cancer cell adhesion kinetics on integrin ligand surface density measured by a high-throughput label-free resonant waveguide grating biosensor,” Sci. Rep., 2014, doi: 10.1038/srep04034.
[20]    M. Maziveyi and S. K. Alahari, “Cell matrix adhesions in cancer: The proteins that form the glue,” Oncotarget. 2017, doi: 10.18632/oncotarget.17265.
[21]    D. Loessner, K. S. Stok, M. P. Lutolf, D. W. Hutmacher, J. A. Clements, and S. C. Rizzi, “Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells,” Biomaterials, 2010, doi: 10.1016/j.biomaterials.2010.07.064.
[22]    S. Pradhan and M. C. Farach-Carson, “Mining the extracellular matrix for tissue engineering applications,” Regenerative Medicine. 2010, doi: 10.2217/rme.10.61.
[23]    Y. H. Tsou, J. Khoneisser, P. C. Huang, and X. Xu, “Hydrogel as a bioactive material to regulate stem cell fate,” Bioactive Materials. 2016, doi: 10.1016/j.bioactmat.2016.05.001.
[24]    J. A. Burdick and K. S. Anseth, “Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering,” Biomaterials, 2002, doi: 10.1016/S0142-9612(02)00176-X.
[25]    J. A. Rowley, G. Madlambayan, and D. J. Mooney, “Alginate hydrogels as synthetic extracellular matrix materials,” Biomaterials, 1999, doi: 10.1016/S0142-9612(98)00107-0.
[26]    H. D. Kim et al., “Extracellular-matrix-based and Arg-Gly-Asp-modified photopolymerizing hydrogels for cartilage tissue engineering,” Tissue Eng. – Part A, 2015, doi: 10.1089/ten.tea.2014.0233.
[27]    L. S. Wang et al., “Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid-tyramine hydrogel system to promote the formation of functional vasculature,” Acta Biomater., 2014, doi: 10.1016/j.actbio.2014.02.022.
[28]    S. A. DeLong, J. J. Moon, and J. L. West, “Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration,” Biomaterials, 2005, doi: 10.1016/j.biomaterials.2004.09.021.
[29]    M. Shachar, O. Tsur-Gang, T. Dvir, J. Leor, and S. Cohen, “The effect of immobilized RGD peptide in alginate scaffolds on cardiac tissue engineering,” Acta Biomater., 2011, doi: 10.1016/j.actbio.2010.07.034.[30]    A. C. Brown, J. A. Rowe, and T. H. Barker, “Guiding epithelial cell phenotypes with engineered integrin-specific recombinant fibronectin fragments,” Tissue Eng. – Part A, 2011, doi: 10.1089/ten.tea.2010.0199.
[31]    Y. D. Park, N. Tirelli, and J. A. Hubbell, “Photopolymerized hyaluronic acid-based hydrogels and interpenetrating networks,” in The Biomaterials: Silver Jubilee Compendium, 2002.
[32]    X. Z. Shu et al., “Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel,” J. Biomed. Mater. Res. – Part A, 2004, doi: 10.1002/jbm.a.20002.
[33]    D. L. Hern and J. A. Hubbell, “Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing,” J. Biomed. Mater. Res., 1998, doi: 10.1002/(SICI)1097-4636(199802)39:2<266::AID-JBM14>3.0.CO;2-B.
[34]    E. J. Kim et al., “The new biocompatible material for mouse ovarian follicle development in three-dimensional in vitro culture systems,” Theriogenology, 2020, doi: 10.1016/j.theriogenology.2019.12.009.
[35]    F. Yang, C. G. Williams, D. A. Wang, H. Lee, P. N. Manson, and J. Elisseeff, “The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells,” Biomaterials, 2005, doi: 10.1016/j.biomaterials.2005.03.018.
[36]    X. He, J. Ma, and E. Jabbari, “Effect of grafting RGD and BMP-2 protein-derived peptides to a hydrogel substrate on osteogenic differentiation of marrow stromal cells,” Langmuir, 2008, doi: 10.1021/la802447v.
[37]    K. M. Park, Y. K. Joung, K. D. Park, S. Y. Lee, and M. C. Lee, “RGD-conjugated chitosan-pluronic hydrogels as a cell supported scaffold for articular cartilage regeneration,” Macromol. Res., 2008, doi: 10.1007/BF03218553.
[38]    H. Lee et al., “Chondrocyte 3D-culture in RGD-modified crosslinked hydrogel with temperature-controllable modulus,” Macromol. Res., 2012, doi: 10.1007/s13233-012-0074-6.
[39]    N. Zhao, M. R. Battig, M. Xu, X. Wang, N. Xiong, and Y. Wang, “Development of a Dual-Functional Hydrogel Using RGD and Anti-VEGF Aptamer,” Macromol. Biosci., 2017, doi: 10.1002/mabi.201700201.
[40]    X. Wang, C. Yan, K. Ye, Y. He, Z. Li, and J. Ding, “Effect of RGD nanospacing on differentiation of stem cells,” Biomaterials, 2013, doi: 10.1016/j.biomaterials.2013.01.021.
[41]    L. Schukur, P. Zorlutuna, J. M. Cha, H. Bae, and A. Khademhosseini, “Directed Differentiation of Size-Controlled Embryoid Bodies Towards Endothelial and Cardiac Lineages in RGD-Modified Poly(Ethylene Glycol) Hydrogels,” Adv. Healthc. Mater., 2013, doi: 10.1002/adhm.201200194.
[42]    D. Guarnieri et al., “Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters,” Acta Biomater., 2010, doi: 10.1016/j.actbio.2009.12.050.
[43]    Y. Lei, S. Gojgini, J. Lam, and T. Segura, “The spreading, migration and proliferation of mouse mesenchymal stem cells cultured inside hyaluronic acid hydrogels,” Biomaterials, 2011, doi: 10.1016/j.biomaterials.2010.08.103.
[44]    F. Z. Cui, W. M. Tian, S. P. Hou, Q. Y. Xu, and I. S. Lee, “Hyaluronic acid hydrogel immobilized with RGD peptides for brain tissue engineering,” in Journal of Materials Science: Materials in Medicine, 2006, doi: 10.1007/s10856-006-0615-7.
[45]      S. Woerly, E. Pinet, L. De Robertis, D. Van Diep, and M. Bousmina, “Spinal cord repair with PHPMA hydrogel containing RGD peptides (NeuroGelTM),” Biomaterials, 2001, doi: 10.1016/S0142-9612(00)00354-9.