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 16–18. 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
|Goat bone marrow stromal cells||Promoted osteogenic differentiation|
|Rat bone marrow stromal cells||Promoted osteogenic differentiation|
|Rat osteoblasts||Increased cell attachment and spreading|
|Biphasic synovial sarcoma SYO-1||Cell proliferation and cell matrix interactions|
|Bone OSA 1777||Cell proliferation and cell matrix interactions|
|Breast 4T1||Cell proliferation, division, migration, and invasion|
|Breast AU-565||Cell proliferation and cell matrix interactions|
|Breast Cancer MCF-7||Cell proliferation, intercellular connections|
|Breast E0771||Cell proliferation and cell matrix interactions|
|Breast MDA-MB-231||Cell proliferation, division, migration, and invasion|
|Breast T47D||Cell proliferation, division, migration, and invasion|
|Colorectal adenocarcinoma DLD-1 cells||Cell proliferation and cell matrix interactions|
|Epithelial ovarian OV-MZ-6||Promoted spheroid formation and proliferation|
|Epithelial ovarian SKOV-3||Promoted spheroid formation and proliferation|
|Fuji Cells||Cell proliferation and cell matrix interactions|
|Glioblastoma SF 268||Cell proliferation and cell matirx interaction|
|Glioblastoma SF 295||Cell proliferation and cell matirx interaction|
|Glioblastoma SNB75||Cell proliferation and cell matirx interaction|
|Glioblastoma U-251 MG||Cell proliferation and cell matirx interaction|
|Glioma U87-MG||Increased cell spreading and actin stress fiber assembly|
|Glioma U87-MG||Cell proliferation and cell matirx interaction|
|Glioma U373-MG||Increased cell adhesion duration and migration (on higher stiffness)|
|HEK 293||Cell proliferation and cell matrix interactions|
|Huaman colon carcinoma HCT-8||Cell proliferation and cell matirx interaction|
|Human colorectal carcinoma HCT 116||cell proliferation, cell survival, and intercelluar networking|
|Human pancreatic cancer PANC-1||cell proliferation and cellular interactions|
|Insulinoma ins-1 (Rat)||Cell proliferation and cell matrix interactions|
|Liver carcinoma HepG2||Cell proliferation and cell matirx interaction|
|Melanoma Cells||Cell proliferation and cell matrix interactions|
|Ovarian carcinoma OVCAR-3||Cell proliferation and invasion|
|Primary glioblastom U87||cell proliferation and cellular interactions|
|Prostate adenocarcinoma LNCaP||Increased cell attachment|
|Prostate CRPC||Cell proliferatin and invasion|
|Prostate DU145||Cell proliferation and invasion|
|Prostate PC3||Cell proliferation and invasion|
|Bovine chondrocytes||Increased cell viability and proliferation|
|Bovine chondrocytes||Promoted cell attachment, viability, and stress fiber formation|
|Human chondrocytes||Promoted cell viability and proliferation|
|Fibroblast NIH3T3||Promoted cell spreading|
|Fibroblasts NIH3T3||Increased directional cell migration toward gradient|
|Human dermal fibroblasts||Promoted cell survival and spreading|
|Human dermal fibroblasts||Increased cell adhesion and proliferation|
|Human foreskin fibroblasts||Promoted cell spreading|
|Chick dorsal root ganglion cells||Increased neurite length, neurite outgrowth, and neurite number|
|In vivo lesioned rat cortex||Supported angiogenesis and inhibited glial scars|
|In vivo lesioned rat spinal cord||Supported angiogenesis and axon regeneration|
|Human embryonic stem cells||Increased retinal pigmented epithelium and optic vesicle development|
|Human iPSC||Cell proliferation, and cell matrix interactions|
|Human mesenchymal stem cells||Increased cell viability|
|Mouse embryonic stem cells||Promoted endothelial cell differentiation|
|Mouse mesenchymal stem cells||Promoted cell spreading and migration|
|Rat mesenchymal stem cells||Increased cell adhesion and spreading|
|Rat mesenchymal stem cells||Promoted cell attachment and differentiation|
|Human aortic smooth muscle cells||Promoted cell attachment|
|Human umbilical vein endothelial cells||Increased cell adhesion, proliferation, migration, and angiogenesis|
|Human umbilical vein endothelial cells||Increased cell adhesion and proliferation|
|Rat neonatal cardiac||Promoted cell attachment and tissue regeneration and prevented apoptosis|
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