A self-assembling peptide matrix used to control stiffness and binding site density supports the formation of microvascular networks in three dimensions

Abstract

A three-dimensional (3-D) cell culture system that allows control of both substrate stiffness and integrin binding density was created and characterized. This system consisted of two self-assembling peptide (SAP) sequences that were mixed in different ratios to achieve the desired gel stiffness and adhesiveness. The specific peptides used were KFE ((acetyl)-FKFEFKFE-CONH2), which has previously been reported not to support cell adhesion or MVN formation, and KFE-RGD ((acetyl)-GRGDSP-GG-FKFEFKFE-CONH2), which is a similar sequence that incorporates the RGD integrin binding site. Storage modulus for these gels ranged from ~60 to 6000 Pa, depending on their composition and concentration. Atomic force microscopy revealed ECM-like fiber microarchitecture of gels consisting of both pure KFE and pure KFE-RGD as well as mixtures of the two peptides. This system was used to study the contributions of both matrix stiffness and adhesiveness on microvascular network (MVN) formation of endothelial cells and the morphology of human mesenchymal stem cells (hMSC). When endothelial cells were encapsulated within 3-D gel matrices without binding sites, little cell elongation and no network formation occurred, regardless of the stiffness. In contrast, matrices containing the RGD binding site facilitated robust MVN formation, and the extent of this MVN formation was inversely proportional to matrix stiffness. Compared with a matrix of the same stiffness with no binding sites, a matrix containing RGD-functionalized peptides resulted in a ~2.5-fold increase in the average length of network structure, which was used as a quantitative measure of MVN formation. Matrices with hMSC facilitated an increased number and length of cellular projections at higher stiffness when RGD was present, but induced a round morphology at every stiffness when RGD was absent. Taken together, these results demonstrate the ability to control both substrate stiffness and binding site density within 3-D cell-populated gels and reveal an important role for both stiffness and adhesion on cellular behavior that is cell-type specific.

Advancing biomaterials of human origin for tissue engineering

“…In contrast, the absence of RGD induced round morphology at all rigidities. Overall, these findings reveal the potential to control both the binding site density and the substrate stiffness within 3D cell-populated hydrogels and demonstrate the crucial influence of both adhesion and stiffness on cell type-specific cellular behaviors [433]. …”

Tuning of hydrogel stiffness using a two‐component peptide system for mammalian cell culture

“…Several studies have shown peptide hydrogel systems with tuneable stiffness, where tuning is mainly achieved by varying peptide concentrations in the hydrogels . Some attempts to change hydrogel stiffness by varying amino acid composition in peptide amphiphilic systems has been explored, highlighting the key role of hydrophobic interactions in gel stiffness .…”

Effect of self-assembled peptide–mesenchymal stem cell complex on the progression of osteoarthritis in a rat model

“…Even though several studies were conducted on the effect of SAP on MSCs in in vitro studies, [49][50][51] the fate of MSCs injected into the joint cavity will be different according to the microenvironments, as stem cells have high susceptibility to diverse microenvironments, including mechanical strain, shear stress, and cell-cell interaction, and will show altered differentiation and paracrine effects. 52,53 Wang 54 suggested the migration of injected MSCs from the injection site to the cartilage defect through the evidence of Prussian blue-positive and bromodeoxyuridine-labeled cells entering into the defect, which can be applied to our study.…”