The development of advanced biomaterials capable of mimicking the dynamic and heterogeneous nature of the extracellular matrix (ECM) is critical for next-generation cell culture systems. This study presents a modular hydrogel platform based on engineered bacterial fimbriae protein Caf1, which enables precise control over both mechanical and biochemical properties to guide human dermal fibroblast (hDFB) behavior in two-dimensional environments. Caf1, originally isolated from *Yersinia pestis*, forms highly stable, fibrillar polymers through non-covalent interactions between immunoglobulin-like subunits. Its structural robustness, combined with ease of genetic engineering, makes it an ideal candidate for designing synthetic ECM analogs.
In this work, Caf1 was modified by inserting the Arg-Gly-Asp-Ser (RGDS) peptide motif into loop 5 of its structure, enabling integrin-mediated cell adhesion without chemical modification. By combining wild-type Caf1WT and RGDS-modified Caf1RGDS subunits in varying ratios, researchers created copolymers with tunable bioactivity. These were then crosslinked with 8-arm PEG to form transparent hydrogels at concentrations of 2.5% or 5.0% w/v. The resulting gels exhibited a wide range of stiffness, from ~240 Pa to over 3000 Pa, depending on Caf1 concentration. Importantly, the inclusion of RGDS motifs did not alter mechanical properties, allowing independent tuning of biological signals and physical cues.
Rheological characterization revealed that hydrogels derived from thermally refolded Caf1 exhibited faster stress relaxation than those from native polymers. This behavior stems from shorter polymer chains formed during thermal cycling, leading to reduced network entanglement and increased viscoelasticity. The 2.5%-refolded-Caf1RGDS hydrogel displayed rapid stress relaxation within seconds—comparable to soft biological tissues—and was associated with significantly higher metabolic activity and DNA content in hDFBs compared to stiffer counterparts.
Cellular responses were strongly influenced by these material properties. On RGDS-functionalized gels, cells adhered efficiently, spread extensively, and proliferated robustly. The 2.5%-refolded-Caf1RGDS formulation induced the most favorable phenotype: high metabolic activity, extensive cytoskeletal organization, and elevated collagen I deposition. In contrast, stiffer 5%-native-Caf1RGDS gels promoted elongated, aligned morphologies consistent with high mechanical tension, resembling behavior on tissue culture plastic. Live/dead assays confirmed excellent cytocompatibility across all RGDS-containing gels, with minimal cell death observed.
Scanning electron microscopy showed interconnected porous networks in all hydrogels, facilitating nutrient diffusion and waste removal—key features for long-term culture.Filamin 1 Antibody Epigenetics Notably, the presence of Caf1WT subunits in mixed gels did not impair cell adhesion or viability, indicating a neutral rather than inhibitory effect.Cadherin-17 Proteinmedchemexpress This allows for dilution of expensive bioactive subunits with highly expressed wild-type variants, reducing production costs while maintaining biological function.PMID:33932162
These findings demonstrate that Caf1-based hydrogels offer unprecedented design flexibility. Their ability to independently modulate stiffness, stress relaxation, and ligand density enables fine-tuning of cellular functions such as proliferation, morphology, and ECM secretion. As a scalable, animal-free alternative to conventional ECM materials, this system holds strong potential for applications in drug screening, disease modeling, regenerative medicine, and personalized therapies.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
