RGD Peptides

Titanium nanotube arrays promote the activity of anastomotic healing-related cells by increasing fibronectin adsorption and activating the RGD-integrin pathway.

Pengyu Chen, Bang Liu, Yijia Li +2 more

The smooth titanium staples of stapling devices cannot reduce the incidence of gastrointestinal anastomotic leakage due to their bioinert nature and lack of active wound-healing promotion capability. This study aims to investigate whether titanium nanotube arrays (TNTs) can enhance the activity of cells involved in gastrointestinal anastomotic healing and further explore the potential mechanisms. TNTs were fabricated on pure titanium sheets via anodic oxidation, and characterized using scanning electron microscopy, roughness analysis, contact angle measurement, and x-ray photoelectron spectroscopy. Cell adhesion, proliferation, spreading, collagen secretion, and integrin expression were evaluated using methods such as CCK-8, immunofluorescence, qPCR, enzyme-linked immunosorbent assay (ELISA), and Western blot. Fibronectin (FN) adsorption and Arg-Gly-Asp tripeptide sequence (RGD domain) exposure were detected via bicinchoninic acid assay, fluorescent staining, and ELISA. The role of the RGD-integrin pathway was further investigated by supplementing serum-reduced medium with exogenous FN and using RGD-specific antagonists. The results showed that TNTs increased the roughness, hydrophilicity, and surface free energy of titanium surfaces. Compared with smooth pure titanium, TNTs promoted the adhesion, proliferation, spreading, and integrin expression of gastric mucosal epithelial cells and fibroblasts, while enhancing the collagen secretion capacity of fibroblasts. Moreover, TNTs adsorbed more FN and exposed more RGD domains, thereby upregulating integrinα5β1 expression. The RGD antagonist could reverse these enhanced cellular responses, confirming the pivotal role of the FN-RGD-integrin pathway. The conclusion indicates that TNTs enhance the adhesion, proliferation, and functional activity of gastrointestinal anastomosis-related cells by promoting FN adsorption and activating the RGD-integrin pathway, which demonstrates that TNT-modified titanium materials hold significant potential for developing bioactive anastomotic devices and promoting tissue healing.

Integrin α5β1 in head and neck squamous cell carcinoma: expression, mechanisms, and clinical implications.

Bo Wu, Hong-Zhi Ma, Jun-Jun Shang +3 more

Head and neck squamous cell carcinoma (HNSCC) represents a significant global health challenge associated with high mortality. A major obstacle in its management is therapeutic resistance, which limits the efficacy of existing treatment modalities. Altered expression of integrins, a family of cell adhesion receptors, has been shown to influence tumor proliferation, migration, and invasion. Among them, integrin α5β1, a member of the RGD (Arg-Gly-Asp)-recognizing integrin subfamily, has emerged as a potentially critical mediator of HNSCC progression and therapeutic resistance, according to a growing body of research. In this review, we assess the evidence regarding the aberrant expression of integrin α5β1 in HNSCC, with a particular focus on common subtypes such as oral squamous cell carcinoma (OSCC), nasopharyngeal carcinoma (NPC), and laryngeal squamous cell carcinoma (LSCC). We then summarize its potential value as a diagnostic and prognostic marker. Furthermore, we discuss the molecular mechanisms that regulate integrin α5β1 and its downstream signaling, especially in the context of therapy resistance. Finally, we outline the potential clinical applications and future research directions related to targeting integrin α5β1. Collectively, this paper aims to synthesize the current knowledge of integrin α5β1 in HNSCC, providing a foundation for the development of personalized, tumor-specific diagnostic tools and targeted therapies.

Bioactive peptides-incorporated photo-crosslinking hydrogel for suture-free repair of corneal injuries.

Wei Wu, Shuo Huai, Chenhao Li +9 more

The development of new biomaterials for suture-free repair of corneal stromal injuries holds promise, especially given the shortage of donor corneas and the limitations of current tissue adhesives. Here, we developed a biocompatible hydrogel via ultrafast in situ photo-crosslinking between di-o-nitrobenzyl-modified Arg-Gly-Asp (RGD) peptide and carboxymethyl chitosan. This hydrogel exhibits shape adaptability, high transparency, and robust bioadhesion, making it suitable for suture-free corneal repair. In vitro experiments confirm that the hydrogel promotes corneal epithelial cell migration. In a rabbit model of penetrating corneal injury, the hydrogel achieves rapid sealing within 60 s while maintaining anterior chamber integrity. In a deep corneal defect model, it effectively seals the stromal wound and supports corneal regeneration, enhancing epithelial barrier restoration with upregulated expression of zonula occludens-1 (ZO-1) and cytokeratin 3 (CK3). Moreover, incorporation of the neuropeptide CGRP into the hydrogel reduces stromal fibrosis, as indicated by decreased expression of α-smooth muscle actin (α-SMA) and vimentin. This bioactive peptides-integrated photo-crosslinking hydrogel represents a promising alternative for suture-free repair of severe corneal injuries and offers a new, effective strategy for clinical corneal wound management.

Dynamic Slide-Ring Hydrogels with Dityrosine-Driven pH-Responsive Fluorescence: Enabling 3D Printing, Enhanced Drug Delivery and Regenerative Therapy.

Yong Li, Maoya Xu, Hongkui Li +6 more

The development of intelligent hydrogels with multifunctional capabilities holds great promise for advancing medical applications. In this work, we developed a dynamic slide-ring supramolecular hydrogel with dual molecular innovations: a dityrosine (DY)-driven pH-responsive fluorescence core in four-arm PEG (4A-PD) and Arginine-Glycine-Aspartic acid peptide (RGD) peptide-functionalized hyperbranched polyglycerol (HPG)-modified α-cyclodextrin (CHR) for enhanced cell adhesion and drug loading. The host-guest interactions between 4A-PD and CHR form a network with shear-thinning, rapid self-healing, and UV-triggered covalent stabilization, enabling room-temperature extrusion-based 3D printing. The printed constructs exhibit high shape fidelity and porous architectures that promote RGD-mediated cell adhesion and proliferation. The DY core imparts the hydrogel with pH-dependent blue fluorescence (emission peak: 400 nm), allowing real-time microenvironmental monitoring. HPG modification of α-CD significantly enhances the drug-loading capacity, demonstrated by the sustained release of tobramycin (TOB) for effective infection control. In a full-thickness infected rat wound model, the TOB-loaded hydrogel accelerates wound closure (97.67% ± 0.56% by day 14), promotes collagen deposition, and modulates inflammation (reduced IL-6, increased IL-10), while enhancing angiogenesis. This multifunctional hydrogel integrates 3D printing, drug delivery, and responsive sensing, offering a versatile platform for regenerative medicine.

Bone sialoprotein: a multifunctional regulator of bone remodelling and tumour progression.

Valentina Kottmann, Philipp Drees, Erol Gercek +1 more

Bone sialoprotein (BSP) is a major non-collagenous protein of the bone extracellular matrix and an important regulator of bone formation and resorption. BSP is produced by bone cells and chondrocytes and present in the bone matrix, cells, dentin and cartilage. However, its aberrant expression in primary tumour tissues and the sera of cancer patients with metastases implicates BSP in tumour biology and progression. The Arg-Gly-Asp (RGD) motif of BSP may be crucial not only for the attachment of metastasising cells to the bone surface but also for tumour growth, survival and activity. This review examines the structure and functions of BSP, including its roles in angiogenesis, bone formation, osteoclast differentiation and activity and cancer cell proliferation, survival, complement evasion, adhesion, migration and invasion. Growing evidence highlights BSP as a key mediator of tumour pathophysiology, skeletal metastasis development and associated bone remodelling. These processes are driven through RGD-integrin binding, the integrin/BSP/matrix metalloproteinase axis, integrin-independent signalling pathways, epithelial-to-mesenchymal transition and potentially post-translational modifications. A deeper understanding of BSP's role in tumour progression may reinforce its potential as a prognostic and diagnostic tumour biomarker and aid the development of anti-BSP antibodies or targeted inhibitors for skeletal metastases and bone diseases.

Incorporation of Visible Light-Responsive Push-Pull Azobenzene into Polymer Networks toward the Construction of Photodynamic Hydrogel Scaffolds.

Itsuki Miyaguni, Kenta Homma, Michiya Matsusaki

Forces play vital roles in regulating cellular behavior, and integrins are prime examples that cells use to sense forces. Designer scaffolds have been developed to trigger integrin-mediated mechanotransduction to control cellular functions. However, current scaffolds lack spatiotemporal control of integrin mechanostimulation in a three-dimensional matrix. In this study, a photoresponsive hydrogel scaffold in which a cell-adhesive push-pull azobenzene was covalently loaded onto the hydrogel was synthesized. The cis-trans photoisomerization of azobenzene is expected to mechanostimulate the interaction of integrins with the cell-adhesive peptides (RGD peptide; arginine-glycine-aspartic acid) bound to azobenzene. The photoresponsive behavior of the synthesized azobenzene exhibited a photoresponse immediately after the on-off switching of blue light. The efficient cross-linking of azobenzene-bearing PEG through a click reaction allowed successful cell encapsulation in the azobenzene-bearing hydrogel. Taken together, the photoresponsive hydrogel scaffold is expected to find applications in controlling cellular behaviors in four dimensions via integrin-mediated mechanotransduction.

3D Printing of Chitosan Scaffolds and Films with Varying Roughness for Cultivation of Human Retinal Progenitor Cells.

Amalie Solberg, Natalia Robles-Anda, Eva Pasquier +4 more

Chitosan was used for three-dimensional (3D) printing of films and well-resolved scaffolds. Three different molecular weights with a comparable degree of de-N-acetylation were studied for 3D printing; inks were characterized using rheology, and the resulting two-dimensional (2D) and 3D architectures were characterized by scanning electron microscopy (SEM). Printing and fixation were optimized for retention of shape fidelity. The films and scaffolds were functionalized with a short peptide containing the integrin-binding arginylglycylaspartic acid (Arg-Gly-Asp, RGD) sequence using a postprinting grafting approach. The 2D films and 3D scaffolds prepared were studied as support materials for human retinal progenitor cells (hRPCs), and the properties of native and RGD-modified chitosan were compared as support materials for hRPCs. The adhesion and proliferation of hRPCs were studied over a period of 6 or 18 days, and Matrigel was used as a positive control. Grafting with the RGD-containing peptide generally improved the biocompatibility of the materials. When comparing films with varying surface roughness resulting from the method used for drying, the cellular response differed significantly. The best performing material was air-dried chitosan films, which resulted in the formation of axons and larger cell clusters with observable live cells after 18 days of culture time. This work demonstrates effective methods for the preparation of 3D printed architectures and the promise of these materials for cell therapies and bioengineering applications.

The emerging role of human transmembrane RGD-based counter-receptors of integrins in health and disease.

Carlos Cabañas, Elisa Rossi, Ruben A Bartolomé +4 more

Most of the canonical Arg-Gly-Asp (RGD)-containing integrin ligands are extracellular matrix proteins, such as fibronectin, vitronectin and fibrinogen, which regulate cell-ECM adhesion processes. However, during the last years, several reports have demonstrated the existence of non-canonical RGD-containing integrin ligands that are cell surface transmembrane proteins. At variance with the canonical extracellular matrix integrin ligands, the RGD-containing cell surface integrin ligands are involved in cell-cell adhesion processes and function as "integrin counter-receptors". We propose in this review grouping these transmembrane proteins, which include endoglin, cadherin-5, cadherin-6, cadherin-17, ADAM15, and L1CAM, under the newly coined acronym RGD-ICRs (RGD-containing Integrin Counter-Receptors). We present and discuss the structure of RGD-ICRs, their RGD-based interactions with integrins, the specific signaling pathways triggered in different cell types, as well as their pathophysiological involvement. It can be postulated that RGD-ICRs constitute an emerging group of non-canonical RGD-based integrin counter-receptors. In spite of being encoded by different and independent genes and involved in different pathophysiological processes, all of them appear to have undergone a strong evolutionary convergence in order to acquire the same functional capacity to bind integrins via the RGD motif. Importantly, these RGD-ICRs are also emerging as novel biomarkers and therapeutic targets, with promising clinical potential in a wide array of pathologies.

Engineered basement membrane mimetic hydrogels to study mammary epithelial morphogenesis and invasion.

Jane A Baude, Megan D Li, Sabrina M Jackson +3 more

Reconstituted basement membrane products, like Matrigel, suffer from variability and xenogenic contaminants, hindering three-dimensional cell culture models. To overcome these challenges, we developed engineered basement membranes (eBMs) using peptide-conjugated alginate hydrogels with independently tunable mechanics. Ile-Lys-Val-Ala-Val (IKVAV)-modified eBMs, with fast stress relaxation and low stiffness, supported normal mammary acinus formation. Both increased stiffness and slow relaxation were required to induce invasion in IKVAV-modified eBMs, differing from the invasive phenotype observed in Arg-Gly-Asp (RGD)-modified eBMs regardless of the mechanical properties. Mechanistic studies revealed the balance of β1 and β4 integrin signaling, hemidesmosome formation, and laminin production were influenced by eBM properties. Inhibiting focal adhesion kinase or hemidesmosome signaling disrupted acinus formation in IKVAV-modified eBMs. This defined, xenogenic-free eBM system offers a modular platform for tissue engineering and disease modeling.

Robotic manipulations of single cells using a large-volume piezoelectric micropipette with nanoliter precision.

Boglarka Kovacs, Szabolcs Novak, Igor Sallai +5 more

Single-cell manipulations are a limiting factor in single-cell omics (genomics, transcriptomics, proteomics), in vitro fertilization, and cloning. Cellular adhesion force often plays a pivotal role in various biological contexts, spanning from lower organisms to the human body. Investigating the mechanism of adhesive interactions at the individual cell level holds significant importance. We used a computer-controlled piezoelectric micropipette (NanoPick) built onto an inverted microscope, offering subnanoliter precision liquid handling in the range of 0.1-600 nanoliters with a temporal resolution of 1 millisecond. In contrast to previous pipette-based cell manipulations, in our device, phase contrast and fluorescent imaging of the microscope was not limited by the micropipette. Moreover, this compact setup efficiently enabled single-cell detection, targeting, picking, and isolation without fluidic tubes and syringes. We investigated the integrin-mediated adhesion between an RGD (Arg-Gly-Asp) motif displaying surface and the HeLa Fucci tumor cell line. Using a 70 µm inner diameter micropipette, we found that increasing the pipetting speed (voltage ramp rate applied on the piezoelectric head) improved the cell picking success rate to almost 100 %. Although the more strongly attached unmodified HeLa cells could not be picked up even at the highest flow rates. However, vibrating the fluid in the micropipette successfully detached fully flattened cells without any biochemical treatment. This vibration micropipetting method enabled the detachment of 79.9 % of the strongly adherent HeLa cells, preserving mechanical integrity for downstream omics analyses despite a loss in viability. Compared to valve-controlled systems, NanoPick demonstrated higher efficiency and precision, particularly in handling THP-1 cells. Its rigid design minimized transient delays, allowing time-dependent flow profiles and enhanced detachment at lower flow rates. Our method allows adhesion measurements on hundreds of cells and offers precise control over fluid volume and timing, suitable for manipulating adherent cells or larger objects such as organoids, spheroids, oocytes, or larvae. The introduced vibration micropipetting method could be employed for the mechanical stimulation of single cells.

De Novo Design of Integrin α5β1 Modulating Proteins to Enhance Biomaterial Properties.

Xinru Wang, Jordi Guillem-Marti, Saurav Kumar +23 more

Integrin α5β1 is crucial for cell attachment and migration in development and tissue regeneration, and α5β1 binding proteins can have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5β1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5β1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin (FN) and RGD peptides in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed FN- and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.

Kinetic Analysis of SARS-CoV-2 S1-Integrin Binding Using Live-Cell, Label-Free Optical Biosensing.

Nicolett Kanyo, Krisztina Borbely, Beatrix Peter +6 more

The SARS-CoV-2 spike (S1) protein facilitates viral entry through binding to angiotensin-converting enzyme 2 (ACE2), but it also contains an Arg-Gly-Asp (RGD) motif that may enable interactions with RGD-binding integrins on ACE2-negative cells. Here, we provide quantitative evidence for this alternative binding pathway using a live-cell, label-free resonant waveguide grating (RWG) biosensor. RWG technology allowed us to monitor real-time adhesion kinetics of live cells to RGD-displaying substrates, as well as cell adhesion to S1-coated surfaces. To characterize the strength of the integrin-S1 interaction, we determined the dissociation constant using two complementary approaches. First, we performed a live-cell competitive binding assay on RGD-displaying surfaces, where varying concentrations of soluble S1 were added to cell suspensions. Second, we recorded the adhesion kinetics of cells on S1-coated surfaces and fitted the data using a kinetic model based on coupled ordinary differential equations. By comparing the results from both methods, we estimate that approximately 33% of the S1 molecules immobilized on the Nb2O5 biosensor surface are capable of initiating integrin-mediated adhesion. These findings support the existence of an alternative integrin-dependent entry route for SARS-CoV-2 and highlight the effectiveness of label-free RWG biosensing for quantitatively probing virus-host interactions under physiologically relevant conditions without the need of the isolation of the interaction partners from the cells.

An Enzyme-Cleavable Cage on Integrin Adhesive Ligand Regulates Stem Cell Fate in An External Stimulus-Free Manner.

Shuhou Yang, Jiacheng Lei, Kaikai Zheng +8 more

The interaction between integrins and their receptors is essential for cell adhesion, acting as a critical link for cells to sense and respond to signals from the extracellular matrix (ECM). While various stimuli-responsive systems, such as electrical, optical, enzymatic, and magnetic systems, have been employed to modulate the binding of arginine-glycine-aspartic acid (RGD) to integrins, these triggers often pose risks of cellular damage. Here, the cRGD ligands are passivated by conjugating amide cages with varying electron densities on the side chain of aspartic acid in cRGD peptides in order to undergo spontaneous hydrolysis of the cages via MSC exocrine enzymes during cell adhesion. This modification allows precise control over enzyme-mediated cage degradation and cRGD activation as the varying electron densities affect the stability of the amide cages. Our approach enables controllable spatiotemporal integrin activation, thereby regulating cell spreading and differentiation while minimizing the risks associated with external stimuli.

Structural and Functional Differences of Rhodostomin and Echistatin in Integrin Recognition and Biological Implications.

Yi-Chun Chen, Chun-Hao Huang, Yao-Tsung Chang +5 more

Rhodostomin (Rho) and Echistatin (Ech) are RGD-containing disintegrins with different sizes, disulfide bond patterns, and amino acid sequences in their RGD loops and C-termini. Cell adhesion analyzes showed that Rho exhibited a 5.2-, 18.9-, 2.2-, and 1.7-fold lower inhibitory activity against integrins αvβ3, α5β1, αIIbβ3, and αvβ5 in comparison with those of Ech. In contrast, Rho exhibited an 8.8-fold higher activity than Ech in inhibiting integrin αvβ6. The swapping of Ech's RGD loop and C-terminal sequences into those of Rho cannot increase its integrins' inhibitory activities. Interestingly, the mutation of Ech into Rho's RGD loop PRGDMP sequence and C-terminal YH sequence caused an 8.2-fold higher activity in inhibiting integrin αvβ6. Structural analyzes of Rho and Ech showed that they have similar conformations in their RGD loop and different conformations in their C-terminal regions. Molecular docking found that not only the RGD loop but also the C-terminal region of Rho and Ech interacted with integrins, showing that the C-terminal region is also important for integrin recognition. The docking of Rho into integrin αvβ6 showed that the C-terminal H68 residue of Rho interacted with D129 of β6. In contrast, the docking of Ech into integrin α5β1 showed that the C-terminal H44 residue of Ech interacted with Q191 of β1. Ech exhibited 78.5- and 10.9-fold higher activities in inhibiting HUVEC proliferation and A375 melanoma cell migration than those of Rho. These findings demonstrate that the disulfide bond pattern, RGD loop, and C-terminal region of disintegrins may cause their functional differences. The functional and structural differences between Rho and Ech support their potential as scaffolds to design drugs targeting their respective integrins.

Biomimetic Dynamics of Nanoscale Groove and Ridge Topography for Stem Cell Regulation.

Hyunsik Hong, Dahee Kim, Hwapyung Jung +6 more

Native extracellular matrix exhibits multiscale groove and ridge structures that continuously change, such as collagen fibril-based nanogrooves in bone tissue, and regulate cellular responses. However, dynamic switching between groove and ridge nanostructures at the molecular level has not been demonstrated. Herein, materials capable of dynamic groove-ridge switching at tens-of-nanometers scale are developed by flexibly conjugating RGD-magnetically activatable nanoridges (MANs) to non-magnetic nanogrooves with independently tuned widths comparable to the sizes of integrin-presenting filopodia by modulating hydrophobicity in bicontinuous microemulsion, allowing for cyclic modulation of RGD accessibility and cellular adhesion. Nanogrooves with medium width restrict RGD accessibility in the "groove" state in which the RGD-MANs are buried, which is reversed by magnetically raising them to protrude and form the "ridge" state that fully exposes the RGDs. This reversibly stimulates integrin recruitment, focal adhesion complex assembly, mechanotransduction, and differentiation of stem cells in vivo. This is the first demonstration of molecular-level groove and ridge nanostructures that exhibit unprecedented switchability between groove and ridge nanostructures. Versatile tuning of the width, height, pitch, and shape of intricate nanogroove structures with remote manipulability can enlighten the understanding of molecular-scale cell-ligand interactions for stem cell engineering-based treatment of aging, injuries, and stress-related diseases.

Tunable Integrin-Ligand Coupling Strength Modulates Cellular Adaptive Mechanosensing.

Zheng Zhang, Xiaoxi Liu, Baoyong Sha +10 more

Cells sense and respond to the matrix by exerting traction force through binding of integrins to an integrin-specific ligand. Here, Arg-Gly-Asp (RGD) peptide is covalently conjugated to the double-stranded DNA (dsDNA) and stem-loop DNA (slDNA) tethers with a tension tolerance of 43pN and immobilized on a PEG substrate. Unlike dsDNA, which is ruptured under high tension, leading to the removal of RGD, slDNA remains bound even when ruptured. Our results suggest that cells adapt their adhesion state by modulating actin filament polymerization and cofilin phosphorylation, effectively balancing the talin conformation to prevent dsDNA rupture and maintain normal adhesion. This phenomenon, termed integrin-ligand coupling strength, mediated cellular adaptive mechanosensing. Furthermore, we demonstrate that positive durotaxis can shift to negative durotaxis, depending on the integrin-ligand coupling strength. This study highlights the significance of the coupling strength in cell-extracellular matrix (ECM) interactions and offers new insights into designing biomaterials with tunable adhesive properties for cell-based applications.

Charge microenvironment and bioactivity of in situ-formed PEG-RGD dual hydrogel dressings promote wound healing.

Chuanjie He, Yulin Wang, Xinyu Fang +10 more

Healing of large skin wounds involves a complex biological process with overlapping phases, facing challenges from fibroblast proliferation, immune response, and extracellular matrix (ECM) remolding. Hydrogel dressings serve as temporary barriers protecting injured tissue from exogenous infections while providing an advantageous microenvironment for cellular regeneration. However, traditionally molded hydrogels through catalyzed or triggered crosslinking into fixed size and strength prior to treatment struggle to integrate tightly with irregular wound surfaces, leading to dressing detachment and wound exposure in areas with high curvature and mobility. Here, we designed CGRGDGC peptide enantiomers, incorporating with 4 arm-PEG-maleimide, to in situ form functional and morphologically matching dual-phasic hydrogel dressing. In situ elastic hydrogel dressing forms within 10 min after applying, with a storage modulus of 1300 Pa and internal porous networks. The peptide incorporation increased the surface potential to ∼370 mV, twice that of PEG hydrogels. The bioactive L-peptide hydrogel exhibited strongest immunomodulation and skin regeneration enhancement, while the non-bioactive D-peptide hydrogel also showed significant promotion compared to the PEG hydrogel. We demonstrated that both the charge microenvironment and bioactivity of hydrogel dressing regulate the immune response and promote wound healing after skin injury. This research provides novel insights and strategies showing that non-ligand peptide sequences achieve biological functions by modulating molecular potential and that adjusting the charge microenvironment and incorporating bioactive peptides through peptide phase introduction enhance skin regeneration.

Cell/Surface Interactions and Osseointegration of Ti-6AI-4V: Effects of Laser Microgrooves, Hydroxyapatite Nanorods, and Arginyl-Glycyl-Aspartic Acid (RGD) on Ti-6Al-4V.

Precious O Etinosa, Ali A Salifu, Sarah A Osafo +3 more

This work presents the results of an experimental study of surface-modified Ti-6Al-4V designed to enhance implant integration with human fetal osteoblast (hFOB) cells. Three surface profiles-laser-grooved (LG), Hydroxyapatite (HA)-coated laser-grooved (LGH), and arginyl glycyl aspartic acid (RGD)-functionalized HA-coated laser-grooved (LGHR)-were developed and evaluated for their effects on hFOB cell attachment, spreading, proliferation, and ECM formation over a 28-day period. Cell-laden surfaces were analyzed using scanning electron and fluorescence microscopies, and cell proliferation was quantified using the Alamar Blue assay to provide additional insights. The surface characterization revealed that the LG substrate facilitated contact guidance, promoting directional cell alignment and attachment. The LGH substrate additionally created a bioactive interface by mimicking natural bone tissue, releasing calcium and phosphate ions that enhanced cell attachment and spreading. The LGHR substrate provided specific biological cues, further improving early cell attachment, accelerating proliferation, and promoting extracellular matrix (ECM) formation. Quantitative analysis confirmed that LGHR surfaces exhibited the highest cell density, areal coverage, and metabolic activity, particularly during the initial stages of culture, emphasizing the synergistic effects of HA and RGD coatings in accelerating osseointegration. This novel approach offers robust improvements in implant-tissue integration, accelerating wound healing and enhancing tissue compatibility, with promising implications for orthopedic and dental applications.

Hydrogels with multiple RGD presentations increase cell adhesion and spreading.

Abolfazl Salehi Moghaddam, Katelyn Dunne, Wendy Breyer +2 more

A key challenge in designing hydrogels for cell culture is replicating the cell-matrix interactions found in tissues. Cells use integrins to bind their local matrix and form adhesions in which integrins dynamically move on the cell membrane while applying significant forces to the local matrix. Identifying the important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands, and the ability of these ligands to support cellular forces. In this work, we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tune the viscoelasticity of the hydrogel by modulating the composition of both networks. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the arginine-glycine-aspartic acid (RGD) cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. We find that endothelial cell adhesion formation and spreading is maximized in soft gels in which adhesion ligands are present on both the covalent and non-covalent networks. The dynamic nature of adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or capable of withstanding significant forces is needed to mimic different aspects of complex cell-matrix adhesions. These results will contribute to the design of hydrogels that better recapitulate physiological cell-matrix interactions. STATEMENT OF SIGNIFICANCE: Creating artificial environments that accurately mimic how cells interact with their surrounding matrix in natural tissues remains a fundamental challenge in biomaterials science. This study introduces a dual-network hydrogel platform that independently controls mechanical properties and adhesion ligand mobility by combining stable and dynamic polymer networks. A significant body of work has shown that matrix viscoelasticity and adhesion ligand mobility are important for cell adhesion and spreading. Our work builds on this by showing that endothelial cells function optimally when they can simultaneously engage with both mobile adhesion sites and force-resistant anchoring points, independent of matrix viscoelasticity. These insights will guide the design of more physiologically relevant hydrogels for tissue engineering applications and disease modeling.

Scrambled RGD Hexameric Peptide Hydrogel Supports Efficient Self-Assembly and Cell Activity.

Karrar Al Taief, Stephanie Nemec, Isis A Middleton +2 more

The amino acid sequence is crucial in controlling peptide-based hydrogel formation, whereby changing the position of a single amino acid can significantly alter the gel's properties. Herein, we report the gelation kinetics and cell viability of scrFmoc-GFFRDG (where we have scrambled the RGD-based gel hexapeptide; Fmoc-GFFRGD). The scrambled sequence showed improved gelation properties compared to the original Fmoc-GFFRGD sequence, with scrFmoc-GFFRDG forming a gel in under 10 min, significantly faster than the 2-h gelation time, and at a concentration eight times lower than the original Fmoc-GFFRGD sequence. We also examined the combination of the two gelators in a ratio of 1:1, final concentration of 0.4% (w/v). Interestingly, the stiffness of the hybrid hydrogel was ∼3 kPa, whereas individually, neither gelator at the same concentration exceeded 0.5 kPa. The cell-adhesion motif RGD improves the ability of the peptides to promote attachment of cells due to integrin recognition. However, when fibroblasts were cultured on the hydrogels, scrFmoc-GFFRDG yielded a higher level of α-SMA expression in cells than those cultured on Fmoc-GFFRGD, suggesting a microenvironment conducive to myofibroblast transitions. This study provides a new outlook on how a well-known scrambled peptide motif (RDG) can fine-tune hydrogel assembly and cell culture applications.

Spatial regulation of mitochondrial membrane potential by α5β1 integrin engagement in collective cell migration.

Gustavo G Pacheco, Bette J Dzamba, Wakako Endo +9 more

The mechanistic links between mechanical forces and bioenergetics remain elusive. We report an increase in mitochondrial membrane potential (MMP) along the leading row of collectively migrating Xenopus laevis mesendoderm cells at sites where fibronectin-α5β1 integrin substrate traction stresses are greatest. Real-time metabolic analyses reveal α5β1 integrin-dependent increases in respiration efficiency in cells on fibronectin substrates. Elevation of metabolic activity is reduced following pharmacologic inhibition of focal adhesion kinase (FAK; also known as PTK2) signaling. Attachment of mesendoderm cells to fibronectin fragments that support differing α5β1 integrin conformational and ligand-binding affinity states, increases MMP when both the Arg-Gly-Asp (RGD) and Pro-Pro-Ser-Arg-Asn (PPSRN) synergy sites of fibronectin are engaged by the receptor. Cell stretch on deformable fibronectin substrates also results in a FAK-dependent increase in MMP. Inhibition of MMP or ATP-synthase activity slows collective cell migration velocity in vivo, further suggesting that integrin-dependent adhesion and signaling contribute to metabolic changes. These data highlight an underexplored link between extracellular matrix (ECM)-integrin adhesion and metabolic activity in embryonic cell migration. We propose that fibronectin-integrin adhesion and signaling help shape the metabolic landscape of collectively migrating cells.

Nanoscale distribution of bioactive ligands on biomaterials regulates cell mechanosensing through translocation of actin into the nucleus.

Xiaojing Liu, Man Zhang, Peng Wang +8 more

Cells respond to adhesive ligands such as arginine-glycine-aspartate (RGD) through integrins, which regulates cellular activities via influencing cytoskeleton assembly. Herein, we report that the nanoscale distribution of active ligands on biomaterials regulates cells through not only cytoplasmic tension but also nuclear tension. This is particularly related to translocation of actin into nucleus and highlighted in our interpretation of an "abnormal" phenomenon that large RGD nanospacing (>70 nm) disassembles integrin clusters, inhibits cell adhesion, but promotes osteogenic differentiation of mesenchymal stem cells. Our studies reveal that the unstable adhesion at the 150 nm RGD distance increases actin dynamics, resulting in the nuclear translocation of globular (G) actin. The compartment polymerization of more G-actins to filamentous actins in nucleus increases nuclear tension, facilitating transcription activity and releasing calcium ions from the endoplasmic reticulum. This noncanonical mechanotransduction process sheds insight into mechanotransduction pertinent to cell-material interactions.

Antheraea pernyi silk nanofibrils with inherent RGD motifs accelerate diabetic wound healing: A novel drug-free strategy to promote hemostasis, regulate immunity and improve re-epithelization.

Lian Duan, Ga Liu, Fuying Liao +7 more

The chronic inflammation and matrix metalloprotease (MMP)-induced tissue degradation significantly disrupt re-epithelization and delay the healing process of diabetic wounds. To address these issues, we produced nanofibrils from Antheraea pernyi (Ap) silk fibers via a facile and green treatment of swelling and shearing. The integrin receptors on the cytomembrane could specifically bind to the Ap nanofibrils (ApNFs) due to their inherent Arg-Gly-Asp (RGD) motifs, which activated platelets to accelerate coagulation and promoted fibroblast migration, adhesion and spreading. These degradable nanofibrils served as effective competitive substrates to reduce MMP-induced tissue degradation. ApNFs and their enzymatic hydrolysates could modulate macrophage polarization due to their RGD motifs. RNA sequencing further revealed that ApNFs treatment activated the JAK2-STAT5b and PI3K-Akt signaling pathways while suppressed the NF-κB, IL-17 and TNF signaling pathways in macrophages. The full-thickness skin wound experiments confirmed that ApNFs significantly accelerated wound healing in both diabetic and non-diabetic rats. Notably, in diabetic wound, ApNFs and their enzymatic hydrolysates polarized the accumulated M1-type macrophages into M2-type, which promoted the wound to get rid of the inflammatory stage and transition to the following proliferative stage, improving the wound healing percentage on day 14 from 74.9 % to 93.2 % by facilitating collagen deposition, angiogenesis and re-epithelization. These results demonstrate that ApNFs are promising drug-free diabetic wound dressings with favorable inherent immunoregulatory properties for biomedical translation.

The novel ECM protein SNED1 mediates cell adhesion via the RGD-binding integrins α5β1 and αvβ3.

Dharma Pally, Nandini Kapoor, Alexandra Naba

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays crucial roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by the RGD integrins α5β1 and αvβ3. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

Ligand Inter-Relation Analysis Via Graph Theory Predicts Macrophage Response.

Nayeon Kang, Jangsun Hwang, Daun Jeong +23 more

Graph theory has been widely used to quantitatively analyze complex networks of molecules, materials, and cells. Analyzing the dynamic complex structure of extracellular matrix can predict cell-material interactions but has not yet been demonstrated. In this study, graph theory-based mathematical modeling of RGD ligand graph inter-relation is demonstrated by differentially cutting off RGD-to-RGD interlinkages with flexibly conjugated magnetic nanobars (MNBs) with tunable aspect ratio. The RGD-to-RGD interlinkages are less effectively cut off by MNBs with a lower aspect ratio, which decreases the shortest path while increasing the number of instances thereof, thereby augmenting RGD nano inter-relation. This facilitates integrin recruitment of macrophages and thus actin fiber assembly and vinculin expression, which mediates pro-regenerative polarization, involving myosin II, actin polymerization, and rho-associated protein kinase. Unidirectional pre-aligning or reversibly lifting highly elongated MNBs both increase RGD nano inter-relation, which promotes host macrophage adhesion and switches their polarization from pro-inflammatory to pro-regenerative phenotype. The latter approach produces nano-spaces through which macrophages can penetrate and establish RGD links thereunder. Using graph theory, this study presents the example of mathematically modeling the functionality of extracellular-matrix-mimetic materials, which can help elucidate complex dynamics of the interactions occurring between host cells and materials via versatile geometrical nano-engineering.

Mictlan-D3: A novel medium sized RGD-Disintegrin obtained from Crotalus mictlantecuhtli venom, in vitro tested against human breast Cancer and endothelial cells.

E Rivas-Mercado, E Neri-Castro, V Zarzosa +4 more

Disintegrins are small non-enzymatic proteins present often at low concentration in the venom of viperid snakes. Isolated disintegrins are known for their lack of toxicity as well as their capacity to antagonize integrin receptors. Integrins are a major family of heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. Integrins regulate key functions in cancer pathology and also tumor development. The aim of this study consisted in the isolation and characterization of disintegrins from rattlesnake new species Crotalus mictlantecuhtli venom. A disintegrin fraction obtained by RP-HPLC and named mictlan-D3, consist in two isoforms of 7439 and 7509 Da with 72 amino acid sequence containing the RGD binding motif. Mictlan-D3 inhibited MDA-MB-231 and HMEC-1 cell adhesion to laminin (LN), fibronectin (FN) and vitronectin (VN), highest inhibition was on MDA-MB-231 cell adhesion to LN by 81 % at 1 μM. The blockade of ⍺Vβ3 integrin was evaluated by wound healing migration assay. Mictlan-D3 inhibited MDA-MB-231 cell migration by 80 % and 38 % after 24 and 72 h of incubation respectively. HMEC-1 cell migration was inhibited by 67.6 % and 27.9 % after 24 and 72 h of incubation. Additionally, mictlan-D3. This work represent the first characterization of disintegrins from the Crotalus mictlantecuhtli venom.

Importance of integrin transmembrane helical interactions for antagonistic versus agonistic ligand behavior: Consequences for medical applications.

Ute Reuning, Vincenzo Maria D'Amore, Kairbaan Hodivala-Dilke +2 more

Integrins are well-characterized receptors involved in cell adhesion and signaling. With six approved drugs, they are recognized as valuable therapeutic targets. Here, we explore potential activation mechanisms that may clarify the agonist versus antagonist behavior of integrin ligands. The reorganization of the transmembrane domain (TMD) in the integrin receptor, forming homooligomers within focal adhesions, could be key to the understanding of the agonistic properties of integrin ligands at substoichiometric concentrations. This has significant implications for medical applications. While we focus on the RGD peptide-recognizing integrin subfamily, we propose that these mechanistic insights may also apply to other integrin subtypes. For application of integrin ligands in medicine it is essential to consider this mechanism and its consequences for affinity and bioavailability.

Role of RGD-binding Integrins in ovarian cancer progression, metastasis and response to therapy.

Vipin Ranga, Tikam Chand Dakal, Pawan Kumar Maurya +3 more

Integrins are transmembrane receptors that play a crucial role in cell adhesion and signaling by connecting the extracellular environment to the intracellular cytoskeleton. After binding with specific ligands in the extracellular matrix (ECM), integrins undergo conformational changes that transmit signals across the cell membrane. The integrin-mediated bidirectional signaling triggers various cellular responses, such as changes in cell shape, migration and proliferation. Irregular integrin expression and activity are closely linked to tumor initiation, angiogenesis, cell motility, invasion, and metastasis. Thus, understanding the intricate regulatory mechanism is essential for slowing cancer progression and preventing carcinogenesis. Among the four classes of integrins, the arginine-glycine-aspartic acid (RGD)-binding integrins stand out as the most crucial integrin receptor subfamily in cancer and its metastasis. Dysregulation of almost all RGD-binding integrins promotes ECM degradation in ovarian cancer, resulting in ovarian carcinoma progression and resistance to therapy. Preclinical studies have demonstrated that targeting these integrins with therapeutic antibodies and ligands, such as RGD-containing peptides and their derivatives, can enhance the precision of these therapeutic agents in treating ovarian cancer. Therefore, the development of novel therapeutic agents is essential for treating ovarian cancer. This review mainly discusses genes and their importance across different ovarian cancer subtypes, the involvement of RGD motif-containing ECM proteins in integrin-mediated signaling in ovarian carcinoma, ongoing, completed, partially completed, and unsuccessful clinical trials of therapeutic agents, as well as existing limitations and challenges, advancements made so far, potential strategies, and directions for future research in the field. Insight Box Integrin-mediated signaling regulates cell migration, proliferation and differentiation. Dysregulated integrin expression and activity promote tumor growth and dissemination. Thus, a proper understanding of this complex regulatory mechanism is essential to delay cancer progression and prevent carcinogenesis. Notably, integrins binding to RGD motifs play an important role in tumor initiation, evolution, and metastasis. Preclinical studies have demonstrated that therapeutic agents, such as antibodies and small molecules with RGD motifs, target RGD-binding integrins and disrupt their interactions with the ECM, thereby inhibiting ovarian cancer proliferation and migration. Altogether, this review highlights the potential of RGD-binding integrins in providing new insights into the progression and metastasis of ovarian cancer and how these integrins have been utilized to develop effective treatment plans.

Magnetic Nanoactuator-Protein Fiber Coated Hydrogel Dressing for Well-Balanced Skin Wound Healing and Tissue Regeneration.

Chenlong He, Ming Yin, Han Zhou +8 more

Despite significant progress in skin wound healing, it is still a challenge to construct multifunctional bioactive dressings based on a highly aligned protein fiber coated hydrogel matrix for antifibrosis skin wound regeneration that is indistinguishable to native skin. In this study, a "dual-wheel-driven" strategy is adopted to modify the surface of methacrylated gelatin (GelMA) hydrogel with highly aligned magnetic nanocomposites-protein fiber assemblies (MPF) consisting of photothermal responsive antibacteria superparamagnetic nanocomposites-fibrinogen (Fg) complexes as the building blocks. Whole-phase healing properties of the modified hydrogel dressing, GelMA-MPF (GMPF), stem from the integration of Fg protein with RGD peptide activity decorated on the surface of the antibacterial magnetic nanoactuator, facilitating facile and reproducible dressing preparation by self-assembly and involving biochemical, morphological, and biophysical cues. Payload and substantial release of copper ions for in situ catalytic production of nitric oxide (NO) from the fiber inorganic skeleton adsorbed by Fg molecules collectively regulate the proliferation, migration, reorganization, and transdifferentiation behavior of fibroblasts and fulfill antifibrosis in the process of skin wound healing and subcutaneous appendage regeneration. In full-thickness skin lesion mouse models, the complete regeneration of skin tissue with regenerated hair follicle cells and capillary blood vessels is realized in a temporally and spatially ordered manner.

Self-assembled Arginine-Glycine-Aspartic Acid Mimic Peptide Hydrogels as Multifunctional Biomaterials for Wound Healing.

Zeba Ahmadi, Diksha Jha, Santosh Yadav +5 more

Clinical management of nonhealing ulcers requires advanced materials that can enhance wound closure rates without relying on the release of drugs or other growth factors to obviate systemic deleterious side effects. In our previous work, we synthesized an integrin-binding cell adhesive MNH2 {Fmoc-FFβAR(K)βA-NH2 consisting of an RGD mimic, [R(K)], with an amide terminus}, MOH {Fmoc-FFβAR(K)βA-OH consisting of an RGD mimic, [R(K)], with acid terminus}, and MR (Fmoc-FFβARGDβA-NH2 consisting of an RGD peptide, reference) with multifunctional activity. Here, we reported the synthesis, characterization, and performance of a reversed derivative, R-MNH2 (Fmoc-FFβA(K)RβA-NH2 consisting of an RGD mimic, [K(R)], with an amide terminus) of an antimicrobial cell adhesive peptide, MNH2. Both peptides (MNH2 and R-MNH2) were found to interact with αvβ3 integrin, as shown by docking studies; however, they differed in cell adhesive properties, hydrogel formation, and antimicrobial efficacy. Later, the wound healing ability of a series of RGD/RGD peptide mimics (MR, R-MNH2, MNH2, and MOH) was studied in a methicillin-resistant Staphylococcus aureus (MRSA)-infected Balb/c mouse model. All studied peptides showed cell adhesion and wound healing properties; however, only the amide-terminal RGD peptide mimic, MNH2, and its reversed derivative, R-MNH2, showed antimicrobial activity in both in vitro and in vivo studies. Of these, MNH2 showed the highest integrin-mediated spreading, migration, and proliferation of dermal cells in vitro as well as in vivo. Therefore, the MNH2 peptide mimic represents a paradigm shift in the development of dermoconductive strategies to treat chronic wounds.