Cell Adhesion Ligands Research Articles

Tuning pro-survival effects of human induced pluripotent stem cell-derived exosomes using elastin-like polypeptides

Exosome-based regenerative therapies are potentially easier to manufacture and safer to apply compared to cell-based therapies. However, many questions remain about how to bio-manufacture reproducible and potent exosomes using animal-free reagents. Here we evaluate the hypothesis that designer biomaterial substrates can be used to alter the potency of exosomes secreted by human induced pluripotent stem cells (iPSCs). Two animal-free designer matrices were fabricated based on recombinant elastin-like polypeptides (ELPs): one including a cell-adhesive RGD ligand and a second with a non-adhesive RDG peptide. While iPSCs cultured on these two substrates and Matrigel-coated controls had similar levels of proliferation, the RDG-ELP substrate significantly increased protein expression of stemness markers OCT4 and SOX2 and suppressed spontaneous differentiation compared to those on RGD-ELP. The pro-survival potency of iPSC-derived exosomes was evaluated using three distinct stress tests: serum starvation in murine fibroblasts, hypoxia in human endothelial cells, and hyperosmolarity in canine kidney cells. In all three cases, exosomes produced by iPSCs grown on RDG-ELP substrates had similar pro-survival effects to those produced using iPSCs grown on Matrigel, while use of RGD-ELP substrates led to significantly reduced exosome potency. These data demonstrate that recombinant substrates can be designed for the robust bio-manufacturing of iPSC-derived, pro-survival exosomes.

Directed Conformational Switching of a Zinc Finger Analogue Regulates the Mechanosensing and Differentiation of Stem Cells.

The dynamic conformational changes in the secondary structures of proteins are essential to their functions and can regulate diverse cellular events. Herein we report the design of a synthetic polymer-based secondary structure analogue of a zinc finger (ZnF) by introducing a zinc coordination motif to overcome the free energy barrier predicted by theoretical calculations and fold-free polymer chains. The conformational switching between unfolded and folded state of the ZnF analogue can be triggered in situ to drastically manipulate the accessibility of conjugated cell adhesive ligands to the cell membrane receptors, thereby effectively controlling the adhesion, spreading, mechanosensing, and differentiation of stem cells. We believe that emulating the dynamic secondary structures of proteins via rational design of a folded synthetic polymer-cation complex is a promising strategy for developing bioactive materials to mediate desired cellular functions.

Directed Conformational Switching of a Zinc Finger Analogue Regulates the Mechanosensing and Differentiation of Stem Cells

The dynamic conformational changes in the secondary structures of proteins are essential to their functions and can regulate diverse cellular events. Herein we report the design of a synthetic polymer-based secondary structure analogue of a zinc finger (ZnF) by introducing a zinc coordination motif to overcome the free energy barrier predicted by theoretical calculations and fold-free polymer chains. The conformational switching between unfolded and folded state of the ZnF analogue can be triggered in situ to drastically manipulate the accessibility of conjugated cell adhesive ligands to the cell membrane receptors, thereby effectively controlling the adhesion, spreading, mechanosensing, and differentiation of stem cells. We believe that emulating the dynamic secondary structures of proteins via rational design of a folded synthetic polymer–cation complex is a promising strategy for developing bioactive materials to mediate desired cellular functions.

Application of Bio-Active Elastin-like Polypeptide on Regulation of Human Mesenchymal Stem Cell Behavior

Regenerative medicine using stem cells offers promising strategies for treating a variety of degenerative diseases. Regulation of stem cell behavior and rejuvenate senescence are required for stem cells to be clinically effective. The extracellular matrix (ECM) components have a significant impact on the stem cell’s function and fate mimicking the local environment to maintain cells or generate a distinct phenotype. Here, human elastin-like polypeptide-based ECM-mimic biopolymer was designed by incorporating various cell-adhesion ligands, such as RGD and YIGSR. The significant effects of bioactive fusion ELPs named R-ELP, Y-ELP, and RY-ELP were analyzed for human bone-marrow-derived stem cell adhesion, proliferation, maintenance of stemness properties, and differentiation. Multivalent presentation of variable cell-adhesive ligands on RY-ELP polymers indeed promote efficient cell attachment and proliferation of human fibroblast cells dose-dependently. Similarly, surface modified with RY-ELP promoted strong mesenchymal stem cell (MSCs) attachment with greater focal adhesion (FA) complex formation at 6 h post-incubation. The rate of cell proliferation, migration, population doubling time, and collagen I deposition were significantly enhanced in the presence of RY-ELP compared with other fusion ELPs. Together, the expression of multipotent markers and differentiation capacity of MSCs remained unaffected, clearly demonstrating that stemness properties of MSCs were well preserved when cultured on a RY-ELP-modified surface. Hence, bioactive RY-ELP offers an anchorage support system and effectively induces stimulatory response to support stem cell proliferation.

Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis.

Aim: To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis. Materials & methods: Previously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acidwere used as cell culture substrates to control cell adhesion at the nanoscale. In vitro chondrogenesis of mesenchymal stem cells was conductedon the nanopatterns. Cohesion and GJIC were evaluatedin cell condensates. Results: Mechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70nm, thus providing an onset for cell signaling. Conclusion: Cell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.

DNA-Based Daisy Chain Rotaxane Nanocomposite Hydrogels as Dual-Programmable Dynamic Scaffolds for Stem Cell Adhesion.

Interlocked DNA nanostructures perform programmable movements in nanoscales such as sliding, contraction, and expansion. However, utilizing nanoscaled interlocked movements to regulate the functions of larger length scaled matrix and developing their applications has not yet been reported. Herein we describe the assembly of DNA-based daisy chain rotaxane nanostructure (DNA-DCR) composed of two hollow DNA nanostructures as macrocycles, two interlocked axles and two triangular prism-shaped DNA structures as stoppers, in which three mechanical states─fixed extended state (FES), sliding state (SS), and fixed contracted state (FCS)─are characterized by using toehold-mediated strand displacement reaction (SDR). The DNA-DCRs are further used as nanocomposites and introduced into hydrogel matrix to produce interlocked hydrogels, which shows modulable stiffness by elongating the interlocked axles to regulate the hydrogel swelling with hybridization chain reaction (HCR) treatment. Then the DCR-hydrogels are employed as dynamic biointerfaces for human mesenchymal stem cells (hMSCs) adhesion studies. First, hMSCs showed lower cell density on bare DCR-hydrogel treated with HCR-initiated swelling for stiffness decreasing. Second, the cell adhesion ligand (RGD) modified DNA-DCRs are constructed for hydrogel functionalization. DCR(RGD) hydrogel endows the mobility of RGDs by switching the mechanical states of DNA-DCR. HMSCs showed increased cell density on DCRSS(RGD) hydrogel than on DCRFCS(RGD) hydrogel. Therefore, our DNA-DCR nanocomposite hydrogel exhibit dual-programmable performances including swelling adjustment and offering sliding for incorporated ligands, which can be both utilized as dynamic scaffolds for regulating the stem cell adhesion. The dual-programmable cross-scale regulation from interlocked DNA nanostructures to hydrogel matrix was achieved, demonstrating a new pathway of DNA-based materials.

Manipulating Nanoparticle Aggregates Regulates Receptor-Ligand Binding in Macrophages.

The receptor-ligand interactions in cells are dynamically regulated by modulation of the ligand accessibility. In this study, we utilize size-tunable magnetic nanoparticle aggregates ordered at both nanometer and atomic scales. We flexibly anchor magnetic nanoparticle aggregates of tunable sizes over the cell-adhesive RGD ligand (Arg-Gly-Asp)-active material surface while maintaining the density of dispersed ligands accessible to macrophages at constant. Lowering the accessible ligand dispersity by increasing the aggregate size at constant accessible ligand density facilitates the binding of integrin receptors to the accessible ligands, which promotes the adhesion of macrophages. In high ligand dispersity, distant magnetic manipulation to lift the aggregates (which increases ligand accessibility) stimulates the binding of integrin receptors to the accessible ligands available under the aggregates to augment macrophage adhesion-mediated pro-healing polarization both in vitro and in vivo. In low ligand dispersity, distant control to drop the aggregates (which decreases ligand accessibility) repels integrin receptors away from the aggregates, thereby suppressing integrin receptor-ligand binding and macrophage adhesion, which promotes inflammatory polarization. Here, we present "accessible ligand dispersity" as a novel fundamental parameter that regulates receptor-ligand binding, which can be reversibly manipulated by increasing and decreasing the ligand accessibility. Limitless tuning of nanoparticle aggregate dimensions and morphology can offer further insight into the regulation of receptor-ligand binding in host cells.

3D Bioprinting of Multifunctional Dynamic Nanocomposite Bioinks Incorporating Cu-Doped Mesoporous Bioactive Glass Nanoparticles for Bone Tissue Engineering.

Bioprinting has seen significant progress in recent years for the fabrication of bionic tissues with high complexity. However, it remains challenging to develop cell-laden bioinks exhibiting superior physiochemical properties and bio-functionality. In this study, a multifunctional nanocomposite bioink is developed based on amine-functionalized copper (Cu)-doped mesoporous bioactive glass nanoparticles (ACuMBGNs) and a hydrogel formulation relying on dynamic covalent chemistry composed of alginate dialdehyde (oxidized alginate) and gelatin, with favorable rheological properties, improved shape fidelity, and structural stability for extrusion-based bioprinting. The reversible dynamic microenvironment in combination with the impact of cell-adhesive ligands introduced by aminated particles enables the rapid spreading (within 3 days) and high survival (>90%) of embedded human osteosarcoma cells and immortalized mouse bone marrow-derived stroma cells. Osteogenic differentiation of primary mouse bone marrow stromal stem cells (BMSCs) and angiogenesis are promoted in the bioprinted alginate dialdehyde-gelatin (ADA-GEL or AG)-ACuMBGN scaffolds without additional growth factors in vitro, which is likely due to ion stimulation from the incorporated nanoparticles and possibly due to cell mechanosensing in the dynamic matrix. In conclusion, it is envisioned that these nanocomposite bioinks can serve as promising platforms for bioprinting complex 3D matrix environments providing superior physiochemical and biological performance for bone tissue engineering.

Facile and Versatile Method for Micropatterning Poly(acrylamide) Hydrogels Using Photocleavable Comonomers.

We here present a micropatterning strategy to introduce small molecules and ligands on patterns of arbitrary shapes on the surface of poly(acrylamide)-based hydrogels. The main advantages of the presented approach are the ease of use, the lack of need to prefabricate photomasks, the use of mild UV light and biocompatible bioconjugation chemistries, and the capacity to pattern low-molecular-weight ligands, such as peptides, peptidomimetics, or DNA fragments. To achieve the above, a monomer containing a caged amine (NVOC group) was co-polymerized in the hydrogel network; upon UV light illumination using a commercially available setup, primary amines were locally deprotected and served as reactive groups for further functionalization. Cell patterning on various cell adhesive ligands was demonstrated, with cells responding to a combination of pattern shape and substrate elasticity. The approach is compatible with standard traction force microscopy (TFM) experimentation and can further be extended to reference-free TFM.

Integrating Soft Hydrogel with Nanostructures Reinforces Stem Cell Adhesion and Differentiation

Biophysical cues can regulate stem cell behaviours and have been considered as critical parameters of synthetic biomaterials for tissue engineering. In particular, hydrogels have been utilized as promising biomimetic and biocompatible materials to emulate the microenvironment. Therefore, well-defined mechanical properties of a hydrogel are important to direct desirable phenotypes of cells. Yet, limited research pays attention to engineering soft hydrogel with improved cell adhesive property, which is crucial for stem cell differentiation. Herein, we introduce silica nanoparticles (SiO2 NPs) onto the surface of methacrylated hyaluronic (MeHA) hydrogel to manipulate the presentation of cell adhesive ligands (RGD) clusters, while remaining similar bulk mechanical properties (2.79 ± 0.31 kPa) to that of MeHA hydrogel (3.08 ± 0.68 kPa). RGD peptides are either randomly decorated in the MeHA hydrogel network or on the immobilized SiO2 NPs (forming MeHA–SiO2). Our results showed that human mesenchymal stem cells exhibited a ~1.3-fold increase in the percentage of initial cell attachment, a ~2-fold increase in cell spreading area, and enhanced expressions of early-stage osteogenic markers (RUNX2 and alkaline phosphatase) for cells undergoing osteogenic differentiation with the osteogenic medium on MeHA–SiO2 hydrogel, compared to those cultured on MeHA hydrogel. Importantly, the cells cultivated on MeHA–SiO2 expressed a ~5-fold increase in nuclear localization ratio of the yes-associated protein, which is known to be mechanosensory in stem cells, compared to the cells cultured on MeHA hydrogel, thereby promoting osteogenic differentiation of stem cells. These findings demonstrate the potential use of nanomaterials into a soft polymeric matrix for enhanced cell adhesion and provide valuable guidance for the rational design of biomaterials for implantation.

Immunohistochemical evaluation of forkhead box A1 and EphA5 markers in serous ovarian carcinomas, and their impact on the clinical outcome of patients.

Ovarian cancer is the most lethal gynaecological neoplasm in females. In ovarian cancer, forkhead box A1 (FOXA1) aids transcription of YAP-associated protein mediated by the cyclic adenosine monophosphate response element-binding protein. As aresult, cellular proliferation and migration increased. The roles of erythropoietin-producing human hepatocellular carcinoma cell (Eph) receptors and ephrin ligands in cell adhesion, migration, cell proliferation regulation in various cancers, and angiogenesis are well characterized. This study included formalin-fixed, paraffin-embedded tissue specimens from 41 patients with ovarian serous cystadenocarcinoma, including both low- and high-grade tumours. For each case, aparaffin block with tumour tissue was chosen for an immunohistochemical procedure using primary antibodies against EphA5 and FOXA1. By the end of 2017, patients finished their chemotherapy and were followed for the next 3 years. Positive FOXA1 and EphA5 results were presented in 68.3% and 39% of patients, respectively. Astatistically significant correlation was detected between FOXA1 expression and each of CA-125 level, tumour stage, tumour grade, and the presence of lymph node metastasis. In our work, the overall survival was positively correlated with EphA5 expression and inversely correlated to FOXA1 immunoreactivity. The estimated disease-free survival (DFS) and EphA5 immunoreactivity had asignificant positive association, whereas DFS and FOXA1 protein expression had asignificant inverse link. FOXA1 and EphA5 expression play arole in ovarian cancer progression and prognosis prediction.

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

Probing Allosteric Modulation of Membrane Receptor in the Native State by Data Mining-Integrated Tracking Microscopy

3D bioprinting of molecularly engineered PEG-based hydrogels utilizing gelatin fragments

Three-dimensional (3D) bioprinting is an additive manufacturing process in which the combination of biomaterials and living cells, referred to as a bioink, is deposited layer-by-layer to form biologically active 3D tissue constructs. Recent advancements in the field show that the success of this technology requires the development of novel biomaterials or the improvement of existing bioinks. Polyethylene glycol (PEG) is one of the well-known synthetic biomaterials and has been commonly used as a photocrosslinkable bioink for bioprinting; however, other types of cell-friendly crosslinking mechanisms to form PEG hydrogels need to be explored for bioprinting and tissue engineering. In this work, we proposed micro-capillary based bioprinting of a novel molecularly engineered PEG-based bioink that transiently incorporates low molecular weight gelatin (LMWG) fragments. The rheological properties and release profile of the LMWG fragments were characterized, and their presence during hydrogel formation had no effect on the swelling ratio or sol fraction when compared to PEG hydrogels formed without the LMWG fragments. For bioprinting, PEG was first functionalized with cell-adhesive RGD ligands and was then crosslinked using protease-sensitive peptides via a Michael-type addition reaction inside the micro-capillary. The printability was assessed by the analysis of extrudability, shape fidelity, and printing accuracy of the hydrogel filaments after the optimization of the gelation conditions of the PEG-based bioink. The LMWG fragments supplemented into the bioink allowed the extrusion of smooth and uniform cylindrical strands of the hydrogel and improved shape fidelity and printing accuracy. Encapsulated cells in both bioprinted and non-bioprinted PEG-based hydrogels showed high viability and continued to proliferate over time in culture with a well-defined cell morphology depending on the presence of the cell adhesive peptide RGD. The presented micro-capillary based bioprinting process for a novel PEG-based bioink can be promising to construct complex 3D structures with micro-scale range and spatiotemporal variations without using any cytotoxic photoinitiator, UV light, or polymer support.

Enhanced mechanosensing of cells in synthetic 3D matrix with controlled biophysical dynamics

3D culture of cells in designer biomaterial matrices provides a biomimetic cellular microenvironment and can yield critical insights into cellular behaviours not available from conventional 2D cultures. Hydrogels with dynamic properties, achieved by incorporating either degradable structural components or reversible dynamic crosslinks, enable efficient cell adaptation of the matrix and support associated cellular functions. Herein we demonstrate that given similar equilibrium binding constants, hydrogels containing dynamic crosslinks with a large dissociation rate constant enable cell force-induced network reorganization, which results in rapid stellate spreading, assembly, mechanosensing, and differentiation of encapsulated stem cells when compared to similar hydrogels containing dynamic crosslinks with a low dissociation rate constant. Furthermore, the static and precise conjugation of cell adhesive ligands to the hydrogel subnetwork connected by such fast-dissociating crosslinks is also required for ultra-rapid stellate spreading (within 18 h post-encapsulation) and enhanced mechanosensing of stem cells in 3D. This work reveals the correlation between microscopic cell behaviours and the molecular level binding kinetics in hydrogel networks. Our findings provide valuable guidance to the design and evaluation of supramolecular biomaterials with cell-adaptable properties for studying cells in 3D cultures.

Laminin β2 variants associated with isolated nephropathy that impact matrix regulation.

Mutations in LAMB2, encoding laminin β2, cause Pierson syndrome and occasionally milder nephropathy without extrarenal abnormalities. The most deleterious missense mutations that have been identified affect primarily the N-terminus of laminin β2. On the other hand, those associated with isolated nephropathy are distributed across the entire molecule, and variants in the β2 LEa-LF-LEb domains are exclusively found in cases with isolated nephropathy. Here we report the clinical features of mild isolated nephropathy associated with 3 LAMB2 variants in the LEa-LF-LEb domains (p.R469Q, p.G699R, and p.R1078C) and their biochemical characterization. Although Pierson syndrome missense mutations often inhibit laminin β2 secretion, the 3 recombinant variants were secreted as efficiently as WT. However, the β2 variants lost pH dependency for heparin binding, resulting in aberrant binding under physiologic conditions. This suggests that the binding of laminin β2 to negatively charged molecules is involved in glomerular basement membrane (GBM) permselectivity. Moreover, the excessive binding of the β2 variants to other laminins appears to lead to their increased deposition in the GBM. Laminin β2 also serves as a potentially novel cell-adhesive ligand for integrin α4β1. Our findings define biochemical functions of laminin β2 variants influencing glomerular filtration that may underlie the pathogenesis of isolated nephropathy caused by LAMB2 abnormalities.

Biomimetic cell-adhesive ligand-functionalized peptide composite hydrogels maintain stemness of human amniotic mesenchymal stem cells.

In vivo, stem cells reside in a three-dimensional (3D) extracellular microenvironment in which complicated biophysical and biochemical factors regulate their behaviors. Biomimicking of the stem cell−matrix interactions is an ideal approach for controlling the stem cell fate. This study investigates the effects of the incorporation of cell-adhesive ligands in 3D self-assembling peptide hydrogels to modulate stem cell survival, proliferation, maintenance of stemness, and osteogenic differentiation. The results show that the composite hydrogels were non-cytotoxic and effective for maintaining human amniotic mesenchymal stem cell (hAMSC) survival, proliferation and phenotypic characterization. The expression levels of pluripotent markers were also upregulated in the composite hydrogels. Under inductive media conditions, mineral deposition and mRNA expression levels of osteogenic genes of hAMSCs were enhanced. The increasing expression of integrin α- and β-subunits for hAMSCs indicates that the ligand−integrin interactions may modulate the cell fate for hAMSCs in composite hydrogels.

Photodegradable Polyacrylamide Gels for Dynamic Control of Cell Functions.

Cross-linked polyacrylamide hydrogels are commonly used in biotechnology and cell culture applications due to advantageous properties, such as the precise control of material stiffness and the attachment of cell adhesive ligands. However, the chemical and physical properties of polyacrylamide gels cannot be altered once fabricated. Here, we develop a photodegradable polyacrylamide gel system that allows for a dynamic control of polyacrylamide gel stiffness with exposure to light. Photodegradable polyacrylamide hydrogel networks are produced by copolymerizing acrylamide and a photocleavable ortho-nitrobenzyl (o-NB) bis-acrylate cross-linker. When the hydrogels are exposed to light, the o-NB cross-links cleave and the stiffness of the photodegradable polyacrylamide gels decreases. Further examination of the effect of dynamic stiffness changes on cell behavior reveals that in situ softening of the culture substrate leads to changes in cell behavior that are not observed when cells are cultured on presoftened gels, indicating that both dynamic and static mechanical environments influence cell fate. Notably, we observe significant changes in nuclear localization of YAP and cytoskeletal organization after in situ softening; these changes further depend on the type and concentration of cell adhesive proteins attached to the gel surface. By incorporating the simplicity and well-established protocols of standard polyacrylamide gel fabrication with the dynamic control of photodegradable systems, we can enhance the capability of polyacrylamide gels, thereby enabling cell biologists and engineers to study more complex cellular behaviors that were previously inaccessible using regular polyacrylamide gels.

Thymosin β4 released from functionalized self-assembling peptide activates epicardium and enhances repair of infarcted myocardium.

The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin β4 (Tβ4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium.Methods: The functionalized SAP was constituted with self-assembling motif, Tβ4-binding site, and cell adhesive ligand. Myocardial infarction (MI) models of the transgenic mice were established by ligation of the left anterior descending coronary artery. At one week after intramyocardial injection of Tβ4-conjugated SAP, the activation of the epicardium was assessed. At four weeks after implantation, the migration and differentiation of epicardium-derived cells (EPDCs) as well as angiogenesis, lymphangiogenesis and myocardial regeneration were examined.Results: We found that the designer RADA-RPR bound Tβ4 and adhered to EPDCs and that Tβ4 released from the functionalized SAP could effectively activate the epicardium and induce EPDCs to differentiate towards cardiovascular cells as well as lymphatic endothelial cells. Moreover, SAP-released Tβ4 (SAP-Tβ4) promoted proliferation of cardiomyocytes. Furthermore, angiogenesis, lymphangiogenesis and myocardial regeneration were enhanced in the MI models at 4 weeks after delivery of SAP-Tβ4 along with attenuation of adverse myocardial remodeling and significantly improved cardiac function.Conclusions: These results demonstrate that sustained release of Tβ4 from the functionalized SAP can activate the epicardium and effectively enhance the repair of infarcted myocardium. We believe the delivery of SAP-Tβ4 may be a promising strategy for MI therapy.

Structural, mechanical, and biological characterization of hierarchical nanofibrous Fmoc-phenylalanine-valine hydrogels for 3D culture of differentiated and mesenchymal stem cells.

Fmoc-dipeptides are a class of short aromatic peptides featuring eminent supramolecular self-assembly, which is due to the aromaticity of the Fmoc group, which improves the association of peptide building blocks. This study aimed to introduce a new dipeptide hydrogel scaffold, Fmoc-phenylalanine-valine (Fmoc-FV), for 3D culture of various cells. Peptide hydrogel scaffolds were prepared by the pH-titration method in various concentrations and temperatures, and characterized by spectroscopic methods, including circular dichroism, attenuated total reflection FT-IR and fluorimetry. Mechanical behaviors such as thixotropy and temperature-sensitivity were investigated by oscillatory rheology. The Fmoc-FV hydrogels were then applied in 3D-culture of WJ-MSCs (mesenchymal stem cells), HUVECs (normal endothelial cells), and MDA-MB231 (tumor cell line) by live-dead fluorescence microscopy and Alamar blue viability assay experiments. The results confirmed that the β-sheet structure is principally interlocked by π-π stacking of the Fmoc groups and entangled nanofibrous morphologies as revealed by FE-SEM. Fmoc-FV self-assembly in physiologic conditions resulted in a thermo-sensitive and shear-thinning hydrogel. Notably, the Fmoc-FV hydrogel exhibited cell type-dependent biological activity, so higher cell proliferation was attained in HUVEC or MDA-MB231 cells than WJ-MSCs, indicating a possible need for incorporating cell-adhesion ligands in the Fmoc-FV hydrogel matrix. Therefore, the structural and biological properties of the Fmoc-dipeptide hydrogels are inter-related and can affect their applications in 3D cell culture and regenerative medicine.

Self-Strengthening Adhesive Force Promotes Cell Mechanotransduction.

The extracellular matrix (ECM) undergoes dynamic remodeling and progressive stiffening during tissue regeneration and disease progression. However, most of the artificial ECMs and in vitro disease models are mechanically static. Here, a self-strengthening polymer coating mimicking the dynamic nature of native ECM is designed to study the cellular response to dynamic biophysical cues and promote cell mechanical sensitive response. Spiropyran (SP) is utilized as dynamic anchor group to regulate the strength of cell adhesive peptide ligands. Benefiting from spontaneous thermal merocyanine-to-spiropyran (MC-SP) isomerization, the resulting self-responsive coating displays dynamic self-strengthening of interfacial interactions. Comparing with the static and all of the previous dynamic artificial ECMs, cells on this self-responsive surface remodel the weakly bonded MC-based coatings to activate α5β1 integrin and Rac signaling in the early adhesion stage. The subsequent MC-to-SP conversion strengthens the ligand-integrin interaction to further activate αvβ3 integrin and RhoA/ROCK signaling in the latter stage. This sequential process enhances cellular mechanotransduction as well as the osteogenic differentiation of mesenchymal stem cells (MSCs). It is worth emphasizing that the self-strengthening occurs spontaneously in the absence of any stimulus, making it especially useful for implanted scaffolds in regenerative medicine.