Nanoengineering Hybrid Supramolecular Multilayered Biomaterials Using Polysaccharides and Self‐Assembling Peptide Amphiphiles

Developing complex supramolecular biomaterials through highly dynamic and reversible noncovalent interactions has attracted great attention from the scientific community aiming key biomedical and biotechnological applications, including tissue engineering, regenerative medicine, or drug delivery. In this study, the authors report the fabrication of hybrid supramolecular multilayered biomaterials, comprising high‐molecular‐weight biopolymers and oppositely charged low‐molecular‐weight peptide amphiphiles (PAs), through combination of self‐assembly and electrostatically driven layer‐by‐layer (LbL) assembly approach. Alginate, an anionic polysaccharide, is used to trigger the self‐assembling capability of positively charged PA and formation of 1D nanofiber networks. The LbL technology is further used to fabricate supramolecular multilayered biomaterials by repeating the alternate deposition of both molecules. The fabrication process is monitored by quartz crystal microbalance, revealing that both materials can be successfully combined to conceive stable supramolecular systems. The morphological properties of the systems are studied by advanced microscopy techniques, revealing the nanostructured dimensions and 1D nanofibrous network of the assembly formed by the two molecules. Enhanced C2C12 cell adhesion, proliferation, and differentiation are observed on nanostructures having PA as outermost layer. Such supramolecular biomaterials demonstrate to be innovative matrices for cell culture and hold great potential to be used in the near future as promising biomimetic supramolecular nanoplatforms for practical applications.     

Stimuli‐Responsive Thin Coatings Using Elastin‐Like Polymers for Biomedical Applications

Smart thin coatings using a recombinant elastin‐like polymer (ELP) containing the cell attachment sequence arginine–glycine–(aspartic acid) (RGD) are fabricated for the first time through simple deposition of the ELP dissolved in aqueous‐based solutions. The biopolymer is produced and characterized using electrophoresis and mass spectroscopy. The temperature and pH responsiveness are assessed by aggregate size measurements and differential scanning calorimetry. The deposition of the studied ELP onto chitosan is followed in situ with a quartz‐crystal microbalance with dissipation monitoring (QCM‐D). Contact angle measurements are performed at room temperature and at 50 °C, showing reversible changes from a moderate hydrophobic behavior to an extremely wettable surface. AFM analysis performed at room temperature reveals a smooth surface and no organized structure. At 50 °C, the surface presents spherical nanometer‐sized structures of collapsed biopolymer chains. Such results suggest that the ELP chains, when collapsed, aggregate into micelle‐like structures at the surface of the substrate, increasing its water affinity. Cell adhesion tests on the developed coatings are conducted using a SaOS‐2 cell line. Enhanced cell adhesion could be observed in the H‐RGD6‐coated surfaces, as compared with the original chitosan monolayer. An intermediate behavior is found in chitosan coated with the corresponding ELP without the RGD sequence. Therefore, the developed films have great potential as biomimetic coatings of biomaterials for different biomedical applications, including tissue engineering and controlled delivery of bioactive agents. Their thermo‐responsive behavior can also be exploited for tunable cell adhesion and controlled protein adsorption.     

A Novel Hydrogel Surface Grafted With Dual Functional Peptides for Sustaining Long‐Term Self‐Renewal of Human Induced Pluripotent Stem Cells and Manipulating Their Osteoblastic Maturation

Realizing the clinical potential of human induced pluripotent stem cells (hiPSCs) in bone regenerative medicine requires the development of safe and chemically defined biomaterials for expansion of hiPSCs followed by directing their lineage commitment to osteoblasts. In this study, novel multipurpose peptide‐presenting hydrogel surfaces are prepared on common tissue culture plates via carboxymethyl chitosan grafting and subsequent immobilization of two functional peptides allowing for in vitro feeder‐free culture, long‐term self‐renewal, and osteogenic induction of hiPSCs. After vitronectin (VN) peptide modification, the engineered surfaces facilitate adhesion, proliferation, colony formation, and the maintenance of pluripotency of hiPSCs up to passage 10 under fully defined conditions without Matrigel or protein coating. Further, this synthetic niche exhibits an appealing regulatory effect on the osteogenic conversion of hiPSCs to osteoblastic phenotype without an embryoid body formation step by co‐decoration of different ratios of VN and bone‐forming peptide. Such a well‐defined, xeno‐free 2D engineered microenvironment not only helps to accelerate the clinical development of hiPSCs, but also provides a safe and robust platform for the generation of osteoblast‐like cells or bone‐like tissues at different maturation levels. Thus, the strategy may hold great potential for application in cell therapy and bone tissue engineering.     

Manipulation of Stem Cells Fates: The Master and Multifaceted Roles of Biophysical Cues of Biomaterials

Owing to their self-renewal and differentiation ability, stem cells are conducive for repairing injured tissues, making them a promising source of seed cells for tissue engineering. The extracellular microenvironment (ECM) is under dynamic mechanical control, which is closely related to stem cell behaviors. During the design and fabrication of biomaterials for regenerative medicine, the physiochemical properties of the natural ECM should be closely mimicked, which can reinforce stem cell lineage choice and tissue engineering. By reproducing the biophysical stimulations that stem cells may experience in vivo, many studies have highlighted the key role of biophysical cues in regulation of cell fate. Optimization of biophysical factors leads to desirable stem cell functions, which can maximize the effectiveness of regenerative treatment. In this review, the main biophysical cues of biomaterials, including stiffness, topography, mechanical force, and external physical fields are summarized, and their individual and synergistic influence on stem cell behavior is discussed. Subsequently, the current progress in tissue regeneration using biomaterials is presented, which directs the design and fabrication of functional biomaterial. The mechanisms via which biophysical cues activate cellular responses are also analyzed. Finally, the challenges in basic research as well as for clinical translation in this field are discussed.     

Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients

The profound effects that nanoscale surface topography exerts on cell behavior are highly relevant to the development of advanced biomaterials and to advances in tissue engineering and regenerative medicine. Here, an asymmetric anodization procedure is used to produce n‐type porous silicon (pSi) gradients with pore sizes ranging from tens to hundreds of nanometers in diameter and changes in the ridge nanoroughness from a few to tens nanometers. Rat mesenchymal stem cells (rMSCs) adhere poorly at the regions with small pore size but high ridge roughness. Cell adhesion is increased gradually towards the large pore size but low ridge roughness end of the pSi gradients. Surface topography influences cell differentiation, but not cell proliferation. Osteogenesis of rMSCs is enhanced by porous topography with a ridge roughness lower than 10 nm, while adipogenesis of rMSCs is enhanced on the entire pSi gradient compared with flat Si substrates. The results demonstrate that the gradient format allows in‐depth screening of surface parameters that are important for the control of mammalian cell behavior, thereby advancing the development of new and improved biomaterials for orthopaedic and tissue engineering applications.     

Length‐Scale Mediated Differential Adhesion of Mammalian Cells and Microbes

Surfaces of implantable biomedical devices are increasingly engineered to promote their interactions with tissue. However, surfaces that stimulate desirable mammalian cell adhesion, spreading, and proliferation also enable microbial colonization. The biomaterials‐associated infection that can result is now a critical clinical problem. We have identified an important mechanism to create a surface that can simultaneously promote healing while reducing the probability of infection. Surfaces are created with submicrometer‐sized, non‐adhesive microgels patterned on an otherwise cell‐adhesive surface. Quantitative force measurements between a staphylococcus and a patterned surface show that the adhesion strength decreases significantly at inter‐gel spacings comparable to bacterial dimensions. Time‐resolved flow‐chamber measurements show that the microbial deposition rate dramatically decreases at these same spacings. Importantly, the adhesion and spreading of osteoblast‐like cells is preserved despite the sub‐cellular non‐adhesive surface features. Since such length‐scale‐mediated differential interactions do not rely on antibiotics, this mechanism can be particularly significant in mitigating biomaterials‐associated infection by antibiotic‐resistant bacteria such as MRSA.     

Hierarchical Intrafibrillar Nanocarbonated Apatite Assembly Improves the Nanomechanics and Cytocompatibility of Mineralized Collagen

Nanoscale replication of the hierarchical organization of minerals in biogenic mineralized tissues is believed to contribute to the better mechanical properties of biomimetic collagen scaffolds. Here, an intrafibrillar nanocarbonated apatite assembly is reported, which has a bone‐like hierarchy, and which improves the mechanical and biological properties of the collagen matrix derived from fibril‐apatite aggregates. A modified biomimetic approach is used, which based on the combination of poly(acrylic acid) as sequestration and sodium tripolyphosphate as templating matrix‐protein analogs. With this modified dual‐analog‐based biomimetic approach, the hierarchical association between collagen and the mineral phase is discerned at the molecular and nanoscale levels during the process of intrafibrillar collagen mineralization. It is demonstrated by nanomechanical testing, that intrafibrillarly mineralized collagen features a significantly increased Young's modulus of 13.7 ± 2.6 GPa, compared with pure collagen (2.2 ± 1.7 GPa) and extrafibrillarly‐mineralized collagen (7.1 ± 1.9 GPa). Furthermore, the hierarchy of the nanocarbonated apatite assembly within the collagen fibril is critical to the collagen matrix's ability to confer key biological properties, specifically cell proliferation, differentiation, focal adhesion, and cytoskeletal arrangement. The availability of the mineralized collagen matrix with improved nanomechanics and cytocompatibility may eventually result in novel biomaterials for bone grafting and tissue‐engineering applications.     

Tuning the Cell Adhesion on Biofunctionalized Nanoporous Organic Frameworks

The ability to control the structure and surface chemistry of biomaterials on a molecular level is crucial for optimizing their performance. Here, a novel type of nanoporous organic framework that is suited for the fabrication of thin films is described. These surface‐grafted gels (SURGELs) are prepared and functionalized using two orthogonal, metal‐free click chemistries. The SURGELs are shown to be cytocompatible and to efficiently mediate adhesion of osteoblast‐like cells. This process can be further enhanced by surface modification. In addition, the use of light‐triggered reactions in combination with photomasks allows a patterned functionalization of the substrates. The potential to vary and exactly adjust the parameters within the SURGEL polymer network (including porosity and exact network topology on the nanometer scale as well as addressable functional groups) combined with the ability to functionalize their surfaces with any clickable biomolecule of choice in any desired pattern allow the targeted design of novel SURGEL‐based biomaterials for applications in nanomedicine, tissue engineering scaffolds, wound dressing,and medical implants.     

Connective‐Tissue Fibroblasts Established on Micropillar Interfaces are Pivotal for Epithelial‐Tissue Morphogenesis

Polydimethylsiloxane (PDMS) pillar arrays are applied as a biomechanical microenvironment to establish gingival connective‐tissue fibroblasts (GCTFs) and to further analyze the pivotal role of GCTFs in epithelial‐tissue morphogenesis. GCTFs are known to exert successful adhesion and growth on fibronectin immobilized on pillar heads, over time, concomitant with the increased gene expression of vimentin and collagen type‐I. GCTF‐populated pillar arrays clearly reveal that epithelial‐tissue morphogenesis of immortalized human gingival keratinocytes (IHGKs), co‐cultured for 7 and 14 days, parallels the in vivo phenotype more closely, when compared with GCTF‐free control arrays. This in vivo‐like phenotype is substantiated by higher mRNA levels for keratin 1, involucrin and filaggrin differentiation markers. Furthermore, it is reflected by a tissue‐specific protein orientation of the aforementioned molecules, and also of the cell‐to‐cell contact forming desmoplakin and the basement membrane constituents, laminin‐5, laminin‐1/10, and collagen type‐IV. These experiments suggest that the in vivo‐like phenotype of the IHGK is governed by the GCTFs growing on the micropillar interfaces. Moreover, they form the basis for the optimization or neogeneration of biomaterials by varying predefined microenvironmetal parameters to achieve an in vivo‐like cell growth and differentiation, indispensable for tissue morphogenesis during regeneration.     

Direct‐Write Assembly of Microperiodic Silk Fibroin Scaffolds for Tissue Engineering Applications

Three–dimensional, microperiodic scaffolds of regenerated silk fibroin have been fabricated for tissue engineering by direct ink writing. The ink, which consisted of silk fibroin solution from the Bombyx mori silkworm, was deposited in a layer‐by‐layer fashion through a fine nozzle to produce a 3D array of silk fibers of diameter 5 µm. The extruded fibers crystallized when deposited into a methanol‐rich reservoir, retaining a pore structure necessary for media transport. The rheological properties of the silk fibroin solutions were investigated and the crystallized silk fibers were characterized for structure and mechanical properties by infrared spectroscopy and nanoindentation, respectively. The scaffolds supported human bone marrow‐derived mesenchymal stem cell (hMSC) adhesion, and growth. Cells cultured under chondrogenic conditions on these scaffolds supported enhanced chondrogenic differentiation based on increased glucosaminoglycan production compared to standard pellet culture. Our results suggest that 3D silk fibroin scaffolds may find potential application as tissue engineering constructs due to the precise control of their scaffold architecture and their biocompatibility.     

Wireless Manipulation of Magnetic/Piezoelectric Micromotors for Precise Neural Stem‐Like Cell Stimulation

Precise neural electrical stimulation, which is a means of promoting neuronal regeneration, is a promising solution for patients with neurotrauma and neurodegenerative diseases. In this study, wirelessly controllable targeted motion and precise stimulation at the single‐cell level using S.platensis@Fe₃O₄@tBaTiO₃ micromotors are successfully demonstrated for the first time. A highly versatile and multifunctional biohybrid soft micromotor is fabricated via the integration of S.platensis with magnetic Fe₃O₄ nanoparticles and piezoelectric BaTiO₃ nanoparticles. The results show that this micromotor system can achieve navigation in a highly controllable manner under a low‐strength rotating magnetic field. The as‐developed system can achieve single‐cell targeted motion and then precisely induce the differentiation of the targeted neural stem‐like cell by converting ultrasonic energy to an electrical signal in situ owing to the piezoelectric effect. This new approach toward the high‐precision stimulation of neural stem‐like cells opens up new applications for micromotors and has excellent potential for precise neuronal regenerative therapies.     

Surface Topography and Electrical Signaling: Single and Synergistic Effects on Neural Differentiation of Stem Cells

Incomplete regeneration and restoration of function in damaged nerves is a major clinical challenge. In this regard, stem cells hold much promise in nerve tissue engineering, with advantages such as prevention of scar‐tissue ingrowth and guidance of axonal regrowth. Engineering 3D and patterned microenvironments using biomaterials with chemical and mechanical characteristics close to those of normal nervous tissue has enabled new approaches for guided differentiation of various stem cells toward neural cells and possible treatment of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Differentiation of stem cells in a neurogenic lineage is largely affected by signals from the surrounding microenvironment (niche). The stem cell niche refers to a specific microenvironment around the stem cells, which provides specific biochemical (soluble factors) and biophysical signals (topography, electrical, and mechanical). This specified niche regulates the stem cells' behavior and fate. While the role of chemical cues in neural differentiation is well appreciated, recently, the cues presented by the physical microenvironment are increasingly documented to be important regulators of nerve cell differentiation. The single and synergistic effects of surface topography and electrical signals on neural differentiation of stem cells are reviewed.     

Ascidian‐Inspired Fast‐Forming Hydrogel System for Versatile Biomedical Applications: Pyrogallol Chemistry for Dual Modes of Crosslinking Mechanism

Exploitation of unique biochemical and biophysical properties of marine organisms has led to the development of functional biomaterials for various biomedical applications. Recently, ascidians have received great attention, owing to their extraordinary properties such as strong underwater adhesion and rapid self‐regeneration. Specific polypeptides containing 3,4,5‐trihydroxyphenylalanine (TOPA) in the blood cells of ascidians are associated with such intrinsic properties generated through complex oxidative processes. In this study, a bioinspired hydrogel platform is developed, demonstrating versatile applicability for tissue engineering and drug delivery, by conjugating pyrogallol (PG) moiety resembling ascidian TOPA to hyaluronic acid (HA). The HA–PG conjugate can be rapidly crosslinked by dual modes of oxidative mechanisms using an oxidant or pH control, resulting in hydrogels with different mechanical and physical characteristics. The versatile utility of HA–PG hydrogels formed via different crosslinking mechanisms is tested for different biomedical platforms, including microparticles for sustained drug delivery and tissue adhesive for noninvasive cell transplantation. With extraordinarily fast and different routes of PG oxidation, ascidian‐inspired HA–PG hydrogel system may provide a promising biomaterial platform for a wide range of biomedical applications.     

Influence of Direct Laser Written 3D Topographies on Proliferation and Differentiation of Osteoblast‐Like Cells: Towards Improved Implant Surfaces

The influence of truly three‐dimensional microstructures on osteoblast‐like cells is reported. Well defined templates are fabricated by direct laser writing and biocompatibly coated with titanium dioxide, resembling implant surfaces. The influence of structural parameters on proliferation, morphology, adhesion, and differentiation is studied. A significantly higher proliferation (170%) is observed on particular topographies compared to unstructured surfaces. Additionally, an influence of structural parameters on the morphology of osteoblast‐like cells is obtained, whereas all cells possess the osteoblastic marker alkaline phosphatase within all structures.     

Silk Biomaterials with Vascularization Capacity

Functional vascularization is critical for the clinical regeneration of complex tissues such as kidney, liver, or bone. The immobilization or delivery of growth factors has been explored to improve vascularization capacity of tissue‐engineered constructs; however, the use of growth factors has inherent problems such as the loss of signaling capability and the risk of complications including immunological responses and cancer. Here, a new method of preparing water‐insoluble silk protein scaffolds with vascularization capacity using an all‐aqueous process is reported. Acid is added temporally to tune the self‐assembly of silk in the lyophilization process, resulting in water‐insoluble scaffold formation directly. These biomaterials are mainly noncrystalline, offering improved cell proliferation than previously reported silk materials. These systems also have an appropriate softer mechanical property that could provide physical cues to promote cell differentiation into endothelial cells, and enhance neovascularization and tissue ingrowth in vivo without the addition of growth factors. Therefore, silk‐based degradable scaffolds represent an exciting biomaterial option, with vascularization capacity for soft tissue engineering and regenerative medicine.     

Regulating Electrical Cue and Mechanotransduction in Topological Gradient Structure Modulated Piezoelectric Scaffolds to Predict Neural Cell Response

Neural cells respond to topographical cues with alterations in cell growth and neurite sprouting mediated by changes in cell behavior. The interaction of fiber topography with cell adhesion receptors affects how the cells adhere to the surface of fibers and defines cell fate through alterations in the biochemistry, physiology, and morphology of neural cells. Although previous studies suggest topographical features influence neural cell proliferation and neurite sprouting, only a few studies have attempted to assess the use of both electrical and topological cues in piezoelectric scaffolds for nerve regeneration. In this study, variations in the shape‐modified collectors enable tunable surface topographic constructs, from micropatterns to fiber bundle structure. The crystallinity, chemical composition, and quantitative analysis confirm that the interplay between the topological structures of the fibers and the blending of nanocomposite materials is critical for the formation of the β‐phase. It is found that the topographical features and induced electrical characteristics affect cell growth. Also, the intracellular signaling pathway is induced that can provide clues as to how neural cells respond to the topological gradient structure modulated piezoelectric scaffolds. An analysis of the neuron‐specific cytoskeletal related markers further reveals that the specific topographical features piezoelectric fibrous scaffold reinforces neuron‐specific cytoskeletal proteins and microtubule assembly.     

Capillary Force Lithography: A Versatile Tool for Structured Biomaterials Interface Towards Cell and Tissue Engineering

This Feature Article aims to provide an in‐depth overview of the recently developed molding technologies termed capillary force lithography (CFL) that can be used to control the cellular microenvironment towards cell and tissue engineering. Patterned polymer films provide a fertile ground for controlling various aspects of the cellular microenvironment such as cell–substrate and cell–cell interactions at the micro‐ and nanoscale. Patterning thin polymer films by molding typically involves several physical forces such as capillary, hydrostatic, and dispersion forces. If these forces are precisely controlled, the polymer films can be molded into the features of a polymeric mold with high pattern fidelity and physical integrity. The patterns can be made either with the substrate surface clearly exposed or unexposed depending on the pattern size and material properties used in the patterning. The former (exposed substrate) can be used to adhere proteins or cells on pre‐defined locations of a substrate or within a microfluidic channel using an adhesion‐repelling polymer such as poly(ethylene glycol) (PEG)‐based polymer and hyaluronic acid (HA). Also, the patterns can be used to co‐culture different cells types with molding‐assisted layer‐by‐layer deposition. In comparison, the latter (unexposed substrate) can be used to control the biophysical surrounding of a cell with tailored mechanical properties of the material. The surface micropatterns can be used to engineer cellular and multi‐cellular architecture, resulting in changes of the cell shape and the cytoskeletal structures. Also, the nanoscale patterns can be used to affect various aspects of the cellular behavior, such as adhesion, proliferation, migration, and differentiation.     

Surface Engineered Carboxymethylchitosan/Poly(amidoamine) Dendrimer Nanoparticles for Intracellular Targeting

Novel highly branched biodegradable macromolecular systems have been developed by grafting carboxymethylchitosan (CMCht) onto low generation poly(amidoamine) (PAMAM) dendrimers. Such structures organize into sphere‐like nanoparticles that are proposed to be used as carriers to deliver bioactive molecules aimed at controlling the behavior of stem cells, namely their proliferation and differentiation. The nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 1 mg mL^?1, and fluorescent probe labeled nanoparticles were found to be internalized with highly efficiency by both human osteoblast‐like cells and rat bone marrow stromal cells, under fluorescence‐activated cell sorting and fluorescence microscopy analyses. Dexamethasone (Dex) has been incorporated into CMCht/PAMAM dendrimer nanoparticles and release rates were determined by high performance liquid chromatography. Moreover, the biochemical data demonstrates that the Dex‐loaded CMCht/PAMAM dendrimer nanoparticles promote the osteogenic differentiation of rat bone marrow stromal cells, in vitro. The nanoparticles exhibit interesting physicochemical and biological properties and have great potential to be used in fundamental cell biology studies as well as in a variety of biomedical applications, including tissue engineering and regenerative medicine.     

Modulation of Mechanical Interactions by Local Piezoelectric Effects

Piezoelectricity is a well‐established property of biological materials, yet its functional role has remained unclear. Here, a mechanical effect of piezoelectric domains resulting from collagen fibril organisation is demonstrated, and its role in tissue function and application to material design is described. Using a combination of scanning probe and nonlinear optical microscopy, a hierarchical structuring of piezoelectric domains in collagen‐rich tissues is observed, and their mechanical effects are explored in silico. Local electrostatic attraction and repulsion due to shear piezoelectricity in these domains modulate fibril interactions from the tens of nanometre (single fibril interactions) to the tens of micron (fibre interactions) level, analogous to modulated friction effects. The manipulation of domain size and organisation thus provides a capacity to tune energy storage, dissipation, stiffness, and damage resistance.     

A Single Component Conducting Polymer Hydrogel as a Scaffold for Tissue Engineering

Conducting polymers (CPs) have exciting potential as scaffolds for tissue engineering, typically applied in regenerative medicine applications. In particular, the electrical properties of CPs has been shown to enhance nerve and muscle cell growth and regeneration. Hydrogels are particularly suitable candidates as scaffolds for tissue engineering because of their hydrated nature, their biocompatibility, and their tissue‐like mechanical properties. This study reports the development of the first single component CP hydrogel that is shown to combine both electro‐properties and hydrogel characteristics. Poly(3‐thiopheneacetic acid) hydrogels were fabricated by covalently crosslinking the polymer with 1,1′‐carbonyldiimidazole (CDI). Their swelling behavior was assessed and shown to display remarkable swelling capabilities (swelling ratios up to 850%). The mechanical properties of the networks were characterized as a function of the crosslinking density and were found to be comparable to those of muscle tissue. Hydrogels were found to be electroactive and conductive at physiological pH. Fibroblast and myoblast cells cultured on the hydrogel substrates were shown to adhere and proliferate. This is the first time that the potential of a single component CP hydrogel has been demonstrated for cell growth, opening the way for the development of new tissue engineering scaffolds.