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Articular cartilage has limited regeneration capacity because of its acellular and avascular nature. Although tissue engineering has been shown to be life-saving, reforming cartilage zones required by the appropriate tissue functions are challenging. Herein, the need is addressed by designing and producing a nano-engineered structure mimicking the superficial zone (SZ) of articular cartilage. The substrate is based on silk with good mechanical properties in conjunction with nano-topographical and biochemical cues. Nanopillar arrays are produced on the silk surface to regulate the stem cell morphology rendering them with a flattened ellipsoidal shape that is similar to that of chondrocytes in the SZ of natural cartilage. The cell interactions are enhanced by nitrogen ion implantation and the biomolecule, kartogenin (KGN), is loaded to promote chondrogenesis of the stem cells and furthermore, a thermosensitive chitosan hydrogel is formed on the nanopatterned silk to produce rheological properties similar to those of a synovial fluid. Based on the in vitro results and mechanical properties, it is a desirable implantable smart structure mimicking the cartilage SZ with the ability of continuous drug release for cartilage regeneration., (© 2020 Wiley-VCH GmbH.)

For tissue engineering applications, it is important to develop fabrication strategies for building models with controlled cell distributions in defined structures. Here, a simple, flexible approach (named the μ-eraser strategy) is developed to construct multicell micropatterns. This approach involves pressing a poly(dimethylsiloxane) stamp to erase cells growing on substrates, and seeding other types of cells. The pressing/seeding process can be conducted in any designed pattern at desired time point. In a proof of concept, multicell micropatterns of human lung adenocarcinoma epithelial A549 cells, murine fibroblast (FB) cells and murine osteoblast (OB) cells are achieved on Petri dishes and electrospun sheets. Besides forming multicell micropatterns, the cell orientation can be regulated by microstripes and alignment of nanofibers. On Petri dishes and random fiber sheets, FB and OB cells align along microstripes, while A549 cells do not. However, when growing on aligned fiber sheets, no matter whether solo-cultured or co-cultured, all cells in micropatterns orient along the fibers. Based on this technique, a platform is built up to investigate rates of cell migration and interinvasion under solo-culture and co-culture systems. It is believed that this μ-eraser strategy has promise for biological, pharmaceutical, and biomedical applications., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Elaborating a bone replacement using tissue-engineering strategies for bone repair seems to be a promising remedy. However, previous platforms are limited in constructing three-dimensional porous scaffolds and neglected the critical importance of periosteum (a pivotal source of osteogenic cells for bone regeneration). We report here an innovative method using the periosteum as a template to replicate its exquisite morphologies onto the surfaces of biomaterials. The precise topographic cues (grooved micropatterns) on the surface of collagen membrane inherited from the periosteum effectively directed cell alignment as the way of natural periosteum. Besides, we placed the stem-cell and endothelial-cell-laden collagen membrane (pseudo-periosteum) onto a three-dimensional porous scaffold. The pseudo-periosteum-covered scaffolds showed remarkable osteogenesis when compared with the pseudo-periosteum-free scaffolds, indicating the significant importance of pseudo-periosteum on bone regeneration. This study gives a novel concept for the construction of bone tissue engineering scaffold and may provide new insight for periosteum research., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Controlling the cellular microenvironment can be used to direct the cellular organization, thereby improving the function of synthetic tissues in biosensing, biorobotics, and regenerative medicine. In this study, we were inspired by the microstructure and biological properties of the extracellular matrix to develop freestanding ultrathin polymeric films (referred as "nanomembranes") that were flexible, cell adhesive, and had a morphologically tailorable surface. The resulting nanomembranes were exploited as flexible substrates on which cell-adhesive micropatterns were generated to align C2C12 skeletal myoblasts and embedded fibril carbon nanotubes enhanced the cellular elongation and differentiation. Functional nanomembranes with tunable morphology and mechanical properties hold great promise in studying cell-substrate interactions and in fabricating biomimetic constructs toward flexible biodevices.

Injury to articular cartilage, especially the defects induced by degenerative diseases has presented insurmountable challenges. Elaborating a replacement of articular cartilage using biomimic tissue-engineering strategies provides a promising remedy. However, none of the previous osteo/chondrogenic methodologies can not only simultaneously induce osteo/chondrogenesis of stem cells in one scaffolding niche, but also generate a biomimic interface between the formed osteogenic and chondrogenic zones. We report here an innovative method using biomicrofluidic techniques to simultaneously steer distinct specialized differentiation of stem cells into chondrocytes and osteoblasts in one hydrogel slab. Importantly, a gradient that mimics the interface of bone-to-cartilage was generated in the middle of the hydrogel slab. We compared this format with the conventional method for osteochondrogenesis; this format using the gradient-generating microfluidic device indicated outstanding superiorities in stem cell culture and differentiation. Our findings will have a major impact on the design of versatile biomicrofluidic devices for interfacial tissue regeneration., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although biomimetic stimuli, such as microgroove-induced alignment (μ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined μ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-β signaling was downregulated; the TGF-β receptor agonist and antagonist TGF-β1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

A universal and facile approach to modifying proteins so that they can rapidly form hydrogel upon mixing with crosslinkers is presented. The concept of it is to introduce maleimide, which is highly reactive with dithiol-containing crosslinkers via thiol-ene click chemistry, onto proteins. Bovine serum albumin (BSA) is used as a model protein due to its good stability and low cost. The results here show that a protein hydrogel can be readily formed by blending modified BSA and resilin-related peptide crosslinker solutions at a proper ratio. The hydrogel exhibits good elasticity and tunable mechanical as well as biochemical properties. Moreover, it allows convenient 3D cell encapsulation and shows good biocompatibility. Muscle cells embedded in the hydrogel are promoted to spread by incorporating arginyl-glycyl-aspartic acid (RGD)-containing peptide into the system, thus warranting a bright future of it in regenerative medicine. [ABSTRACT FROM AUTHOR]

Engineering living tissues that simulate their natural counterparts is a dynamic area of research. Among the various models of biological tissues being developed, fiber-shaped cellular architectures, which can be used as artificial blood vessels or muscle fibers, have drawn particular attention. However, the fabrication of continuous microfiber substrates for culturing cells is still limited to a restricted number of polymers (e.g., alginate) having easy processability but poor cell-material interaction properties. Moreover, the typical smooth surface of a synthetic fiber does not replicate the micro- and nanofeatures observed in vivo, which guide and regulate cell behavior. In this study, a method to fabricate photocrosslinkable cell-responsive methacrylamide-modified gelatin (GelMA) fibers with exquisite microstructured surfaces by using a microfluidic device is developed. These hydrogel fibers with microgrooved surfaces efficiently promote cell encapsulation and adhesion. GelMA fibers significantly promote the viability of cells encapsulated in/or grown on the fibers compared with similar grooved alginate fibers used as controls. Importantly, the grooves engraved on the GelMA fibers induce cell alignment. Furthermore, the GelMA fibers exhibit excellent processability and could be wound into various shapes. These microstructured GelMA fibers have great potential as templates for the creation of fiber-shaped tissues or tissue microstructures. [ABSTRACT FROM AUTHOR]

We report a chitosan-based nanocomposite thermogel with superior shear modulus resembling that of cartilage and dual pro-chondrogenic and anti-inflammatory functions. Two therapeutic agents, kartogenin (KGN) and diclofenac sodium (DS), are employed to promote chondrogenesis of stem cells and suppress inflammation, respectively. To extend the release time in a controlled manner, KGN is encapsulated in the uniform-sized starch microspheres and DS is loaded into the halloysite nanotubes. Both drug carriers are doped into the maleimide-modified chitosan hydrogel to produce a shear modulus of 167 ± 5 kPa that is comparable to that of articular cartilage (50–250 kPa). Owing to the hydrogel injectability and relatively suitable gelation time (5 ± 0.5 min) at 37 °C, this system potentially constitutes a manageable platform for clinical practice. Moreover, sustained linear drug release for over a month boosts chondro-differentiation of stem cells to eliminate the necessity for multiple administrations. Considering virtues such as thermogel strength and ability to co-deliver anti-inflammatory and chondro-inductive biomolecules continuously, the materials and strategy have promising potential in functional cartilage tissue engineering. [Display omitted] • Novel injectable chitosan thermogel with high shear modulus comparable to cartilage • Development of kartogenin encapsulated starch microspheres by droplet microfluidics. • Extended linear kartogenin release from the thermogel for cartilage regeneration • Sustained diclofenac sodium release from the thermogel for pain reduction [ABSTRACT FROM AUTHOR]

• New thermosensitive chitosan-based hydrogels with tunable rheological properties. • Modeling of hydrogel rheological and swelling properties by statistical models. • Kartogenin sustained release from gel interface for cartilage tissue engineering. • Improvement of chitosan hydrogel shear modulus by 55 times compared to pure gel. Limited regeneration capacity of cartilage can be addressed by tissue engineering approaches including localized delivery of bioactive agents using biomaterials. Although chitosan hydrogels have been considered as appropriate candidates for these purposes, however, their poor mechanical properties limit their real applications. Here, we develop in situ forming chitosan hydrogels with enhanced shear modulus by chemical modification of chitosan using N -(β-maleimidopropyloxy) succinimide ester (BMPS). Moreover, we utilize β-Glycerophosphate (β-GP) in the hydrogels for achieving thermosensitivity. We investigate the effects of BMPS, β-GP and chitosan concentration on rheological and swelling properties of the hydrogels. Accordingly, we generate significant statistical models by response surface method to predict these properties. These models provide us beneficial tools to tune the hydrogel properties depending on the cartilage defect location and properties. Finally, we incorporate a recently discovered small biomolecule, kartogenin (KGN), for promoting chondrogenesis of stem cells into the optimized hydrogel. The hydrogel's shear modulus is 78 ± 5 kPa which covers a wide range of human articular cartilage shear modulus (50–250 kPa). It can be injected to the defects non-invasively at room temperature which gels at 37 °C within minutes. Additionally, it provides a sustained KGN release for ∼40 days that may eliminate the need of multiple injections. In vitro chondrogenic results confirm enhanced chondrogenic differentiation of human adipose mesenchymal stem cells (hAMSCs) treated with KGN-loaded hydrogel, compared to pure KGN. Based on the enhanced hydrogel shear modulus, injectability, gelation behavior, long-term drug release and in vitro results, this thermosensitive hydrogel looks promising for cartilage tissue engineering. [ABSTRACT FROM AUTHOR]