Natural resilin, the rubber-like protein that exists in specialized compartments of most arthropods, possesses excellent mechanical properties such as low stiffness, high resilience and effective energy storage. Recombinantly-engineered resilin-like polypeptides (RLPs) that possess the favorable attributes of native resilin would be attractive candidates for the modular design of biomaterials for engineering mechanically active tissues. Based on our previous success in creating a novel RLP-based hydrogel and demonstrating useful mechanical and cell-adhesive properties, we have produced a suite of new RLP-based constructs, each equipped with 12 repeats of the putative resilin consensus sequence and a single, distinct biologically active domain. This approach allows independent control over the concentrations of cell-binding, MMP-sensitive, and polysaccharide-sequestration domains in hydrogels comprising mixtures of the various RLPs. The high purity, molecular weight and correct compositions of each new polypeptide have been confirmed via high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and amino acid analysis. These RLP-based polypeptides exhibit largely random-coil conformation, both in solution and in the cross-linked hydrogels, as indicated by circular dichroic and infrared spectroscopic analyses. Hydrogels of various compositions, with a range of elastic moduli (1kPa to 25kPa) can be produced from these polypeptides, and the activity of the cell-binding and matrix metalloproteinase (MMP) sensitive domains was confirmed. Tris(hydroxymethyl phosphine) cross-linked RLP hydrogels were able to maintain their mechanical integrity as well as the viability of encapsulated primary human mesenchymal stem cells (MSCs). These results validate the promising properties of these RLP-based elastomeric biomaterials.

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(e-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindleshaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.

Derived from any somatic cell type and possessing unlimited selfrenewal and differentiation potential, induced pluripotent stem cells (iPSCs) are poised to revolutionize stem cell biology and regenerative medicine research, bringing unprecedented opportunities for treating debilitating human diseases. To overcome the limitations associated with safety, efficiency, and scalability of traditional iPSC derivation, expansion, and differentiation protocols, biomaterials have recently been considered. Beyond addressing these limitations, the integration of biomaterials with existing iPSC culture platforms could offer additional opportunities to better probe the biology and control the behavior of iPSCs or their progeny in vitro and in vivo. Herein, we discuss the impact of biomaterials on the iPSC field, from derivation to tissue regeneration and modeling. Although still exploratory, we envision the emerging combination of biomaterials and iPSCs will be critical in the successful application of iPSCs and their progeny for research and clinical translation.

We are interested in the in vitro engineering of artificial vocal fold tissues via the strategic combination of multipotent mesenchymal stem cells (MSCs), physiologically relevant mechanical stimulations, and biomimetic artificial matrices. We have constructed a vocal fold bioreactor that is capable of imposing vibratory stimulations on the cultured cells at human phonation frequencies. Separately, fibrous poly (e-caprolactone) (PCL) scaffolds emulating the ligamentous structure of the vocal fold were prepared by electrospinning, were incorporated in the vocal fold bioreactor, and were driven into a wave-like motion in an axisymmetrical fashion by the oscillating air. MSC-laden PCL scaffolds were subjected to vibrations at 200 Hz with a normal center displacement of *40 mm for a total of 7 days. A continuous (CT) or a 1 h-on-1 h-off (OF) regime with a total dynamic culture time of 12 h per day was applied. The dynamic loading did not cause any physiological trauma to the cells. Immunohistotochemical staining revealed the reinforcement of the actin filament and the enhancement of a 5 b 1 integrin expression under selected dynamic culture conditions. Cellular expression of essential vocal fold extracellular matrix components, such as elastin, hyaluronic acid, and matrix metalloproteinase-1, was significantly elevated as compared with the static controls, and the OF regime is more conducive to matrix production than the CT vibration mode. Analyses of genes of typical fibroblast hallmarks (tenascin-C, collagen III, and procollagen I) as well as markers for MSC differentiation into nonfibroblastic lineages confirmed MSCs' adaptation of fibroblastic behaviors. Overall, the high-frequency vibratory stimulation, when combined with a synthetic fibrous scaffold, serves as a potent modulator of MSC functions. The novel bioreactor system presented here, as a versatile, yet well-controlled model, offers an in vitro platform for understanding vibration-induced mechanotransduction and for engineering of functional vocal fold tissues.

Both the incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally; in the industrialized world up to 0.5% of the population are affected and around 4.2 million individuals suffer from IBD in Europe and North America combined. Successful engraftment in experimental colitis models suggests that intestinal stem cell transplantation could constitute a novel treatment strategy to re‐establish mucosal barrier function in patients with severe disease. Intestinal stem cells can be grown in vitro in organoid structures, though only a fraction of the cells contained are stem cells with regenerative capabilities. Hence, techniques to enrich stem cell populations are being pursued through the development of multiple two‐dimensional and three‐dimensional culture protocols, as well as co‐culture techniques and multiple growth medium compositions. Moreover, research in support matrices allowing for efficient clinical application is in progress. In vitro culture is accomplished by modulating the signaling pathways fundamental for the stem cell niche with a suitable culture matrix to provide additional contact‐dependent stimuli and structural support. The aim of this review was to discuss medium compositions and support matrices for optimal intestinal stem cell culture, as well as potential modifications to advance clinical use in IBD.

The superior elasticity, excellent resilience at high-frequency, and hydrophilic capacity of natural resilin have motivated investigations of recombinant resilin-based biomaterials as a new class of bio-elastomers in the engineering of mechanically active tissues. Accordingly, we report here the comprehensive characterization of modular resilin-like polypeptide (RLP) hydrogels and introduce their suitability as a novel biomaterial for in vivo applications. Oscillatory rheology confirmed that a full suite of the RLPs can be rapidly crosslinked upon addition of the tris(hydroxymethyl phosphine) (THP) cross-linker, achieving similar in situ shear storage moduli (20k±3.5Pa) across various material compositions. Uniaxial stress relaxation tensile testing of hydrated RLP hydrogels under cyclic loading and unloading showed negligible stress reduction and hysteresis, superior reversible extensibility, and high resilience with Young’s moduli of 30±7.4kPa. RLP hydrogels containing MMP-sensitive domains are susceptible to enzymatic degradation by MMP-1. Cell culture studies revealed that RLP-based hydrogels supported the attachment and spreading (2D) of human mesenchymal stem cells (hMSCs) and did not activate cultured macrophages. Subcutaneous transplantation of RLP hydrogels in a rat model, which to our knowledge is the first such reported in vivo analysis of RLP-based hydrogels, illustrated that these materials do not elicit a significant inflammatory response, suggesting their potential as materials for tissue engineering applications with targets of mechanically demanding tissues such as vocal fold and cardiovascular tissues.

SUMMARY Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to ICAM-1, such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in-vitro, and enabled targeted delivery of systemically administered MSCs to inflamed sites in-vivo in a CD11a (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a/ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

Synthetic hydrogels containing covalently-integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically crosslinkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic crosslinkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently-applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair.