Coacervation-Mediated Cytocompatible Formation of Supramolecular Hydrogels with Self-Evolving Macropores for 3D Multicellular Spheroid Culture.

Coacervation driven liquid-liquid phase separation of biopolymers has aroused considerable attention for diverse applications, especially for the construction of microstructured polymeric materials. Herein, a coacervate-to-hydrogel transition strategy is developed to create macroporous hydrogels (MPH), which are formed via the coacervation process of supramolecular assemblies (SA) built by the host-guest complexation between γ-cyclodextrin and anthracene dimer. The weak and reversible supramolecular crosslinks endow the SA with liquid-like rheological properties, which facilitate the formation of SA-derived macroporous coacervates and the subsequent transition to MPH (pore size ≈ 100 µm). The excellent structural dynamics (derived from SA) and the cytocompatible void-forming process of MPH can better accommodate the dramatic volumetric expansion associated with colony growth of encapsulated multicellular spheroids compared with the non-porous static hydrogel with similar initial mechanical properties. The findings of this work not only provide valuable guidance to the design of biomaterials with self-evolving structures but also present a promising strategy for 3D multicellular spheroid culture and other diverse biomedical applications.

Dynamic gelatin-based hydrogels promote the proliferation and self-renewal of embryonic stem cells in long-term 3D culture.

Long-term maintenance of embryonic stem cells (ESCs) in the undifferentiated state is still challenging. Compared with traditional 2D culture methods, 3D culture in biomaterials such as hydrogels is expected to better support the long-term self-renewal of ESCs by emulating the biophysical and biochemical properties of the extracellular matrix (ECM). Although prior studies showed that soft and degradable hydrogels favor the 3D growth of ESCs, few studies have examined the impact of the structural dynamics of the hydrogel matrix on ESC behaviors. Herein, we report a gelatin-based structurally dynamic hydrogel (GelCD hydrogel) that emulates the intrinsic structural dynamics of the ECM. Compared with covalently crosslinked gelatin hydrogels (GelMA hydrogels) with similar stiffness and biodegradability, GelCD hydrogels significantly promote the clonal expansion and viability of encapsulated mouse ESCs (mESCs) independent of MMP-mediated hydrogel degradation. Furthermore, GelCD hydrogels better maintain the pluripotency of encapsulated mESCs than do traditional 2D culture methods that use MEF feeder cells or medium supplementation with GSK3ß and MEK 1/2 inhibitors (2i). When cultured in GelCD hydrogels for an extended period (over 2 months) with cell passaging every 7 days, mESCs preserve their normal morphology and maintain their pluripotency and full differentiation capability. Our findings highlight the critical role of the structural dynamics of the hydrogel matrix in accommodating the volume expansion that occurs during clonal ESC growth, and we believe that our dynamic hydrogels represent a valuable tool to support the long-term 3D culture of ESCs.

Biomaterial-mediated presentation of wnt5a mimetic ligands enhances chondrogenesis and metabolism of stem cells by activating non-canonical Wnt signaling.

The presentation of development-relevant bioactive cues by biomaterial scaffolds is essential to the guided differentiation of seeded human mesenchymal stem cells (hMSCs) and subsequent tissue regeneration. Wnt5a is a critical non-canonical Wnt signaling ligand and plays a key role in the development of musculoskeletal tissues including cartilage. Herein we investigate the efficacy of biofunctionalizing the hyaluronic acid hydrogel with a synthetic Wnt5a mimetic ligand (Foxy5 peptide) to promote the chondrogenesis of hMSCs and the potential underlying molecular mechanism. Our findings show that the conjugation of Foxy5 peptide in the hydrogels activates non-canonical Wnt signaling of encapsulated hMSCs via the upregulation expression of PLCE1, CaMKII-ß, and downstream NFATc1, leading to enhanced expression of chondrogenic markers such as SOX9. The decoration of Foxy5 peptide also promotes the metabolic activities of encapsulated hMSCs as evidenced by upregulated gene expression of mitochondrial complex components and glucose metabolism biomarkers, leading to enhanced ATP biosynthesis. Furthermore, the conjugation of Foxy5 peptide activates the non-canonical Wnt, PI3K-PDK-AKT and IKK/NF-κB signaling pathways, thereby inhibiting the hypertrophy of the chondrogenically induced hMSCs in the hydrogels under both in vitro and in vivo conditions. This enhanced chondrogenesis and attenuated hypertrophy of hMSCs by the biomaterial-mediated bioactive cue presentation facilitates the potential clinical translation of hMSCs for cartilage regeneration. Our work provides valuable guidance to the rational design of bio-inductive scaffolds for various applications in regenerative medicine.

Biomaterial-Mediated Presentation of Jagged-1 Mimetic Ligand Enhances Cellular Activation of Notch Signaling and Bone Regeneration.

The development from stem cells to adult tissues requires the delicate presentation of numerous crucial inductive cues and the activation of associated signaling pathways. The Notch signaling pathways triggered by ligands such as Jagged-1 have been demonstrated to be essential in various development processes especially in osteogenesis and ossification. However, few studies have capitalized on the osteoinductivity of the Jagged-1 mimetic ligands to enhance the osteogenesis and skeleton regeneration. In this study, we conjugate the porous hyaluronic acid hydrogels with a Jagged-1 mimetic peptide ligand (Jagged-1) and investigate the efficacy of such biomimetic functionalization to promote the mechanotransduction and osteogenesis of human mesenchymal stem cells by activating the Notch signaling pathway. Our findings indicate that the immobilized Jagged-1 mimetic ligand activates Notch signaling via the upregulation of NICD and downstream MSX2, leading to the enhanced mechanotransduction and osteogenesis of stem cells. We further demonstrate that the functionalization of the Jagged-1 ligand in the porous scaffold promotes angiogenesis, regulates macrophage recruitment and polarization, and enhances in situ regeneration of rat calvarial defects. Our findings provide valuable guidance to the design of development-inspired bioactive biomaterials for diverse biomedical applications.

Biomimetic Presentation of Cryptic Ligands via Single-Chain Nanogels for Synergistic Regulation of Stem Cells.

Dynamic controlling the nanoscale presentation of synergistic ligands to stem cells by biomimetic single-chain materials can provide critical insights to understand the molecular crosstalk underlying cells and their extracellular matrix. Here, a stimuli-responsive single-chain macromolecular nanoregulator with conformational dynamics is fabricated based on an advanced scale-up single polymeric chain nanogel (SCNG). Such a carefully designed SCNG is capable of mediating a triggered copresentation of the master and cryptic ligands in a single molecule to elicit the synergistic crosstalk between different intracellular signaling pathways, thereby considerably boosting the bioactivity of the presented ligands. This controllable nanoswitching-on of cell-adhesive ligands' presentation allows the regulation of cell adhesion and fate from molecular scale. The modular nature of this synthetic macromolecular nanoregulator makes it a versatile nanomaterial platform to assist basic and fundamental studies in a wide array of research topics.

Synthetic presentation of noncanonical Wnt5a motif promotes mechanosensing-dependent differentiation of stem cells and regeneration.

Noncanonical Wnt signaling in stem cells is essential to numerous developmental events. However, no prior studies have capitalized on the osteoinductive potential of noncanonical Wnt ligands to functionalize biomaterials in enhancing the osteogenesis and associated skeleton formation. Here, we investigated the efficacy of the functionalization of biomaterials with a synthetic Wnt5a mimetic ligand (Foxy5 peptide) to promote the mechanosensing and osteogenesis of human mesenchymal stem cells by activating noncanonical Wnt signaling. Our findings showed that the immobilized Wnt5a mimetic ligand activated noncanonical Wnt signaling via the up-regulation of Disheveled 2 and downstream RhoA-ROCK signaling, leading to enhanced intracellular calcium level, F-actin stability, actomyosin contractility, and cell adhesion structure development. This enhanced mechanotransduction in stem cells promoted the in vitro osteogenic lineage commitment and the in vivo healing of rat calvarial defects. Our work provides valuable guidance for the developmentally inspired design of biomaterials for a wide array of therapeutic applications.