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Self-assembling peptide (SAP) nanofiber hydrogel scaffolds have become increasingly important in tissue engineering due to their outstanding bioactivity and biodegradability. However, there is an initial concern on their long-term clinical use, since SAPs made of L-form amino acid sequences are sensitive to enzymatic degradation. In this study, we present a designer SAP, D-RADA16, made of all D-amino acid. We investigated the nanofiber morphology of D-RADA16, its potential for the culture of bone marrow-derived mesenchymal stem cells (BMSCs), and the proteolytic resistance of the biomaterial. The results revealed that D-RADA16 exhibited stable β-sheets and formed interwoven nanofiber scaffolds in water. D-RADA16 and L-RADA16 hydrogel scaffolds were both found to promote the proliferation and migration of rat BMSCs in the 3D cell culture microenvironment. Furthermore, the D-RADA16 scaffolds exhibited a higher proteolytic resistance against proteinase K than the L-RADA16 scaffolds. These observations indicate that D-RADA16 hydrogel scaffolds have excellent bioactivity, biocompatibility and biostability, and thus may serve as promising candidates for long-term application in vivo.

Background: Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical. Methods: We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro. Results: NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids. Conclusions: We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration. [ABSTRACT FROM AUTHOR]

[Display omitted] • A biocompatible, injectable, and lesion-localized hydrogel loaded with extracellular matrix (ECM) was developed. • Stimulation of mesenchymal stem cells with TNF-α, IL-6, and IFN-γ enhanced the quality and biological function of the ECM. • The engineered ECM hydrogels effectively maintained chondrocyte homeostasis. • The engineered ECM hydrogels preserved cellular homeostasis by augmenting mitochondrial energy metabolism. • ECM derived from various inflammatory cytokine pre-treatments displayed distinct functional strengths and weaknesses, with IFN-γ-ECM demonstrating superior efficacy. Cartilage damage and degeneration are fundamental pathological features of osteoarthritis (OA), attributed largely to disruptions in chondrocyte homeostasis. Tissue engineering employing functional bioactive materials presents a promising avenue for cartilage regeneration and repair. Particularly, extracellular matrix (ECM) produced by mesenchymal stem cells (MSCs) is gaining traction in the field of tissue engineering and scaffold-based regenerative therapies due to its robust bioactivity and excellent biocompatibility. In this study, we utilized specific inflammatory cytokines associated with OA as stimulators to optimize the MSC-derived secretome. Our in vitro experiments demonstrated that the engineered ECM effectively inhibited early chondrocyte apoptosis, reversed cellular senescence, and maintained anabolic-catabolic equilibrium. Moreover, we developed aldehyde- and methacrylic anhydride-modified hyaluronic acid hydrogels incorporated with ECM (ECM@AH) using photo-cross-linking techniques. The affinity of these ECM-functionalized hydrogels for degraded cartilage was confirmed through in situ cartilage lesion localization experiments in the knee joints of rat OA models. Further in vitro analysis revealed that the engineered ECM@AH, especially IFN-γ-ECM@AH, sustains chondrocyte homeostasis by restoring mitochondrial energy metabolism. Animal studies demonstrated that ECM@AH, localized to cartilage lesions, could reverse cartilage matrix degradation and promote cartilage repair, underscoring the potential of this biomaterial in OA treatment. [ABSTRACT FROM AUTHOR]