Biomimetic Dual-Network Collagen Fibers with Porous and Mechanical Cues Reconstruct Neural Stem Cell Niche via AKT/YAP Mechanotransduction after Spinal Cord Injury.

Tissue engineering scaffolds can mediate the maneuverability of neural stem cell (NSC) niche to influence NSC behavior, such as cell self-renewal, proliferation, and differentiation direction, showing the promising application in spinal cord injury (SCI) repair. Here, dual-network porous collagen fibers (PCFS) are developed as neurogenesis scaffolds by employing biomimetic plasma ammonia oxidase catalysis and conventional amidation cross-linking. Following optimizing the mechanical parameters of PCFS, the well-matched Young's modulus and physiological dynamic adaptability of PCFS (4.0 wt%) have been identified as a neurogenetic exciter after SCI. Remarkably, porous topographies and curving wall-like protrusions are generated on the surface of PCFS by simple and non-toxic CO2 bubble-water replacement. As expected, PCFS with porous and matched mechanical properties can considerably activate the cadherin receptor of NSCs and induce a series of serine-threonine kinase/yes-associated protein mechanotransduction signal pathways, encouraging cellular orientation, neuron differentiation, and adhesion. In SCI rats, implanted PCFS with matched mechanical properties further integrated into the injured spinal cords, inhibited the inflammatory progression and decreased glial and fibrous scar formation. Wall-like protrusions of PCFS drive multiple neuron subtypes formation and even functional neural circuits, suggesting a viable therapeutic strategy for nerve regeneration and functional recovery after SCI.

Baicalein-functionalized collagen scaffolds direct neuronal differentiation toward enhancing spinal cord injury repair.

Spinal cord injury (SCI) repair remains a major challenge in clinics. Though neural stem cells (NSCs) have shown great potentials in SCI treatment, their applications were hampered since they primarily differentiate into astrocytes rather than neurons in the injured area, indicating a high demand for effective strategies to direct neuronal differentiation. Baicalein is a clinical drug with multiple pharmacological activities, while its effects on NSCs have rarely been reported. In the current work, inspired by a similarity of the metabolic reprogramming required in neuronal differentiation and that involved in chemoresistance reversal of cancer cells induced by baicalein, we studied the role of baicalein in NSC differentiation and discovered its promotion effects on neuronal differentiation. Based on this observation, baicalein-functionalized collagen scaffolds (BFCSs) were developed and applied for SCI treatment. The BFCSs released the payload in a sustained way and possessed comparable physical properties to the commonly used collagen. Both in vitro studies with primary NSCs and in vivo studies in SCI rats showed that the BFCSs containing a low amount of baicalein can facilitate not only neurogenesis and axon extension, but also reduce astrocyte production and glial scar formation. More importantly, the BFCS implantation led to improvement in the motor functional recovery of SCI rats. Thus, the BFCSs provided a potential strategy to induce neuronal differentiation towards facilitating SCI repair, as well as for the treatment of other central nervous system injuries.

Long-term stability, high strength, and 3D printable alginate hydrogel for cartilage tissue engineering application.

Cartilage damage is one of the main causes of disability, and 3D bioprinting technology can produce complex structures that are particularly suitable for constructing a customized and irregular tissue engineering scaffold for cartilage repair. Alginate is an attractive biomaterial for bioinks because of its good biological safety profile and fast ionic gelation. However, ionically crosslinked alginate hydrogels are recognized as lacking enough mechanical property and long-term stability due to ion exchange. Here, we developed a double crosslinked alginate (DC-Alg) hydrogel for 3D bioprinting, and human umbilical cord mesenchymal stem cells (huMSCs) could differentiate into chondrocytes on its printed 3D scaffold after 4 weeks' culture. We performed sequential modification of alginate with L-cysteine and 5-norbornene-2-methylamine, and the DC-Alg hydrogels were obtained in the presence of CaCl2and ultraviolet light with stronger mechanical properties than those of the single ionic crosslinked alginate hydrogels, which was similar to natural cartilage. They also had better stability and could be maintained in DMEM medium for over 1 month, as well good viability for huMSCs. Moreover, the DC-Alg as 3D printing inks demonstrated a better printing accuracy (∼200 µm). After 4 weeks culture of huMSCs in the 3D printed DC-Alg scaffolds, the expressions of chondrogenic genes such asaggrecan (agg), collagen II (col II), and SRY-box transcription factor9(sox-9) were obviously observed, indicating the differentiation of huMSCs into cartilage. Immumohistochemical staining analysis further exhibited cartilage tissue developed well in the 3D printed scaffolds. Our study is the first demonstration of DC-Alg in 3D printing for MSC differentiation into cartilage, which shows a potential application in cartilage defect repair.

High strength pure chitosan hydrogels via double crosslinking strategy.

Chitosan (CS) hydrogels have been widely used throughout basic tissue engineering and regenerative medicine research and it is very desirable to develop advanced CS materials with superior mechanical and topographical properties for more extensive applications. Herein, we present the design of a double crosslinking pure CS hydrogel material via the synergic effect of the chemical covalent network, hydrophobic interactions, enhanced intermolecular hydrogen bonding and the formation of the CS crystallite. The resultant pure CS hydrogel possesses increases in strength and toughness by two orders of magnitude (fracture energy ∼7.733 J m-2; maximal compression stress ∼10.81 MPa, elastic modulus ∼1.33 MPa). We utilize1H NMR and FT-IR to prove the success of chemical modification. The results of Raman spectra and WXRD have proved the existence of physical interaction between CS hydrogels and microcrystals, thus explaining the enhancement mechanism of mechanical strength of CS hydrogel. The live and death results also show that MSCs can grow well on CS hydrogels, and the results of CCK-8 indicate low cytotoxicity of CS hydrogels. This CS hydrogel shows great potential applications in tissue engineering and regenerative medicine.

NSCs Migration Promoted and Drug Delivered Exosomes-Collagen Scaffold via a Bio-Specific Peptide for One-Step Spinal Cord Injury Repair.

Spinal cord injury (SCI) is plaguing medical professionals globally due to the complexity of injury progression. Based on tissue engineering technology, there recently emerges a promising way by integrating drugs with suitable scaffold biomaterials to mediate endogenous neural stem cells (NSCs) to achieve one-step SCI repair. Herein, exosomes extracted from human umbilical cord-derived mesenchymal stem cells (MExos) are found to promote the migration of NSCs in vitro/in vivo. Utilizing MExos as drug delivery vehicles, a NSCs migration promoted and paclitaxel (PTX) delivered MExos-collagen scaffold is designed via a novel dual bio-specificity peptide (BSP) to effectively retain MExos within scaffolds. By virtue of the synergy that MExos recruit endogenous NSCs to the injured site, and PTX induce NSCs to give rise to neurons, this multifunctional scaffold has shown superior performance for motor functional recovery after complete SCI in rats by enhancing neural regeneration and reducing scar deposition. Besides, the dual bio-specific peptide demonstrates the capacity of tethering other cells-derived exosomes on collagen scaffold, such as erythrocytes-derived or NSCs-derived exosomes on collagen fibers or membranes. The resulting exosomes-collagen scaffold may serve as a potential multifunctional therapy modality for various disease treatments including SCI.

Collagen/Heparin Bi-Affinity Multilayer Modified Collagen Scaffolds for Controlled bFGF Release to Improve Angiogenesis In Vivo.

Basic fibroblast growth factor (bFGF) is an important protein for wound healing and angiogenesis in tissue engineering, but the lack of a viable delivery system hampers its clinical application. This study aims to maintain the long-term controlled release of bFGF by utilizing a collagen/heparin bi-affinity multilayer delivery system (CHBMDS), which is fabricated by the alternate deposition of negatively charged heparin, positively charged collagen, and CBD-bFGF (a collagen-binding domain [CBD] was fused into the native bFGF) via specific or electrostatic interaction. The results show that CHBMDS not only support localized and prolonged release of CBD-bFGF(over 35 days) but also lead to enhanced angiogenesis (higher density and larger diameter (≈70 µm) of newly formed blood vessels in subcutaneous tissue of SD rat after 5 weeks). This system could act as a versatile approach for bFGF delivery and further improve therapeutic efficacy for injured tissues.

Lower fluidity of supported lipid bilayers promotes neuronal differentiation of neural stem cells by enhancing focal adhesion formation.

Extensive studies have been performed to understand how the mechanical properties of a stem cell's microenvironment influence its behaviors. Supported lipid bilayers (SLBs), a well-known biomimetic platform, have been used to mimic the dynamic characteristics of the extracellular matrix (ECM) because of their fluidity. However, the effect of the fluidity of SLBs on stem cell fate is unknown. We constructed SLBs with different fluidities to explore the influence of fluidity on the differentiation of neural stem cells (NSCs). The results showed that the behavior of NSCs was highly dependent on the fluidity of SLBs. Low fluidity resulted in enhanced focal adhesion formation, a dense network of stress fibers, stretched and elongated cellular morphology and increased neuronal differentiation, while high fluidity led to less focal adhesion formation, immature stress fibers, round cellular morphology and more astrocyte differentiation. Mechanistic studies revealed that low fluidity may have enhanced focal adhesion formation, which activated FAK-MEK/ERK signaling pathways and ultimately promoted neuronal differentiation of NSCs. This work provides a strategy for manipulating the dynamic matrix surface for the development of culture substrates and tissue-engineered scaffolds, which may aid the understanding of how the dynamic ECM influences stem cell behaviors as well as improve the efficacy of stem cell applications.