Remodeling of the Intra-Conduit Inflammatory Microenvironment to Improve Peripheral Nerve Regeneration with a Neuromechanical Matching Protein-Based Conduit.

Peripheral nerve injury (PNI) remains a challenging area in regenerative medicine. Nerve guide conduit (NGC) transplantation is a common treatment for PNI, but the prognosis of NGC treatment is unsatisfactory due to 1) neuromechanical unmatching and 2) the intra-conduit inflammatory microenvironment (IME) resulting from Schwann cell pyroptosis and inflammatory-polarized macrophages. A neuromechanically matched NGC composed of regenerated silk fibroin (RSF) loaded with poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (P:P) and dimethyl fumarate (DMF) are designed, which exhibits a matched elastic modulus (25.1 ± 3.5 MPa) for the peripheral nerve and the highest 80% elongation at break, better than most protein-based conduits. Moreover, the NGC can gradually regulate the intra-conduit IME by releasing DMF and monitoring sciatic nerve movements via piezoresistive sensing. The combination of NGC and electrical stimulation modulates the IME to support PNI regeneration by synergistically inhibiting Schwann cell pyroptosis and reducing inflammatory factor release, shifting macrophage polarization from the inflammatory M1 phenotype to the tissue regenerative M2 phenotype and resulting in functional recovery of neurons. In a rat sciatic nerve crush model, NGC promoted remyelination and functional and structural regeneration. Generally, the DMF/RSF/P:P conduit provides a new potential therapeutic approach to promote nerve repair in future clinical treatments.

Key Process Parameters Study for the Fill Finish of Vaccines Containing Aluminum Hydroxide Adjuvant.

Vaccine manufacturing is one of the most challenging and complex processes in pharmaceutical industry, and the process control strategy is critical for the safety, effectiveness, and consistency of a vaccine. The efficacy of aluminum salt adjuvant on vaccines strongly depends on its physicochemical properties, such as size, structure, surface charge, etc. However, stresses during the vaccine manufacturing may affect the stability of adjuvant. In this study, the impacts of cold/thermal stress, autoclaving, pumping, mixing, and filling shear stress on the physicochemical properties of aluminum hydroxide (AH) adjuvant were evaluated as part of the manufacturing process development. The results showed that the autoclaving process would slightly influence the structure and properties of the investigated AH adjuvant, but thermal incubation at 2-8 °C, 25 °C and 40 °C for 4 weeks did not. However, -20 °C freezing AH adjuvant led to the adjuvant agglomeration and rapid sedimentation. For the high shear stress study with mixing at 500 rpm in a 1-L mixing bag and pumping at 220 rpm for up to 24 h, the average particle dimension of the bulk AH adjuvant decreased, along with decreasing protein adsorption ratio. The studies indicate that various stresses during manufacturing process could affect the structure and physicochemical properties of AH adjuvant, which calls for more attention on the control of adjuvant process parameters during manufacturing.

Biomimetic Structural Protein Based Magnetic Responsive Scaffold for Enhancing Bone Regeneration by Physical Stimulation on Intracellular Calcium Homeostasis.

The bone matrix has distinct architecture and biochemistry which present a barrier to synthesizing bone-mimetic regenerative scaffolds. To mimic the natural structures and components of bone, biomimetic structural decellularized extracellular matrix (ECM)/regenerated silk fibroin (RSF) scaffolds incorporated with magnetic nanoparticles (MNP) are prepared using a facile synthetic methodology. The ECM/RSF/MNP scaffold is a hierarchically organized and interconnected porous structure with silk fibroin twined on the collagen nanofibers. The scaffold demonstrates saturation magnetization due to the presence of MNP, along with good cytocompatibility. Moreover, the ß-sheet crystalline domain of RSF and the chelated MNP could mimic the deposition of hydroxyapatite and enhance compressive modulus of the scaffold by ≈20%. The results indicate that an external static magnetic field (SMF) with a magnetic responsive scaffold effectively promotes cell migration, osteogenic differentiation, neogenesis of endotheliocytes in vitro, and new bone formation in a critical-size femur defect rat model. RNA sequencing reveals that the molecular mechanisms underlying this osteogenic effect involve calsequestrin-2-mediated Ca2+ release from the endoplasmic reticulum to activate Ca2+ /calmodulin/calmodulin-dependent kinase II signaling axis. Collectively, bionic magnetic scaffolds with SMF stimulation provide a potent strategy for bone regeneration through internal structural cues, biochemical composition, and external physical stimulation on intracellular Ca2+ homeostasis.

Pro-Inflammatory of PRDM1/SIRT2/NLRP3 Axis in Monosodium Urate-Induced Acute Gouty Arthritis.

PR domain-containing 1 with zinc finger domain (PRDM1) has been reported as a promoter of inflammation, which is a critical process involved in the pathogenesis of acute gouty arthritis. Herein, we sought to ascertain the function of PRDM1 in the development of acute gouty arthritis and related mechanisms. At first, peripheral blood-derived monocytes from patients with acute gouty arthritis and healthy individuals were collected as experimental samples. Then, macrophages were induced from monocytes using phorbol myristate acetate (PMA). The expression patterns of PRDM1, sirtuin 2 (SIRT2), and NLR family, pyrin domain-containing 3 (NLRP3) were characterized by RT-qPCR and Western blot assay. PMA-induced macrophages were stimulated by monosodium urate (MSU) for in vitro experimentation. Meanwhile, a murine model of MSU-induced acute gouty arthritis was established for in vivo validation. PRDM1 was highly expressed while SIRT2 poorly expressed in patients with acute gouty arthritis. Loss of PRDM1 could reduce NLRP3 inflammasome and mature IL-1ß levels and downregulate inflammatory cytokines in macrophages, which contributed to protection against acute gouty arthritis. Furthermore, results showed that PRDM1 could inhibit SIRT2 expression via binding to the deacetylase SIRT2 promoter. Finally, the in vivo experiments demonstrated that PRDM1 increased NLRP3 inflammasome and mature IL-1ß through transcriptional inhibition of SIRT2, whereby aggravating MSU-induced acute gouty arthritis. To sum up, PRDM1 increased NLRP3 inflammasome through inhibiting SIRT2, consequently aggravating MSU-induced acute gouty arthritis.

Piezoresistive MXene/Silk fibroin nanocomposite hydrogel for accelerating bone regeneration by Re-establishing electrical microenvironment.

The electrical microenvironment plays an important role in bone repair. However, the underlying mechanism by which electrical stimulation (ES) promotes bone regeneration remains unclear, limiting the design of bone microenvironment-specific electroactive materials. Herein, by simple co-incubation in aqueous suspensions at physiological temperatures, biocompatible regenerated silk fibroin (RSF) is found to assemble into nanofibrils with a ß-sheet structure on MXene nanosheets, which has been reported to inhibit the restacking and oxidation of MXene. An electroactive hydrogel based on RSF and bioencapsulated MXene is thus prepared to promote efficient bone regeneration. This MXene/RSF hydrogel also acts as a piezoresistive pressure transducer, which can potentially be utilized to monitor the electrophysiological microenvironment. RNA sequencing is performed to explore the underlying mechanisms, which can activate Ca2+/CALM signaling in favor of the direct osteogenesis process. ES is found to facilitate indirect osteogenesis by promoting the polarization of M2 macrophages, as well as stimulating the neogenesis and migration of endotheliocytes. Consistent improvements in bone regeneration and angiogenesis are observed with MXene/RSF hydrogels under ES in vivo. Collectively, the MXene/RSF hydrogel provides a distinctive and promising strategy for promoting direct osteogenesis, regulating immune microenvironment and neovascularization under ES, leading to re-establish electrical microenvironment for bone regeneration.

Injectable Mussel-Inspired highly adhesive hydrogel with exosomes for endogenous cell recruitment and cartilage defect regeneration.

In the early stage of osteoarthritis (OA), cartilage degradation in the surface region leads to superficial cartilage defect. However, enhancing the regeneration of cartilage defect remains a great challenge for existing hydrogel technology because of the weak adhesion to wet tissue. In the present study, an injectable mussel-inspired highly adhesive hydrogel with exosomes was investigated for endogenous cell recruitment and cartilage defect regeneration. The hydrogel with high bonding strength to the wet surface was prepared using a crosslinked network of alginate-dopamine, chondroitin sulfate, and regenerated silk fibroin (AD/CS/RSF). Compared with commercial enbucrilate tissue adhesive, the AD/CS/RSF hydrogel provided a comparative lap shear strength of 120 kPa, with a similar gelation time and a higher capacity for maintaining adhesive strength. The AD/CS/RSF/EXO hydrogel with encapsulated exosomes recruited BMSCs migration and inflation, promoted BMSCs proliferation and differentiation. Most importantly, the AD/CS/RSF/EXO hydrogel accelerated cartilage defect regeneration in situ, and extracellular matrix remodeling after injection in rat patellar grooves. The exosomes released by the hydrogels could recruit BMSCs into the hydrogel and neo-cartilage via the chemokine signaling pathway. Our findings reveal an injectable and adhesive hydrogel for superficial cartilage regeneration, which is a promising approach for minimally treating cartilage defect with arthroscopic assistance.

Fabrication of Air-Stable and Conductive Silk Fibroin Gels.

Owing to their promising applications in flexible electronics, researchers have extensively explored flexible and conductive gels. However, these gels have unsatisfactory strength and flexibility as well as easily dry in air. Herein, a rationally designed robust regenerated silk fibroin (RSF)-based gel with significant flexibility and strength, favorable conductivity, and excellent air stability is fabricated by inducing the conformation transition of RSF from random coil to ß-sheet in ionic liquid (IL)/water mixtures. We found that such RSF-based gels have a unique homogeneous network structure of RSF nanofibers, which is likely formed because of evenly distributed cross-links dominated by small-sized ß-sheet domains created during the conformation transition of RSF. Although the unique homogeneous nanostructure/network contributes toward improving the mechanical properties of these gels, it also provides pathways for ionic transport to help the gels preserve high conductivity of ILs. The prepared RSF-based gels display a remarkable air stability and reversible loss/absorption water capability in a wide humidity range environment primarily because of the distinguished combination of the IL and water. Therefore, the novel RSF-based gels hold a great potential in various applications as multifunctional, flexible, conductive materials, which are dispensed with encapsulation.

Enhancing Mechanical Properties of Silk Fibroin Hydrogel through Restricting the Growth of ß-Sheet Domains.

Usually, regenerated silk fibroin (RSF) hydrogels cross-linked by chemical agents such as horseradish peroxide (HRP)/H2O2 perform elastic properties, while display unsatisfactory strength for practical applications especially as load-bearing materials, and inadequate stability when incubated in a simulated in vivo environment. Here, the RSF hydrogel with both excellent strength and elasticity was prepared by inducing the conformation transition from random coil to ß-sheet in a restricted RSF network precross-linked by HRP/H2O2. Such "dual-networked" hydrogels, regarding the one with 10 wt % RSF (Mw: 220 kDa) as a representative, show around 100% elongation, as well as the compressive modulus and tensile modulus up to 3.0 and 2.5 MPa respectively, which are much higher than those of physically cross-linked natural polymer hydrogels (commonly within 0.01-0.1 MPa at the similar solid content). It has been shown that the enhanced comprehensive mechanical properties of RSF hydrogels derive from the formation of small-sized and uniformly distributed ß-sheet domains in the hydrogel during the conformation transition of RSF whose size is limited by the first network formed by cross-linkers with HRP/H2O2. Importantly, the tough RSF hydrogel changes the normally weak recognition of various RSF hydrogels and holds a great potential to be the material in biomedical field because it seems to be very promising regarding its biocompatibility, biodegradability, etc.

Enhancing the Gelation and Bioactivity of Injectable Silk Fibroin Hydrogel with Laponite Nanoplatelets.

Regenerated silk fibroin (RSF) of Bombyx mori silk fiber is a promising natural material for bone defect repair. However, a lack of specific integrin and growth factor for osteoinduction significantly hinders its application in this area. In this study, the role of Laponite nanoplatelet (LAP), a bioactive clay that can promote osteoblast growth, in the formation of RSF hydrogel, as well as the various properties of RSF/LAP hybrid hydrogel, was closely investigated. The results indicate that LAP could serve as a medium to accelerate hydrophobic interaction among the RSF molecules and a disruptor to limit the growth of ß-sheet domain during the gelation of RSF. Rheological measurement suggests that the RSF/LAP hydrogel is injectable as it displays thixotropy in the room temperature. Proliferation and differentiation results of the primary osteoblasts encapsulated in hydrogel show that RSF/LAP hydrogel can promote the cell proliferation and enhance the osteogenic differentiation. The transcript levels for alkaline phosphatase, osteocalcin, osteopontin, and collagen type I osteogenic markers obviously improve with RSF/LAP hydrogel compared to the controls at 14 days, especially with the higher contents of LAP. Overall, the results suggest that the RSF/LAP hydrogel have great potential to be utilized as an injectable biomaterial for irregular bone defect repair.