Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation.

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

Human hair keratins promote the regeneration of peripheral nerves in a rat sciatic nerve crush model.

Axon regeneration and functional recovery after peripheral nerve injury remains a clinical challenge. Injury leads to axonal disintegration after which Schwann cells (SCs) and macrophages re-engage in the process of regeneration. At present, biomaterials are regarded as the most promising way to repair peripheral nerve damage. As a natural material, keratin has a wide range of sources and has good biocompatibility and biodegradability. Here, a keratin was extracted from human hair by reducing method and a keratin sponge with porous structure was obtained by further processing. The results suggested that keratin can promote cell adhesion, proliferation, migration as well as the secretion of neurotrophic factors by SCs and the regulation of the expression of macrophage inflammatory cytokines in vitro. We report for the first time that human hair keratin can promote the extension of axon in DRG neurons. The motor deficits caused by a sciatic nerve crush injury were alleviated by keratin sponge dressing in vivo. Thus, keratin has been identified as a valuable biomaterial that can enhance peripheral nerve regeneration.

Effects of Nanoscale Spatial Arrangement of Arginine-Glycine-Aspartate Peptides on Dedifferentiation of Chondrocytes.

Cell dedifferentiation is of much importance in many cases such as the classic problem of dedifferentiation of chondrocytes during in vitro culture in cartilage tissue engineering. While cell differentiation has been much investigated, studies of cell dedifferentiation are limited, and the nanocues of cell dedifferentiation have little been reported. Herein, we prepared nanopatterns and micro/nanopatterns of cell-adhesive arginine-glycine-aspartate (RGD) peptides on nonfouling poly(ethylene glycol) (PEG) hydrogels to examine the effects of RGD nanospacing on adhesion and dedifferentiation of chondrocytes. The relatively larger RGD nanospacing above 70 nm was found to enhance the maintainence of the chondrocyte phenotype in two-dimensional culture, albeit not beneficial for adhesion of chondrocytes. A unique micro/nanopattern was employed to decouple cell spreading, cell shape, and cell-cell contact from RGD nanospacing. Under given spreading size and shape of single cells, the large RGD nanospacing was still in favor of preserving the normal phenotype of chondrocytes. Hence, the nanoscale spatial arrangement of cell-adhesive ligands affords a new independent regulator of cell dedifferentiation, which should be taken into consideration in biomaterial design for regenerative medicine.

Matrix Stiffness and Nanoscale Spatial Organization of Cell-Adhesive Ligands Direct Stem Cell Fate.

One of the breakthroughs in biomaterials and regenerative medicine in the latest decade is the finding that matrix stiffness affords a crucial physical cue of stem cell differentiation. This statement was recently challenged by another understanding that protein tethering on material surfaces instead of matrix stiffness was the essential cue to regulate stem cells. Herein, we employed nonfouling poly(ethylene glycol) (PEG) hydrogels as the matrix to prevent nonspecific protein adsorption, and meanwhile covalently bound cell-adhesive arginine-glycine-aspartate (RGD) peptides onto the hydrogel surfaces in the form of well-defined nanoarrays to control specific cell adhesion. This approach enables the decoupling of the effects of matrix stiffness and surface chemistry. Mesenchymal stem cells (MSCs) were cultured on four substrates (two compressive moduli of the PEG hydrogels multiplied by two RGD nanospacings) and incubated in the mixed osteogenic and adipogenic medium. The results illustrate unambiguously that matrix stiffness is a potent regulator of stem cell differentiation. Moreover, we reveal that RGD nanospacing affects spreading area and differentiation of rat MSCs, regardless of the hydrogel stiffness. Therefore, both matrix stiffness and nanoscale spatial organization of cell-adhesive ligands direct stem cell fate.

Liposome Nanomedicine Based on Tumor Cell Lysate Mitigates the Progression of Lynch Syndrome-Associated Colon Cancer.

Treatment with immune checkpoint inhibitors (ICIs) has shown efficacy in some patients with Lynch syndrome-associated colon cancer, but some patients still do not benefit from it. In this study, we adopted a combination strategy of tumor vaccines and ICIs to maximize the benefits of immunotherapy. Here, we obtained tumor-antigen-containing cell lysate (TCL) by lysing MC38Mlh1 KD cells and prepared liposome nanoparticles (Lipo-PEG) with a typical spherical morphology by thin-film hydration. Anti-PD-L1 was coupled to the liposome surface by the amidation reaction. As observed, anti-PD-L1/TCL@Lipo-PEG was not significantly toxic to mouse intestinal epithelial cells (MODE-K) in the safe concentration range and did not cause hemolysis of mouse red blood cells. In addition, anti-PD-L1/TCL@Lipo-PEG reduced immune escape from colon cancer cells (MC38Mlh1 KD) by the anti-PD-L1 antibody, restored the killing function of CD8+ T cells, and targeted more tumor antigens to bone marrow-derived dendritic cells (BMDCs), which also expressed PD-L1, to stimulate BMDC antigen presentation. In syngeneic transplanted Lynch syndrome-associated colon cancer mice, the combination of anti-PD-L1 and TCL provided better cancer suppression than monoimmunotherapy, and the cancer suppression effect of anti-PD-L1/TCL@Lipo-PEG treatment was even better than that of the free drug. Meanwhile anti-PD-L1/TCL@Lipo-PEG enhanced the immunosuppressive tumor microenvironment. In vivo fluorescence imaging and H&E staining showed that the nanomedicine was mainly retained in the tumor site and had no significant toxic side effects on other major organs. The anti-PD-L1/TCL@Lipo-PEG prepared in this study has high efficacy and good biosafety in alleviating the progression of Lynch syndrome-associated colon cancer, and it is expected to be a therapeutic candidate for Lynch syndrome-associated colon cancer.

Dual-layer conduit containing VEGF-A - Transfected Schwann cells promotes peripheral nerve regeneration via angiogenesis.

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair. This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration.

Microvesicles from Schwann-Like Cells as a New Biomaterial Promote Axonal Growth.

Schwann cells promote axonal regeneration following peripheral nerve injury. However, in terms of clinical treatment, the therapeutic effects of Schwann cells are limited by their source. The transmission of microvesicles from neuroglia cells to axons is a novel communication mechanism in axon regeneration.To evaluate the effect of microvesicles released from Schwann-like cells on axonal regeneration, neural stem cells derived from human embryonic stem cells differentiated into Schwann-like cells, which presented a typical morphology and characteristics similar to those of schwann cells. The glial markers like MBP, P0, P75NTR, PMP-22, GFAP, HNK-1 and S100 were upregulated, whereas the neural stem markers like NESTIN, SOX1 and SOX2 were significantly downregulated in schwann-like cells. Microvesicles enhanced axonal growth in dorsal root ganglia neurons and regulated GAP43 expression in neuron-like cells (N2A and PC12) through the PTEN/PI3 K/Akt signaling pathway. A 5 mm section of sciatic nerve was transected in Sprague-Dawley rats. With microvesicles transplantation, regenerative nerves were evaluated after 6 weeks. Microvesicles increased sciatic function index scores, delayed gastrocnemius muscle atrophy and elevated ßIII-tubulin-labeled axons in vivo. Schwann-like cells serve as a convenient source and promote axonal growth by secreting microvesicles, which may potentially be used as bioengineering materials for nerve tissue repair.

Biological sealing and integration of a fibrinogen-modified titanium alloy with soft and hard tissues in a rat model.

Percutaneous or transcutaneous devices are important and unique, and the corresponding biological sealing at the skin-implant interface is the key to their long-term success. Herein, we investigated the surface modification to enhance biological sealing, using a metal sheet and screw bonded by biomacromolecule fibrinogen mediated via pre-deposited synthetic macromolecule polydopamine (PDA) as a demonstration. We examined the effects of a Ti-6Al-4V titanium alloy modified with fibrinogen (Ti-Fg), PDA (Ti-PDA) or their combination (Ti-PDA-Fg) on the biological sealing and integration with skin and bone tissues. Human epidermal keratinocytes (HaCaT), human foreskin fibroblasts (HFF) and preosteoblasts (MC3T3-E1), which are closely related to percutaneous implants, exhibited better adhesion and spreading on all the three modified sheets compared with the unmodified alloy. After three-week subcutaneous implantation in Sprague-Dawley (SD) rats, the Ti-PDA-Fg sheets could significantly attenuate the soft tissue response and promote angiogenesis compared with other groups. Furthermore, in the model of percutaneous tibial implantation in SD rats, the Ti-PDA-Fg screws dramatically inhibited epithelial downgrowth and promoted new bone formation. Hence, the covalent immobilization of fibrinogen through the precoating of PDA is promising for enhanced biological sealing and osseointegration of metal implants with soft and hard tissues, which is critical for an orthopedic percutaneous medical device.

Cell migration regulated by RGD nanospacing and enhanced under moderate cell adhesion on biomaterials.

While nanoscale modification of a biomaterial surface is known to influence various cell behaviors, it is unclear whether there is an optimal nanospacing of a bioactive ligand with respect to cell migration. Herein, we investigated the effects of nanospacing of arginine-glycine-aspartate (RGD) peptide on cell migration and its relation to cell adhesion. To this end, we prepared RGD nanopatterns with varied nanospacings (31-125 nm) against the nonfouling background of poly(ethylene glycol), and employed human umbilical vein endothelial cells (HUVECs) to examine cell behaviors on the nanopatterned surfaces. While HUVECs adhered well on surfaces of RGD nanospacing less than 70 nm and exhibited a monotonic decrease of adhesion with the increase of RGD nanospacing, cell migration exhibited a nonmonotonic change with the ligand nanospacing: the maximum migration velocity was observed around 90 nm of nanospacing, and slow or very slow migration occurred in the cases of small or large RGD nanospacings. Therefore, moderate cell adhesion is beneficial for fast cell migration. Further molecular biology studies revealed that attenuated cell adhesion and activated dynamic actin rearrangement accounted for the promotion of cell migration, and the genes of small G proteins such as Cdc42 were upregulated correspondingly. The present study sheds new light on cell migration and its relation to cell adhesion, and paves a way for designing biomaterials for applications in regenerative medicine.

Degradation rate affords a dynamic cue to regulate stem cells beyond varied matrix stiffness.

While various static cues such as matrix stiffness have been known to regulate stem cell differentiation, it is unclear whether or not dynamic cues such as degradation rate along with the change of material chemistry can influence cell behaviors beyond simple integration of static cues such as decreased matrix stiffness. The present research is aimed at examining effects of degradation rates on adhesion and differentiation of mesenchymal stem cells (MSCs) in vitro on well-defined synthetic hydrogel surfaces. Therefore, we synthesized macromers by extending both ends of poly(ethylene glycol) (PEG) with oligo(lactic acid) and then acryloyl, and the corresponding hydrogels that were obtained after photopolymerization of the macromers were biodegradable. Combining the unique techniques of block copolymer micelle nanolithography with transfer lithography, we prepared a nanoarray of cell-adhesive arginine-glycine-aspartate peptides on this nonfouling biodegradable hydrogel. The biodegradation is caused by hydrolysis of the ester bonds, and different degradation rates in the cell culture medium were achieved by different stages of accelerated pre-hydrolysis in an acidic medium. For the following cell culture and induction, both the matrix stiffness and degradation rate varied among the examined groups. While adipogenic differentiation of MSCs can be understood by the lowered stiffness, the osteogenic differentiation was contradictory with common sense because we found enhanced osteogenesis on soft hydrogels. Higher degradation rates were suggested to account for this interesting phenomenon in the sole osteogenic/adipogenic induction and even more complicated trends in the co-induction. Hence, the degradation rate is a dynamic cue influencing cell behaviors, which should be paid attention to for degradable biomaterials.

Subcellular cell geometry on micropillars regulates stem cell differentiation.

While various material factors have been shown to influence cell behaviors, recent studies started to pay attention to the effects of some material cues on "subcellular" geometry of cells, such as self-deformation of cell nuclei. It is particularly interesting to examine whether a self deformation happens discontinuously like a first-order transition and whether subcellular geometry influences significantly the extent of stem cell differentiation. Herein we prepared a series of micropillar arrays of poly(lactide-co-glycolide) and discovered a first-order transition of nuclear shape as a function of micropillar height under the examined section area and interspacing of the pillars. The deformed state of the nuclei of mesenchymal stem cells (MSCs) was well maintained even after osteogenic or adipogenic induction for several days. The nuclear deformation on the micropillar arrays was accompanied with smaller projected areas of cells, but led to an enhanced osteogenesis and attenuated adipogenesis of the MSCs, which is different from the previously known relationship between morphology and differentiation of stem cells on flat substrates. Hence, the present study reveals that the geometry of cell nuclei may afford a new cue to regulate the lineage commitment of stem cells on the subcellular level.

Adhesion, proliferation, and differentiation of mesenchymal stem cells on RGD nanopatterns of varied nanospacings.

The present report is an extension of our preceding publication in Biomaterials (2013) entitled "Effect of RGD nanospacing on differentiation of stem cells." Cell-adhesive peptide arginine-glycine-aspartate (RGD) was nanopatterned on a non-fouling poly(ethylene glycol) (PEG) hydrogel, and mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured on the patterned surfaces at nanospacings from 37 to 124 nm. Cell adhesion parameters such as spreading areas varied with RGD nanospacings significantly. The differences were well observed at both the first and eighth days, which confirmed the persistence of this nanospacing effect on our nanopatterns. The proliferation rate also varied with the nanospacings. Osteogenic and adipogenic inductions were undertaken, and a significant influence of RGD nanospacing on stem cell differentiation was found. The effect on differentiation cannot be simply interpreted by differences in cell adhesion and proliferation. We further calculated the fractions of single, coupled, and multiple cells on those nanopatterns, and ruled out the possibility that the extent of cell-cell contact determined the different differentiation fractions. Accordingly, we reinforced the idea that RGD nanospacing might directly influence stem cell differentiation.