A Surface-Mediated Biomimetic Porous Polyether-Ether-Ketone Scaffold for Regulating Immunity and Promoting Osteogenesis.

The repair of critical-sized bone defects remains a major challenge for clinical orthopedic surgery. Here, we develop a surface biofunctionalized three-dimensional (3D) porous polyether-ether-ketone (PEEK) scaffold that can simultaneously promote osteogenesis and regulate macrophage polarization. The scaffold is created using polydopamine (PDA)-assisted immobilization of silk fibroin (SF) and the electrostatic self-assembly of nanocrystalline hydroxyapatite (nano-HA) on a 3D-printed porous PEEK scaffold. The SF/nano-HA functionalized surface provides a bone-like microenvironment for osteoblastic cells' adhesion, proliferation, mineralization and osteogenic differentiation. Moreover, the biofunctionalized surface can effectively drive macrophages polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Integrin ß1-specific cell-matrix binding and the activation of Ca2+ receptor-mediated signaling pathway play critical roles in the regulation of macrophage polarization. Compared with the as-printed scaffold, the SF/nano-HA functionalized porous PEEK scaffold induces minimal inflammatory response, enhanced angiogenesis, and substantial new bone formation, resulting in improved osseointegration in vivo. This study not only develops a promising candidate for bone repair but also demonstrates a facile surface biofunctionalization strategy for orthopedic implants to improve osseointegration by stimulating osteogenesis and regulating immunity.

Fully Biodegradable Packaging Films for Fresh Food Storage Based on Oil-Infused Bacterial Cellulose.

Fully biodegradable packaging materials are demanded to resolve the issue of plastic pollution. However, the fresh food storage performance of biodegradable materials is generally much lower than that of plastics due to their high permeability, microbial friendliness, and limited stretchability and transparency. Here a biodegradable packaging material is reported with high fresh food storage performance based on an oil-infused bacterial cellulose (OBC) porous film. The oil infusion significantly improved cellulose's food-keeping performance by reducing its gas permeability, increasing its stretchability and transparency, and enabling the active release of green vapor-phase preservative molecules, while maintaining its intrinsically high degradability. Strawberries stored in a container with the OBC lid at 23 °C after 5 days exhibited a moldy rate of 0%, in contrast to the 100% moldy rate of those stored by poly(ethylene). Enhanced storage performance is also obtained on tomatoes, pork, and shrimp. The OBC film is naturally degraded after being buried in wet soil at 30 °C for 9 days, identical to the degradation rate of bacterial cellulose. The liquid seal strategy broadly applies to different celluloses, providing a general option for developing cellulose-based biodegradable packaging materials.

Scaffold-based non-viral CRISPR delivery platform for efficient and prolonged gene activation to accelerate tissue regeneration.

Clustered regularly interspaced short palindromic repeat activation (CRISPRa) technology has emerged as a precise genome editing tool for activating endogenous transgene expression. While it holds promise for precise cell modification, its translation into tissue engineering has been hampered by biosafety concerns and suboptimal delivery methods. To address these challenges, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. The Gel/NF scaffold facilitates the controlled and sustained release of CRISPRa complexes and also promotes cell recruitment to the scaffold for efficient and localized transfection. As a proof of concept, we employed this CRISPRa delivery platform to activate the vascular endothelial growth factor (VEGF) gene in a rat model with full-thickness skin defects. Our results demonstrate sustained upregulation of VEGF expression even at 21 days post-implantation, resulting in enhanced angiogenesis and improved skin regeneration. These findings underscore the potential of the Gel/NF scaffold-based CRISPRa delivery platform as an efficient and durable strategy for gene activation, offering promising prospects for tissue regeneration. STATEMENT OF SIGNIFICANCE: Translation of clustered regularly interspaced short palindromic repeat activation (CRISPRa) therapy to tissue engineering is limited by biosafety concerns and unsatisfactory delivery strategy. To solve this issue, we have developed a CRISPRa non-viral gene delivery platform by immobilizing non-viral CRISPRa complexes into a biocompatible hydrogel/nanofiber (Gel/NF) composite scaffold. This scaffold enables controlled and sustained release of CRISPRa and can induce cell recruitment for localized transfection. As a proof of concept, we activated vascular endothelial growth factor (VEGF) in a rat model with full-thickness skin defects, leading to sustained upregulation of VEGF expression, enhanced angiogenesis and improved skin regeneration in vivo. These findings demonstrate the potential of this platform for gene activation, thereby offering promising prospects for tissue regeneration.

Spatiotemporal Delivery of pBMP2 and pVEGF by a Core-Sheath Structured Fiber-Hydrogel Gene-Activated Matrix Loaded with Peptide-Modified Nanoparticles for Critical-Sized Bone Defect Repair.

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects remains a grand challenge. The gene-activated matrix (GAM) combining gene therapy and tissue engineering scaffold offers a promising strategy for the restoration of structure and function of damaged or dysfunctional tissues. Herein, a gene-activated biomimetic composite scaffold consisting of an electrospun poly(ε-caprolactone) fiber sheath and an alginate hydrogel core which carried plasmid DNA encoding bone morphogenetic protein 2 (pBMP2) and vascular endothelial growth factor (pVEGF), respectively, is developed. A peptide-modified polymeric nanocarrier with low cytotoxicity and high efficiency serves as the nonviral DNA delivery vector. The obtained GAM allows spatiotemporal release of pVEGF and pBMP2 and promotes osteogenic differentiation of preosteoblasts in vitro. In vivo evaluation using a critical-sized segmental femoral defect model in rats shows that the dual gene delivery system can significantly accelerate bone healing by activating angiogenesis and osteogenesis. These findings demonstrate the effectiveness of the developed dual gene-activated core-sheath structured fiber-hydrogel composite scaffold for critical-sized bone defect regeneration and the potential of cell-free scaffold-based gene therapy for tissue engineering.

Mouse Strain- and Charge-Dependent Vessel Permeability of Nanoparticles at the Lower Size Limit.

Remarkable advancement has been made in the application of nanoparticles (NPs) for cancer therapy. Although NPs have been favorably delivered into tumors by taking advantage of the enhanced permeation and retention (EPR) effect, several physiological barriers present within tumors tend to restrict the diffusion of NPs. To overcome this, one of the strategies is to design NPs that can reach lower size limits to improve tumor penetration without being rapidly cleared out by the body. Several attempts have been made to achieve this, such as selecting appropriate nanocarriers and modifying surface properties. While many studies focus on the optimal design of NPs, the influence of mouse strains on the effectiveness of NPs remains unknown. Therefore, this study aimed to assess whether the vascular permeability of NPs near the lower size limit differs among mouse strains. We found that the vessel permeability of dextran NPs was size-dependent and dextran NPs with a size below 15 nm exhibited leakage from postcapillary venules in all strains. Most importantly, the leakage rate of 8-nm fluorescein isothiocyanate dextran was significantly higher in the BALB/c mouse strain than in other strains. This strain dependence was not observed in slightly positive TRITC-dextran with comparable sizes. Our results indicate that the influence on mouse strains needs to be taken into account for the evaluation of NPs near the lower size limit.

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing.

Regulating cell function and tissue formation by combining gene delivery with functional scaffolds to create gene-activated matrices (GAMs) is a promising strategy for tissue engineering. However, fabrication of GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery properties remains a challenge. In this study, a non-viral DNA delivery nanocomplex was developed by modifying poly (D, L-lactic-co-glycolic acid)/polyethylenimine (PLGA/PEI) nanoparticles with the cell-penetrating peptide KALA. Subsequently, the nanocomplex carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF) was immobilized onto a polydopamine-coated electrospun alginate nanofibrous scaffold, resulting in a GAM for enhanced skin wound healing. The nanocomplex exhibited much lower cytotoxicity and comparable or even higher transfection efficiency compared with PEI. The GAM enabled sustained gene release and long-tern transgene expression of VEGF in vitro. In an excisional full-thickness skin wound rat model, the GAM could accelerate wound closure, promote complete re-epithelization, reduce inflammatory response, and enhance neovascularization, ultimately enhancing skin wound healing. The current GAM comprising a low-toxic gene delivery nanocomplex and a biocompatible 3D nanofibrous scaffold demonstrates great potential for mediating long-term cell functions and may become a powerful tool for gene delivery in tissue engineering. STATEMENT OF SIGNIFICANCE: Gene delivery is a promising strategy in promoting tissue regeneration as an effective alternative to growth factor delivery, but the study on three-dimensional gene-activated scaffolds remains in its infancy. Herein, a biodegradable nanofibrous gene-activated matrix integrating non-viral nanoparticle vector was designed and evaluated both in vitro and in vivo. The results show that the nanoparticle vector provided high transfection efficiency with minimal cytotoxicity. After surface immobilization of the nanocomplexes carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF), the nanofibrous scaffold enabled sustained DNA release and long-term transgene expression in vitro. In a rat full-thickness skin wound model, the scaffold could accelerate wound healing. This innovative gene-activated matrix can be a promising candidate for tissue regeneration.

Bioadhesive injectable hydrogel with phenolic carbon quantum dot supported Pd single atom nanozymes as a localized immunomodulation niche for cancer catalytic immunotherapy.

Immunotherapy is a powerful way to treat cancer, however, systemic treatment-associated adverse effects remain a major concern. In this study, a bioadhesive injectable hydrogel is developed to provide localized immune niches for tumor microenvironment immunomodulation and cancer catalytic immunotherapy. First, a phenolic single atom nanozyme (SAN) was developed by in situ synthesis of Pd single atom on catechol-grafted carbon-quantum-dot (DA-CQD@Pd) templates. Then, the bioadhesive injectable hydrogel consisting of DA-CQD@Pd SAN and immune adjuvant CpGODN was formed through SAN-catalyzed free-radical polymerization. The SAN exhibited peroxidase-like activity to generate ROS and kill tumor cells through catalytic therapy. The hydrogel locally released CpGODN in a sustained manner, which limited the risk of systemic exposure, reducing the impact of CpGODN toxicity, and protecting CpGODN from degradation. The bioadhesive hydrogel immobilized around solid tumor to provide an immune response site after injection. When combined it with the administration of immune checkpoint inhibitor anti-PD-L1, the hydrogel realized localized immunomodulation, maximized therapeutic efficacy and prevents tumor metastasis via a catalytic immunotherapy.

Mussel-Inspired Redox-Active and Hydrophilic Conductive Polymer Nanoparticles for Adhesive Hydrogel Bioelectronics.

Conductive polymers (CPs) are generally insoluble, and developing hydrophilic CPs is significant to broaden the applications of CPs. In this work, a mussel-inspired strategy was proposed to construct hydrophilic CP nanoparticles (CP NPs), while endowing the CP NPs with redox activity and biocompatibility. This is a universal strategy applicable for a series of CPs, including polyaniline, polypyrrole, and poly(3,4-ethylenedioxythiophene). The catechol/quinone contained sulfonated lignin (LS) was doped into various CPs to form CP/LS NPs with hydrophilicity, conductivity, and redox activity. These CP/LS NPs were used as versatile nanofillers to prepare the conductive hydrogels with long-term adhesiveness. The CP/LS NPs-incorporated hydrogels have a good conductivity because of the uniform distribution of the hydrophilic NPs in the hydrogel network, forming a well-connected electric path. The hydrogel exhibits long-term adhesiveness, which is attributed to the mussel-inspired dynamic redox balance of catechol/quinone groups on the CP/LS NPs. This conductive and adhesive hydrogel shows good electroactivity and biocompatibility and therefore has broad applications in electrostimulation of tissue regeneration and implantable bioelectronics.

Experimental and simulation studies of strontium/fluoride-codoped hydroxyapatite nanoparticles with osteogenic and antibacterial activities.

Multiple ions codoping may effectively modulate physicochemical and biological properties of hydroxyapatite (HA) for diverse biomedical applications. This study synthesized strontium (Sr)-, fluorine (F)- doped, and Sr/F-codoped HA nanoparticles by a hydrothermal method, and investigated the effect of ion doping on characteristics of HA, including crystallinity, crystal size, lattice parameters, and substitution sites by experiments and simulation with density functional theory (DFT) methods. It was found that, Sr doping increased the lattice parameters of HA whereas F doping decreased these parameters. Additionally, F doping enhanced the structural stability of the Sr-doped HA. F doping created excellent antibacterial properties to effectively inhibit growth of Streptococcus mutans. An appropriate Sr doping level endowed HA with optimum osteogenic ability to promote osteoblastic differentiation of bone marrow stem cells. These suggest that, Sr/F codoping is an effective approach to synthesizing HA-based materials with both antibacterial and osteogenic properties. More broadly, HA nanomaterials with specific characteristics may be designed for meeting diverse requirements from biomedical applications.

A Mussel-Inspired Persistent ROS-Scavenging, Electroactive, and Osteoinductive Scaffold Based on Electrochemical-Driven In Situ Nanoassembly.

Conductive polymers are promising for bone regeneration because they can regulate cell behavior through electrical stimulation; moreover, they are antioxidative agents that can be used to protect cells and tissues from damage originating from reactive oxygen species (ROS). However, conductive polymers lack affinity to cells and osteoinductivity, which limits their application in tissue engineering. Herein, an electroactive, cell affinitive, persistent ROS-scavenging, and osteoinductive porous Ti scaffold is prepared by the on-surface in situ assembly of a polypyrrole-polydopamine-hydroxyapatite (PPy-PDA-HA) film through a layer-by-layer pulse electrodeposition (LBL-PED) method. During LBL-PED, the PPy-PDA nanoparticles (NPs) and HA NPs are in situ synthesized and uniformly coated on a porous scaffold from inside to outside. PDA is entangled with and doped into PPy to enhance the ROS scavenging rate of the scaffold and realize repeatable, efficient ROS scavenging over a long period of time. HA and electrical stimulation synergistically promote osteogenic cell differentiation on PPy-PDA-HA films. Ultimately, the PPy-PDA-HA porous scaffold provides excellent bone regeneration through the synergistic effects of electroactivity, cell affinity, and antioxidative activity of the PPy-PDA NPs and the osteoinductivity of HA NPs. This study provides a new strategy for functionalizing porous scaffolds that show great promise as implants for tissue regeneration.

Cancellous-Bone-like Porous Iron Scaffold Coated with Strontium Incorporated Octacalcium Phosphate Nanowhiskers for Bone Regeneration.

The repair of large bone defects poses a grand challenge in tissue engineering. Thus, developing biocompatible scaffolds with mechanical and structural similarity to human cancellous bone is in great demand. Herein, we fabricated a three-dimensional (3D) porous iron (Fe) scaffold with interconnected pores via a template-assisted electrodeposition method. The porous Fe scaffold with a skeleton diameter of 143 µm had the porosity >90%, an average pore size of 345 µm, and a yield strength of 3.5 MPa. Such structure and mechanical strength were close to those of cancellous bone. In order to enhance the biocompatibility of the scaffold, strontium incorporated octacalcium phosphate (Sr-OCP) was coated on the skeletons of the porous Fe scaffold. The coated Sr-OCP was in the form of nanowhiskers with a mean diameter of 300 nm and length of 30 µm. Such Sr-OCP coating could effectively reduce the release rate of the Fe ions to a level which was safe for the human body. Both in vitro cytotoxicity tests by extraction method and direct contact assay confirmed that the Sr-OCP coating could promote the cell adhesion and substantially enhance the biocompatibility of the porous Fe scaffolds. Thus, the cancellous-bone-like porous structure with compatible mechanical properties and excellent biocompatibility enables the present Sr-OCP coated porous Fe scaffold to be a promising candidate for bone repair and regeneration.

Mussel-inspired graphene oxide nanosheet-enwrapped Ti scaffolds with drug-encapsulated gelatin microspheres for bone regeneration.

Graphene oxide (GO) attracts considerable attention for biomedical applications owing to its unique nanostructure and remarkable physicochemical characteristics. However, it is challenging to uniformly deposit GO on chemically inert Ti scaffolds, which have good biocompatibility and wide applications in bone engineering. In this study, a GO-functionalized Ti porous scaffold (GO/Ti scaffold) was prepared by depositing GO onto polydopamine (PDA) modified Ti scaffolds. The mussel-inspired PDA modification facilitated the interaction between GO and Ti surfaces, leading to a uniform coverage of GO on Ti scaffolds. BMP2 and vancomycin (Van) were separately encapsulated into gelatin microspheres (GelMS). Then, drug-containing GelMS were assembled on GO/Ti scaffolds and anchored by the functional groups of GO. The modified scaffold independently delivered multiple biomolecules with different physiochemical properties, without interfering with each other. Thus, the GO/Ti scaffold has the dual functions of inducing bone regeneration and preventing bacterial infection. In summary, this mussel-inspired GO/Ti hybrid scaffold combined the good mechanical properties of Ti scaffolds and the advantages of GO nanosheets. GO nanosheets with their unique nanostructure and functional groups, together with GelMS on Ti scaffolds, are suitable carriers for drug delivery and provide adhesive sites for cell adhesion and create nanostructured environments for bone regeneration.

Mussel-inspired nano-building block assemblies for mimicking extracellular matrix microenvironments with multiple functions.

The assembly of nano-building blocks is an effective way to produce artificial extracellular matrix microenvironments with hierarchical micro/nano structures. However, it is hard to assemble different types of nano-building blocks, to form composite coatings with multiple functions, by traditional layer-by-layer (LbL) self-assembly methods. Inspired by the mussel adhesion mechanism, we developed polydopamine (PDA)-decorated bovine serum albumin microspheres (BSA-MS) and nano-hydroxyapatite (nano-HA), and assembled them to form bioactive coatings with micro/nano structures encapsulating bone morphogenetic protein-2 (BMP-2). First, PDA-decorated nano-HA (nano-pHA) was obtained by oxidative polymerization of dopamine on nano-HA. Second, BMP-2-encapsulated BSA microspheres were prepared through desolvation, and then were also decorated by PDA (pBSA-MS). Finally, the nano-pHA and pBSA-MS were assembled using the adhesive properties of PDA. Bone marrow stromal cell cultures and in vivo implantation, showed that the pHA/pBSA (BMP-2) coatings can promote cell adhesion, proliferation, and benefited for osteoinductivity. PDA decoration was also applied to assemble various functional nanoparticles, such as nano-HA, polystyrene, and Fe3O4 nanoparticles. In summary, this study provides a novel strategy for the assembly of biofunctional nano-building blocks, which surpasses traditional LbL self-assembly of polyelectrolytes, and can find broad applications in bioactive agents delivery or multi-functional coatings.

Polydopamine mediated assembly of hydroxyapatite nanoparticles and bone morphogenetic protein-2 on magnesium alloys for enhanced corrosion resistance and bone regeneration.

Magnesium alloys have the great potential to be used as orthopedic implants due to their biodegradability and mechanical resemblance to human cortical bone. However, the rapid degradation in physiological environment with the evolution of hydrogen gas release hinders their clinical applications. In this study, we developed a novel functional and biocompatible coating strategy through polydopamine mediated assembly of hydroxyapatite nanoparticles and growth factor, bone morphogenetic protein-2 (BMP-2), onto the surface of AZ31 Mg alloys. Such functional coating has strong bonding with the substrate and can increase surface hydrophilicity of magnesium alloys. In vitro electrochemical corrosion and hydrogen evolution tests demonstrate that the coating can significantly enhance the corrosion resistance and therefore slow down the degradation of AZ31 Mg alloys. In vitro cell culture reveals that immobilization of HA nanoparticles and BMP-2 can obviously promote cell adhesion and proliferation. Furthermore, in vivo implantation tests indicate that with the synergistic effects of HA nanoparticles and BMP-2, the coating does not cause obvious inflammatory response and can significantly reduce the biodegradation rate of the magnesium alloys and induce the new bone formation adjacent to the implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2750-2761, 2017.

Biomimetic Mineralized Hierarchical Graphene Oxide/Chitosan Scaffolds with Adsorbability for Immobilization of Nanoparticles for Biomedical Applications.

Biomimetic calcium phosphate mineralized graphene oxide/chitosan (GO/CS) scaffolds with hierarchical structures were developed. First, GO/CS scaffolds with large micropores (∼300 µm) showed high mechanical strength due to the electrostatic interaction between the oxygen-containing functional groups of GO and the amine groups of CS. Second, octacalcuim phosphate (OCP) with porous structures (∼1 µm) was biomimetically mineralized on the surfaces of the GO/CS scaffolds (OCP-GO/CS). The hierarchical microporous structures of OCP-GO/CS scaffolds provide a suitable environment for cell adhesion and growth. The scaffolds have exceptional adsorbability of nanoparticles. Bone morphogenetic protein-2 (BMP-2)-encapsulated bovine serum albumin (BSA) nanoparticles and Ag nanoparticles (Ag-NPs) were adsorbed in the scaffolds for enhancement of osteoinductivity and antibacterial properties, respectively. Antibacterial tests showed that the scaffolds exhibited high antibacterial properties against both Escherichia coli and Staphylococcus epidermidis. In vitro and in vivo experiments revealed that the scaffolds have good biocompatibility, enhanced bone marrow stromal cells proliferation and differentiation, and induced bone tissue regeneration. Thus, the biomimetic OCP-GO/CS scaffolds with immobilized growth factors and antibacterial agents might be excellent candidates for bone tissue engineering.