The current treatments for wounds often fail to induce adequate healing, leaving wounds vulnerable to persistent infections and development of drug-resistant microbial biofilms. New natural-derived nanoparticles were studied to impair bacteria colonization and hinder the formation of biofilms in wounds. The nanoparticles were fabricated through polyelectrolyte complexation of chitosan (CS, polycation) and hyaluronic acid (HA, polyanion). UV-induced photo-crosslinking was used to enhance the stability of the nanoparticles. To achieve this, HA was methacrylated (HAMA, degree of modification of 20 %). Photo-crosslinked nanoparticles obtained from HAMA and CS had a diameter of 478 nm and a more homogeneous size distribution than nanoparticles assembled solely through complexation (742 nm). The nanoparticles were loaded with the antimicrobial agent bacitracin (BC), resulting in nanoparticles with a diameter of 332 nm. The encapsulation of BC was highly efficient (97 %). The BC-loaded nanoparticles showed significant antibacterial activity against gram-positive bacteria Staphylococcus aureus, Methicillin-resistant S. aureus and S. epidermidis. Photo-crosslinked HAMA/CS nanoparticles loaded with BC demonstrated inhibition of biofilm formation and a positive effect on the proliferation of mammalian cells (L929). These crosslinked nanoparticles have potential for the long-term treatment of wounds and controlled antibiotic delivery at the location of a lesion.

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

The dynamic nature of the extracellular matrix (ECM), particularly its stiffness, plays a pivotal role in cellular behavior, especially after myocardial infarction (MI), where cardiac fibroblasts (cFbs) are key in ECM remodeling. This study explores the effects of dynamic stiffness changes on cFb activation and ECM production, addressing a gap in understanding the dynamics of ECM stiffness and their impact on cellular behavior. Utilizing gelatin methacrylate (GelMA) hydrogels, we developed a model to dynamically alter the stiffness of cFb environment through a two-step photocrosslinking process. By inducing a quiescent state in cFbs with a TGF-β inhibitor, we ensured the direct observation of cFbs-responses to the engineered mechanical environment. Our findings demonstrate that the mechanical history of substrates significantly influences cFb activation and ECM-related gene expression. Cells that were initially cultured for 24 h on the soft substrate remained more quiescent when the hydrogel was stiffened compared to cells cultured directly to a stiff static substrate. This underscores the importance of past mechanical history in cellular behavior. The present study offers new insights into the role of ECM stiffness changes in regulating cellular behavior, with significant implications for understanding tissue remodeling processes, such as in post-MI scenarios.

Critical size bone defects cannot heal without aid and current clinical approaches exhibit some limitations, underling the need for novel solutions. Silk fibroin, derived from silkworms, is widely utilized in tissue engineering and regenerative medicine due to its remarkable properties, making it a promising candidate for bone tissue regeneration in vitro and in vivo. However, the clinical translation of silk-based materials requires refinements in 3D architecture, stability, and biomechanical properties. In earlier research, improved mechanical resistance and stability of chemically crosslinked methacrylate silk fibroin (Sil-Ma) sponges over physically crosslinked counterparts were highlighted. Furthermore, the influence of photo-initiator and surfactant concentrations on silk properties was investigated. However, the characterization of sponges with Sil-Ma solution concentrations above 10 % (w/V) was hindered by production optimization challenges, with only cell viability assessed. This study focuses on the evaluation of methacrylate sponges\' suitability as temporal bone tissue regeneration scaffolds. Sil-Ma sponge fabrication at a fixed concentration of 20 % (w/V) was optimized and the impact of photo-initiator (LAP) concentrations and surfactant (Tween 80) presence/absence was studied. Their effects on pore formation, silk secondary structure, mechanical properties, and osteogenic differentiation of hBM-MSCs were investigated. We demonstrated that, by tuning silk sponges\' composition, the optimal combination boosted osteogenic gene expression, offering a strategy to tailor biomechanical properties for effective bone regeneration. Utilizing Design of Experiment (DoE), correlations between sponge composition, porosity, and mechanical properties are established, guiding tailored material outcomes. Additionally, correlation matrices elucidate the microstructure\'s influence on gene expressions, providing insights for personalized approaches in bone tissue regeneration.

Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan-protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan-protein networks and is poised to help elucidate the glycan-protein interactome, providing novel targets for therapeutic intervention.

Osteochondral tissue (OC) repair remains a significant challenge in the field of musculoskeletal tissue engineering. OC tissue displays a gradient structure characterized by variations in both cell types and extracellular matrix components, from cartilage to the subchondral bone. These functional gradients observed in the native tissue have been replicated to engineer OC tissuein vitro. While diverse fabrication methods have been employed to create these microenvironments, emulating the natural gradients and effective regeneration of the tissue continues to present a significant challenge. In this study, we present the design and development of CMC-silk interpenetrating (IPN) hydrogel with opposing dual biochemical gradients similar to native tissue with the aim to regenerate the complete OC unit. The gradients of biochemical cues were generated using an in-house-built extrusion system. Firstly, we fabricated a hydrogel that exhibits a smooth transition of sulfated carboxymethyl cellulose (sCMC) and TGF-β1 (SCT gradient hydrogel) from the upper to the lower region of the IPN hydrogel to regenerate the cartilage layer. Secondly, a hydrogel with a hydroxyapatite (HAp) gradient (HAp gradient hydrogel) from the lower to the upper region was fabricated to facilitate the regeneration of the subchondral bone layer. Subsequently, we developed a dual biochemical gradient hydrogel with a smooth transition of sCMC + TGF-β1 and HAp gradients in opposing directions, along with a blend of both biochemical cues in the middle. The results showed that the dual biochemical gradient hydrogels with biochemical cues corresponding to the three zones (i.e. cartilage, interface and bone) of the OC tissue led to differentiation of bone-marrow-derived mesenchymal stem cells to zone-specific lineages, thereby demonstrating their efficacy in directing the fate of progenitor cells. In summary, our study provided a simple and innovative method for incorporating gradients of biochemical cues into hydrogels. The gradients of biochemical cues spatially guided the differentiation of stem cells and facilitated tissue growth, which would eventually lead to the regeneration of the entire OC tissue with a smooth transition from cartilage (soft) to bone (hard) tissues. This promising approach is translatable and has the potential to generate numerous biochemical and biophysical gradients for regeneration of other interface tissues, such as tendon-to-muscle and ligament-to-bone.

Limited bone regeneration, uncontrollable degradation rate, mismatched defect zone and poor operability have plagued the reconstruction of irregular bone defect by tissue-engineered materials. A combination of biomimetic scaffolds with hydroxyapatite has gained great popularity in promoting bone regeneration. Therefore, we designed an injectable, photocurable and in-situ curing hydrogel by methacrylic anhydride -modified carboxymethyl cellulose (CMC-MA) loading with spherical hydroxyapatite (HA) to highly simulate the natural bony matrix and match any shape of damaged tissue. The prepared carboxymethyl cellulose-methacrylate/ hydroxyapatite(CMC-MA/HA) composite presented good rheological behavior, swelling ratio and mechanical property under light illumination. Meanwhile, this composite hydrogel promoted effectively proliferation, supported adhesion and upregulated the osteogenic-related genes expression of MC3T3-E1 cells in vitro, as well as the activity of the osteogenic critical protein, Integrin α1, β1, Myosin 9, Myosin 10, BMP-2 and Smad 1 in Integrin/BMP-2 signal pathway. Together, the composite hydrogels realized promotion of bone regeneration, deformity improvement, and the enhanced new bone strength in skull defect. It also displayed a good histocompatibility and stability of subcutaneous implantation in vivo. Overall, this study laid the groundwork for future research into developing a novel biomaterial and a minimally invasive therapeutic strategies for reconstructing bone defects and contour deficiencies.

Versatile and efficient regulation of the mechanical properties of the extracellular matrix is crucial not only for understanding the dynamic changes in biological systems, but also for obtaining precise and effective cellular responses in drug testing. In this study, we developed a well plate-based hydrogel photo-crosslinking system to effectively control the mechanical properties of hydrogels and perform high-throughput assays. We improved cell biocompatibility by using gelatin methacryloyl (GelMA) with a visible light photo-crosslinking method. Multiple cell-laden GelMA hydrogels were simultaneously and uniformly created using multi-arrayed 520 nm light-emitting diodes in a well plate format. The elastic modulus of the hydrogels can be widely adjusted (0.5-30 kPa) using a photo-crosslinking system capable of independently controlling the light intensity or exposure time for multiple samples. We demonstrate the feasibility of our system by observing enhanced bone differentiation of human mesenchymal stem cells (hMSCs) cultured on stiffer hydrogels. Additionally, we observed that the osteogenic fate of hMSCs, affected by the different mechanical properties of the gel, was regulated by parathyroid hormone (PTH). Notably, in response to PTH, hMSCs in a high-stiffness microenvironment upregulate osteogenic differentiation while exhibiting increased proliferation in a low-stiffness microenvironment. Overall, the developed system enables the generation of multiple cell-laden three-dimensional cell culture models with diverse mechanical properties and holds significant potential for expansion into drug testing.

We herein report a method for site-selective photo-crosslinking of a DNA duplex. A stilbene pair was introduced into a DNA duplex and a ruthenium complex was conjugated with a triplex-forming oligonucleotide. We demonstrated that [2+2] photocycloaddition of the stilbene pair occurred upon irradiation with visible light when the ruthenium complex was in close proximity due to triplex formation. No reaction occurred when the ruthenium complex was not in proximity to the stilbene pair. The wavelength of visible light used was of lower energy than the wavelength of UV light necessary for direct excitation of stilbene. Quantum chemical calculation indicated that ruthenium complex catalyzed the photocycloaddition via triplet-triplet energy transfer. Site selectivity of this photo-crosslinking system was evaluated using a DNA duplex bearing two stilbene pairs as a substrate; we showed that the site of crosslinking was precisely regulated by the sequence of the oligonucleotide linked to the ruthenium complex. Since this method does not require orthogonal photoresponsive molecules, it will be useful in construction of complex photoresponsive DNA circuits, nanodevices and biological tools.

Porous hydrogels as scaffolds have great potential in tissue engineering. However, there are still challenges in preparing porous hydrogels with tunable pore size and controlled porosity. Here, we successfully established a photoinduced gas-foaming method of porous hydrogels with controlled macro-micro-nano multiscale. A diazirine (DZ)-modified gelatin (GelDZ) biomaterial was prepared by introducing photocrosslinked DZ group into gelatin. Upon exposure to 365 nm UV light, DZ could be converted to the active group carbene, which could randomly insert into OH, NH, or CH bonds to form covalent crosslinks. GelDZ generated N2 by photodegradation and formed gas-induced porous hydrogels by intermolecular crosslinking without initiator. The loose porous structure of the hydrogel can promote the infiltration of host cells and blood vessels, which was conducive to tissue repair. The interfacial crosslinking of photoactivated GelDZ with tissue proteins imparted adhesion properties to the hydrogel. GelDZ also possessed photoreduction ability, which can reduce silver ions from metal precursors to silver nanoparticles (Ag NPs) in situ, and showed great antibacterial activity due to the sustained release of Ag NPs. GelDZ-Ag NPs prepared by in situ photoreaction can effectively inhibit wound infection and promote skin wound healing, providing a new strategy for designing porous hydrogel in tissue engineering.