The fibroblast is one of the ideal target cell candidates for cell-based gene therapy approaches to promote tissue repair. Gene delivery to fibroblasts by viral transfection has been confirmed to have high transfection efficiency. However, in addition to immunogenic effects of viruses, the random integration of viral genes may damage the genome, affect the cell phenotype or even cause cancerous mutations in the transfected cells. Due to these potential biohazards and unknown long-term risks, the clinical use of viral transfection has been very limited. In contrast, initial non-viral transfection methods have been simple and safe to implement, with low immunogenicity, insertional mutagenesis, and risk of carcinogenesis, but their transfection efficiency has been relatively low. Nucleofection, a more recent non-viral transfection method, now combines the advantages of high transfection efficiency and direct nucleic acid delivery to the nucleus with a high safety.Here, we reviewed recent articles on fibroblast nucleofection, summarized different research points, improved methods and application scopes, and opened up ideas for promoting the further improvement and development of fibroblast nucleofection to meet the needs of a variety of disease research and clinical applications.

Osteoporosis is a systemic and endocrine bone disorder distinguished by declined bone mineral density, compromised bone strength, and destruction of trabecular structure. The abnormally excessive osteoclastogenesis and bone erosion play imperative roles in the progression of osteoporosis. However, treatment of osteoporosis is far from satisfactory due to poor adherence to existing medications and adverse reactions, there is an urgent to develop novel therapies for osteoporosis. Probucol, a synthetic compound with two characteristic phenolic rings, owns anti-inflammatory and antioxidant properties. Accumulating evidence have indicated that intracellular reactive oxygen species (ROS) is closely related to osteoclastogenesis. Hence, we investigated the potential effects of probucol on osteoclastogenesis in vivo and in vitro. In this study, TRAP staining and bone slice resorption assay showed that probucol suppressed RANKL-induced osteoclast formation and function. The mRNA and protein levels of osteoclastogenesis marker genes were reduced by probucol in a concentration-dependent manner. Besides, probucol suppressed osteoclast differentiation by inhibiting ROS production, MAPKs and NF-κB signaling pathways, while Nrf2 silencing reversed the inhibitory effect of probucol on osteoclast formation and function. Consistent with the above findings, in vivo experiments demonstrated that probucol visibly alleviated bone loss caused by estrogen deficiency. In brief, these results showed the potential of anti-oxidant compound probucol in the treatment of osteoporosis, highlighting Nrf2 as a promising target in osteoclast-related disease.

The treatment of infected bone defects remains challenging due to the dynamic coexistence of bacterial infection, dysregulated inflammation, and impaired tissue regeneration, which demands spatiotemporally coordinated immunomodulatory interventions. Here, we develop a bilayer self-assembling polypeptide hydrogel that enables the spatiotemporal delivery of distinct hepcidin forms to reprogram macrophage responses during different phases of infection and repair. The upper layer, consisting of a PC₁₀A-Hamp hydrogel loaded with hepcidin-overexpressing mesenchymal stem cells (PHMH), undergoes rapid degradation to provide early-stage antimicrobial immune activation, effectively eliminating bacteria, inhibiting biofilm formation, and scavenging excessive reactive oxygen species. In contrast, the lower layer, a PC₁₀A-RGD hydrogel loaded with hepcidin-engineered extracellular vesicles (PREH), enables sustained release to promote M2 macrophage polarization and support osteogenic and angiogenic regeneration. In vitro studies reveal that hepcidin enhances osteogenic differentiation of MSCs via activation of the p38/ERK pathway and promotes angiogenesis through the PI3K/AKT pathway. In vivo, the PHMH/PREH hydrogel demonstrates robust therapeutic efficacy in an infected bone defect model, achieving effective infection control and accelerated bone repair. Mechanistically, spatiotemporal hepcidin delivery orchestrates phase-adaptive macrophage reprogramming by maintaining M1 polarization during the infectious phase while driving Twist1-mediated M2 polarization during the resolution phase. Collectively, this bilayer hydrogel platform establishes a phase-adaptive immunomodulatory delivery strategy and offers a promising therapeutic approach for infected bone defect repair.