Large segmental bone regeneration remains a great challenge due to the lack of vascularization in newly formed bone. Conventional strategies primarily combine bone scaffolds with seed cells and growth factors to modulate osteogenesis and angiogenesis. Nevertheless, cell-based therapies have some intrinsic issues regarding immunogenicity, tumorigenesis, bioactivity and off-the-shelf transplantation. Exosomes are nano-sized (50-200 nm) extracellular vesicles with a complex composition of proteins, nucleic acids and lipids, which are attractive as therapeutic nanoparticles for disease treatment. Exosomes also have huge potential as desirable drug/gene delivery vectors in the field of regenerative medicine due to their excellent biocompatibility and efficient cellular internalization. Methods: We developed a cell-free tissue engineering system using functional exosomes in place of seed cells. Gene-activated engineered exosomes were constructed by using ATDC5-derived exosomes to encapsulate the VEGF gene. The specific exosomal anchor peptide CP05 acted as a flexible linker and effectively combined the engineered exosome nanoparticles with 3D-printed porous bone scaffolds. Results: Our findings demonstrated that engineered exosomes play dual roles as an osteogenic matrix to induce the osteogenic differentiation of mesenchymal stem cells and as a gene vector to controllably release the VEGF gene to remodel the vascular system. In vivo evaluation further verified that the engineered exosome-mediated bone scaffolds could effectively induce the bulk of vascularized bone regeneration. Conclusion: In our current work, we designed specifically engineered exosomes based on the requirements of vascularized bone repair in segmental bone defects. This work simultaneously illuminates the potential of functional exosomes in acellular tissue engineering.

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FLOWERING LOCUS T (FT) can promote early flowering in annual species, but such role has not been well demonstrated in woody species. We produced self and reciprocal grafts involving non-transgenic blueberry (NT) and transgenic blueberry (T) carrying a 35S-driven blueberry FT (VcFT-OX). We demonstrated that the transgenic VcFT-OX rootstock promoted flowering of non-transgenic blueberry scions in the NT (scion):T (rootstock) grafts. We further analyzed RNA-Seq profiles and six groups of phytohormones in both NT:T and NT:NT plants. We observed content changes of several hormone metabolites, in a descending order, in the transgenic NT:T, non-transgenic NT:T, and non-transgenic NT:NT leaves. By comparing differential expression transcripts (DETs) of these tissues in relative to their control, we found that the non-transgenic NT:T leaves had many DETs shared with the transgenic NT:T leaves, but very few with the transgenic NT:T roots. Interestingly, a number of these shared DETs belong to hormone pathway genes, concurring with the content changes of hormone metabolites in both transgenic and non-transgenic leaves of the NT:T plants. These results suggest that phytohormones induced by VcFT-OX in the transgenic leaves might serve as part of the signals that resulted in early flowering in both transgenic plants and the non-transgenic NT:T scions.

The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants. Mu-legacy is a valuable blueberry mutant, in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2). Mu-legacy plants, compared with nontransgenic ‘Legacy’ plants, show dwarfing, promotion of flower bud formation, and can flower under nonchilling conditions. We conducted transcriptomic comparisons in leaves, chilled and nonchilled flowering buds, and late-pink buds, and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and ‘Legacy’ plants. These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENT-COPALYL DIPHOSPHATE SYNTHETASE 1 (VcGA1), which induces new homeostasis of hormones, including increased gibberellin 4 (GA4) levels in Mu-legacy leaves. Consequently, increased expression of VcRR2 and VcGA1, which function in cytokinin responses and gibberellin synthesis, respectively, initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches. In nonchilled flower buds, 29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and ‘Legacy’ plants, of which 22 were chilling responsive. Thus, these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions. We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.

Zoysia matrella [L.] Merr. is a widely cultivated warm-season turf grass in subtropical and tropical areas. Dwarf varieties of Z. matrella are attractive to growers because they often reduce lawn mowing frequencies. In this study, we describe a dwarf mutant of Z. matrella induced from the 60Co-γ-irradiated calluses. We conducted morphological test and physiological, biochemical and transcriptional analyses to reveal the dwarfing mechanism in the mutant. Phenotypically, the dwarf mutant showed shorter stems, wider leaves, lower canopy height, and a darker green color than the wild type (WT) control under the greenhouse conditions. Physiologically, we found that the phenotypic changes of the dwarf mutant were associated with the physiological responses in catalase, guaiacol peroxidase, superoxide dismutase, soluble protein, lignin, chlorophyll, and electric conductivity. Of the four endogenous hormones measured in leaves, both indole-3-acetic acid and abscisic acid contents were decreased in the mutant, whereas the contents of gibberellin and brassinosteroid showed no difference between the mutant and the WT control. A transcriptomic comparison between the dwarf mutant and the WT leaves revealed 360 differentially-expressed genes (DEGs), including 62 up-regulated and 298 down-regulated unigenes. The major DEGs related to auxin transportation (e.g., PIN-FORMED1) and cell wall development (i.e., CELLULOSE SYNTHASE1) and expansin homologous genes were all down-regulated, indicating their potential contribution to the phenotypic changes observed in the dwarf mutant. Overall, the results provide information to facilitate a better understanding of the dwarfing mechanism in grasses at physiological and transcript levels. In addition, the results suggest that manipulation of auxin biosynthetic pathway genes can be an effective approach for dwarfing breeding of turf grasses.

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