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The technique bottleneck of repairing large bone defects with tissue engineered bone is the vascularization of tissue engineered grafts. Although some studies have shown that extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs) promote bone healing and repair by accelerating angiogenesis, the effector molecules and the mechanism remain unclear, which fail to provide ideas for the future research and development of cell-free interventions. Here, we found that Nidogen1-enriched EV (EV-NID1) derived from BMSCs interferes with the formation and assembly of focal adhesions (FAs) by targeting myosin-10, thereby reducing the adhesion strength of rat arterial endothelial cells (RAECs) to the extracellular matrix (ECM), and enhancing the migration and angiogenesis potential of RAECs. Moreover, by delivery with composite hydrogel, EV-NID1 is demonstrated to promote angiogenesis and bone regeneration in rat femoral defects. This study identifies the intracellular binding target of EV-NID1 and further elucidates a novel approach and mechanism, thereby providing a cell-free construction strategy with precise targets for the development of vascularized tissue engineering products.

The prevascularization of tissue-engineered bone grafts (TEBGs) has been shown to accelerate capillary vessel ingrowth in bone defect remodeling and to enhance new bone formation. However, the exact mechanisms behind this positive effect remain unknown. Here, we report that basic fibroblast growth factor (FGF2)-Ras homolog gene family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling functions as a molecular switch to regulate the lineage fate of bone mesenchymal stem cells (BMSCs) and that prevascularization promotes the cell fate switch, which contributes to increased bone regeneration with the use of prevascularized TEBGs compared with control TEBGs. Prevascularized TEBGs enhanced the in vivo endothelial differentiation of BMSCs by inhibiting RhoA/ROCK signaling. In vitro data more clearly showed that BMSCs differentiated into von Willebrand factor (vWF)-positive endothelial cells, and FGF2-induced inhibition of RhoA/ROCK signaling played a key role. Our novel findings uncovered a new mechanism that stimulates the increased vascularization of engineered bone and enhanced regeneration by promoting the endothelial differentiation of BMSCs implanted in TEBGs. These results offer a new molecular target to regulate TEBG-induced bone regeneration.

Yue Song,1,* Kaifeng Lin,2,* Shu He,3,* Chunmei Wang,1 Shuaishuai Zhang,1 Donglin Li,1 Jimeng Wang,4 Tianqing Cao,1 Long Bi,1 Guoxian Pei1 1Department of Orthopedics, Xijing Hospital, The Fourth Military Medical University, Xi’an, China; 2Second Department of Orthopedics and Traumatology, Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA, Fuzhou, China; 3Department of Orthopedics, Xi’an Hong Hui Hospital, Xi’an, China; 4Department of Orthopedics, The 251st Hospital of Chinese PLA, Zhangjiakou, China *These authors contributed equally to this work Background and aim: As a newly emerging three-dimensional (3D) printing technology, low-temperature robocasting can be used to fabricate geometrically complex ceramic scaffolds at low temperatures. Here, we aimed to fabricate 3D printed ceramic scaffolds composed of nano-biphasic calcium phosphate (BCP), polyvinyl alcohol (PVA), and platelet-rich fibrin (PRF) at a low temperature without the addition of toxic chemicals.Methods: Corresponding nonprinted scaffolds were prepared using a freeze-drying method. Compared with the nonprinted scaffolds, the printed scaffolds had specific shapes and well-connected internal structures.Results: The incorporation of PRF enabled both the sustained release of bioactive factors from the scaffolds and improved biocompatibility and biological activity toward bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. Additionally, the printed BCP/PVA/PRF scaffolds promoted significantly better BMSC adhesion, proliferation, and osteogenic differentiation in vitro than the printed BCP/PVA scaffolds. In vivo, the printed BCP/PVA/PRF scaffolds induced a greater extent of appropriate bone formation than the printed BCP/PVA scaffolds and nonprinted scaffolds in a critical-size segmental bone defect model in rabbits.Conclusion: These experiments indicate that low-temperature robocasting could potentially be used to fabricate 3D printed BCP/PVA/PRF scaffolds with desired shapes and internal structures and incorporated bioactive factors to enhance the repair of segmental bone defects. Keywords: three-dimensional printing, nano-biphasic calcium phosphate, polyvinyl alcohol, platelet-rich fibrin, bone substitutes, tissue engineering

Background Vascularization is one of the most important processes in tissue-engineered bone graft (TEBG)-mediated regeneration of large segmental bone defects. We previously showed that prevascularization of TEBGs promoted capillary vessel formation within the defected site and accelerated new bone formation. However, the precise mechanisms and contribution of endogenous cells were not explored. Methods We established a large defect (5 mm) model in the femur of EGFP+ transgenic rats and implanted a β-tricalcium phosphate (β-TCP) scaffold seeded with exogenous EGFP− cells; the femoral vascular bundle was inserted into the scaffold before implantation in the prevascularized TEBG group. Histopathology and scanning electron microscopy were performed and connective tissue growth factor (CTGF) and fibrin expression, exogenous cell survival, endogenous cell migration and behavior, and collagen type I and III deposition were assessed at 1 and 4 weeks post implantation. Results We found that the fibrinogen content can be increased at the early stage of vascular bundle transplantation, forming a fibrin reticulate structure and tubular connections between pores of β-TCP material, which provides a support for cell attachment and migration. Meanwhile, CTGF expression is increased, and more endogenous cells can be recruited and promote collagen synthesis and angiogenesis. By 4 weeks post implantation, the tubular connections transformed into von Willebrand factor-positive capillary-like structures with deposition of type III collagen, and accelerated angiogenesis of endogenous cells. Conclusions These findings demonstrate that prevascularization promotes the recruitment of endogenous cells and collagen deposition by upregulating fibrinogen and CTGF, directly resulting in new blood vessel formation. In addition, this molecular mechanism can be used to establish fast-acting angiogenesis materials in future clinical applications. Electronic supplementary material The online version of this article (10.1186/s13287-018-0925-y) contains supplementary material, which is available to authorized users.

Massive bone defects are a challenge in orthopaedic research. Defective regeneration leads to bone atrophy, non-union of bone, and physical morbidity. Large animals are important models, however, production costs are high, nursing is complex, and evaluation methods are limited. A suitable laboratory animal model is required to explore the underlying molecular mechanism and cellular process of bone tissue engineering. We designed a stainless steel plate with 8 holes; the middle 2 holes were used as a guide to create a standardized critical size defect in the femur of anaesthetized rats. The plate was fixed to the bone using 6 screws, serving as an inner fixed bracket to secure a tricalcium phosphate implant seeded with green fluorescent protein-positive rat bone marrow mesenchymal stem cells within the defect. In some animals, we also grafted a vessel bundle into the lateral side of the implant, to promote vascularized bone tissue engineering. X-ray, microcomputed tomography, and histological analyses demonstrated the stainless steel plate resulted in a stable large segmental defect model in the rat femur. Vascularization significantly increased bone formation and implant degradation. Moreover, survival and expansion of green fluorescent protein-positive seeded cells could be clearly monitored in vivo at 1, 4, and 8 weeks postoperation via fluorescent microscopy. This standardized large segmental defect model in a small animal may help to advance the study of bone tissue engineering. Furthermore, availability of antibodies and genetically modified rats could help to dissect the precise cellular and molecular mechanisms of bone repair.