Engineering 3D-Printed Strontium-Titanium Scaffold-Integrated Highly Bioactive Serum Exosomes for Critical Bone Defects by Osteogenesis and Angiogenesis

AUTHORS

Hao Liu, Ranli Gu, Wei Li, Lijun Zeng, Yuan Zhu, Boon Chin Heng, Yunsong Liu, and Yongsheng Zhou

ABSTRACT

Currently, healing of large bone defects faces significant challenges such as a bulk of bone regeneration and revascularization on the bone defect region. Here, a “cell-free scaffold engineering” strategy that integrates strontium (Sr) and highly bioactive serum exosomes (sEXOs) inside a three-dimensional (3D)-printed titanium (Ti) scaffold (Sc) is first developed. The constructed SrTi Sc can serve as a sophisticated biomaterial platform for maintaining bone morphological characteristics of the radius during the period of critical bone defect (CBD) repair and further accelerating bone formation and fibroblastic suppression via the controlled release of Sr from the superficial layer of the scaffold. Moreover, compared with sEXO from healthy donors, the sEXO extracted from the serum of the femoral fracture rabbit model at the stage of fracture healing, named BF EXO, is robustly capable of facilitating osteogenesis and angiogenesis. In addition, the underlying therapeutic mechanism is elucidated, whereby altering miRNAs shuttled by BF EXO enables osteogenesis and angiogenesis. Further, the in vivo study revealed that the SrTi Sc + BF EXO composite dramatically accelerated bone repair via osteoconduction, osteoinduction, and revascularization in radial CBD of rabbits. This study broadens the source and biomedical potential of specifically functionalized exosomes and provides a comprehensive clinically feasible strategy for therapeutics on large bone defects.

3D-printed Strontium-Titanium Scaffolds Incorporated with Highly Bioactive Serum Exosomes Promotes Critical Bone Defect Repair by Enhancing Osteogenesis and Angiogenesis

AUTHORS

Hao Liu, Ranli Gu, Wei Li, Lijun Zeng, Yuan Zhu, Siyi Wang, Xuenan Liu, Boon Chin Heng, Yunsong Liu, Yongsheng Zhou

ABSTRACT

Background

Large bone defect healing faces significant challenges because of inadequate bone regeneration and revascularization. Serum exosomes (sEXO) during bone defect repair are rich in osteogenic factors. Titanium (Ti) scaffolds and low dose strontium (Sr) can promote bone regeneration. Here, a “cell-free scaffold engineering” strategy that incorporates strontium and highly bioactive sEXO within a 3D-printed Ti scaffold is developed.

Methods

Sr-Ti-sEXO composite was prepared by ion implantation and ultra-high-speed centrifugation. Alkaline phosphatase (ALP), Alizarin red (ARS), immunofluorescence (IF) staining, and polymerase chain reaction (PCR) were used to detect the osteogenic effect of Sr-Ti-sExo on bone marrow mesenchymal stem cells (BMSCs). Tartrate-resistant acid phosphatase (TRAP) staining, and PCR were used to detect the osteoclast effect of Sr-Ti-sEXO on RAW264.7. The vascularization effect of Sr-Ti-sEXO on human umbilical vein endothelial cells (HUVECs) was investigated by scratch and migration experiments. Micro-CT and histological staining were used to study the osteogenic and vasculogenic effects of Sr-Ti-sEXO implanted in rabbit large radius defect at 6 and 12 weeks in vivo. RNA-seq was used to explore the potential mechanism.

Results

Sr-Ti-sEXO composite promoted early osteogenesis and inhibited osteoclast formation through the combined release of Sr ions and sEXO, and sustained release of Sr ions enhanced bone conduction, bone induction and inhibited fibroblasts. sEXO can promote the vascular reconstruction of CBD in fracture stage, which has the dual effect of promoting bone and promoting angiogenesis in critical bone defect repair. These effects are regulated by multiple miRNAs that shuttle in sEXO.

Conclusions

Sr-Ti-sEXO has favourable sustained release performance, osteogenic and vasogenic effects, which is a biocompatible and clinically feasible critical bone defect repair strategy. This study also broadens the biomedical potential of exosomes with specific functions such as sEXO in fracture stage. Based on the relative abundance of sEXO, a sEXO library for clinical treatment can be established.

Neutralizing antibody evasion and transduction with purified extracellular vesicle-enveloped AAV vectors.

AUTHORS

Dr. Ming Cheng, Ms. Laura Dietz, Dr. Yi Gong, Dr. Florian Eichler, Ms. Josette Nammour, Miss Carrie Ng, Dr. Dirk Grimm, and Dr. Casey A Maguire

ABSTRACT

Adeno-associated virus (AAV) is classified as a non-enveloped DNA virus. However, several years ago we discovered that in media of packaging cells producing recombinant AAV vectors, AAV capsids can associate with the interior and surface of extracellular vesicles (EVs), sometimes referred to as exosomes. Since then we and others have demonstrated that exosome-enveloped AAV, exo-AAV, can enhance transduction in vivo as well as evade neutralizing antibodies. While promising, these data were generated with differential centrifugation to pellet the exo-AAV. This method results in a heterogeneous mixture of exo-AAV, co-precipitating proteins, as well as free AAV capsids. To define the properties of exo-AAV more accurately, here we used a density gradient method to purify exo-AAV. We next performed head-to-head comparisons of standard AAV1, differential centrifuged exo-AAV1, and gradient purified exo-AAV1 for antibody evasion and transgene expression in the murine brain. We found purified exo-AAV1 to be more resistant to neutralizing antibodies than the other AAV preparations. Direct intracranial injection of purified exo-AAV1 into mice resulted in robust transduction, which transduced a larger area of brain than standard AAV1. We also identified the recently described membrane-associated accessory protein (MAAP) by mass spectrometry of purified exo-AAV1 preparations. Finally, we used a scalable method, size-exclusion chromatography to isolate exo-AAV1, and demonstrated functional transduction in cultured cells and increased antibody resistance. Together, these data suggest that higher purity exo-AAV will have beneficial characteristics for gene delivery and also may lead to mechanistic insights into the incorporation of AAV into EVs.