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.

Mitochondrial fragmentation and donut formation enhance mitochondrial secretion to promote osteogenesis

AUTHORS

Joonho Suh, Na-Kyung Kim, Wonn Shim, Jae Hyuck Jang, Jung-Eun Kim, Yun-Sil Lee

ABSTRACT

Mitochondrial components have been abundantly detected in bone matrix, implying that they are somehow transported extracellularly to regulate osteogenesis. Here, we demonstrate that mitochondria and mitochondrial-derived vesicles (MDVs) are secreted from mature osteoblasts to promote differentiation of osteoprogenitors. We show that osteogenic induction stimulates mitochondrial fragmentation, donut formation, and secretion of mitochondria through CD38/cADPR signaling. Enhancing mitochondrial fission and donut formation through Opa1 knockdown or Fis1 overexpression increases mitochondrial secretion and accelerates osteogenesis. We also show that mitochondrial fusion promoter M1, which induces Opa1 expression, impedes osteogenesis, whereas osteoblast-specific Opa1 deletion increases bone mass. We further demonstrate that secreted mitochondria and MDVs enhance bone regeneration in vivo. Our findings suggest that mitochondrial morphology in mature osteoblasts is adapted for extracellular secretion, and secreted mitochondria and MDVs are critical promoters of osteogenesis.

Apoptotic Vesicles Regulate Bone Metabolism via the miR1324/SNX14/SMAD1/5 Signaling Axis

AUTHORS

Yuan Zhu, Kunkun Yang, Yawen Cheng, Yaoshan Liu, Ranli Gu, Xuenan Liu, Hao Liu, Xiao Zhang, Yunsong Liu

ABSTRACT

Mesenchymal stem cells (MSCs) are widely used in the treatment of diseases. After their in vivo application, MSCs undergo apoptosis and release apoptotic vesicles (apoVs). This study investigates the role of apoVs derived from human bone marrow mesenchymal stem cells (hBMMSCs) in bone metabolism and the molecular mechanism of the observed effects. The results show that apoVs can promote osteogenesis and inhibit osteoclast formation in vitro and in vivo. ApoVs may therefore attenuate the bone loss caused by primary and secondary osteoporosis and stimulate bone regeneration in areas of bone defect. The mechanisms responsible for apoV-induced bone regeneration include the release of miR1324, which inhibit expression of the target gene Sorting Nexin 14 (SNX14) and thus activate the SMAD1/5 pathway in target cells. Given that MSC-derived apoVs are easily obtained and stored, with low risks of immunological rejection and neoplastic transformation, The findings suggest a novel therapeutic strategy to treat bone loss, including via cell-free approaches to bone tissue engineering.

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.

VEGFA from osteoblasts is not required for lamellar bone formation following tibial loading

AUTHORS

Jennifer A. McKenzie, Ian M. Galbreath, Andre F. Coello, Katherine R. Hixon, Matthew J.Silva

ABSTRACT

The relationship between osteogenesis and angiogenesis is complex. Normal bone development requires angiogenesis, mediated by vascular endothelial growth factor A (VEGFA). Studies have demonstrated through systemic inhibition or genetic modification that VEGFA is indispensable for several types of bone repair, presumably via its role in supporting angiogenesis. But a direct role for VEGFA within osteoblasts, in the absence of angiogenesis, has also been suggested. To address the question of whether VEGFA from osteoblasts supports bone formation directly, we applied anabolic loading to induce lamellar bone formation in mice, a process shown to be independent of angiogenesis. We hypothesized that VEGFA from osteoblasts is required for lamellar bone formation. To test this hypothesis, we applied axial tibial compression to inducible Cre/LoxP mice from three lines. Vegfafl/fl mice were crossed with Ubiquitin C (UBC), Osterix (Osx) and Dentin-Matrix Protein 1 (DMP1) Cre-ERT2 mice to target all cells, (pre)osteoblast-lineage cells, and mature osteoblasts and osteocytes, respectively. Genotype effects were determined by comparing control (Vegfafl/fl) and Cre+ (VegfaΔ) mice for each line. At 5 months of age tamoxifen was injected for 5 days followed by a 3-week clearance prior to loading. Female and male mice (N = 100) were loaded for 5 days to peak forces to engender −3100 με peak compressive strain and processed for dynamic histomorphometry (day 12). Percent MS/BS increased 20–70 % as a result of loading, with no effect of genotype in Osx or Dmp1 lines. In contrast, the UBC groups had a significant decrease in relative periosteal BFR/BS in VegfaΔ vs. Vegfafl/fl mice. The UBC line did not have any cortical bone phenotype in non-loaded femurs. In summary, dynamic histomorphometry data confirmed that tibial loading induces lamellar bone formation. Contrary to our hypothesis, there was no decrease in loading-induced bone formation in the Osx or Dmp1 lines in the absence of VEGFA. There was a decrease in bone formation in the UBC line where all cells were targeted. This result indicates that VEGFA from a non-osteoblast cell source supports loading-induced lamellar bone formation, although osteoblast/osteocyte VEGFA is dispensable. These findings support a paracrine model whereby non-osteoblast VEGFA supports lamellar bone formation, independent of angiogenesis.

Effect of simvastatin on osteogenesis of the extremity bones in aging rats

AUTHORS

Mengran Wang, Haowei Li, Jiaxin Tang, Yue Xi, Shiyi Chen & Ming Liu

ABSTRACT

Purpose

Simvastatin is a prodrug of the potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. The main purpose of the current study is to assess the accurate function of simvastatin on osteoporosis of extremity bones in aging rats.

Materials and methods

Fifty 15-month-old SD rats were divided into five groups (four simvastatin groups and one control group). The rats in four simvastatin groups were fed with different doses of simvastatin (5, 10, 20, and 40 mg/kg/d, respectively) for 3 months, whereas the rats in control group were fed the equal physiological saline. Calcium (Ca), phosphorus (P), and the lipid spectrum in serum were measured. Biochemical markers of bone metabolism, osteocalcin (OC), and tartrate-resistant acid phosphatase (Trap-5b), were analyzed using ELISA. The content of adipocytes in bone marrow was analyzed by histological staining. Finally, the bone quality of the femur and tibia were evaluated using dual-energy X-ray absorptiometry (DEXA), peri-quantity CT (pQCT), and the 3-point bending biomechanical test.

Results

Simvastatin reduced serum triglycerides (TG), and 10 mg/kg/d of simvastatin significantly reduced the content of adipocytes in bone marrow compared to the control group. However, statistically significant differences between the simvastatin groups and the control group were not found in the CA, P, OC, Trap-5b, or the evaluation indexes of bone quality from DEXA, pQCT, and biomechanical tests.

Conclusion

Simvastatin could not prevent osteoporosis of the extremity bones in aging rats.