3D-printed biodegradable magnesium alloy scaffolds with zoledronic acid-loaded ceramic composite coating promote osteoporotic bone defect repair.

Osteoporotic fracture is one of the most serious complications of osteoporosis. Most fracture sites have bone defects, and restoring the balance between local osteogenesis and bone destruction is difficult during the repair of osteoporotic bone defects. In this study, we successfully fabricated three-dimensional (3D)-printed biodegradable magnesium alloy (Mg-Nd-Zn-Zr) scaffolds and prepared a zoledronic acid-loaded ceramic composite coating on the surface of the scaffolds. The osteogenic effect of Mg and the osteoclast inhibition effect of zoledronic acid were combined to promote osteoporotic bone defect repair. In vitro degradation and drug release experiments showed that the coating significantly reduced the degradation rate of 3D-printed Mg alloy scaffolds and achieved a slow release of loaded drugs. The degradation products of drug-loaded coating scaffolds can promote osteogenic differentiation of bone marrow mesenchymal stem cells as well as inhibit the formation of osteoclasts and the bone resorption by regulating the expression of related genes. Compared with the uncoated scaffolds, the drug-coated scaffolds degraded at a slower rate, and more new bone grew into these scaffolds. The healing rate and quality of the osteoporotic bone defects significantly improved in the drug-coated scaffold group. This study provides a new method for theoretical research and clinical treatment using functional materials for repairing osteoporotic bone defects.

3D-printed vascularized biofunctional scaffold for bone regeneration.

3D-printed biofunctional scaffolds have promising applications in bone tissue regeneration. However, the development of bioinks with rapid internal vascularization capabilities and relatively sustained osteoinductive bioactivity is the primary technical challenge. In this work, we added rat platelet-rich plasma (PRP) to a methacrylated gelatin (GelMA)/methacrylated alginate (AlgMA) system, which was further modified by a nanoclay, laponite (Lap). We found that Lap was effective in retarding the release of multiple growth factors from the PRP-GelMA/AlgMA (PRP-GA) hydrogel and sustained the release for up to 2 weeks. Our in vitro studies showed that the PRP-GA@Lap hydrogel significantly promoted the proliferation, migration, and osteogenic differentiation of rat bone marrow mesenchymal stem cells, accelerated the formation of endothelial cell vascular patterns, and promoted macrophage M2 polarization. Furthermore, we printed hydrogel bioink with polycaprolactone (PCL) layer-by-layer to form active bone repair scaffolds and implanted them in subcutaneous and femoral condyle defects in rats. In vivo experiments showed that the PRP-GA@Lap/PCL scaffolds significantly promoted vascular inward growth and enhanced bone regeneration at the defect site. This work suggests that PRP-based 3D-bioprinted vascularized scaffolds will have great potential for clinical translation in the treatment of bone defects.

Effects of astragaloside IV on glucocorticoid-induced avascular necrosis of the femoral head via regulating Akt-related pathways.

We investigated the role of astragaloside IV (AS-IV) in preventing glucocorticoid-induced avascular necrosis of the femoral head (ANFH) and the underlying molecular mechanisms. Network pharmacology was used to predict the molecular targets of AS-IV. Molecular dynamic simulations were performed to explore the binding mechanism and interaction mode between AS-IV and Akt. Rat models of glucocorticoid-induced ANFH with AS-IV intervention were established, and osteogenesis, angiogenesis, apoptosis and oxidative stress were evaluated before and after blocking the PI3K/Akt pathway with LY294002. The effects of glucocorticoid and AS-IV on bone marrow mesenchymal stem cells and human umbilical vein endothelial cells incubated with and without LY294002 were determined. Downregulated p-Akt expression could be detected in the femoral heads of glucocorticoid-induced ANFH patients and rats. AS-IV increased trabecular bone integrity and vessel density of the femoral head in the model rats. AS-IV increased Akt phosphorylation and upregulated osteogenesis-, angiogenesis-, apoptosis- and oxidative stress-related proteins and mRNA and downregulated Bax, cleaved caspase-3 and cytochrome c levels. AS-IV promoted human umbilical vein endothelial cell migration, proliferation and tube formation ability; bone marrow mesenchymal stem cell proliferation; and osteogenic differentiation under glucocorticoid influence. AS-IV inhibited apoptosis. LY294002 inhibited these effects. AS-IV prevented glucocorticoid-induced ANFH by promoting osteogenesis and angiogenesis via the Akt/Runx2 and Akt/HIF-1α/VEGF pathways, respectively, and suppressing apoptosis and oxidative stress via the Akt/Bad/Bcl-2 and Akt/Nrf2/HO-1 pathways, respectively.

The first 3D-bioprinted personalized active bone to repair bone defects: A case report.

The repair and reconstruction of bone defects are still major problems to be solved in the field of orthopedics. Meanwhile, 3D-bioprinted active bone implants may provide a new and effective solution. In this case, we used bioink prepared from the patient's autologous platelet-rich plasma (PRP) combined with polycaprolactone/ß-tricalcium phosphate (PCL/ß-TCP) composite scaffold material to print personalized PCL/ß-TCP/PRP active scaffolds layer by layer through 3D bioprinting technology. The scaffold was then applied in the patient to repair and reconstruct bone defect after tibial tumor resection. Compared with traditional bone implant materials, 3D-bioprinted personalized active bone will have significant clinical application prospects due to its advantages of biological activity, osteoinductivity, and personalized design.

Mechanosensitive miR-99b mediates the regulatory effect of matrix stiffness on bone marrow mesenchymal stem cell fate both in vitro and in vivo.

Mechanical signals from extracellular matrix stiffness are important cues that regulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the incorporation of BMSCs into soft hydrogels and the dominance of soft matrices for BMSC growth and differentiation limit the directed differentiation of BMSCs incorporated into hydrogels for tissue engineering, especially osteogenesis. Here, we found that the expression of miR-99b increased with increasing hydrogel stiffness and that miR-99b regulated the proliferation and differentiation of BMSCs seeded on the surface of substrates with different stiffnesses. Furthermore, miR-99b significantly promoted the migration of BMSCs in 3D hydrogels. Mechanistically, we demonstrated that matrix stiffness-sensitive miR-99b targets the mammalian target of the rapamycin signaling pathway to regulate the adipogenic and osteogenic differentiation of BMSCs. In addition, by modulating the expression of miR-99b, the osteogenic differentiation of BMSCs in soft 3D hydrogels was promoted. Consistently, the flexible BMSC-GelMA hydrogel transfected with miR-99b significantly promoted bone regeneration in the rat calvarial defect area. These results suggest that miR-99b plays a key role in the mechanotransduction and phenotypic transformation of BMSCs and may inspire new tissue engineering applications with MSCs as key components.

Niobium promotes fracture healing in rats by regulating the PI3K-Akt signalling pathway: An in vivo and in vitro study.

Background: Stable fixation is crucial in fracture treatment. Currently, optimal fracture fixation devices with osteoinductivity, mechanical compatibility, and corrosion resistance are urgently needed for clinical practice. Niobium (Nb), whose mechanical properties are similar to those of bone tissue, has excellent biocompatibility and corrosion resistance, so it has the potential to be the most appropriate fixation material for internal fracture treatment. However, not much attention has been paid to the use of Nb in the area of clinical implants. Yet its role and mechanism of promoting fracture healing remain unclear. Hence, this study aims at elucidating on the effectiveness of Nb by systematically evaluating its osteogenic performance via in vivo and ex vivo tests. Methods: Systematic in vivo and in vitro experiments were conducted to evaluate the osteogenic properties of Nb. In vitro experiments, the biocompatibility and osteopromoting activity of Nb were assessed. And the osteoinductive activity of Nb was assessed by alizarin red, ALP staining and PCR test. In vivo experiments, the effectiveness and biosafety of Nb in promoting fracture healing were evaluated using a rat femoral fracture model. Through the analysis of gene sequencing results of bone scab tissues, the upregulation of PI3K-Akt pathway expression was detected and it was verified by histochemical staining and WB experiments. Results: Experiments in this study had proved that Nb had excellent in-vitro cell adhesion and proliferation-promoting effects without cytotoxicity. In addition, ALP activity, alizarin red staining and semi-quantitative analysis in the Nb group had indicated its profound impact on enhancing osteogenic differentiation of MC3T3-E1 cells. We also found that the use of Nb implants can accelerate fracture healing compared to that with Ti6Al4V using an animal model of femur fracture in rats, and the biosafety of Nb was confirmed in vivo via histological evaluation. Furthermore, we found that the osteogenic effects of Nb were achieved through activation of the PIK/Akt3 signalling pathway. Conclusion: As is shown in the present research, Nb possessed excellent biosafety in clinical implants and accelerated fracture healing by activating the PI3K-Akt signalling pathway, which had good prospects for clinical translation, and it can replace titanium alloy as a material for new functional implants.

Comprehensive Analysis of Regulatory Networks of m6A Regulators and Reveals Prognosis Biomarkers in Sarcoma.

Sarcomas are rare malignant tumors that may arise from anywhere of the body, such as bone, adipose, muscle and vascular. However, the conventional pathogenesis of sarcomas has not been found. Therefore, there is an urgent need to identify novel therapeutic strategies and improve prognosis effects for sarcomas. Methylation of N6 adenosine (m6A) regulation is a novel proposed regulatory pattern that works in post-transcription level, which was also the most widely distributed methylation modification in eukaryotic mRNA. Growing evidences have demonstrated that m6A modification played an indispensable role in tumorigenesis. Here, we integrated multi-omics data including genetic alterations, gene expression and epigenomics regulation to systematically analysis the regulatory atlas of 21 m6A regulators in sarcoma. Firstly, we investigated the genetic alterations of m6A regulators and found that ~44% TCGA sarcoma patients have genetic mutations. We also investigated the basic annotation of 21 regulators, such as expression correlation and PPI interactions. Then we identified the upstream and downstream regulatory networks of between transcription factors (TFs)/non-coding RNAs and m6A regulators in sarcoma based on motif analysis and gene expression. These results implied that m6A regulator mediated regulatory axes could be used as prognostic biomarkers in sarcoma. Knockdown experiment results revealed that m6A regulators, YTHDF2 and HNRNPA2B1 participated in the cancer cell invasion and metastasis. Moreover, we also found that the expression levels of m6A regulators were related to immune cell infiltration of sarcoma patients.

MicroRNA-365 promotes the contractile phenotype of venous smooth muscle cells and inhibits neointimal formation in rat vein grafts.

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA-365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus-mediated miR-365 was able to attenuate neointimal formation in rat vein grafts. We found that miR-365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced grafting-induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR-365 in vein grafts. Specially, to increase the efficiency of miR-365 gene transfection, a 30% poloxamer F-127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus-mediated miR-365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR-365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019.

Dedicator of cytokinesis 2 silencing therapy inhibits neointima formation and improves blood flow in rat vein grafts.

OBJECTIVE: The high rate of vein graft failure due to neointimal hyperplasia is a major challenge for cardiovascular surgery. Finding novel approaches to prevent neointimal hyperplasia is important. Thus, the purpose of this study was to investigate whether dedicator of cytokinesis 2 (DOCK2) plays a role in the development of neointima formation in the vein grafts. METHODS AND RESULTS: We found that DOCK2 levels were significantly elevated in the vein grafts following grafting surgery. In addition, overexpression of DOCK2 promoted venous smooth muscle cell (SMC) proliferation and migration. Conversely, knocking-down endogenous DOCK2 expression in venous SMCs inhibited SMC proliferation and migration. Consistent with this, knocking-down DOCK2 expression in the grafted veins significantly reduced neointimal formation compared with the controls 28 days after vein transplantation. Moreover, DOCK2 silencing treatment improved hemodynamics in the vein grafts. Mechanistically, knockdown of DOCK2 significantly alleviated the vein graft-induced down regulation of SMC contractile protein expression and impeded the vein graft-induction of both Cyclin D1 and PCNA expression. In particular, to ensure high efficiency when transferring the DOCK2 short hairpin RNA (shDOCK2) into the grafted veins, a 30% poloxamer F-127 gel incorporated with 0.25% trypsin was smeared around the vein grafts to increase the adenovirus contact time and penetration. CONCLUSIONS: DOCK2 silencing gene therapy effectively attenuates neointimal hyperplasia in vein grafts. Knock-down of DOCK2 would be a potential therapeutic approach for the treatment of vein graft failure.

MicroRNA-146a sponge therapy suppresses neointimal formation in rat vein grafts.

The long-term failure of vein grafts due to neointimal hyperplasia remains a difficult problem in cardiovascular surgery. Exploring novel approaches to prevent neointimal hyperplasia is important. MicroRNA-146a (miR-146a) plays an essential role in promoting vascular smooth muscle cell (VSMC) proliferation. Thus, the aim of the present study is to investigate whether adenovirus-mediated miR-146a sponge (Ad-miR-146a-SP) gene therapy could attenuate neointimal formation in rat vein grafts. (Ad-miR-146a-SP) was constructed to transfect cultured VSMCs and grafted veins. To improve the efficiency of transferring the miR-146a sponge gene into the grafted veins, 20% poloxamer F-127 gel incorporated with 0.25% trypsin was used to increase adenovirus contact time and penetration. miR-146a-SP transduction significantly reduced the expression of miR-146a both in cultured VSMCs and vein grafts. miR-146a sponge markedly attenuated VSMC proliferation and migration. Consistent with this, miR-146a sponge gene therapy significantly attenuated neointimal formation and also improved blood flow in the vein grafts. Mechanistically, we identified the Krüppel-like factor 4(KLF4) as a potential downstream target gene of miR-146a in vein grafts. Our data show that miR-146a sponge gene therapy could effectively reduce miR-146a activity and attenuate neointimal formation in vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. © 2018 IUBMB Life, 71(1):125-133, 2019.