Dynamic degradation patterns of porous polycaprolactone/ß-tricalcium phosphate composites orchestrate macrophage responses and immunoregulatory bone regeneration.

Biodegradable polycaprolactone/ß-tricalcium phosphate (PT) composites are desirable candidates for bone tissue engineering applications. A higher ß-tricalcium phosphate (TCP) ceramic content improves the mechanical, hydrophilic and osteogenic properties of PT scaffolds in vitro. Using a dynamic degradation reactor, we established a steady in vitro degradation model to investigate the changes in the physio-chemical and biological properties of PT scaffolds during degradation.PT46 and PT37 scaffolds underwent degradation more rapidly than PT scaffolds with lower TCP contents. In vivo studies revealed the rapid degradation of PT (PT46 and PT37) scaffolds disturbed macrophage responses and lead to bone healing failure. Macrophage co-culture assays and a subcutaneous implantation model indicated that the scaffold degradation process dynamically affected macrophage responses, especially polarization. RNA-Seq analysis indicated phagocytosis of the degradation products of PT37 scaffolds induces oxidative stress and inflammatory M1 polarization in macrophages. Overall, this study reveals that the dynamic patterns of biodegradation of degradable bone scaffolds highly orchestrate immune responses and thus determine the success of bone regeneration. Therefore, through evaluation of the biological effects of biomaterials during the entire process of degradation on immune responses and bone regeneration are necessary in order to develop more promising biomaterials for bone regeneration.

Magnesium surface-activated 3D printed porous PEEK scaffolds for in vivo osseointegration by promoting angiogenesis and osteogenesis.

Polyetheretherketone (PEEK) has been an alternative material for titanium in bone defect repair, but its clinical application is limited by its poor osseointegration. In this study, a porous structural design and activated surface modification were used to enhance the osseointegration capacity of PEEK materials. Porous PEEK scaffolds were manufactured via fused deposition modeling and a polydopamine (PDA) coating chelated with magnesium ions (Mg2+) was utilized on the surface. After surface modification, the hydrophilicity of PEEK scaffolds was significantly enhanced, and bioactive Mg2+ could be released. In vitro results showed that the activated surface could promote cell proliferation and adhesion and contribute to osteoblast differentiation and mineralization; the released Mg2+ promoted angiogenesis and might contribute to the formation of osteogenic H-type vessels. Furthermore, porous PEEK scaffolds were implanted in rabbit femoral condyles for in vivo evaluation of osseointegration. The results showed that the customized three-dimensional porous structure facilitated vascular ingrowth and bone ingrowth within the PEEK scaffolds. The PDA coating enhanced the interfacial osseointegration of porous PEEK scaffolds and the released Mg2+ accelerated early bone ingrowth by promoting early angiogenesis during the coating degradation process. This study provides an efficient solution for enhancing the osseointegration of PEEK materials, which has high potential for translational clinical applications.

Electrical stimulation of piezoelectric BaTiO3 coated Ti6Al4V scaffolds promotes anti-inflammatory polarization of macrophages and bone repair via MAPK/JNK inhibition and OXPHOS activation.

Bone regeneration is a highly synchronized process that requires multiple biochemical, bioelectrical, mechanical, and other physiological cues. The restoration and delivery of electrical cues locally through piezoelectric materials have been demonstrated to facilitate osteogenesis in vitro and bone repair in vivo. However, the underlying mechanism by which piezoelectric stimulation promotes osteogenesis and bone repair remains unclear yet, limiting the design and clinical application of piezoelectric materials for bone repair. Herein, a piezoelectric BaTiO3/Ti6Al4V (BT/Ti) scaffold was prepared by hydrothermal synthesis of a uniform BaTiO3 layer on three dimensionally printed Ti6Al4V scaffold. The BT/Ti scaffolds exhibited piezoelectricity and favorable biocompatibility with RAW264.7 macrophages after polarization. In vitro results demonstrated that the piezoelectric effects of the poled BT/Ti scaffolds promoted M2 polarization of macrophages and immunoregulatory osteogenesis of MC-3T3 osteoblasts. In a subcutaneous implantation model, a higher proportion of CD68+ CD206+ M2 macrophages was observed in the tissues around the poled BT/Ti scaffolds under low intensity pulsed ultrasound (LIPUS) stimulation. Improvements in macrophage M2 polarization and bone regeneration were further identified in a sheep cervical corpectomy model. RNA sequencing and mechanistic investigation revealed that the piezoelectric BT/Ti (poled) scaffolds inhibited the inflammatory MAPK/JNK signaling cascade and activated oxidative phosphorylation (OXPHOS) and ATP synthesis in macrophages. Collectively, our study provides a promising method for regulating the immune microenvironment and enhancing bone regeneration using polarized piezoelectric BT/Ti scaffolds.

Protective Effect of Photobiomodulation against Hydrogen Peroxide-Induced Oxidative Damage by Promoting Autophagy through Inhibition of PI3K/AKT/mTOR Pathway in MC3T3-E1 Cells.

Photobiomodulation (PBM) has been repeatedly reported to play a major role in the regulation of osteoblast proliferation and mineralization. Autophagy is closely associated with various pathophysiological processes in osteoblasts, while its role in oxidative stress is even more critical. However, there is still no clear understanding of the mechanism of the role of autophagy in the regulation of osteoblast mineralization and apoptosis under oxidative stress by PBM. It was designed to investigate the impact of 808 nm PBM on autophagy and apoptosis in mouse preosteoblast MC3T3-E1 treated with hydrogen peroxide (H2O2) through PI3K/AKT/mTOR pathway. PBM could inhibit MC3T3-E1 cell apoptosis under oxidative stress and promote the expression of osteogenic proteins, while enhancing the level of autophagy. In contrast, 3-methyladenine (3-MA) inhibited the expression of osteoblast autophagy under oxidative stress conditions, increased apoptosis, and plus counteracted the effect of PBM on osteoblasts. We also found that PBM suppressed the activated PI3K/AKT/mTOR pathway during oxidative stress and induced autophagy in osteoblasts. PBM promoted autophagy of MC3T3 cells and was further blocked by 740 Y-P, which reversed the effect of PBM on MC3T3 cells with H2O2. In conclusion, PBM promotes autophagy and improves the level of osteogenesis under oxidative stress by inhibiting the PI3K/AKT/mTOR pathway. Our results can lay the foundation for the clinical usage of PBM in the treatment of osteoporosis.

Corrigendum: Sestrin2-mediated autophagy contributes to drug resistance via endoplasmic reticulum stress in human osteosarcoma.

[This corrects the article DOI: 10.3389/fcell.2021.722960.].

Increased BMSC exosomal miR-140-3p alleviates bone degradation and promotes bone restoration by targeting Plxnb1 in diabetic rats.

BACKGROUND: Diabetes mellitus (DM) is considered to be an important factor for bone degeneration disorders such as bone defect nonunion, which is characterized by physical disability and tremendous economy cost to families and society. Exosomal miRNAs of BMSCs have been reported to participate in osteoblastogenesis and modulating bone formation. However, their impacts on the development of bone degeneration in DM are not yet known. The role of miRNAs in BMSCs exosomes on regulating hyperglycemia bone degeneration was investigated in the present study. RESULTS: The osteogenic potential in bone defect repair of exosomes derived from diabetes mellitus BMSCs derived exosomes (DM-Exos) were revealed to be lower than that in normal BMSCs derived exosomes (N-Exos) in vitro and in vivo. Here, we demonstrate that miR-140-3p level was significantly altered in exosomes derived from BMSCs, ADSCs and serum from DM rats. In in vitro experiments, upregulated miR-140-3p exosomes promoted DM BMSCs differentiation into osteoblasts. The effects were exerted by miR-140-3p targeting plxnb1, plexin B1 is the receptor of semaphoring 4D(Sema4D) that inhibited osteocytes differentiation, thereby promoting bone formation. In DM rats with bone defect, miR-140-3p upregulated exosomes were transplanted into injured bone and accelerated bone regeneration. Besides, miR-140-3p in the exosomes was transferred into BMSCs and osteoblasts and promoted bone regeneration by targeting the plexin B1/RohA/ROCK signaling pathway. CONCLUSIONS: Normal-Exos and miR-140-3p overexpressed-Exos accelerated diabetic wound healing by promoting the osteoblastogenesis function of BMSCs through inhibition plexin B1 expression which is the receptor of Sema4D and the plexin B1/RhoA/ROCK pathway compared with diabetes mellitus-Exos. This offers a new insight and a new therapy for treating diabetic bone unhealing.

The anti-infective outcomes of the distal femoral replacement coated with antibiotic cement in limb salvage surgery: A randomized clinical trial.

BACKGROUND: The aim of this study was to observe the anti-infective effect of the distal femoral tumor prosthesis coated with antibiotic cement during limb salvage treatment, and evaluate its potential prospect in clinic. METHODS: In this randomized controlled trial, the en bloc resection and reconstruction were performed in 36 patients with distal femoral primary bone tumor. Patients were divided into 2 groups randomly according to the application of antibiotic bone cement coating, which included antibiotic cement coating group (16 cases) and control group (18 cases). There were 10 men and 6 women in anti-infection group, aged from 18 to 54 years (23.47 ±â€Š3.53), and there were 12 men and 6 women in control group, aged from 19 to 56 years (24.16 ±â€Š4.32). The tumor type, age, sex, and Enneking stage were enrolled with well-matched of the 2 groups of patients. There was no difference between bundles and routine standard care for each group. The antibiotic cement was coated on the surface of polyethylene jacket with punched holes during operation. The peri-prosthetic infection, local recurrence and distant metastasis were followed up and limb functions were evaluated by Musculoskeletal Tumor Society 93 (MSTS93) scoring system. RESULTS: Patients were followed up till 34.7 months (range 18∼62 months). There was no periprosthetic infection in anti-infection group. Four cases in control group showed deep infection. Infection rate had significant differences between the 2 groups (P < .05). Infection-related prosthesis mortality was 0% (0/16) in anti-infection group and 16.67% (3/18) in control group. Local recurrence and distant metastasis occurred in 7 of 34 patients with primary malignant bone tumor, wherein 2 cases of local recurrence and 1 cases of distant metastasis occurred in anti-infective group; 2 cases of local recurrence and 2 cases of distant metastasis occurred in the control group. During a latest follow-up, MSTS93 function scoring revealed a mean of 25.6 ±â€Š4.2 in anti-infection group and 18.5 ±â€Š3.3 in control group. The survival rate of anti-infective group is 75%, and the survival rate of control group is 61.11%. CONCLUSION: The antibiotic cement-coated technique on the surface of the polyethylene jacket of custom-made distal femoral prosthesis is simple and effective in controlling the periprosthetic infection after tumor prosthesis reconstruction.

Three-Dimensionally Printed Ti2448 With Low Stiffness Enhanced Angiogenesis and Osteogenesis by Regulating Macrophage Polarization via Piezo1/YAP Signaling Axis.

Previous studies have found that the novel low-elastic-modulus Ti2448 alloy can significantly reduce stress shielding and contribute to better bone repair than the conventional Ti6Al4V alloy. In this study, the promotion of osteogenesis and angiogenesis by three-dimensionally printed Ti2448 were also observed in vivo. However, these were not significant in a series of in vitro tests. The stiffness of materials has been reported to greatly affect the response of macrophages, and the immunological regulation mediated by macrophages directly determines the fate of bone implants. Therefore, we designed more experiments to explore the role of three-dimensionally printed Ti2448 in macrophage activation and related osteogenesis and angiogenesis. As expected, we found a significant increase in the number of M2 macrophages around Ti2448 implants, as well as better osteogenesis and angiogenesis in vivo. In vitro studies also showed that macrophages pre-treated with Ti2448 alloy significantly promoted angiogenesis and osteogenic differentiation through increased PDGF-BB and BMP-2 secretion, and the polarization of M2 macrophages was enhanced. We deduced that Ti2448 promotes angiogenesis and osteogenesis through Piezo1/YAP signaling axis-mediated macrophage polarization and related cytokine secretion. This research might provide insight into the biological properties of Ti2448 and provide a powerful theoretical supplement for the future application of three-dimensionally printed Ti2448 implants in orthopaedic surgery.

The Synergistic Reducing Drug Resistance Effect of Cisplatin and Ursolic Acid on Osteosarcoma through a Multistep Mechanism Involving Ferritinophagy.

Increasing evidence suggests that traditional Chinese medicine strategies are obviously beneficial for cancer treatment, but scientific research on the underlying molecular mechanisms is lacking. We report that ursolic acid, a bioactive ingredient isolated from Radix Actinidiae chinensis, has strong antitumour effects on osteosarcoma cells. Functional studies showed that ursolic acid inhibited tumour cell proliferation and promoted the apoptosis of a variety of osteosarcoma cells. Ursolic acid had a synergistic cytotoxic effect with cisplatin on osteosarcoma cells. In a mouse osteosarcoma xenograft model, low-dose cisplatin combined with ursolic acid significantly reduced tumour growth. Notably, ursolic acid reversed weight loss in mice treated with cisplatin. Mechanistic studies showed that ursolic acid degraded ferritin by activating autophagy and induced intracellular overload of ferrous ions, leading to ferroptosis. In addition, ursolic acid enhanced the DNA-damaging effect of cisplatin on osteosarcoma cells. Taken together, these findings suggest that ursolic acid is a nontoxic adjuvant that may enhance the effectiveness of chemotherapy in osteosarcoma.

Sestrin2-Mediated Autophagy Contributes to Drug Resistance via Endoplasmic Reticulum Stress in Human Osteosarcoma.

One contributor to the high mortality of osteosarcoma is its reduced sensitivity to chemotherapy, but the mechanism involved is unclear. Improving the sensitivity of osteosarcoma to chemotherapy is urgently needed to improve patient survival. We found that chemotherapy triggered apoptosis of human osteosarcoma cells in vitro and in vivo; this was accompanied by increased Sestrin2 expression. Importantly, autophagy was also enhanced with increased Sestrin2 expression. Based on this observation, we explored the potential role of Sestrin2 in autophagy of osteosarcoma. We found that Sestrin2 inhibited osteosarcoma cell apoptosis by promoting autophagy via inhibition of endoplasmic reticulum stress, and this process is closely related to the PERK-eIF2α-CHOP pathway. In addition, our study showed that low Sestrin2 expression can effectively reduce autophagy of human osteosarcoma cells after chemotherapy, increase p-mTOR expression, decrease Bcl-2 expression, promote osteosarcoma cell apoptosis, and slow down tumour progression in NU/NU mice. Sestrin2 activates autophagy by inhibiting mTOR via the PERK-eIF2α-CHOP pathway and inhibits apoptosis via Bcl-2. Therefore, our results explain one underlying mechanism of increasing the sensitivity of osteosarcoma to chemotherapy and suggest that Sestrin2 is a promising gene target.

Biological Effects of a Three-Dimensionally Printed Ti6Al4V Scaffold Coated with Piezoelectric BaTiO3 Nanoparticles on Bone Formation.

Bone defect repair at load-bearing sites is a challenging clinical problem for orthopedists. Defect reconstruction with implants is the most common treatment; however, it requires the implant to have good mechanical properties and the capacity to promote bone formation. In recent years, the piezoelectric effect, in which electrical activity can be generated due to mechanical deformation, of native bone, which promotes bone formation, has been increasingly valued. Therefore, implants with piezoelectric effects have also attracted great attention from orthopedists. In this study, we developed a bioactive composite scaffold consisting of BaTiO3, a piezoelectric ceramic material, coated on porous Ti6Al4V. This composite scaffold showed not only appropriate mechanical properties, sufficient bone and blood vessel ingrowth space, and a suitable material surface topography but also a reconstructed electromagnetic microenvironment. The osteoconductive and osteoinductive properties of the scaffold were reflected by the proliferation, migration, and osteogenic differentiation of mesenchymal stem cells. The ability of the scaffold to support vascularization was reflected by the proliferation and migration of human umbilical vein endothelial cells and their secretion of VEGF and PDGF-BB. A well-established sheep spinal fusion model was used to evaluate bony fusion in vivo. Sheep underwent implantation with different scaffolds, and X-ray, micro-computed tomography, van Gieson staining, and elemental energy-dispersive spectroscopy were used to analyze bone formation. Isolated cervical angiography and visualization analysis were used to assess angiogenesis at 4 and 8 months after transplantation. The results of cellular and animal studies showed that the piezoelectric effect could significantly reinforce osteogenesis and angiogenesis. Furthermore, we also discuss the molecular mechanism by which the piezoelectric effect promotes osteogenic differentiation and vascularization. In summary, Ti6Al4V scaffold coated with BaTiO3 is a promising composite biomaterial for repairing bone defects, especially at load-bearing sites, that may have great clinical translation potential.

Correction: A vessel subtype beneficial for osteogenesis enhanced by strontium-doped sodium titanate nanorods by modulating macrophage polarization.

Correction for 'A vessel subtype beneficial for osteogenesis enhanced by strontium-doped sodium titanate nanorods by modulating macrophage polarization' by Shuo Guo et al., J. Mater. Chem. B, 2020, 8, 6048-6058. DOI: .

Fabrication of piezoelectric porous BaTiO3 scaffold to repair large segmental bone defect in sheep.

Porous titanium scaffolds can provide sufficient mechanical support and bone growth space for large segmental bone defect repair. However, they fail to restore the physiological environment of bone tissue. Barium titanate (BaTiO3) is considered a smart material that can produce an electric field in response to dynamic force. Low-intensity pulsed ultrasound stimulation (LIPUS), as a kind of micromechanical wave, can not only promote bone repair but also induce BaTiO3 to generate an electric field. In our studies, BaTiO3 was coated on porous Ti6Al4V and LIPUS was externally applied to observe the influence of the piezoelectric effect on the repair of large bone defects in vitro and in vivo. The results show that the piezoelectric effect can effectively promote the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro as well as bone formation and growth into implants in vivo. This study provides an optional alternative to the conventional porous Ti6Al4V scaffold with enhanced osteogenesis and osseointegration for the repair of large bone defects.

A vessel subtype beneficial for osteogenesis enhanced by strontium-doped sodium titanate nanorods by modulating macrophage polarization.

Early vascularization plays an important role in bone healing, especially in interfacial bone formation. Many modifications have been made to titanium surfaces to promote angiogenesis. In addition, cytokines secreted by osteoblasts have been reported to enhance early angiogenesis, however, the effect is limited because osteoblasts arise after inflammation subsides. We fabricated a newly sustained release system consisting of Sr ion-loaded sodium titanate nanorods (STSr) and studied its effect on angiogenesis by regulating macrophage subtypes. In an in vitro study, STSr significantly promoted the angiogenesis and formation of CD31hiEmcnhi vessels by modulating the transformation of M1 macrophages toward M2 macrophages. After incubation on STSr surfaces, macrophages (RAW264.7) polarized toward M2 subtypes and expressed high levels of PDGF-BB. Furthermore, the conditioned medium from RAW264.7 cells enhanced the ability of tubule formation and migration of HUVECs and their differentiation into pro-osteogenesis vessels (CD31hiEmcnhi vessels). In vivo studies showed high expression levels of CD31hiEmcnhi surrounding implants. Accompanied with enhanced vascularization, improved bone formation and osseointegration were observed. Our study serves as a basis for the clinical application of novel functional topography surfaces fabricated on titanium, which can be applied in new orthopedic implants for the better prognosis of patients.

Magnesium promotes bone formation and angiogenesis by enhancing MC3T3-E1 secretion of PDGF-BB.

Although magnesium and its alloys are candidates for orthopedic implants, they can effectively promote osteogenesis and angiogenesis. However, due to the degradability of magnesium, different concentrations of magnesium ions have different effects on cells, which affects the safety of magnesium implants. The cellular and molecular mechanisms by which magnesium promotes osteogenesis and angiogenesis are still unclear, which further affects its clinical use. In this study, HUVECs were treated with different concentrations of magnesium ions, and the concentration between 1 and 5 mM, especially 5 mM, was most suitable for cultivation of HUVECs. Using gene sequencing, RT-PCR, ELISA and Western blot analysis, we found that magnesium can promote MC3T3-E1 expression and secretion of PDGF-BB. Then, osteogenesis-related tests and angiogenesis-related tests found that magnesium promotes the secretion of PDGF-BB by MC3T3-E1 not only to improve the osteogenic differentiation ability of osteoblasts but also to effectively promote the angiogenic ability of HUVECs.

[Effect of hepatitis B virus C protein on function of natural killer cell in NK-92 cells].

OBJECTIVE: To investigate the influence of hepatitis B virus C protein on the function of natural killer cell. METHODS: Recombinant eukaryotic expression plasmid pHBI-CMV-HBC was constructed and confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with lipofectamine encapsuled. The transfected NK-92 cells containing expressive HBV C protein was confirmed by Western Blot analysis. ELISA was employed to determine the IFN-gamma level secreted by NK-92 cells. And finally the cytotoxicities of NK cells were analysed by MTT colorimetry, with the hepatoblastoma cell line (HepG2) as target cell. RESULTS: Western blotting confirmed the expression of HBV C protein in the NK-92 cells transfected with pHBI-CMV-HBC. NK cytotoxicities and IFN-gamma secretion level of NK-92 cells transfected with recombinant plasmid significantly increased compared to control NK-92 cells transfected with blank plasmid (P < 0.01) and untransfected NK-92 cells(P < 0.01). CONCLUSION: Transient expression of HBC can increase IFN-gamma secretion and cytotoxicities of NK-92 cells.