Injectable and high-strength PLGA/CPC loaded ALN/MgO bone cement for bone regeneration by facilitating osteogenesis and inhibiting osteoclastogenesis in osteoporotic bone defects.

Osteoporosis (OP) can result in slower bone regeneration than the normal condition due to the imbalance between osteogenesis and osteoclastogenesis, making osteoporotic bone defects healing a significant clinical challenge. Calcium phosphate cement (CPC) is a promising bone substitute material due to its good osteoinductive activity, however, the drawbacks such as fragility, slow degradation rate and incapability to control bone loss restrict its application in osteoporotic bone defects treatment. Currently, we developed the PLGA electrospun nanofiber sheets to carry alendronate (ALN) and magnesium oxide nanoparticle (nMgO) into CPC, therefore, to obtain a high-strength bone cement (C/AM-PL/C). The C/AM-PL/C bone cement had high mechanical strength, anti-washout ability, good injection performance and drug sustained release capacity. More importantly, the C/AM-PL/C cement promoted the osteogenic differentiation of bone marrow mesenchymal stem cells and neovascularization via the release of Mg2+ (from nMgO) and Ca2+ (during the degradation of CPC), and inhibited osteoclastogenesis via the release of ALN in vitro. Moreover, the injection of C/AM-PL/C cement significantly improved bone healing in an OP model with femur condyle defects in vivo. Altogether, the injectable C/AM-PL/C cement could facilitate osteoporotic bone regeneration, demonstrating its capacity as a promising candidate for treatment of osteoporotic bone defects.

Picein alleviates oxidative stress and promotes bone regeneration in osteoporotic bone defect by inhibiting ferroptosis via Nrf2/HO-1/GPX4 pathway.

Osteoporosis (OP) can result in slower bone regeneration than the normal condition due to abnormal oxidative stress and high levels of reactive oxygen species (ROS), a condition detrimental for bone formation, making the OP-related bone healing a significant clinical challenge. As the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) is closely related to bone regeneration; currently, this study assessed the effects of Picein on BMSCs in vitro and bone regeneration in osteoporotic bone defect in vivo. Cell viability was determined by CCK-8 assay. The production of (ROS), malonaldehyde, superoxide dismutase activities, and glutathione was evaluated by using commercially available kits, and a flow cytometry analysis was adopted to detect macrophage polarization. Osteogenic capacity of BMSCs was evaluated by alkaline phosphatase (ALP) activity, ALP staining, and Alizarin red S staining. The expression of osteogenic-related proteins (OPN, Runx-2, OCN) and osteogenic-related genes (ALP, BMP-4, COL-1, and Osterix) were evaluated by Western blotting and real-time PCR (RT-PCR). In addition, proliferation, migration ability, and angiogenic capacity of human umbilical vein endothelial cells (HUVECs) were evaluated by EdU staining, scratch test, transwell assay, and tube formation assay, respectively. Angiogenic-related genes (VEGF, vWF, CD31) were also evaluated by RT-PCR. Results showed that Picein alleviated erastin-induced oxidative stress, enhanced osteogenic differentiation capacity of BMSCs, angiogenesis of HUVECs, and protects cells against ferroptosis through Nrf2/HO-1/GPX4 axis. Moreover, Picein regulate immune microenvironment by promoting the polarization of M2 macrophages in vitro. In addition, Picein also reduce the inflammation levels and promotes bone regeneration in osteoporotic bone defect in OP rat models in vivo. Altogether, these results suggested that Picein can promote bone regeneration and alleviate oxidative stress via Nrf2/HO-1/GPX4 pathway, offering Picein as a novel antioxidant agent for treating osteoporotic bone defect.

Punicalagin attenuates TNF-α-induced oxidative damage and promotes osteogenic differentiation of bone mesenchymal stem cells by activating the Nrf2/HO-1 pathway.

Oxidative stress is one of the most important factors in changing bone homeostasis. Redox homeostasis plays a key role in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and the angiogenesis ability of human umbilical vein endothelial cells (HUVECs) for bone regeneration. Currently, this study assessed the effects of punicalagin (PUN) on BMSCs and HUVECs. Cell viability was determined by CCK-8 assay. A flow cytometry analysis was adopted to detect macrophage polarization. The production of reactive oxygen stress (ROS), glutathione (GSH), malonaldehyde (MDA) and superoxide dismutase (SOD) activities were evaluated by using commercially-available kits. Osteogenic capacity of BMSCs was evaluated by ALP activity, ALP staining and ARS staining. The expression of osteogenic-related proteins (OCN, Runx-2, OPN) and Nrf/HO-1 levles were evaluated by Western blotting. Osteogenic-related genes (Osterix, COL-1, BMP-4, ALP) were evaluated by RT-PCR. Migration ability and invasion ability of HUVECs were evaluated by wound healing assay and Transwell assay. Angiogenic ability was detected by tube formation assay and the expression of angiogenic-related genes (VEGF, vWF, CD31) were evaluated by RT-PCR. Results showed that PUN alleviated oxidative stress by TNF-α, enhanced osteogenic differentiation in BMSCs and angiogenesis in HUVECs. Moreover, PUN regulate immune microenvironment by promoting the polarization of M2 macrophages and reduce the oxidative stress related products by activating Nrf2/HO-1 pathway. Altogether, these results suggested that PUN can promote osteogenic capacity of BMSCs, angiogenesis of HUVECs, alleviate oxidative stress via Nrf2/HO-1 pathway, offering PUN as a novel antioxidant agent for treating bone loss diseases.

Inhibition of Schwann Cell Pyroptosis Promotes Nerve Regeneration in Peripheral Nerve Injury in Rats.

Background: Peripheral nerve injury (PNI) is one of the most debilitating injuries, but therapies for PNI are still far from satisfactory. Pyroptosis, a recently identified form of cell death, has been demonstrated to participate in different diseases. However, the role of pyroptosis of Schwann cells in PNI remains unclear. Methods: We established a rat PNI model, and western blotting, transmission electron microscopy, and immunofluorescence staining were used to confirm pyroptosis of Schwann cells in PNI in vivo. In vitro, pyroptosis of Schwann cells was induced by lipopolysaccharides (LPS)+adenosine triphosphate disodium (ATP). An irreversible inhibitor of pyroptosis, acetyl (Ac)-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), was used to attenuate Schwann cell pyroptosis. Moreover, the influence of pyroptotic Schwann cells on the function of dorsal root ganglion neurons (DRGns) was analyzed by a coculture system. Finally, the rat PNI model was intraperitoneally treated with Ac-YVAD-cmk to observe the effect of pyroptosis on nerve regeneration and motor function. Results: Schwann cell pyroptosis was notably observed in the injured sciatic nerve. LPS+ATP treatment effectively induced Schwann cell pyroptosis, which was largely attenuated by Ac-YVAD-cmk. Additionally, pyroptotic Schwann cells inhibited the function of DRGns by secreting inflammatory factors. A decrease in pyroptosis in Schwann cells promoted regeneration of the sciatic nerve and recovery of motor function in rats. Conclusion: Given the role of Schwann cell pyroptosis in PNI progression, inhibition of Schwann cell pyroptosis might be a potential therapeutic strategy for PNI in the future.

Engeletin alleviates erastin-induced oxidative stress and protects against ferroptosis via Nrf2/Keap1 pathway in bone marrow mesenchymal stem cells.

Ferroptosis is a novel form of cell death, which is a unique modality of cell death and closely associated with iron concentrations, generation of reactive oxygen species (ROS), and accumulation of the lipid reactive oxygen species. In the present study, the anti-ferroptosis effects of Engeletin was studied in erastin-induced bone marrow mesenchymal stem cells (BMSCs). After treatment with Engeletin, cell viability was determined by CCK-8 assay. The production of ROS, malonaldehyde (MDA), Superoxide dismutase (SOD) activities and glutathione peroxidase (GSH) were detected by using commercially-available kits. Ferroptosis-related proteins (GPX4, SLC7A11, TFR1, FPN1, Nrf2, Keap1) were evaluated by Western blotting. Osteogenic capacity was evaluated by ALP staining and ARS staining. The expression of osteogenic-related proteins (OPN, Runx2, OCN) were evaluated by Western blotting and changes in mRNA (ALP, BMP-2, COL-1, Osterix) were evaluated by RT-PCR. Consistent improvements in angiogenesis are observed with Engeletin in the presence of erastin. Engeletin significantly alleviated erastin-induced oxidative damage and protected against ferroptosis in BMSCs. Ferroptosis was inhibited by Engeletin, leading to decreasing reducing accumulation of ROS and lipid peroxidation products. Moreover, Engeletin promoted osteogenic differentiation in BMSCs and angiogenesis in human umbilical vein endothelial cells (HUVECs). Taken together, these findings indicate that Engeletin can protect BMSCs from erastin-induced ferroptosis through the Nrf2/Keap1 antioxidant pathway and identify Engeletin as a novel ferroptosis inhibitor, suggesting that Engeletin may promote resistance to ferroptosis and enable osteogenic function of BMSCs.

Polydopamine-Decorated PLCL Conduit to Induce Synergetic Effect of Electrical Stimulation and Topological Morphology for Peripheral Nerve Regeneration.

Due to the limited self-repairing capacity after peripheral nerve injuries (PNI), artificial nerve conduits are widely applied to facilitate neural regeneration. Exogenous electrical stimulation (ES) that is carried out by the conductive conduit regulates the biological behavior of Schwann cells (SCs). Meanwhile, a longitudinal surface structure counts to guide axonal growth to accelerate the end-to-end connection. Currently, there are no conduits equipped with both electrical conduction and axon-guiding surface structure. Herein, a biodegradable, conductive poly(l-lactide-co-caprolactone)/graphene (PLCL/GN) composite conduit is designed. The conduit with 20.96 ± 1.26 MPa tensile strength has a micropatterned surface of 20 µm groove fabricated by microimprint technology and self-assembled polydopamine (PDA). In vitro evaluation shows that the conduits with ES effectively stimulate the directional cell migration, adhesion, and elongation, and enhance neuronal expression of SCs. The rat sciatic nerve crush model demonstrates that the conductive micropatterned conduit with ES promotes the growth of myelin sheath, faster nerve regeneration, and 20-fold functional recovery in vivo. These discoveries prove that the PLCL(G)/PDA/GN composite conduit is a promising tool for PNI treatment by providing the functional integration of physical guidance, biomimetic biological regulation, and bioelectrical stimulation, which inspires a novel therapeutic approach for nerve regeneration in the future.

Injectable nanofiber-reinforced bone cement with controlled biodegradability for minimally-invasive bone regeneration.

Injectable materials show their special merits in regeneration of damaged/degenerated bones in minimally-invasive approach. Injectable calcium phosphate bone cement (CPC) has attracted broad attention for its bioactivity, as compared to non-degradable polymethyl methacrylate cement. However, its brittleness, poor anti-washout property and uncontrollable biodegradability are the main challenges to limit its further clinical application mainly because of its stone-like dense structure and fragile inorganic-salt weakness. Herein, we developed a kind of injectable CPC bone cement with porous structure and improved robustness by incorporating poly(lactide-co-glycolic acid) (PLGA) nanofiber into CPC, with carboxymethyl cellulose (CMC) to offer good injectability as well as anti-wash-out capacity. Furthermore, the introduction of PLGA and CMC also enabled a formation of initial porous structure in the cements, where PLGA nanofiber endowed the cement with a dynamically controllable biodegradability which provided room for cell movement and bone ingrowth. Interestingly, the reinforced biodegradable cement afforded a sustainable provision of Ca2+ bioactive components, together with its porous structure, to improve synergistically new bone formation and osteo-integration in vivo by using a rat model of femur condyle defect. Further study on regenerative mechanisms indicated that the good minimally-invasive bone regeneration may come from the synergistic enhanced osteogenic effect of calcium ion enrichment and the improved revascularization capacity contributed from the porosity as well as the lactic acid released from PLGA nanofiber. These results indicate the injectable bone cement with high strength, anti-washout property and controllable biodegradability is a promising candidate for bone regeneration in a minimally-invasive approach.

Endothelial cells promote the proliferation and migration of Schwann cells.

Background: After peripheral nerve injury, Schwann cells proliferate and migrate to the injured site, thereby promoting peripheral nerve regeneration. The process is regulated by various factors. Endothelial cells participate in the process via angiogenesis. However, the effects of endothelial cells on Schwann cells are not yet known. The present study sought to evaluate whether endothelial cells accelerate Schwann cell proliferation and migration. Methods: We established a co-culture model of rat Schwann cells (RSC96s) and rat aortic endothelial cells (RAOECs), and studied the effects of endothelial cells on Schwann cells by evaluating changes in Schwann cell proliferation and migration and related multiple genes and their protein expressions in the co-culture model. Results: The results showed that increasing the proportion of endothelial cells in the co-culture model enhanced the proliferation. At days 1 and 3 following the co-culturing, the relative growth rates of the co-cultured cells were 122.87% and 127.37%, respectively, which showed a significant increase in the viability compared to that of the RSC96s (P<0.05). In this process, the expression of Ki67 increased. The migration ability of Schwann cells was also enhanced. The migration capacity of Schwann cells was detected by wound-healing and Transwell assays. The results of the group with 15% of endothelial cells was significantly higher than the results of the other groups (P<0.0001 and P<0.05, respectively). Further, neuregulin 1 and glial fibrillary acidic protein increased the process of Schwann cell migration. Conclusions: The results showed that endothelial cells can promote the proliferation and migration of Schwann cells and participate in peripheral nerve regeneration.