Microneedle patch with pure drug tips for delivery of liraglutide: pharmacokinetics in rats and minipigs.

Transdermal delivery of peptide drugs is almost impossible with conventional penetration enhancers because of epidermal barrier function. Microneedle (MN) patches can bypass the epidermal barrier and have been developed for trans- and intradermal delivery of peptide drugs and vaccines. However, dissolving MN patches are limited by low drug loading capacities due to their small size and admixture of drug and water-soluble excipients. Furthermore, few in vivo pharmacokinetic studies, especially in large animals such as pigs, have been performed to assess post-application systemic drug exposure. Here, we developed a dissolving MN patch with pure liraglutide at the needle tips. The MN patch could load up to 2.21 ± 0.14 mg of liraglutide in a patch size of 0.9 cm2, which was nearly two orders of magnitude higher than that obtained with conventional MN patches of the same size. Raman imaging confirmed that liraglutide was localized at the MN tips. The MN had sufficient mechanical strength to penetrate the epidermis and could deliver up to 0.93 ± 0.04 mg of liraglutide into skin with a dosing variability of less than 6.8%. The MN patch delivery enabled faster absorption of liraglutide than that provided by subcutaneous (S.C.) injection, and achieved relative bioavailability of 69.8% and 46.3% compared to S.C. injection in rats and minipigs, respectively. The MN patch also exhibited similar patterns of anti-hyperglycemic effect in diabetic rats and individual variability in pharmacokinetic parameters as S.C. injection. The liraglutide MN application was well tolerated; no skin irritation was observed in minipigs except for mild erythema occurring within 4 h after once daily administration for 7 days at the same site. Our preclinical study suggests that MN patch with pure drug needle tips might offer a safe and effective alternative to S.C. injection for administration of liraglutide.

The application of approaches in detecting ferroptosis.

Ferroptosis is a regulatory cell death (RCD) caused by iron-dependent lipid peroxidation, which is the backbone of regulating various diseases such as tumor, nervous system diseases and so on. Despite ferroptosis without specific detection methods currently, there are numerous types of detection technology commonly used, including flow cytometry, cell activity assay, microscopic imaging, western blotting, quantitative polymerase chain reaction (qPCR). In addition, ferroptosis could be detected by quantifying oxygen-free radicals reactive oxygen species (ROS), the lipid metabolite (malondialdehyde ((MDA)), related pathways and observing mitochondrial damage. In the face of numerous detection methods, how to choose appropriate detection methods based on experimental purposes has become a problem that needs to be solved at present. In this review, we summarized the commonly used detection methods of the critical substances in the process of ferroptosis, in the hope of facilitating the comprehensive study of ferroptosis, with a view to providing a guidance for subsequent related research.

Biofilm microenvironment triggered self-enhancing photodynamic immunomodulatory microneedle for diabetic wound therapy.

The treatment of diabetic wounds faces enormous challenges due to complex wound environments, such as infected biofilms, excessive inflammation, and impaired angiogenesis. The critical role of the microenvironment in the chronic diabetic wounds has not been addressed for therapeutic development. Herein, we develop a microneedle (MN) bandage functionalized with dopamine-coated hybrid nanoparticles containing selenium and chlorin e6 (SeC@PA), which is capable of the dual-directional regulation of reactive species (RS) generation, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), in response to the wound microenvironment. The SeC@PA MN bandage can disrupt barriers in wound coverings for efficient SeC@PA delivery. SeC@PA not only depletes endogenous glutathione (GSH) to enhance the anti-biofilm effect of RS, but also degrades GSH in biofilms through cascade reactions to generate more lethal RS for biofilm eradication. SeC@PA acts as an RS scavenger in wound beds with low GSH levels, exerting an anti-inflammatory effect. SeC@PA also promotes the M2-phenotype polarization of macrophages, accelerating wound healing. This self-enhanced, catabolic and dynamic therapy, activated by the wound microenvironment, provides an approach for treating chronic wounds.

Curcumin Attenuates Periodontal Injury via Inhibiting Ferroptosis of Ligature-Induced Periodontitis in Mice.

Periodontitis is a chronic infectious disease characterized by the destruction of connective tissue and alveolar bone that eventually leads to tooth loss. Ferroptosis is an iron-dependent regulated cell death and is involved in ligature-induced periodontitis in vivo. Studies have demonstrated that curcumin has a potential therapeutic effect on periodontitis, but the mechanism is still unclear. The purpose of this study was to investigate the protective effects of curcumin on alleviating ferroptosis in periodontitis. Ligature-induced periodontal-diseased mice were used to detect the protective effect of curcumin. The level of superoxide dismutase (SOD), malondialdehyde (MDA) and total glutathione (GSH) in gingiva and alveolar bone were assayed. Furthermore, the mRNA expression levels of acsl4, slc7a11, gpx4 and tfr1 were measured using qPCR and the protein expression of ACSL4, SLC7A11, GPX4 and TfR1 were investigated by Western blot and immunocytochemistry (IHC). Curcumin reduced the level of MDA and increased the level of GSH. Additionally, curcumin was proven to significantly increase the expression levels of SLC7A11 and GPX4 and inhibit the expression of ACSL4 and TfR1. In conclusion, curcumin plays a protective role by inhibiting ferroptosis in ligature-induced periodontal-diseased mice.

Overview of the main biological mechanisms linked to changes in periodontal ligament stem cells and the inflammatory microenvironment. / 牙周膜干细胞与炎症微环境相互作用的主要生物学机制概述.

Periodontitis is a complex chronic inflammatory disease. The invasion of pathogens induces the inflammatory microenvironment in periodontitis. Cell behavior changes in response to changes in the microenvironment, which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells. It has been confirmed that periodontal ligament stem cells (PDLSCs) are vital in the development of periodontal disease. Moreover, PDLSCs are the most effective cell type to be used for periodontium regeneration. This review focuses on changes in PDLSCs, their basic biological behavior, osteogenic differentiation, and drug effects caused by the inflammatory microenvironment, to provide a better understanding of the influence of these factors on periodontal tissue homeostasis. In addition, we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.

Conditioned media of deer antler stem cells accelerate regeneration of alveolar bone defects in rats.

The destruction of periodontal alveolar bone (AB) caused by periodontitis is regarded as one of the major reasons for tooth loss. The inhibition of bone resorption and regeneration of lost AB are the desirable outcomes in clinical practice but remain in challenge. The use of mesenchymal stem cells (MSCs) is one current approach for achieving true restoration of AB defects (ABD). Antler stem cells (AnSC) are capable of renewing a huge mammalian bony appendage, the deer antler, suggesting an unparalleled potential for bone regeneration. Herein, we investigated the effectiveness of deer AnSCs conditioned medium (CM, AnSC-CM) for repair of surgically-created ABD using a rat model and sought to define the underlying mechanisms. The results showed that AnSC-CM effectively induced regeneration of AB tissue; the outcome was significantly better than human bone marrow mesenchymal stem cell conditioned medium (hBMSC-CM). AnSC-CM treatment upregulated osteogenic factors and downregulated osteoclastic differentiation factors; stimulated proliferation, migration and differentiation of resident MSCs toward osteogenic lineage cells; modulated macrophage polarization toward the M2 phenotype and suppressed osteoclastogenesis. That AnSC-CM resulted in better outcomes than hBMSC-CM in treating ABD was attributed to the cell compatibility as both AnSCs and AB tissue are neural crest-derived. In conclusion, the effects of AnSC-CM on AB tissue regeneration were achieved through both promotion of osteogenesis and inhibition of osteoclastogenesis. We believe that AnSC-CM is a candidate for effective treatment of ABD in dental clinical practice but will require investment in further development.

Inhibitory effect of polysaccharides extracted from Changbai Mountain Ganoderma lucidum on periodontal inflammation.

As the main bioactive substance of Ganoderma lucidum, Ganoderma lucidum polysaccharide (GLP) has anti-inflammatory, antibacterial, and other biological activities. Studies have shown that GLP can regulate the expression of multiple inflammatory cytokines in different inflammatory models and diseases as part of the anti-infection immune response. We extracted crude Changbai Mountain Ganoderma lucidum polysaccharides (CGLPs), analyzed their physical and chemical properties, and then applied them to the periodontitis model to verify whether they have an inhibitory effect on mouse periodontitis. CGLP was determined to be a heteropolysaccharide with dextran as the main component. Its molecular weight was 17.40 kDa. In vivo experiments in mice showed that CGLP can inhibit the alveolar bone loss and reduced inflammation caused of periodontitis by regulating the expression of the inflammatory factors IL-1ß, TNF-α, and IL-10 in a concentration-dependent manner.

Deer Antler Reserve Mesenchyme Cell-Conditioned Medium Reduces the Destruction of Periodontitis in Mice.

Reserve mesenchyme cells (RMCs) are a type of antler stem cells (ASCs) that contribute to the rapid growth of deer antlers, the only known mammalian organ that can fully regenerate annually. Based on the prior evidence, ASC-conditioned medium could improve regenerative cutaneous healing in rats. The purpose of the study was to evaluate the therapeutic effects of RMC-conditioned medium (RMC-CM) on reducing the destruction in the mice periodontitis (PD) model and the underlying mechanisms. The lipopolysaccharide (LPS)-stimulated RAW264.7 cells were used in vitro to verify the effects of RMC-CM. The results revealed that RMC-CM could significantly reduce bone resorption and osteoclast activation, upregulate anti-inflammatory macrophages (M2) related interleukin (IL)-10 and CD206, and downregulate pro-inflammatory macrophages (M1) related tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase in vivo. In vitro, RMC-CM could significantly promote LPS-stimulated RAW264.7 cells migration, reduce osteoclast differentiation, downregulate the expression of TNF-α, IL-6, and IL-1ß, and upregulate the expression of IL-10 and arginase 1. According to the results, we concluded that RMC-CM could significantly reduce alveolar bone resorption and inhibit inflammation in gingival tissue by decreasing the activation of osteoclasts and inducing macrophage polarization toward the M2 phenotype. This study may serve as the experimental foundation for RMC-CM in the treatment of PD.

Advances of exosomes in periodontitis treatment.

Periodontitis is an inflammatory disease initiated by dysbiosis of the local microbial community. Periodontitis can result in destruction of tooth-supporting tissue; however, overactivation of the host immune response is the main reason for alveolar bone loss. Periodontal tissue cells, immune cells, and even further activated osteoclasts and neutrophils play pro-inflammatory or anti-inflammatory roles. Traditional therapies for periodontitis are effective in reducing the microbial quantities and improving the clinical symptoms of periodontitis. However, these methods are non-selective, and it is still challenging to achieve an ideal treatment effect in clinics using the currently available treatments and approaches. Exosomes have shown promising potential in various preclinical and clinical studies, including in the diagnosis and treatment of periodontitis. Exos can be secreted by almost all types of cells, containing specific substances of cells: RNA, free fatty acids, proteins, surface receptors and cytokines. Exos act as local and systemic intercellular communication medium, play significant roles in various biological functions, and regulate physiological and pathological processes in numerous diseases. Exos-based periodontitis diagnosis and treatment strategies have been reported to obtain the potential to overcome the drawbacks of traditional therapies. This review focuses on the accumulating evidence from the last 5 years, indicating the therapeutic potential of the Exos in preclinical and clinical studies of periodontitis. Recent advances on Exos-based periodontitis diagnosis and treatment strategies, existing challenges, and prospect are summarized as guidance to improve the effectiveness of Exos on periodontitis in clinics.

ETC-1002 Attenuates Porphyromonas gingivalis Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells via the AMPK/NF-κB Pathway and Exerts Ameliorative Effects in Experimental Periodontitis in Mice.

Background: Clinically, the failure of periodontal therapy stems largely from an inability to control the inflammatory response. Resolution of inflammation is an active, energy-requiring repair process, not merely a passive termination of inflammation. AMP-activated protein kinase (AMPK), a key energy sensor, has been shown to negatively regulate inflammatory signaling pathways. Thus, there is a crucial need for new therapeutic strategies to modulate AMPK and to promote enhanced resolution of inflammation. This study is aimed at investigating the anti-inflammatory effects of ETC-1002 through modulating AMPK in periodontitis. Methods: RAW264.7 cells were infected with Pg-LPS in the presence or absence of ETC-1002, following which the expression levels of proinflammatory cytokines and inflammation signaling-related proteins were evaluated by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. ETC-1002 was applied in a murine model of periodontitis to determine its anti-inflammatory effect in vivo. Histological changes were investigated by hematoxylin and eosin (H&E) staining, the levels of proinflammatory cytokines were detected using immunohistochemistry, and alveolar bone height was measured using micro-CT imaging. Results: ETC-1002 inhibited the production of proinflammatory cytokines, promoted AMPK phosphorylation, and decreased IκBα and NF-κB p65 phosphorylation levels in Pg-LPS-treated RAW264.7 macrophages. The inhibitory effects of ETC-1002 on the production of proinflammatory mediators were significantly abrogated by siRNA-mediated silencing of AMPKα in RAW264.7 cells. In vivo, ETC-1002 inhibited inflammatory cell infiltration, the expression of proinflammatory cytokines, and the inflammation-mediated destruction of alveolar bone in mice with experimental periodontitis. The anti-inflammatory effect of ETC-1002 in the periodontium could be reversed by the administration of Compound C, an AMPK inhibitor. Conclusions: ETC-1002 exerts anti-inflammatory effects in Pg-LPS-treated RAW264.7 cells via the AMPK/NF-κB pathway in vitro and inhibits the progress of experimental periodontitis in mice in an AMPK signaling-dependent manner in vivo. These results provide evidence for the beneficial effects of ETC-1002 in the treatment of periodontitis.

Research progress on basic fibroblast growth factor in promoting periodontal tissue regeneration / 口腔疾病防治

@#Basic fibroblast growth factor (bFGF) exhibits superior biological functions by improving periodontal inflammation, promoting the migration and proliferation of periodontal-related stem cells, promoting the formation of blood vessels and periodontal ligament-like tissue, and regulating the formation of bone/cementum. It plays an important role in tooth development, repair and regeneration. bFGF can be combined with seed cells and scaffold materials for periodontal tissue regeneration, which has been verified in a number of experimental studies. However, the application of bFGF alone as a drug in clinical treatment requires further research.

Advances in mesenchymal stem cell conditioned medium-mediated periodontal tissue regeneration.

Periodontitis is a chronic inflammatory disease that leads to the destruction of both soft and hard periodontal tissues. Complete periodontal regeneration in clinics using the currently available treatment approaches is still a challenge. Mesenchymal stem cells (MSCs) have shown promising potential to regenerate periodontal tissue in various preclinical and clinical studies. The poor survival rate of MSCs during in vivo transplantation and host immunogenic reaction towards MSCs are the main drawbacks of direct use of MSCs in periodontal tissue regeneration. Autologous MSCs have limited sources and possess patient morbidity during harvesting. Direct use of allogenic MSCs could induce host immune reaction. Therefore, the MSC-based indirect treatment approach could be beneficial for periodontal regeneration in clinics. MSC culture conditioned medium (CM) contains secretomes that had shown immunomodulatory and tissue regenerative potential in pre-clinical and clinical studies. MSC-CM contains a cocktail of growth factors, cytokines, chemokines, enzymes, and exosomes, extracellular vesicles, etc. MSC-CM-based indirect treatment has the potential to eliminate the drawbacks of direct use of MSCs for periodontal tissue regeneration. MSC-CM holds the tremendous potential of bench-to-bed translation in periodontal regeneration applications. This review focuses on the accumulating evidence indicating the therapeutic potential of the MSC-CM in periodontal regeneration-related pre-clinical and clinical studies. Recent advances on MSC-CM-based periodontal regeneration, existing challenges, and prospects are well summarized as guidance to improve the effectiveness of MSC-CM on periodontal regeneration in clinics.

The feasibility of phage therapy for periodontitis.

Periodontitis, a chronic progressive inflammation caused by plaque biofilm, is the main cause of tooth loss in adults. For certain refractory periodontitis cases, it is difficult to achieve a good curative effect using the existing periodontal treatment approaches, which may be due to periodontal pathogenic mechanism in the affected periodontal tissue that the host cannot resist and eliminate. Various pieces of evidence collectively revealed that most studies are focusing on phages in periodontal disease. Several studies have reported periodontitis treatment using phage therapy, highlighting its features including specificity, rapid propagation, and effectiveness on bacteriophage biofilms. In this study, we focus on these reports, aiming to lay the foundation for improved periodontal treatment approaches.

N-Acetyl-l-Leucine-Polyethyleneimine-Mediated Delivery of CpG Oligodeoxynucleotides 2006 Inhibits RAW264.7 Cell Osteoclastogenesis.

INTRODUCTION: CpG oligodeoxynucleotides (CpG ODN) play important roles in resisting inflammation and bone resorption. However, the inherent instability and rapid degradation hinder their wider application. This study aimed to evaluate whether N-acetyl-L-leucine-modified polyethyleneimine (N-Ac-L-Leu-PEI) could effectively deliver CpG ODN 2006 to RAW264.7 cells and and if it can regulate osteoclastogenesis in vitro. MATERIALS AND METHODS: Gel retardation assay was conducted to evaluate whether N- Ac-L-Leu-PEI and CpG ODN could form a stable complex. RAW264.7 cells were divided into four groups of control group, ODN group, phosphorothioate ODN group and N-Ac-L-Leu-PEI/ODN group. Fluorescence assay was conducted to evaluate the transfection rate of ODNs in different groups. Cell viability was determined by MTT assay. Cell apoptosis was determined by live-dead cell staining and flow cytometry assay. Relative expression levels of osteoclastic differentiation factors, including Nfatc, c-fos, receptor activator of nuclear factor κB (RANK), and matrix metalloproteinase 9 (MMP9), were determined by real-time PCR and Western blot. RESULTS: N-Ac-L-Leu-PEI and CpG ODN could form a stable complex at a mass ratio of 1:1 (w:w). MTT assay showed that the cell viability of N-Ac-L-Leu-PEI was relatively high even at a mass ratio of 8 µg/mL. The transfection rate of N-Ac-L-Leu-PEI-ODN complex was higher than 90%. The cell proliferation and apoptosis was significantly enhanced in N-Ac-L-Leu-PEI- CpG ODN group when compared to those in phosphorothioate CpG ODN. The expression levels of Nfatc, c-fos, RANK, and MMP9 were significantly decreased in N-Ac-L-Leu-PEI/ODN complex group. DISCUSSION: N-Ac-L-Leu-PEI could be a potential gene vehicle for the prevention of periodontitis-mediated bone resorption.

The Toll-like receptor ligand, CpG oligodeoxynucleotides, regulate proliferation and osteogenic differentiation of osteoblast.

BACKGROUND: This study aimed to investigate the regulation of CpG oligodeoxynucleotides (ODNs) on proliferation and osteogenic differentiation of MC3T3 cells. METHODS: The laser co-focusing and flow cytometry assay were employed to detect cell uptake of CpG ODN 2006. Twelve ODNs were sythesized, and their effects on proliferation and differentiation were detected by MTT and alkaline phosphatase (ALP) activity assay. Flow cytometry assay was used to examine the regulation of CpG ODN on cell cycle. Quantitative real-time PCR (qRT-PCR) and western blot were used to evaluate the regulation of CpG ODN on mRNA and protein expression of osteogenic differentiation genes. RESULTS: The phosphorothioate CpG ODN 2006 could efficiently enter the MC3T3 cells in 1 h and locate in the cytoplasm. The MTT assay demonstrated CpG ODNs could promote MC3T3 cell proliferation and differentiation in the early stage, and gradually attenuated along with the increase of treating time, except for BW001 and FC001. qRT-PCR assay demonstrated that all the 12 CpG ODNs could promote the relative expression level of osteogenic differentiated genes, SP7 and OCN. In addition, western blot analysis suggested the CpG ODNs of BW001 and FC001 could increase the protein expression of P27Kip1 and Runx2 and decrease the protein expression of cyclin D1. CONCLUSION: The selected CpGODNs may be a potential gene therapy for bone regeneration of periodontitis.

CpG oligodeoxynucleotides inhibit the proliferation and osteoclastic differentiation of RAW264.7 cells.

Clinical prevention and treatment of periodontitis-induced bone absorption remains a challenge. The anti-infection role of CpG oligodeoxynucleotides (CpG ODNs) is well known; however, their effect on osteoclasts is still unclear. Here, we show that some CpG ODNs can regulate osteoclastogenesis of RAW264.7 cells. The phosphorothioate CpG ODN was efficiently taken up by the cells within 1 h and distributed in the cytoplasm. BW006, YW001, YW002, and FC004 CpG ODNs significantly repressed cell proliferation by targeting several cyclin proteins to arrest the cells in the G2 phase and to further initiate cell apoptosis. Regarding differentiation, we selected six CpG ODNs (FC002, BW006, YW002, YW001, FC004, and MT01) that markedly inhibited the gene expression levels of Nfatc, c-fos, RANK, and MMP9. TRAP staining showed that only YW002, YW001, and FC004 suppressed osteoclast generation and maturation. These three CpG ODNs dramatically declined the protein levels of osteoclastogenic proteins by elevating the ratio of OPG/RANKL and also downregulating the inflammatory factors (TNF-α, IL-1ß, IL-6, and IL-17) at different stages. Thus, the selected CpG ODNs may be a potential molecular therapy for the prevention and treatment of periodontitis-mediated bone resorption.