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Copper (Cu) is an essential trace element involved in fundamental physiological processes in the human body. Even slight disturbances in the physiological Cu homeostasis are associated with the manifestation of neurodegenerative diseases. While suggesting a crucial role of Cu in the pathogenesis, the exact mechanisms of Cu neurotoxicity involved in the onset and progression of neurological diseases are far from understood. This study focuses on the molecular and cellular mechanisms of Cu-mediated neurotoxicity in human brain cells. First, the cytotoxic potential of Cu was studied in fully differentiated, human neurons (LUHMES cells). Lysosomal integrity was considerably affected following incubation with 420 µM CuSO 4 for 48 h. Further mechanistic studies revealed mitochondria and neuronal network as most susceptible target organelles (already at 100 µM CuSO 4 , 48 h), while the generation of reactive oxygen species turned out to be a rather later consequence of Cu toxicity. Besides Cu, the homeostasis of other elements might be involved and are likely to contribute to the pathology of Cu-mediated neurological disorders. Besides Cu, also effects on the cellular levels of magnesium, calcium, iron, and manganese were observed in the neurons, presumably aggravating the consequences of Cu neurotoxicity. In conclusion, insights in the underlying mode of action will foster the development of treatment strategies against Cu-mediated neurological diseases. Particularly, the interplay of Cu with other elements might provide a powerful diagnostic tool and might be used as therapeutic approach., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no conflict of interest. Ethical approval: The manuscript does not contain clinical studies or patient data., (© 2024. The Author(s).)

The oncogenic fusion protein promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) is critical for acute promyelocytic leukemia (APL). PML/RARα initiates APL by blocking the differentiation and increasing the self-renewal of leukemic cells. The standard clinical therapies all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), which induce PML/RARα proteolysis, have dramatically improved the prognosis of APL patients. However, the emergence of mutations conferring resistance to ATRA and ATO has created challenges in the treatment of APL patients. Exploring pathways that modulate the oncogenic activity of PML/RARα could help develop novel therapeutic strategies for APL, particularly for drug-resistant APL. Herein, we demonstrated for the first time that palmitoylation of PML/RARα was a critical determinant of its oncogenic activity. PML/RARα palmitoylation was found to be catalyzed mainly by the palmitoyltransferase ZDHHC3. Mechanistically, ZDHHC3-mediated palmitoylation regulated the oncogenic transcriptional activity of PML/RARα and APL pathogenesis. The knockdown or overexpression of ZDHHC3 had respective effects on the expression of proliferation- and differentiation-related genes. Consistently, the depletion or inhibition of ZDHHC3 could significantly arrest the malignant progression of APL, particularly drug-resistant APL, whereas ZDHHC3 overexpression appeared to have a promoting effect on the malignant progression of APL. Thus, our study not only reveals palmitoylation as a novel regulatory mechanism that modulates PML/RARα oncogenic activity but also identifies ZDHHC3 as a potential therapeutic target for APL, including drug-resistant APL., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

Latexin (LXN) is a secreted protein with a molecular weight of 29 KD, which is considered a tumor suppressor and plays an important role in the inflammatory immune response. LXN is highly expressed in macrophages and regulates macrophage polarity and tumor immune escape, demonstrating excellent clinical potential. However, its mechanism is still unclear. In this study, a macrophage-T cell co-culture system is established to clarify the secretion of macrophage LXN into the extracellular through exosomes. The results indicate that LXN in macrophage-derived exosomes is functional, that is, LXN-enriched exosome inhibits CD4 + T cell differentiation into Treg cells in vitro and in vivo, and exhibits good tumor suppressive effects. Based on this discovery, a biomimetic nanoparticle loaded with LXN protein (MØ@LXN-NPS) is designed and synthesized. Furthermore, the MØ@LXN-NPS shows excellent performance in both in vivo and in vitro, especially in enhancing tumor immune surveillance by inhibiting Treg cells in tumor microenvironment, thus exhibiting excellent anti-tumor activity. This study provides a demonstration for the transition of biomolecules from functional research to engineering applications. The excellent performance of MØ@LXN-NPS in tumor immune regulation suggests that the engineered biomimetic nanomedicine has good clinical application prospects., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tooth eruption, a critical stage in tooth development, is related to osteoclastogenesis. Intraperitoneal injection of Shh agonists into neonatal mice promoted tooth eruption at postnatal day (PN) 15, whereas treatment with the Shh inhibitor (LDE225) suppressed this process. When RAW264.7 osteoclast precursor cells were treated with RANKL, NFATc1 translocated from the cytoplasm to the nucleus and induced cell differentiation into TRAP + osteoclasts; this process was activated by Shh but inhibited by LDE225. Treating RAW264.7 cells with the p38 inhibitor, BIRB796, also inhibited NFATc1 nuclear localization. p-p38 expression in the alveolar bone of PN3 and PN5 mice was decreased by treatment with LDE225, and RAW264.7 cell differentiation was reduced by BIRB796, regardless of treatment with Shh. Furthermore, Shh activated p38 signaling pathway in RAW264.7 cells, while p38 phosphorylation was reduced by LDE225, which ultimately inhibited osteoclast precursor differentiation. Therefore, we concluded that Shh promotes osteoclast precursor differentiation via the p38-NFATc1 signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Buserelin has been used to treat central precocious puberty (CPP). However, it could potentially result in immune dysregulation to undermine patients' health. Therefore, it is necessary to elucidate the effects of buserelin on immune cells. Here we explored buserelin-induced impacts on the differentiation and function of macrophage-colony-stimulating factor-producing T helper (ThGM) cells to uncover the immunoregulatory role of buserelin., Methods: Rat CPP was induced by danazol injection followed by buserelin treatment. The frequencies of ThGM cells in the spleen and lymph nodes were evaluated by flow cytometry. ThGM cell generation and function were analyzed in cell culture assays. Cell signaling was measured by Immunoblotting., Results: Buserelin increased the frequencies of splenic and lymph node ThGM cells. Buserelin promoted the in vitro differentiation and proliferation of ThGM cells. Buserelin-treated ThGM cells showed stronger supportive effects on other effector T helper cells. Buserelin induced the activation of the nuclear factor of activated T cells and extracellular signal-regulated kinase 1/2 in ThGM cells., Conclusion: Buserelin enhances the differentiation and function of pro-inflammatory ThGM cells, thus increasing the risk of autoimmune or inflammatory disorders. Therefore, it is necessary to monitor ThGM cells in buserelin-treated children to prevent latent immune dysregulation.

Inorganic nanoparticles are promising materials for bone tissue engineering due to their chemical resemblance to the native bone structure. However, most studies are unable to capture the entirety of the defective environment, providing limited bone regenerative abilities. Hence, this study aims to develop a multifunctional nanoparticle to collectively control the defective bone niche, including immune, angiogenic, and osteogenic systems. The nanoparticles, self-assembled by biomimetic mineralization and tannic acid (TA)-mediated metal-polyphenol network (MPN), are released sustainably after the incorporation within a gelatin cryogel. The released nanoparticles display a reduction in M1 macrophages by means of reactive oxygen species (ROS) elimination. Consequently, osteoclast maturation is also reduced, which is observed by the minimal formation of multinucleated cells (0.4%). Furthermore, the proportion of M2 macrophages, osteogenic differentiation, and angiogenic potential are consistently increased by the effects of magnesium from the nanoparticles. This orchestrated control of multiple systems influences the in vivo vascularized bone regeneration in which 80% of the critical-sized bone defect is regenerated with new bones with mature lamellar structure and arteriole-scale micro-vessels. Altogether, this study emphasizes the importance of the coordinated modulation of immune, osteogenic, and angiogenic systems at the bone defect site for robust bone regeneration., (© 2024 Wiley‐VCH GmbH.)

Mouse embryonic fibroblasts (MEFs) have been widely used as feeder cells in embryonic stem cell cultures because they can mimic the embryonic microenvironment. Milk fat globule-epidermal growth factor 8 (MFGE8) is expressed during mouse gonadal development, 10.5-13.5 embryonic, and is also found in MEF-conditioned medium (MEF-CM). Feeder-less culture of human-induced pluripotent stem cells (iPSCs) with MEF-CM significantly decreased the number of adherent cells when an inhibitory antibody against MFGE8 was used. The concentration of mouse MFGE8 in MEF-CM, as measured by an ELISA (Enzyme-Linked Immunosorbent Assay), was 0.16-1.24 μg/mL. Mouse MFGE8 and human MFGE8 have partially different molecular structures. Both the recombinant mouse MFGE8 and human MFGE8 significantly promoted cell adhesion of human iPSCs at medium-added concentrations of 2 μg/mL. This cell adhesion was also strongly inhibited by Arginylglycylaspartic acid (RGD) inhibitors, suggesting that it is dependent on the RGD sequence. The integrin αVβ5 expressed in iPSCs was thought to be involved in binding to the RGD sequence. MEF-CMs have long been an essential bio-derived material for the feeder culture method of iPSC culture. This study demonstrates that MFGE8 in MEF-CM is a functional factor in the promoting of cell adhesion of human iPSCs. Furthermore, the use of MFGE8-containing media demonstrates that iPSCs can be established and cultured while maintaining pluripotency and inducing three germ layer differentiation. The results of this study suggest the possibility of using MFGE8 as a scaffold material suitable for inducing differentiation when reproducing in vivo maturation in vitro .

Particulate matter (PM) exposure during pregnancy increases the risk of developing asthma in children. However, the placental mechanisms have yet to be elucidated. This study aims to evaluate the mechanisms associated with PM exposure during pregnancy and asthma susceptibility via placental epigenetic dysregulation. We analyzed data from two independent Korean birth cohorts (COCOA, 684 children; PSKC, 818 children). Physician-diagnosed current asthma and bronchial hyperresponsiveness (BHR) via methacholine challenge tests were evaluated at age seven. We estimated PM exposure with a diameter <10 μm (PM 10 ) during pregnancy using land-use regression models. We performed genome-wide methylation profiling in the placenta of 40 samples in the COCOA study and analyzed the gene expression levels. High PM 10 exposure during pregnancy increased the risk of developing current asthma and BHR in the COCOA study (aOR 2.36, 95% CI 1.06-5.22; aOR 2.14, 95% CI 1.40-3.27, respectively) and current asthma in the PSKC (aOR 2.62, 95% CI 1.35-5.09). The genes involved in neuronal differentiation and proliferation and Notch signaling pathways were significantly hypermethylated in children with high PM 10 -exposed asthma. The methylation and expression levels of eight genes (PAX6, REST, OLIG2, GLI1, ZBTB7A, NOTCH4, NOTCH1, and NOTCH3) in these pathways correlated with clinical parameters. This may effectively predict PM-related asthma through a prediction model using degrees of gene-based or CpG-based methylation (AUC = 0.96 and 0.93, respectively). PM 10 exposure during pregnancy impacts asthma development in offspring via placental DNA hypermethylation via neuronal differentiation and proliferation and Notch signaling pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Prevalence of osteoarthritis has been increasing in aging populations, which has necessitated the use of advanced biomedical treatments. These involve grafts or delivering drug molecules entrapped in scaffolds. However, such treatments often show suboptimal therapeutic effects due to poor half-life and off-target effects of drug molecules. As a countermeasure, a 3D printable robust hydrogel-based tissue-repair platform is developed containing decellularized extracellular matrix (dECM) from differentiated mammalian cells as the therapeutic cargo. Here, pre-osteoblastic and pre-chondrogenic murine cells are differentiated in vitro, decellularized, and incorporated into methacrylated gelatin (GelMA) solutions to form osteogenic (GelO) and chondrogenic (GelC) hydrogels, respectively. Integrating the bioactive dECM from differentiated cell sources allows GelO and GelC to induce differentiation in human adipose-derived stem cells (hASCs) toward osteogenic and chondrogenic lineages. Further, GelO and GelC can be covalently adhered using a carbodiimide coupling reaction, forming a multi-layered hydrogel with potential application as a bioactive osteochondral plug. The designed multi-layered hydrogel can also induce differentiation of hASCs in vitro. In conclusion, the bioactive dECM carrying 3D printed robust hydrogel offers a promising new drug and cell-free therapeutic strategy for bone and cartilage repair and future osteoarthritis management., (© 2025 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Clinically, a multitude of factors can contribute to the development of bone defects. In the process of bone healing, the nervous system plays a vital role in bone regeneration. Small molecules from the nervous system, such as neurotrophic factors and neuropeptides, have been found to stimulate osteoblast proliferation and differentiation by activating signaling pathways associated with bone calcification and angiogenesis. These small molecules play a crucial regulatory role at various stages of bone healing. The systematic release mechanism of small molecules within the nervous system through diverse bone tissue engineering materials holds significant clinical implications for the controlled regulation of the bone healing process. This review provides an overview of the involvement of various nervous system small molecules at different stages of bone healing and discusses their regulatory mechanisms, aiming to establish a theoretical foundation for programmed regulation in bone regeneration and design of replacement materials in bone tissue engineering., Competing Interests: No potential conflict of interest was reported by the authors., (© 2025 Wei et al.)

Nonunion is a significant complication in fracture management for surgeons. Salvianolic acid A (SAA), derived from the traditional Chinese plant Salviae miltiorrhizae Bunge (Danshen), exhibits notable anti-inflammatory and antioxidant properties. Although studies have demonstrated its ability to promote osteogenic differentiation, the exact mechanism of action remains unclear. This study investigated the effects of various SAA concentrations on the osteogenic differentiation of mouse-derived bone marrow mesenchymal stem cells (mBMSCs) and the osteoclastic differentiation of bone marrow-derived macrophages. Our findings indicate that SAA promotes the osteogenic differentiation of mBMSCs in a concentration-dependent manner, primarily by inhibiting the Notch1 signaling pathway. Notably, the administration of two Notch1 agonists (Jagged-1 and VPA) inhibited the effects of SAA on osteogenic differentiation. Additionally, SAA facilitated the autophagic degradation of NICD1, further enhancing osteogenic differentiation. Furthermore, SAA also dose-dependently inhibited the osteoclastic differentiation of bone marrow-derived macrophages, which is linked to its suppression of NF-κB signaling pathways. In a fracture model, SAA demonstrated a capacity to promote healing. In conclusion, SAA enhances bone fracture healing by balancing osteoblast and osteoclast differentiation., (© 2025 Federation of American Societies for Experimental Biology.)

Ethnopharmacological Relevance: The Bu Shen Yi Sui capsule (BSYS), a modified version of the classical Chinese medicine formula Liu Wei Di Huang pill, has demonstrated therapeutic efficacy in the treatment of multiple sclerosis (MS). Nevertheless, the precise mechanism through which BSYS facilitates remyelination remains to be elucidated., Aim of the Study: This research investigates the role and potential mechanisms of BSYS-modified exosomes (exos) derived from bone marrow mesenchymal stem cells (BMSCs) in promoting remyelination in a cuprizone (CPZ)-induced demyelination model in mice., Materials and Methods: C57BL/6J mice were administered a 0.2% CPZ-containing diet for 5 weeks to induce demyelination, followed by treatment with exosomes derived from BMSC (BMSC-exos) and BSYS-modified BMSC exosomes (BSYS-BMSC-exos) twice weekly for 2 weeks. Body weight measurements were recorded, and motor function was evaluated using the rotarod test. Pathological changes in myelin and axons were assessed via Luxol fast blue (LFB) staining, transmission electron microscopy (TEM), and immunofluorescence (IF) staining. Oligodendrocyte proliferation, differentiation, and maturation were analyzed using IF double-staining, Western blot (WB), and real-time quantitative reverse transcription PCR (qRT-PCR). Additionally, microRNA (miRNA) sequencing and a luciferase reporter assay were conducted to verify miRNA binding to its target gene. Key markers of the Wnt/β-catenin signaling pathway were examined using WB and qRT-PCR., Results: BSYS-BMSC-exos treatment significantly increased both body weight and rotarod performance in CPZ mice. Moreover, BMSC-exos and BSYS-BMSC-exos reversed myelin loss and axonal damage. These treatments enhanced oligodendrocytes proliferation, differentiation, and maturation, with BSYS-BMSC-exos exhibiting a particularly pronounced effect on the expression of adenomatous polyposis coli clone CC1 (CC1), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP). Sequencing and luciferase assays revealed that miR-15b-5p, enriched in BSYS-BMSC-exos, directly targets Wnt3a. Furthermore, BSYS-BMSC-exos elevated axis inhibition protein 2 (Axin2) expression while markedly reducing Wnt family member 3A (Wnt3a), phospho-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and T-cell specific transcription factor 4/transcription factor 7-like 2 (TCF4/TCF7L2) levels., Conclusions: The findings suggest that BSYS-BMSC-exos alleviate neurological deficits, enhance oligodendrocytes differentiation and maturation, and promote remyelination in CPZ mice. miR-15b-5p, enriched in BSYS-BMSC-exos, targets and downregulates Wnt3a, thereby inhibiting the Wnt/β-catenin signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: People with Hashimoto's thyroiditis (HT) often have low vitamin D3 concentrations. Some research has suggested that vitamin D3 supplementation reduces thyroid inflammation, but this remains controversial., Methods: EAT was induced in female NOD/ShiLtJ mice by giving them water containing 0.05% sodium iodide, and 1μg/kg of 1α,25-(OH) 2 D 3 was injected intraperitoneally every other day. After 8 weeks, the morphological architecture of the mouse thyroid follicles was examined by histological sections, thyroid autoantibodies and thyroid hormone concentrations were determined by enzyme-linked immunosorbent assays (ELISAs), and the major functions and subsets of B- and T-lymphocytes in the mouse thyroid were determined by tissue multiple immunofluorescence technology and ELISA., Results: EAT caused thyroiditis follicle destruction and interfollicular lymphocyte infiltration in mice, increased concentrations of circulating thyroid autoimmune antibodies TG-Ab and TPO-Ab, and abnormal thyroid hormone levels. EAT also increased the number and functionality of CD4+ Tfh, Th17,Th1 and Th2 cells in the thyroid, while decreasing the number and functionality of CD4+ Treg cells and CD19 + B10 cells. Treatment with VD3 reversed these changes., Conclusion: Vitamin D3 supplementation can effectively treat autoimmune thyroiditis in mice. VD3 reduces autoimmune thyroid damage and decreases serum thyroid antibody levels in mice by inhibiting the differentiation and functionality of pro-inflammatory Tfh, Th17, Th1 and Th2 cells and by facilitating the differentiation and functionality of anti-inflammatory B10 cells and Treg., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Wang, Chen, Yang, Yang, Wang, Zeng and Jiang.)

Background: Osteoporosis (OP) is a systemic disease characterized by low bone mass. New progress has been made in the study of OP, such as lipid peroxidation. However, the role of lipid peroxides in osteoclast differentiation is still unclear., Methods: Bone marrow macrophages (BMMs) were extracted from C57BL/6J mice and induced to differentiate into osteoclasts, which were observed via TRAP staining, Phalloidin staining and bone pit assays. Related substances of lipid peroxidation were detected during osteoclastogenesis. The levels of osteoclastogenesis and lipid peroxides were measured by qRT-PCR, Western Blot and immunofluorescence. Activation of the p38/JNK/MAPK pathway was detected by Western Blot. The capacity for osteogenesis and angiogenesis of cells after treatment with supernatant of BMMs was evaluated. Furthermore, Ferrostatin-1 (Fer-1), from which femur and serum samples were comprehensively evaluated, was used in OVX mice., Results: During osteoclastogenesis, the levels of ROS, MDA, ACSL4 and LPCAT3 increased with increasing duration of RANKL stimulation, while there were no significant changes in the levels of GSH or GPX4. Fer-1 inhibited osteoclast differentiation and decreased the level of lipid peroxides. In addition, Fer-1 inhibited osteoclast-related markers by inhibiting the p38/JNK/MAPK pathway. Furthermore, the supernatant of BMMs after Fer-1 treatment promoted osteogenesis and angiogenesis. Finally, Fer-1 successfully alleviated OP in OVX mice by reducing the level of lipid peroxidation in vivo., Conclusion: Fer-1 suppresses osteoclast differentiation by reducing lipid peroxidation levels regulated by ACSL4, which is mediated through the p38/JNK/MAPK signaling pathway. Additionally, Fer-1 enhances the coupling between osteogenesis and angiogenesis and has an anti-OP effect in vivo., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by Ethic Committee of the Second Affiliated Hospital of Harbin Medical University (Ref: YJSDW2023−172). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Ethnopharmacological Relevance: Qing Chang Suppository (QCS), a traditional Chinese medicine formula, has been shown to effectively alleviate mucosal inflammation in patients with ulcerative colitis (UC). While the mechanism of QCS appears to be related to the regulation of CD4 + T cell subset responses, direct evidence demonstrating that QCS inhibits Th1 and Th17 cell activation in UC (particularly based on human data) remains lacking. Additionally, the precise mechanisms through which QCS affects these cells have yet to be fully elucidated., Aim of Study: This study aimed to investigate the effects of QCS on Th1 and Th17 cell responses in UC and to explore the underlying mechanisms., Materials and Methods: Twenty-eight patients with mild-to-moderate UC were recruited and treated with QCS for 12 weeks. Symptoms were assessed every two weeks, with sigmoidoscopies performed at baseline and at week 12. Intestinal mucosal biopsies and peripheral blood (PB) were collected at these time points. At the end of the trial, patients were categorized into responder and non-responder groups based on a modified Mayo disease activity index score. Healthy controls (HCs) were defined as subjects without IBD or colorectal carcinoma but with colon polyps. The frequencies of IFN-γ + CD4 + T cells and IL-17A + CD4 + T cells in PB and colonic mucosa were measured using flow cytometry. The expression levels and localization of T-bet, RORγT, IFN-γ, TNF-α, and IL-17A were determined via immunofluorescence, and JNK signaling activation was assessed through immunoblotting and immunohistochemistry. All parameters were compared across the three groups., Results: At week 12, responders showed a significant reduction in colonic mucosal inflammation compared to baseline, accompanied by decreased frequencies of IFN-γ + CD4 + T and IL-17A + CD4 + T cells in both PB and the colonic epithelial layer. Notably, Th1 and Th17 cell activity around intestinal epithelial cells (IECs) was nearly undetectable, as evidenced by the diminished expression of T-bet, RORγT, IFN-γ, TNF-α, and IL-17A. Additionally, JNK phosphorylation in these cells was significantly reduced. In contrast, non-responders exhibited no meaningful improvement; colonic pathology remained unchanged, and elevated levels of IFN-γ + CD4 + T and IL-17A + CD 4 + T cells persisted in both the PB and colonic epithelial layer. The presence of Th1 and Th17 cells and their associated cytokines around IECs remained substantial, and there was no significant change in JNK activation., Conclusion: QCS attenuates mucosal inflammation in UC patients by inhibiting the differentiation and effector functions of Th1 and Th17 cells, primarily through the regulation of the JNK signaling pathway., Competing Interests: Declaration of competing interest The authors report no conflicts of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: A critical step preceding the potential biomedical application of nanoparticles is the evaluation of their immunomodulatory effects. Such nanoparticles are expected to enter the bloodstream where they can be recognized and processed by circulating monocytes. Despite the required biocompatibility, this interaction can affect intracellular homeostasis and modulate physiological functions, particularly inflammation. This study focuses on titanium dioxide (TiO 2 ) as an example of relatively low cytotoxic nanoparticles with potential biomedical use and aims to evaluate their possible modulatory effects on the inflammasome-based response in human primary monocytes., Methods: Monocyte viability, phenotypic changes, and cytokine production were determined after exposure to TiO 2 (diameter, 25 nm; P25) alone. In the case of the modulatory effects, we focused on NLRP3 activation. The production of IL-1β and IL-10 was evaluated after (a) simultaneous activation of monocytes with bacterial stimuli muramyl dipeptide (MDP), or lipopolysaccharide (LPS), and TiO 2 (co-exposure model), (b) prior activation with TiO 2 alone and subsequent exposure to bacterial stimuli MDP or LPS. The differentiation of TiO 2 -treated monocytes into macrophages and their polarization were also assessed., Results: The selected TiO 2 concentration range (30-120 µg/mL) did not induce any significant cytotoxic effects. The highest dose of TiO 2 promoted monocyte survival and differentiation into macrophages, with the M2 subset being the most prevalent. Nanoparticles alone did not induce substantial production of inflammatory cytokines IL-1β, IL-6, or TNF-α. The immunomodulatory effect on NLRP3 depended on the type of costimulant used. While co-exposure of monocytes to MDP and TiO 2 boosted NLRP3 activity, co-exposure to LPS and TiO 2 inhibited NLRP3 by enhancing IL-10 release. The inhibitory effect of TiO 2 on NLRP3 based on the promotion of IL-10 was confirmed in a post-exposure model for both costimulants., Conclusion: This study confirmed a non-negligible modulatory effect on primary monocytes in their inflammasome-based response and differentiation ability., Competing Interests: The authors report no conflicts of interest in this work., (© 2025 Svadlakova et al.)

Restoration of the intestinal epithelial barrier is crucial for achieving mucosal healing, the therapeutic goal for inflammatory bowel disease (IBD). During homeostasis, epithelial renewal is maintained by crypt stem cells and progenitors that cease to divide as they differentiate into mature colonocytes. Inflammation is a major effector of mucosal damage in IBD and has been found to affect epithelial stemness, regeneration and cellular functions. However, the impact of immune cell-modulating IBD drugs on epithelial homeostasis and repair is poorly understood. It is likely that these drugs will have distinct mechanisms of action (MOA) in intestinal epithelium relevant for homeostasis that will vary among patients. We investigated cellular effects of pan-Janus Kinase (JAK) inhibitor tofacitinib and the corticosteroid budesonide on uninflamed and TNF + Poly(I:C) stimulated human colon organoids (colonoids) from healthy donors and IBD-patients. Our findings reveal that although both tofacitinib and budesonide exhibit anti-inflammatory effects, tofacitinib increased colonoid size and proliferation during differentiation, and promoted epithelial stemness. In contrast, budesonide decreased colonoid size and showed no consistent effect on proliferation or stemness. Our study demonstrates the value of employing human colonoids to investigate how IBD drugs affect intestinal epithelial cells and inter-individual variations relevant to mucosal healing and personalized IBD treatment., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Model organisms are vital for biomedical research and drug testing but face high costs, complexity, and ethical issues. While newer techniques like organoids and assembloids have shown improvements, they still remain inadequate in addressing all research needs. In this study, we present a new method for maintaining the prostate gland of the earthworm, Eudrilus eugeniae ex vivo and examine its potential for regeneration and drug screening. We successfully maintained the earthworm prostate gland in cell culture media for over 200 days, with observed beating behavior confirming its viability. Apoptotic staining and histological analysis show no significant changes, indicating that the prostate gland remains stable. However, significant overexpression of H3 and H2AX on the 10th and 50th days suggests stem cell proliferation and differentiation. Alkaline phosphatase expression analysis confirmed that the stem cell niche is localized to the anterior region. Remarkably, the posterior region of the prostate gland demonstrated significant regenerative capacity, with complete regeneration occurring within 45 days following amputation. Furthermore, treatment with valproic acid enhanced posterior regeneration, leading to full restoration within 12 days. This study confirms the feasibility of maintaining the prostate gland of earthworms in an ex vivo setting, providing a valuable model for studying regeneration and conducting drug screening., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical statement: The ethical statement is not required for earthworms, as they are lower invertebrates. Nonetheless, all research is conducted with appropriate precautions to prevent unnecessary harm to animals., (© 2025. The Author(s).)

Background: The process of B cell activation and plasma cell (PC) formation involves morphological, transcriptional, and metabolic changes in the B cell. Blocking or reducing PC differentiation is one approach to treat autoimmune diseases that are characterized by the presence of pathogenic autoantibodies. Recent studies have suggested the potential of myricetin, a natural flavonoid with anti-inflammatory and antioxidant properties, to block or reduce PC differentiation., Methods: Primary human B cells were purified by using a human B cell isolation kit. B cell subsets such as IgG memory B cells, marginal zone B cells (MZ B cells), and naive B cells were isolated by flow cytometry and activated to induce PC differentiation. Quantification of PCs (CD27 + + , CD38 +) was obtained by flow cytometry. The expression of mRNA was measured by qPCR. Ig secretion in culture supernatant was measured by ELISA., Results: Myricetin treatment significantly reduced PC differentiation in primary human B cells and all B cell subsets. Myricetin exposure reduced Ig production both IgM and IgG, in culture supernatants at day 5. Myricetin treatment led to augmented BACH2 expression and reduced IRF4, BLIMP1, and XBP1 expression compared to control cultures., Conclusion: Myricetin treatment reduced PC differentiation and Ig secretion by primary human B cells. Targeting B cells in this way may be a therapeutic approach for some autoimmune diseases., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: All authors approved the final manuscript and the submission to this journal. Competing interests: Shabirul Haque PhD, is associated with Molecular Medicine and serving as Associate editor and Managing editor. Betty Diamond, MD is associated with Molecular Medicine as Editor-in-Chief., (© 2025. The Author(s).)

The differentiation of human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs) is a promising approach for the treatment of neurodegenerative diseases and regenerative medicine. Dual-SMAD inhibition using small molecules has been identified as a key strategy for directing the differentiation of hiPSCs into NPCs by regulating specific cell signaling pathways. However, conventional culture methods are time-consuming and exhibit low differentiation efficiency in neural differentiation. Nanocarriers can address these obstacles as an efficient platform for the controlled release and accurate delivery of small molecules. In this paper, we developed calcium phosphate-coated mesoporous silica nanoparticles capable of delivering multiple small molecules, including LDN193189 as a bone morphogenetic protein (BMP) inhibitor and SB431542 as a transforming growth factor (TGF)-beta inhibitor, for direct differentiation of hiPSC-mediated NPCs. Our results demonstrated that this nanocarrier-mediated small molecule release system not only enhanced the in vitro formation of neural rosettes but also modulated the expression levels of key markers. In particular, it downregulated OCT4, a marker of pluripotency, while upregulating PAX6, a critical marker for the neuroectoderm. These findings suggest that this controlled small molecule release system holds significant potential for therapeutic applications in neural development and neurodegenerative diseases.

This study examines the biocompatibility, osteogenic potential, and effectiveness of polyether ether ketone (PEEK) composites for treating osteonecrosis, seeking to establish a theoretical basis for clinical application. A range of PEEK composite materials, including sulfonated polyether ether ketone (SPEEK), polydopamine-sulfonated polyether ether ketone (SPEEK-PDA), bone-forming peptide-poly-dopamine-sulfonated polyether ether ketone (SPEEK-PDA-BFP), and vascular endothelial growth factor-poly-dopamine-sulfonated polyether ether ketone (SPEEK-PDA-VEGF), were constructed by concentrated sulfuric acid sulfonation, polydopamine modification and grafting of bioactive factors. The experiments involved adult male New Zealand rabbits aged 24-28 weeks and weighing 2.6-4 kg. The SPEEK-PDA-BFP possesses the smallest water contact angle, indicating the highest hydrophilicity, with its surface characterized by a rich density of clustered BFP particles. The SPEEK-PDA-BFP exhibits superior adhesion, proliferation, and differentiation capabilities, along with pronounced bacteriostatic effects, which are attributed to its dense particle clusters. The SPEEK-PDA-BFP facilitates the formation of regular and dense bone trabeculae. Comparative study on treating osteonecrosis with SPEEK-PDA-VEGF and SPEEK-PDA-BFP highlighted the superior formation of mature bone trabeculae and angiogenic protein CD31 around SPEEK-PDA-VEGF. The PEEK composite materials have good biocompatibility, osteogenic activity and bone repair activity. In particular, SPEEK-PDA-VEGF composite materials have the best effect on bone repair., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Difficult-to-heal wounds management accounts for about 4% of healthcare costs, highlighting the need for innovative solutions. Extracellular signals drive cell proliferation during tissue regeneration, while epigenetic mechanisms regulate stem cell homeostasis, differentiation, and skin repair. Exploring epigenetic regulation in adipose-derived stem cells (ADSCs) holds promise for improving skin injury treatments. We investigated the effects of histone deacetylase inhibitor (SAHA) on ADSCs to better understand its cellular and molecular impacts. ADSCs were treated with SAHA for 72 h, showing no change in cell viability at the studied concentrations. However, the expression of histone deacetylase decreased at 1000 nM, while the cell proliferation marker Ki-67 increased after SAHA treatment, as confirmed by immunofluorescence. CCND1 gene expression increased, whereas protein expression of the proliferating cell nuclear antigen (PCNA) decreased. Cell cycle analysis showed an increase in G2 phase in SAHA-treated cells. Microarray analysis revealed 74 upregulated and 40 downregulated differentially expressed genes, including upregulation of P53 targets, CDKN1A and MDM2. Proteomic analysis identified 631 upregulated and 823 downregulated proteins compared to the vehicle. Pathway enrichment analysis showed cell cycle, ATP-dependent chromatin remodeling and DNA processes were among the affected pathways. This study suggests SAHA modulates ADSCs' biological processes, highlighting its potential for skin regeneration., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Osteoporosis (OP) is a systemic skeletal disorder characterized by decreased bone mineral density and a heightened risk of fractures. Therapies for OP have primarily focused on balancing bone formation and bone resorption, but enhancing the remineralization of osteoporotic bone is also a key strategy for effective repair. Recent insights into biomineralization mechanisms have highlighted the essential role of mineral-containing extracellular vesicles (EVs) secreted by osteoblasts in promoting bone marrow mesenchymal stromal/stem cell (BMSC) differentiation and initiating matrix mineralization. Drawing from these principles, we developed a biomimetic approach to replicate the structure and function of the osteoblast-derived EVs by engineering biomimetic mitochondrial minerals with bone marrow homing cell membranes (CMs). This bone-targeted biomimetic system exhibits excellent biocompatibility, enhancing osteogenic differentiation and stimulating angiogenesis by regulating cellular energy metabolism. Additionally, the CM-coated structure shows affinity for collagen fibrils, effectively enhancing intrafibrillar collagen mineralization, thereby facilitating osteoporotic bone repair. Overall, the biomimetic system offers a safe and efficient therapeutic alternative, positioning it as a platform for bone tissue engineering and regenerative medicine.

Bacterium-triggered carious lesions implicate dental hard tissue destruction and the simultaneous initiation of regenerative events comprising dental stem cell activation. Streptococcus mutans ( S. mutans ) is a prominent pathogen of the oral cavity and the principal cause of caries. S. mutans generates complex products involved in interbacterial interactions, including Mutanobactin-D (Mub-D), which belongs to a group of non-ribosomal cyclic lipopeptides. In the present study, we aimed to analyse the potential role of the synthetic Mub-D peptide in cell populations involved in tissue regenerative processes. To this end, we assessed the in vitro effects of Mub-D in human dental pulp stem cells (hDPSCs) and human bone marrow stem cells (hBMSCs). Our data demonstrated a concentration-dependent effect of Mub-D on their viability and a significant increase in their proliferation and osteogenic/odontogenic differentiation. These events were associated with specific changes in gene expression, where CCDN-1 , RUNX-2 , OSX , OCN , DMP-1 , DSPP , and BMP-2 genes were upregulated. The ability of Mub-D to modulate the osteogenic/odontogenic differentiation of both hDPSCs and hBMSCs and considerably enhance mineralisation in a controlled and concentration-dependent manner opens new perspectives for stem cell-based regenerative approaches in the clinics.

A functional bioink with potential in bone tissue engineering must be subjected to critical investigation throughout its intended lifespan. The aim of this study was to develop alginate-gelatin-based (Alg-Gel) multicomponent bioinks systematically and to assess the short- and long-term exposure responses of human bone marrow stromal cells (hBMSCs) printed within these bioinks with and without crosslinking. The first generation of bioinks was established by incorporating a range of cellulose nanofibrils (CNFs), to evaluate their effect on viscosity, printability and cell viability. Adding CNFs to Alg-Gel solution increased viscosity and printability without compromising cell viability. In the second generation of bioinks , the influence of nano-hydroxyapatite (nHA) on the performance of the optimized Alg-Gel-CNF formulation was investigated. The addition of nHA increased the viscosity and improved printability, and an adjustment in alginate concentration improved the stability of the structures in long-term culture. The third generation bioink incorporated RGD-functionalized alginate to support cell attachment and osteogenic differentiation. The optimized bioink composition exhibited improved printability, structural integrity in long-term culture and high hBMSC viability. In addition, the final bioink composition, RGD-Alg-Gel-CNF-nHA, showed osteogenic potential: production of the osteogenic marker proteins (Runx2, OCN), enzyme (ALP), and gene expression ( Runx2 , OCN ). A further aim of the study was to evaluate the osteogenic functionality of cells released from the structures after bioprinting. Cells were printed in two bioinks with different viscosities and incubated at 37 °C in growth medium without additional CaCl 2 . This caused gelatin to dissolve, releasing the cells to attach to tissue culture plates. The results demonstrated differences in hBMSC osteogenic differentiation. Moreover, the osteogenic differentiation of the released cells was different from that of the embedded cells cultured in 3D. Thus, this systematic investigation into bioink development shows improved results through the generations and sheds light on the biological effects of the bioprinting process., (Creative Commons Attribution license.)

The present study investigated whether dietary n -3 very-long-chain PUFA ( n -3 VLC-PUFA) could increase skin and bone mineralisation in Atlantic salmon ( Salmo salar ) in vivo and examined their potential effects on human osteoblast proliferation and differentiation in vitro . Atlantic salmon were fed different dietary levels of n -3 VLC-PUFA, and changes in tissue n -3 VLC-PUFA composition, skeletal morphology, skin and bone mineral content, bone mineral density (BMD) and gene expression patterns were examined. Additionally, in vitro experiments using human foetal osteoblast cells were conducted to investigate the potential influence of n -3 VLC-PUFA supplementation on cell proliferation, osteogenic differentiation and cytokine expression. The results demonstrated that increasing the dietary levels of n -3 VLC-PUFA increased the mineral content of vertebrae and BMD in salmon, with subtle yet significant impacts on the expression of genes involved in bone-related processes. Furthermore, in vitro experiments showed a potential contextual influence of n -3 VLC-PUFA supplementation on gene expression of osteogenic markers and cytokine expression. Our findings indicate for the first time that n -3 VLC-PUFA may influence processes related to bone mineralisation.

Humans are widely exposed to phthalates, a common chemical plasticizer. Previous cohort studies have revealed that maternal exposure to monobutyl phthalate (MBP), a key metabolite of phthalates, is associated with neurodevelopmental defects. However, the molecular mechanism remains unclear. Here, we demonstrate that maternal exposure to MBP enhances neural stem cell (NSC) differentiation into astrocytes with highly expressed C3 and LCN2 in mouse offspring, resulting in increased synapse phagocytosis and cognitive dysfunction. Mechanistically, we find that MBP exposure activates the IRE1α/XBP1s (spliced XBP1) stress response pathway, which regulates key genes involved in astrocyte differentiation (SOX9 and ATF3) and reactivity (C3 and LCN2). Conditional knockout or pharmacological inhibition of IRE1α markedly inhibits NSC differentiation into astrocytes and astrocyte reactivity, attenuates synapse phagocytosis, and improves cognitive function. This phenotype is further recapitulated in a human brain organoid model. Together, these findings unveil the molecular mechanism underlying the neurodevelopmental deficits caused by a widespread environmental pollutant., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Osteoarthritis (OA) has long been considered a disease of the articular cartilage. Within the past decade it has become increasingly clear that OA is a disease of the entire joint space and that interactions between articular cartilage and subchondral bone likely play an important role in the disease. Driven by this knowledge, we have created a novel microphysiological model of the osteochondral unit containing synovium, cartilage, bone, and vasculature in separate compartments with molecular and direct cell-cell interaction between the cells from the different tissue types. We have characterized the model in terms of differentiation by molecule and matrix secretion and shown that it demonstrates morphology and functionality that mimic the native characteristic of the joint space. Finally, we induced inflammation and subsequently rescued the model constructs by a known compound as proof of concept for anti-inflammatory drug screening applications.

As a prevalent mycotoxin found in cereal foods and feed, deoxynivalenol (DON) disrupts the orderly regeneration of intestinal epithelial tissue by interfering with the intracellular antioxidant defense system. However, the potential of mulberry leaf-derived Morin, a natural flavonoid active substance with clearing reactive oxygen species (ROS), to mitigate DON-induced intestinal oxidative damage remains unclear. Our investigation demonstrates that Morin effectively reverses the decline in growth performance and repairs damaged jejunal structures and barrier function under DON exposure. Furthermore, the proliferation and differentiation of intestinal stem cells (ISCs) is enhanced significantly after Morin intervention. Importantly, Morin increases the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) in the serum and jejunal tissue, while reducing the accumulation of ROS and malondialdehyde (MDA). Molecular interaction analysis further confirms that Morin targets inhibition of Keap1 to activate the Nrf2-mediated antioxidant system. In summary, our results suggest that Morin alleviates the oxidative damage induced by DON by regulating the Keap1/Nrf2 pathway, thereby restoring the proliferation and differentiation activity of ISC, which provides new insights into Morin mitigating DON damage.

Background: Tissue engineering for bone regeneration aims to heal severe bone injuries. This study aimed to prepare and assess the early osteogenic differentiation effects of a gelatin/calcium phosphate- Punica granatum nanocomposite scaffold on stem cells from human exfoliated deciduous (SHED) and human dental pulp stem cells (HDPSCs)., Methods: The electrospinning method was used to prepare a gelatin/calcium phosphate nanocomposite scaffold containing pomegranate (Punica granatum) extract. The physicochemical properties of the scaffold were evaluated. The effect of the scaffold on the selected cells was done by the cell viability evaluation. A special alkaline phosphatase (ALP) kit was utilized to investigate the early osteogenic differentiation effects of the prepared scaffold on HDPSCs and SHED., Results: The results showed that the scaffold had uniformly accumulated in the networked form. Besides, the prepared scaffold did not have beads (structural defects). No new interactions were observed in the spectroscopic spectra of the scaffold and these peaks showed the successful formation of the fibrous nanocomposite as well. Furthermore, cell viability percentage was significantly higher for the scaffold compared with the control group (cells without any material) for both HDPSCs and SHED. Early osteogenic differentiation results specified that the ALP activity was significantly higher for the scaffold compared with the control group (cells without any material) for both HDPSCs and SHED., Conclusion: The appropriate physicochemical assay and cellular results (cell viability and early osteogenic differentiation) for the prepared fibrous nanocomposite showed that the use of this nanocomposite can be considered in the construction of various scaffolds in bone and dental tissue engineering., Competing Interests: Declarations. Ethical approval: The study started after obtaining the code of ethics from the ethics committee of Tabriz University of Medical Sciences. The code of ethics of this project is IR.TBZMED.VCR.REC.1401.370. Consent for publication: No humans/animals were used for studies that are the basis of this research. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

This study aimed to investigate the innate immune response of human macrophages to Porphyromonas gingivalis W83 using a novel in vitro infection model. The growth kinetics of P. gingivalis W83 were analyzed, revealing an exponential growth phase at 8 h (optical density = 0.70). To establish a reliable macrophage model, the differentiation of THP-1 monocytes into macrophages was optimized using low concentrations of phorbol 12-myristate 13-acetate (PMA). This approach induced enhanced adherence and morphological changes, with full differentiation achieved after 48 h of PMA treatment followed by 24 h of rest. Polarization towards the pro-inflammatory M1 phenotype was successfully induced with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), as confirmed using cytokine profiling. Cytokine analysis using Luminex ® technology demonstrated significant increases in interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6, indicating the effective activation of macrophages towards a pro-inflammatory phenotype. Building upon this macrophage model, this study investigated the interactions between macrophages and P. gingivalis W83 during its exponential growth phase. After a one-hour infection period, bacterial DNA quantification in supernatants and lysed macrophages revealed minimal levels of internalized or adherent bacteria, supporting the hypothesis that P. gingivalis effectively evades immune detection. These findings emphasize the utility of this model in uncovering the sophisticated immune evasion strategies employed by P. gingivalis , with significant implications for the development of targeted therapeutic interventions.

There are few in vitro models available to study microglial physiology in a homeostatic context. Recent approaches include the human induced pluripotent stem cell model, but these can be challenging for large-scale assays and may lead to batch variability. To advance our understanding of microglial biology while enabling scalability for high-throughput assays, we developed an inducible immortalized murine microglial cell line using a tetracycline expression system. The addition of doxycycline facilitates rapid cell proliferation, allowing for population expansion. Upon withdrawal of doxycycline, this monoclonal microglial cell line differentiates, resembling in vivo microglial physiology as demonstrated by the expression of microglial genes, innate immune responses, chemotaxis, and phagocytic abilities. We utilized live imaging and various molecular techniques to functionally characterize the clonal 2E11murine microglial cell line. Transcriptomic analysis showed that the 2E11 line exhibited characteristics of immature, proliferative microglia during doxycycline induction, and further differentiation led to a more homeostatic phenotype. Treatment with transforming growth factor-β modified the transcriptome of the 2E11 cell line, affecting cellular immune pathways. Our findings indicate that the 2E11 inducible immortalized cell line is a practical and convenient tool for studying microglial biology in vitro., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Psoriasis is an immune-related inflammatory systemic condition characterized by dysregulated keratinocyte proliferation and chronic inflammation. Tanshinone I (Tan-I) has recently been discovered to have immunomodulatory properties, but its role and mechanisms in treating psoriasis remain unclear., Objective: To evaluate the efficacy of Tan-I in the treatment of psoriasis and to determine the mechanisms involved., Methods: An imiquimod (IMQ)-induced psoriasis-like mouse model was treated topically with Tan-I (7.5 mg/kg/d) or a vehicle. Disease severity was evaluated using the Psoriasis Area and Severity Index (PASI), and histological changes were assessed via H&E staining and Ki67 immunofluorescence. TNF-α-stimulated HaCaT keratinocytes were used for in vitro analyses, including apoptosis, cell cycle progression, and inflammatory gene expression via RT-qPCR. RNA sequencing (RNA-seq) was performed to investigate Tan-I's mechanisms in vivo and in vitro, while keratin expression was analyzed by immunofluorescence and Western blot., Results: Tan-I treatment significantly alleviated psoriasis-like lesions in the IMQ mouse model, improving skin pathology and reducing Ki67-positive cells. RNA-seq revealed that Tan-I modulated immune pathways, keratinocyte differentiation, and barrier function. In TNF-α-stimulated HaCaT cells, Tan-I induced G1-phase cell cycle arrest, reduced apoptosis, and suppressed inflammatory gene expression. RNA-seq further showed that Tan-I normalized cell cycle signaling and apoptosis pathways disrupted by TNF-α. Additionally, Tan-I restored keratin expression patterns, increasing K1 and decreasing K6 and K17 levels in both mouse skin and HaCaT cells., Conclusion: Tan-I is a promising therapeutic candidate for psoriasis, effectively mitigating inflammation, normalizing keratinocyte differentiation, and inhibiting abnormal keratinocyte apoptosis., Competing Interests: The authors report no conflicts of interest in this work., (© 2025 Wang et al.)

Tumor necrosis factor-alpha (TNF-α) is a significant cytokine that regulates bone resorption under inflammatory conditions. However, its mechanism of action in osteocytes remains unclear. In this study, highly purified osteocytes were isolated from dentin matrix protein 1 (DMP1)-Topaz mice using cell sorter. RNA sequencing (RNA-seq) revealed that TNF-α stimulation increased C-X-C motif chemokine ligand 10 (CXCL10) gene expression in osteocytes. Although CXCL10 did not affect osteoclast differentiation in vitro, it enhanced the migration of osteoclast precursors. Additionally, in the transwell co-culture system, TNF-α induced the migration of osteoclast precursors. However, this effect was attenuated by a CXCL10-neutralizing antibody. In vivo, mice were administered supracalvarial injections of TNF-α with or without the CXCL10-neutralizing antibody for 5 days. The percentage of CXCL10-positive osteocytes increased after TNF-α administration. Additionally, osteoclast formation and bone resorption were assessed. CXCL10-neutralizing antibody-treated calvariae exhibited a significantly lower number of osteoclasts and bone resorption than those treated with TNF-α alone. These results indicated that TNF-α-induced CXCL10, which affects the migration of osteocyte-derived osteoclast precursors, may enhance TNF-α-triggered osteoclast formation and bone resorption in vivo., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: All animal experiments were performed following the Regulations for Animal Experiments and Related Activities at Tohoku University (2018DnA-028-06) by the Institutional Animal Care and Use Committee of the Tohoku University Environmental & Safety Committee. This study was reported in accordance with ARRIVE guidelines., (© 2025. The Author(s).)

Polyetheretherketone (PEEK) is widely used in orthopedic and dental implants due to its excellent mechanical properties, chemical stability, and biocompatibility. However, its inherently bioinert nature makes it present weak osteogenic activity, which greatly restricts its clinical adoption. Herein, strontium (Sr) is incorporated onto the surface of PEEK using mussel-inspired polydopamine coating to improve its osteogenic activity. X-ray photoelectron spectroscopy and ion release assay results confirm that different concentrations of Sr are incorporated onto the PEEK substrate surfaces. The strontium-modified PEEK samples show a stable Sr ion release in 35 days of detection. Better results of MC3T3-E1 pre-osteoblasts adhesion, spreading, and proliferation can be observed in strontium-modified PEEK groups, which demonstrates strontium-modified PEEK samples with the improved MC3T3-E1 pre-osteoblasts compatibility. The boosted osteogenic activity of strontium-modified PEEK samples has been demonstrated by the better performed of ALP activity, extracellular matrix mineralization, collagen secretion, and the remarkable up-regulation of ALP, OCN, OPN, Runx2, Col-I, BSP, and OSX of the MC3T3-E1 pre-osteoblasts. Additionally, the strontium-modified PEEK samples exhibit a dose-dependent enhancement of osteoblasts compatibility and osteogenic activity, and the PEEK-Sr10 group shows the best. These findings indicate that strontium-decorated PEEK implants show promising application in orthopedic and dental implants., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Hydroxyapatite (HA) is widely used as a bone graft. However, information on the head-to-head osteoinductivity and in vivo performance of micro- and nanosized natural and synthetic HA is still lacking. Here, we fabricated nanosized bovine HA (nanoBHA) by using a wet ball milling method and compared its in vitro and in vivo performance with microsized BHA, nanosized synthetic HA (nanoHA), and microsized synthetic HA (HA). The results showed that the wet ball milling method successfully reduced the particle size of BHA to 40 nm without changing its natural characteristics. NanoBHA was able to maintain cell viability and induce cell proliferation and calcium deposits. NanoBHA promoted osteogenic differentiation via OPN as a specific regulator, with a 13-fold greater expression level. NanoBHA and HA also activated ERK1/2 indicated corresponding to the proliferation-differentiation and death of cells, respectively. The calvarial bone defect model showed that nanoBHA induced bone growth based on CT images, which is in line with the histological results showing the presence of bone cells and connective tissue at the nanoBHA implantation site. In conclusion, natural HA outperformed synthetic HA. Our findings will attract interest in further research into nanomaterials and their mechanism of action in bone remodeling., Competing Interests: The authors declare no competing interests., (Copyright: © 2025 Gani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Icariin (ICA) serves as the primary biologically active compound in traditional Chinese medicine Epimedium, while Icariside II (ICSII) represents one of its gastrointestinal metabolites. Although ICA and ICSII have demonstrated osteogenic differentiation- promoting effects on BMSCs, there is limited literature comparing their effects and underlying mechanisms. This study aimed to compare the osteogenic effects of Icariin and Icariside II, along with their respective osteogenic mechanisms. In this study, we initially determined the optimal concentrations of Icariin (10 -5 mol/L) and Icariside II (10 -6 mol/L) for inducing rBMSCs osteogenic differentiation using CCK8, ALP activity assay, and flow apoptosis assay. Subsequently, we compared the vascularization and osteogenic capacity of the two groups through alizarin red staining assay, Western Blot, and RT-PCR. Subsequently, we assessed the phosphorylated and non-phosphorylated expression of JNK, ERK1/2, P38, and AKT at different time intervals. We observed their phosphorylated expression and the expression of angiogenic/osteogenic markers after blocking with their corresponding inhibitors. It was observed that both the Icariin and Icariside II groups promoted the expression of osteogenic/angiogenic markers RUNX2, OCN, OPN, VEGF, and ANG1. While there was no significant difference in their osteogenic abilities, ICSII exhibited a stronger promotion of angiogenic differentiation markers, VEGF, compared to ICA. Additionally, it was observed that both ICA and ICSII could activate ERK1/2 phosphorylation, thereby further promoting the osteogenic/angiogenic differentiation of rBMSCs through the activation of the MAPK/ERK1/2 signaling pathway., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Orthodontic relapse, the undesired deviation of teeth from their corrected positions, remains a significant challenge in clinical orthodontics. Incomplete periodontal bone remodeling has been identified as a key factor in this process. Despite decades of research, currently there are no effective strategies to prevent relapse., Methods: We isolated and identified dental pulp stem cell-derived intracellular vesicles (DPSC-IV) from human dental pulp tissue. To investigate its effect, DPSC-IV was added to osteoblast or osteoclast differentiation medium. During the orthodontic retention period, DPSC-IV was administrated to rats by subgingival injection. Relapse distance and relapse rate were calculated to evaluate DPSC-IV's ability to prevent relapse. Additionally, Western blot analysis were used to examine DPSC-IV's inhibitory effect on osteoclast differentiation., Results: DPSC-IV significantly promoted osteoblast differentiation and inhibited osteoclast differentiation. Application of DPSC-IV during retention resulted in a significant reduction in both relapse distance and relapse rate, with improved periodontal structure and decreased osteoclast activity. This effect was mediated by the PI3K/Akt/NF-κB signaling pathway and could be reversed by the PI3K activator insulin-like growth factor-1 (IGF-1)., Conclusion: This study highlights the potential of DPSC-IV as a novel preventive approach against orthodontic relapse, offering a novel strategy for maintaining long-term orthodontic stability., Competing Interests: Declarations. Ethics approval and consent to participate: For animal experiments, the study entitled “Dental pulp stem cell-derived intercellular vesicles prevent orthodontic relapse in a rat model” was approved by the Wuhan University Animal Ethics Committee (S07923060E) on June 19th, 2023, and performed according to the AVMA guidelines. For dental pulp tissues acquisition, the patients provided written informed consent for the use of samples. Third molars with no periodontal disease or dental caries were extracted from healthy volunteers aged between 20 and 30 years from the Department of Oral Surgery, Renmin Hospital of Wuhan University, with ethical approval (Approval Number: WDRY-2022-K025, December 1st, 2021, Wuhan, China). Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2025. The Author(s).)

The burgeoning field of nano-bone regeneration is yet to establish a definitive optimal particle size for nanocarriers. This study investigated the impacts of nanocarrier's particle size on the bone regeneration efficacy of fingolimod (FTY720)-loaded nanoemulsions. Two distinct particle sizes (60 and 190 nm, designated as NF60 and NF190, respectively) were produced using low-energy and high-energy emulsion techniques, maintaining a consistent surfactant, co-surfactant, and oil. In vitro studies using rat mesenchymal stem cells revealed that both NF60 and NF190 exhibited cell viability and reduced lactate dehydrogenase. Interestingly, NF60 demonstrated superior antioxidant properties, significantly reducing nitric oxide and intracellular reactive oxygen species (ROS) levels compared to NF190. Furthermore, NF60 significantly enhanced ALP activity and calcium deposition during osteogenic differentiation, indicating its potential to promote the early stages of bone formation. In vivo studies using a rat calvarial bone defect model demonstrated that both NF60 and NF190 significantly upregulated the expression of key osteogenic genes, including Runx2, Col, ALP, OCN, and BMP2. Notably, NF60 induced significantly higher expression of Runx2 and BMP2. X-ray and histological investigations revealed significantly improved bone regeneration in the NF60 group, highlighting the superior bone healing potential of smaller FTY720 nanoemulsions, without infiltration of inflammatory cells. The smaller particle size demonstrated superior antioxidant properties, enhanced osteogenic differentiation, and improved bone regeneration, suggesting smaller nanoparticles, with their larger surface area, accelerated drug release rate, and lower viscosity, interact more effectively with cells, leading to increased and effective drug delivery and cellular uptake. Findings highlight the importance of nanocarrier size in optimizing drug delivery for bone tissue engineering applications.

Background: Understanding how enteric neural crest cells (ENCCs) differentiate into neurons is crucial for neurogenesis therapy and gastrointestinal disease research. This study explores how magnesium ions regulate the glycolytic pathway to enhance ENCCs differentiation into neurons., Materials and Methods: We used polymerase chain reaction, western blot, immunofluorescence, and multielectrode array techniques to assess magnesium ions' impact on ENCCs differentiation. Non-targeted metabolomic sequencing, cellular acidification rate, oxygen consumption, and western blot analyzed sugar metabolism changes. D-glucose- 13 C6 isotope tracing identified key glucose flux changes. Surface plasmon resonance was used to detect the binding affinity of magnesium ions with key glycolysis genes. The elastic modulus of the hydrogel was measured using a universal testing machine, while pore size and porosity were assessed with scanning electron microscopy. Swelling ratios were determined using gravimetric analysis. In vivo, ENCCs in hydrogels were transplanted into renal capsule and subcutaneously, and magnesium ions' effects on ENCCs differentiation were evaluated., Results: Magnesium ions increased glycolysis levels during ENCCs differentiation into neurons, along with significant upregulation of neuronal markers β-Tubulin and ubiquitin C-terminal hydrolase L1, and enhanced functional neuronal properties. D-glucose- 13 C6 tracing results showed increased carbon flux in the glycolytic pathway after magnesium supplementation. The binding affinity of magnesium ions with the glycolytic key enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 was found to be 1.08 μM. Inhibiting glycolysis suppressed ENCCs differentiation into neurons, emphasizing its crucial role. The double-cross-linked hydrogel gelatin methacryloyl-alginate (gelMA-ALMA), cross-linked with magnesium ions, showed promise in enhancing ENCCs differentiation in vivo without causing systemic hypermagnesemia., Conclusion: Magnesium ions promote ENCCs differentiation into neurons by activating the Warburg effect. The GelMA-ALMA hydrogel serves as an effective localized magnesium delivery system, supporting neuronal differentiation in vivo., Competing Interests: Declarations. Ethics approval and consent to participate: The protocols for animal studies was ethically approved by the Ethics Committee of Qilu Hospital, Shandong University (Approval Nos. DMLL-2023–008). The ethical approval was granted for the study titled "Mechanism of TIMP3 Activation of the Shh/Gli Pathway in Regulating Neuroligins Genes to Induce Hirschsprung's Disease," dated January 1, 2023. This study does not involve any experiments related to human cells or tissues. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: Ferroptosis is an iron-dependent regulatory cell death, which plays an essential role in bone loss. This study investigated whether the mechanism of risperidone (RIS)-induced bone loss is related to ferroptosis., Methods: The schizophrenia mice were induced by administering MK-801. Subsequently, RIS were injected, or ferroptosis inhibitor Ferrostatin-1 (Fer-1) co-injected for 8 weeks. Bone loss of schizophrenia mice were assessed using microCT, H&E staining, ALP staining, ARS staining and WB, respectively. Ferroptosis of schizophrenia mice were detected by Iron Colorimetric Assay Kit and WB, respectively. In addition, ALP staining, ARS staining, and WB were performed to reveal the role of RIS in osteogenic differentiation of MC3T3-E1 and BMSCs cells., Results: RIS treatment facilitates bone loss in schizophrenia mice and inhibit osteogenic differentiation of MC3T3-E1 and BMSCs cells. Moreover, up-regulated ferroptosis was found in vivo and in vitro after RIS treatment. Interesting, the bone loss and inhibition of osteogenic differentiation induced by RIS in schizophrenia mice were reversed by ferroptosis inhibitor Fer-1., Conclusion: Ferroptosis induced by RIS aggravates the bone loss of schizophrenia mice via inhibiting osteogenic differentiation., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The development of effective materials for neural tissue repair remains a major challenge in regenerative medicine. In this study, we present a novel MXene-reinforced composite cryogel scaffold designed for neural tissue regeneration. MXenes, a class of two-dimensional materials with high conductivity and biocompatibility, were integrated into a polyvinyl alcohol (PVA) matrix via cryopolymerization to form a macroporous, mechanically stable scaffold. The morphology, mechanical properties, and swelling behavior of the cryogel with different MXene contents have been assessed. The effects of MXene on the viability/proliferation and differentiation of neural cells (PC-12) cultured in the composite cryogel were elucidated. The MXene/PVA cryogel demonstrated excellent cell-supporting potential, with MXene not only showing no toxicity but also promoting the proliferation of cultured PC-12. Additionally, MXene induced a neuritogenesis-like process in the cells as evidenced by morphological changes and the enhanced expression of the neural marker β-III-tubulin. The neuroprotective properties of the MXene component were revealed by the alleviation of oxidative stress and reduction of intracellular ROS levels. These findings highlight the potential of MXene-embedded PVA cryogel as a promising material that can be further used in conjunction with electrostimulation therapy for advancing strategies in neural tissue engineering.

Background: TGF-β1 is the most abundant cytokine in bone, in which it serves as a vital factor to interdict adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, how TGF-β1 concurrently manipulates differentiation into these two distinct lineages remains elusive., Methods: Treatments with ligands or inhibitors followed by biochemical characterization, reporter assay, quantitative PCR and induced differentiation were applied to MSC line or primary BM-MSCs for signaling dissection. In vivo adipogenesis and ex vivo culture of bone explants were used to verify the functions of different SMAD complexes. Ingenuity Pathway Analysis, and analysis of transcriptomic datasets from human BM-MSCs in combination with hierarchical clustering and STRING assay were used to decipher the interplaying co-repressors. Mouse models of chronic and acute bone loss followed by biochemical assays and micro-computed tomography demonstrated the bone effects when functionally blocking the critical co-repressor HDAC1., Results: Distinct from the TGF-β1 inhibition on adipogenesis through canonical SMAD2/3 signaling, we clarified that TGF-β1 suppresses osteogenesis by inducing the formation of previously unidentified mixed SMADs mainly composed of SMAD1 and SMAD2, in which SMAD2 recruits more TGF-β1-induced co-repressors including HDAC1, TGIF1 and ATF3, whereas SMAD1 allows directing the whole transcriptional suppression complex to the cis-elements of osteogenic genes. Depletion of the cross-activation to the mixed SMADs dismantled specifically the TGF-β1 suppression on osteogenesis without affecting its inhibition on adipogenesis. Such phenomena can be reproduced via knockdown of co-repressors such as Hdac1 or addition of HDAC1 inhibitors in TGF-β1-treated MSCs. In either the chronic or the acute bone loss model, we demonstrated that the TGF-β signaling was augmented in the bone niche during osteolysis, whereas administration of HDAC1 inhibitors significantly improved bone quality., Conclusion: This study identifies a new signal valve through which TGF-β1 can inhibit osteogenesis specifically. Functional interruption of this valve can tilt the seesaw balance of BM-MSC differentiation towards osteogenesis, highlighting the interplaying co-repressors, such as HDAC1, as promising therapeutic targets to combat diverse degenerative orthopedic diseases., Competing Interests: Declarations. Ethics approval and consent to participate: Animals and were approved by the Institutional Animal Care and Use Committee of National Yang Ming Chiao Tung University (Permit Number: 1110216). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Environmental endocrine disruptor chemicals (EDCs) have raised significant concerns due to their potential adverse effects on human health, particularly on the central nervous system (CNS). This study provides a comparative analysis of the effects of 17-alpha ethinyl estradiol (EE2) and diethyl phthalate (DEP) on neuronal cell proliferation and neurotoxicity. Using differentiated SH-SY5Y human neuronal cells, we evaluated cell viability, microRNA (miRNA) regulation, and RNA expression following exposure to subtoxic concentrations of EE2 and DEP. Our results show that both EDCs downregulated specific miRNAs-miR-18b-5p, miR-200a-3p, and miR-653-5p-affecting key processes such as cell proliferation, survival, and apoptosis. Gene expression analysis revealed the upregulation of EGFR, IGF1R, BTG2, and SH3BP4, implicating these miRNAs in the regulation of the Ras and PI3K/Akt/mTOR pathways. Our findings highlight distinct cellular responses: DEP disrupts PTEN activity, while EE2 enhances phosphorylation within the PI3K/Akt/mTOR pathway, promoting pro-survival and anti-apoptotic signals. This study emphasizes the urgent need for regulatory measures to mitigate the neurotoxic effects of EDCs and offers valuable insights into their molecular impacts on brain health., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The hormone and neurotransmitter serotonin regulates numerous physiological functions within the central nervous system and in the periphery upon binding to specific receptors. In the periphery, the serotonin receptor 7 (5-HT7R) is expressed on different immune cells including monocytes and macrophages. To investigate the impact of 5-HT7R-mediated signaling on macrophage properties, we used human THP-1 cells and differentiated them into pro-inflammatory M1- and anti-inflammatory M2-like macrophages. Pharmacological 5-HT7R activation with the specific agonist LP-211 especially modulates morphology of M1-like macrophages by increasing the number of rounded cells. Furthermore, 5-HT7R stimulation results in significantly reduced phagocytic and migratory ability of M1-like macrophages. Noteworthy, LP-211 treatment leads to changes in secretory properties of all macrophage types with the highest effects obtained for M0- and M2c-like macrophages. Finally, the importance of 5-HT7R for regulation of phagocytosis was confirmed in human primary CD14 + cells. These results indicate that 5-HT7R activation selectively impairs basic functions of macrophages and might thus be a new access point for the modulation of macrophage responses in the future treatment of inflammatory diseases., Competing Interests: Declarations. Ethical approval: Not applicable. Consent for publication: The authors hereby consent to publication of the work in Cellular and Molecular Life Sciences. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2025. The Author(s).)

Oxidative stress (OS) refers to the disruption in the balance between free radical generation and antioxidant defenses, leading to potential tissue damage. Reactive oxygen species (ROS) can interact with biological components, triggering processes like protein oxidation, lipid peroxidation, or DNA damage, resulting in the generation of several volatile organic compounds (VOCs). Recently, VOCs provided new insight into cellular metabolism and can serve as potential biomarkers. The objective is to investigate the impact of OS on cell metabolism by analyzing the release or alterations of VOCs in the headspace of differentiated 3T3-L1 adipocytes. An OS model in differentiated 3T3-L1 cell lines was constructed using hydrogen peroxide (H 2 O 2 ) treatment. The effect of OS on cell metabolism was analyzed by detecting VOCs in the headspace of the cells using solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GCMS). Our findings indicate that H 2 O 2 concentrations exceeding 300 µM induce significant OS, leading to adipocyte apoptosis, as evidenced by various assays. Of the twenty VOCs identified, ten were upregulated in the cells. VOCs such as diphenyl ether, 1,3,5-trioxane, 5-methyl tridecane, 2-ethyl-1-hexanol, and 2,4-di-tert-butyl phenol emerged as potential biomarkers for OS. This study demonstrates that elevated OS alters VOC profiles in differentiated 3T3-L1 adipocytes, providing insights into the effects of OS on adipose tissue and identifying potential OS biomarkers., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Neural stem cell (NSCs) transplantation is a promising therapeutic strategy for spinal cord injury (SCI), but its efficacy is greatly limited by the local inhibitory microenvironment. In this study, based on l-arginine (l-Arg)-loaded mesoporous hollow cerium oxide (AhCeO 2 ) nanospheres, we constructed an injectable composite hydrogel (AhCeO 2 -Gel) with microenvironment modulation capability. AhCeO 2 -Gel protected NSCs from oxidative damage by eliminating excess reactive oxygen species while continuously delivering Nitric Oxide to the lesion of SCI in a pathological microenvironment, the latter of which effectively promoted the neural differentiation of NSCs. The process was confirmed to be closely related to the up-regulation of the cAMP-PKA pathway after NO-induced calcium ion influx. In addition, AhCeO 2 -Gel significantly promoted the polarization of microglia toward the M2 subtype as well as enhanced the regeneration of spinal nerves and myelinated axons. The prepared bioactive hydrogel system also efficiently facilitated the integration of transplanted NSCs with host neural circuits, replenished damaged neurons, alleviated neuroinflammation, and inhibited glial scar formation, thus significantly accelerating the recovery of motor function in SCI rats. Therefore, AhCeO 2 -Gel synergized with NSCs transplantation has great potential as an integrated therapeutic strategy to treat SCI by comprehensively reversing the inhibitory microenvironment.

BACKGROUND CERVUS ELAPHUS SIBIRICUS: (CES) has been traditionally used in Korean clinics to promote fracture healing based on its function of tonifying the kidneys and strengthening bones. However, experimental data supporting its efficacy are still insufficient. The aim of this study investigated the bone-union properties of CES in a femoral fracture animal model and its corresponding molecular mechanisms., Methods: Fifty-four C57BL/6 male mice underwent femoral shaft fracture by Bonnarens and Einhorn's method, subsequently receiving a water extract of CES (200 mg/kg/day, daily) for 7 and 14 days. Safranin O staining and immunohistochemistry of the fracture region were conducted against transforming growth factor-β (TGF-β), bone morphogenetic protein 2 (BMP2), and osterix. MG63 cells used to examine the underlying mechanisms of CES focused on BMP2-Smad pathway-related osteogenesis., Results: CES administration led to earlier union of the fractured bones, supported by Safranin O staining of the fracture region, demonstrating significantly increased cartilage formation day on 7 and a rapidly decreased cartilage area due to callus formation day on 14. CES administration also significantly upregulated the expression of TGF-β1 day 7, BMP 2, and osterix day 14 at the fracture site and also up-regulated alkaline phosphatase (ALP) activity, calcium deposition, and the phosphorylation of Smad in MG63 cells., Conclusions: CES promotes fracture healing by promoting osteoblastogenesis via a partial BMP2-Smad pathway., Competing Interests: Declarations. Ethics approval and consent to participate: The animal study was reviewed and approved by The Institutional Animal Care and Use Committee of Daejeon University (Daejeon, Republic of Korea; approval no. DJUARB2018-012). Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Hyaluronic acid (HA) has received considerable attention in the reconstruction of lost periodontal tissues. HA has been proposed to play a role in cell proliferation, differentiation, migration, and cell-matrix as well as cell-cell interactions. Although various studies have been conducted, further research is needed to expand our knowledge based on HA such as its effects on cell proliferation and osteogenic differentiation. The aim of this study is to assess, in single- and multi-culture plate models, the effect of HA on the proliferation, viability, and function of periodontal ligament fibroblasts, osteoblasts, and gingival epithelial cells. A novel multi-culture cell plate was chosen to simulate a cell-cell communication as close as possible to a real clinical condition in an in vitro setting. We found that HA exclusively enhanced epithelial cell proliferation, while intercellular communication stimulated the proliferation and osteogenic potential of the osteoblasts, independently from HA use. The proliferation and function of the periodontal ligament fibroblasts were not changed by HA or the cellular interplay. The use of multi-culture plates could represent a promising method to investigate and compare dental biomaterials in experiments mimicking an in vivo environment.

Aspirin (ASA) is one of the most used medications worldwide and has shown various effects on cellular processes, including stem cell differentiation. However, the effect of ASA on adipogenesis of adipose tissue-derived stem cells (ASCs) remains largely unknown. Considering the potential application of ASCs in regenerative medicine and cell-based therapies, this study investigates the effects of ASA on adipogenic differentiation in human ASCs. ASCs were exposed to varying concentrations of ASA (0 µM, 400 µM, and 1000 µM) and evaluated for changes in morphology, migration, and adipogenic differentiation. While ASA exposure did not affect self-renewal potential, migration ability, or cell morphology, it significantly reduced lipid vacuole formation at 1000 µM after 21 days of adipogenic differentiation ( p = 0.0025). This visible inhibition correlated with decreased expression of adipogenic markers (PPARG, ADIPOQ, and FABP4) and the proliferation marker MKi67 under ASA exposure in comparison to the control (ns). Overall, the findings demonstrate that ASA inhibits adipogenic differentiation of human ASCs in a dose-dependent manner in vitro, contrasting its known role in promoting osteogenic differentiation. This research highlights ASA's complex effects on ASCs and emphasizes the need for further investigation into its mechanisms and potential therapeutic applications in obesity and metabolic diseases. The inhibitory effects of ASA on adipogenesis should be considered in cell-based therapies using ASCs.