Differentiation Of Stromal Cells Research Articles

Nerve-Derived Extracellular Matrix Promotes Neural Differentiation of Bone Marrow Stromal Cells and Enhances Interleukin-4 Efficacy for Advanced Nerve Regeneration.

Facilitating neuronal differentiation of stem cells and microenvironment remodeling are the key challenges in cell-based transplantation strategies for central nervous system regeneration. Herein, the study harnesses the intrinsic pro-neural differentiation potential of nerve-derived extracellular matrix (NDEM) and its specific affinity for cytokines to develop an NDEM-gelatin methacryloyl(gelMA)-based bifunctional hydrogel delivery system for stem cells and cytokines. This system promotes the neural differentiation of bone marrow stromal cells (BMSCs) and optimizes the therapeutic index of Interleukin-4 (IL-4) for spinal cord injury (SCI) treatment. It is observed that incorporating NDEM into the hydrogel system intrinsically promotes BMSC differentiation into neuron-like cells and effectively regulates IL-4 release kinetics to match the neural reconstructing timeframe. Further analysis reveals that trace amounts of endogenous basic fibroblast growth factor (bFGF) detected in NDEM exhibit a potent effect in promoting neural differentiation. The sustained release of IL-4 from the NDEM significantly encourages macrophage polarization toward the M2 phase, optimizing the transplant microenvironment throughout the reconstruction process. This study demonstrates an NDEM-based optimization strategy for hybrid hydrogel to achieve synchronized delivery of stem cells and cytokines in regenerative medicine applications.

Bone Marrow Adipocytes as Novel Regulators of Metabolic Homeostasis: Clinical Consequences of Bone Marrow Adiposity.

Bone marrow adipose tissue is a distinctive fat depot located within the skeleton, with the potential to influence both local and systemic metabolic processes. Although significant strides have been made in understanding bone marrow adipose tissue over the past decade, many questions remain regarding their precise lineage and functional roles. Recent studies have highlighted bone marrow adipose tissue's involvement in continuous cross-talk with other organs and systems, exerting both endocrine and paracrine functions that play a crucial role in metabolic homeostasis, skeletal remodeling, hematopoiesis, and the progression of bone metastases. The advancement of imaging techniques, particularly cross-sectional imaging, has profoundly expanded our understanding of the complexities beyond the traditional view of bone marrow adipose tissue as an inert depot. Notably, marrow adipocytes are anatomically and functionally distinct from brown, beige, and classic white adipocytes. Emerging evidence suggests that bone marrow adipocytes, bone marrow adipose tissue originate from the differentiation of bone marrow mesenchymal stromal cells; however, they appear to be a heterogeneous population with varying metabolic profiles, lipid compositions, secretory properties, and functional responses depending on their specific location within the bone marrow. This review provides an up-to-date synthesis of current knowledge on bone marrow adipocytes, emphasizing the relationships between bone marrow adipogenesis and factors such as aging, osteoporosis, obesity, and bone marrow tumors or metastases, thereby elucidating the mechanisms underlying musculoskeletal pathophysiology.

Developmental exposure to perfluorooctane sulfonate(PFOS) impairs the endometrial receptivity

Perfluorooctane sulfonate (PFOS) is a widely used chemical in industrial production. It can be introduced into the environment through multiple pathways and exhibits resistance to degradation. Recent research has demonstrated a significant correlation between its exposure levels in the human body and the incidence of various diseases. The expression of genes related to endometrial receptivity and the differentiation of human endometrial stromal cells (hESCs) were assessed in this study concerning PFOS. In this study, we investigated the effect of PFOS exposure on endometrial tolerance by cell and animal experiments. The activity against endometrial mesenchymal cells was significantly reduced by PFOS intervention, and the apoptosis flow assay results showed that PFOS significantly promoted cell death in a concentration-dependent manner. Transmission electron microscopy results revealed mitochondrial damage in the PFOS-intervened group, and WB results showed that the expression levels of endometrial tolerance-related proteins Homeobox A10 (HOXA10) and integrin beta3 (ITGB3) were decreased, and the expression level of Forkhead box O1 (FOXO1) protein was increased. Animal studies have shown that PFOS exposure can change uterine morphology, cause obvious damage to pinopodes morphology, change the estrous cycle of mice, and affect endometrial receptivity In the present study, we found that PFOS may synergistically affect the viability of endometrial mesenchymal stromal cells through accumulation in vivo, and that PFOS may contribute to the failure of embryo implantation by affecting mitochondrial function and consequently endometrial permissive sites.

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Foxk2 Enhances Adipogenic Differentiation by Relying on the Transcriptional Activation of Peroxisome Proliferator-Activated Receptor Gamma.

Proper differentiation of bone marrow stromal cells (BMSCs) into adipocytes is crucial for maintaining skeletal homeostasis. However, the underlying regulatory mechanisms remain incompletely understood, posing a challenge for the treatment of age-related osteopenia and osteoporosis. Here, through comprehensive gene expression analysis during BMSC differentiation into adipocytes, we identified the forkhead transcription factor Foxk2 as a key regulator of this process. Foxk2 expression was significantly higher in the inguinal and epididymal white adipose tissues of db/db mice compared to non-obese db/m controls and was induced in BMSCs, C3H/10 T1/2, and ST2 cells following adipogenic stimulation. Overexpression of Foxk2 promoted adipogenic differentiation of C3H/10 T1/2, ST2, and BMSCs, accompanied by increased expression of lipogenic factors. Conversely, Foxk2 silencing inhibited adipogenic differentiation. Moreover, Foxk2 also facilitated lipogenesis of C3H/10 T1/2 and ST2 cells. Adipogenic stimuli triggered the nuclear translocation of Foxk2 through PI3-kinase and mTOR signalling pathways. Once in the nucleus, Foxk2 is directly bound to the promoters of Pparγ1 and Pparγ2, thereby enhancing their transcriptional activity. Notably, PPARγ1 and PPARγ2 reciprocally augmented the transcriptional activity of the Foxk2 promoter, indicating the presence of a Foxk2-PPARγ positive feedback loop that drives adipogenesis.

The Effects of Gender on Mesenchymal Stromal Cell (MSC) Proliferation and Differentiation In Vitro: A Systematic Review.

Mesenchymal stromal cells (MSCs) have the potential for novel treatments of several musculoskeletal conditions due to their ability to differentiate into several cell lineages including chondrocytes, adipocytes and osteocytes. Researchers are exploring whether this could be utilized for novel therapies for joint afflictions. The role of gender in the ability of MSCs to differentiate and proliferate into different cells has not been clearly defined. This systematic review aims to report the current literature on studies, characterized by high quality and in-depth analysis even though quantitatively limited, that have looked at the role of gender in the differentiation and proliferation of MSCs. Sixteen studies were identified during the literature search, reporting 533 patients, of which 202 were male and 331 were female. MSC proliferation, phenotypic analysis and differentiation are reported and contrasted in terms of donor gender. Heterogeneity in methodologies across studies likely contributes to the inconclusive findings presented here, with no discernible statistical disparity observed between genders in differentiation traits. Nevertheless, the proliferation results indicate a notable gender-related impact. Future investigations should aim to ascertain the potential influence of gender on MSC proliferation capacities more conclusively, emphasizing the necessity of standardized protocols for MSC analyses to enhance accuracy and comparability across studies.

USP7 Inhibition Promotes Early Osseointegration in Senile Osteoporotic Mice

Although elderly osteoporotic patients have similar implant survival rates compared with those of normal individuals, they require longer healing periods to achieve proper osseointegration. This may be related to chronic inflammatory responses and impaired stem cell repair functions in the osteoporotic bone microenvironment. Recently, the deubiquitinating enzyme, ubiquitin-specific peptidase 7 (USP7), was found to regulate the macrophage immune response and modulate stem cell osteogenic differentiation. The selective inhibitor of USP7, P5091, has also been found to promote bone repair and homeostasis in osteoporotic conditions. However, the roles of USP7 and P5091 in osteoimmunology and dental implant osseointegration under senile osteoporotic conditions remain unclear. In this study, USP7 depletion and P5091 were shown to inhibit inflammation in senescent bone marrow–derived macrophages (BMDMs) and promote osteogenic differentiation in aged bone marrow mesenchymal stromal cells (BMSCs). Furthermore, mRNA-Seq revealed that USP7 depletion could enhance efferocytosis in senescent BMDMs through the EPSIN1/low-density lipoprotein receptor-related protein 1 (LRP1) pathway and selectively induce apoptosis (senolysis) in aged BMSCs. In senile osteoporotic mice, we found that the osseointegration period was prolonged compared with young mice, and P5091 promoted the early stage of osseointegration, which may be related to macrophage efferocytosis around the implant. Collectively, this study suggests that USP7 inhibition may accelerate the osseointegration process in senile osteoporotic conditions by promoting macrophage efferocytosis and aged BMSCs apoptosis. This has implications for understanding the cellular interactions and signaling mechanisms in the peri-implant bone microenvironment under osteoporotic conditions. It may also provide clinical significance in developing new therapies to enhance osseointegration quality and shorten the edentulous period in elderly osteoporotic patients.

Altered uterine leptin signalling in obese mothers: from impaired decidualisation to pregnancy complications.

Obesity drastically affects maternal health and reproductive outcomes, being often associated with endocrine imbalance, compromised ovarian function, and pregnancy complications. The plastic nature of pregnancy may render the developing fetus particularly vulnerable to oscillations in maternal metabolism, ultimately shaping the health trajectories of the offspring. Presently, we discuss the impact of maternal obesity on decidualisation, a critical step for embryo implantation and placental development. Decidualisation encompasses the differentiation of endometrial stromal cells into specialised decidua. Impaired decidualisation was linked to pregnancy complications, and recent studies suggest that maternal obesity has a detrimental effect on decidualisation. Leptin, an adipokine significantly increased in the circulation of obese women, is known to regulate endometrial function and decidualisation, modulating immune response, angiogenesis, and cell proliferation. Furthermore, hyperleptinaemia in obese mothers was linked to altered leptin signalling in the uterus and compromised endometrial function. In this review, we explore the underlying molecular mechanisms linking altered uterine leptin signalling to impaired decidualisation and early pregnancy complications in obese mothers.

Effect of glucocerebrosidase on human bone marrow multipotent mesenchymal stromal

Introduction. During enzyme replacement therapy in patients with Gaucher disease (GD) with recombinant glucocerebrosidase (GCase), regression of bone manifestations is possible, but with prolonged therapy osteonecrosis may occur. These changes may be due to impaired differentiation of multipotent mesenchymal stromal cells (MSCs).Aim: to study changes in the MSCs of healthy donors and a patient with GD when cultured in the presence of GCase.Material and methods. MSCs were isolated from the bone marrow of 17 healthy donors and a female patient with GD by a standard method and cultured in the presence of various concentrations of GCase after the second passage from 2 to 7 weeks. Cell proliferation and the ability to differentiate were analyzed, including after induction. The assessment was carried out by differential staining, elution, and expression of differentiation marker genes by real-time PCR.Results. Low concentrations of recombinant GCase (0.25–1.5 U/ml) did not affect the proliferative activity of MSCs. Prolonged cultivation of MSCs in the presence of low doses of GCase led to a change in the differentiation potential of these cells in the direction of adipogenesis. Concentrations of GCase of 3–5 U/ml inhibited the proliferation of MSCs and caused significant changes in cell differentiation. High doses of the enzyme (7–10 U/ml) had a cytotoxic effect and led to cell death within one passage. The proliferative and differentiation potential of the MSCs of a patient with GD differed significantly from the cells of healthy donors in all the parameters studied.Conclusion. The cultivation of donor MSCs in the presence of recombinant GCase alters the proliferation and differentiation potential of these cells. These changes depend on the dose of the enzyme in the medium and the duration of cultivation.

Prolonged cultivation enhances the stimulatory activity of hiPSC mesenchymal progenitor-derived conditioned medium

BackgroundHuman induced pluripotent stem cells represent a scalable source of youthful tissue progenitors and secretomes for regenerative therapies. The aim of our study was to investigate the potential of conditioned medium (CM) from hiPSC-mesenchymal progenitors (hiPSC-MPs) to stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (MSCs). We also investigated whether prolonged cultivation or osteogenic pre-differentiation of hiPSC-MPs could enhance the stimulatory activity of CM.MethodsMSCs were isolated from 13 donors (age 20–90 years). CM derived from hiPSC-MPs was added to the MSC cultures and the effects on proliferation and osteogenic differentiation were examined after 14 days and 6 weeks. The stimulatory activity of hiPSC-MP-CM was compared with the activity of MSC-derived CM and with the activity of CM prepared from hiPSC-MPs pre-cultured in growth or osteogenic medium for 14 days. Comparative proteomic analysis of CM was performed to gain insight into the molecular components responsible for the stimulatory activity.ResultsPrimary bone marrow-derived MSC exhibited variability, with a tendency towards lower proliferation and tri-lineage differentiation in older donors. hiPSC-MP-CM increased the proliferation and alkaline phosphatase activity of MSC from several adult/aged donors after 14 days of continuous supplementation under osteogenic conditions. However, CM supplementation failed to improve the mineralization of MSC pellets after 6 weeks under osteogenic conditions. hiPSC-MP-CM showed greater enhancement of proliferation and ALP activity than CM derived from bone marrow-derived MSCs. Moreover, 14-day cultivation but not osteogenic pre-differentiation of hiPSC-MPs strongly enhanced CM stimulatory activity. Quantitative proteomic analysis of d14-CM revealed a distinct profile of components that formed a highly interconnected associations network with two clusters, one functionally associated with binding and organization of actin/cytoskeletal components and the other with structural constituents of the extracellular matrix, collagen, and growth factor binding. Several hub proteins were identified that were reported to have functions in cell-extracellular matrix interaction, osteogenic differentiation and development.ConclusionsOur data show that hiPSC-MP-CM enhances early osteogenic differentiation of human bone marrow-derived MSCs and that prolonged cultivation of hiPSC-MPs enhances CM-stimulatory activity. Proteomic analysis of the upregulated protein components provides the basis for further optimization of hiPSC-MP-CM for bone regenerative therapies.Graphical

Regulatory T cells engineered with polyphenol-functionalized immunosuppressant nanocomplexes for rebuilding periodontal hard tissue under inflammation-challenged microenvironment

Global aging heightens the risk of oral disorders, among which periodontitis is the major cause of tooth loss in the aging population. The regeneration of damaged periodontal hard tissue is highly challenging due to the existence of the refractory local inflammation. Owing to the potent anti-inflammatory capabilities, regulatory T cells hold great promise in immunotherapies for tissue regeneration. However, the transferred regulatory T cells can alter their phenotypes and functions in local inflammatory milieu, significantly impairing their therapeutic efficacy. Herein, we introduce a novel regulatory T cell-based nanobiohybrid system bearing polyphenol-functionalized rapamycin nanocomplexes. The sustained in situ release of immunosuppressant rapamycin from the cell-attached nanocomplexes maintains the anti-inflammatory phenotype of regulatory T cells in the inflammatory milieu. The synergistic actions of the anti-inflammatory cytokines secreted and the immunosuppressant released guide a pro-resolving polarization of macrophages and enhance osteogenic differentiation of bone marrow-derived stromal cells. The stabilized phenotype of the regulatory T cells dramatically promoted the resolution of periodontal inflammation and the repair of the hard tissue (alveolar bone) in vivo. Overall, these studies highlight a potent regulatory T cell-based nanobiohybrid therapy to treat periodontitis by modulating periodontal immune microenvironment.

A bone-targeting delivery platform based on mesoporous silica loaded with piR7472 for the treatment of osteoporosis

Promoting osteogenic differentiation and inhibiting osteoclast formation remain significant challenges in the treatment of osteoporosis. With the growing understanding of osteoporosis, increasing literature has highlighted the regulatory role of m6A methylation in this condition. However, there is currently no reliable method to stably regulate cellular m6A methylation levels. Here, we report a novel approach utilizing alendronate (aln)-modified mesoporous silica nanoparticles (MSNs) to deliver sodium bicarbonate and piR7472, modulating cellular behavior. Our experimental results demonstrate that Aln modification enables the nanoparticles to stably target hydroxyapatite, thereby accumulating in osteoporotic regions. Sodium bicarbonate suppresses osteoclastogenesis, while piR7472 enhances m6A methylation, promoting osteogenic differentiation of bone marrow stromal cells (BMSCs). Computed tomography (CT) and hematoxylin and eosin (HE) staining showed that after 2 weeks of treatment with MSNs-Na@piR7472, cortical bone thickened, trabecular bone density increased, collagen fiber thickness improved, and both the number and staining area of osteoclasts were significantly reduced. These findings indicate a marked improvement in osteoporosis.

Impact of Porosity and Stiffness of 3D Printed Polycaprolactone Scaffolds on Osteogenic Differentiation of Human Mesenchymal Stromal Cells and Activation of Dendritic Cells.

Despite the potential of extrusion-based printing of thermoplastic polymers in bone tissue engineering, the inherent nonporous stiff nature of the printed filaments may elicit immune responses that influence bone regeneration. In this study, bone scaffolds made of polycaprolactone (PCL) filaments with different internal microporosity and stiffness was 3D-printed. It was achieved by combining three fabrication techniques, salt leaching and 3D printing at either low or high temperatures (LT/HT) with or without nonsolvent induced phase separation (NIPS). Printing PCL at HT resulted in stiff scaffolds (modulus of elasticity (E): 403 ± 19 MPa and strain: 6.6 ± 0.1%), while NIPS-based printing at LT produced less stiff and highly flexible scaffolds (E: 53 ± 10 MPa and strain: 435 ± 105%). Moreover, the introduction of porosity by salt leaching in the printed filaments significantly changed the mechanical properties and degradation rate of the scaffolds. Furthermore, this study aimed to show how these variations influence proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBMSC) and the maturation and activation of human monocyte-derived dendritic cells (Mo-DC). The cytocompatibility of the printed scaffolds was confirmed by live-dead imaging, metabolic activity measurement, and the continuous proliferation of hBMSC over 14 days. While all scaffolds facilitated the expression of osteogenic markers (RUNX2 and Collagen I) from hBMSC as detected through immunofluorescence staining, the variation in porosity and stiffness notably influenced the early and late mineralization. Furthermore, the flexible LT scaffolds, with porosity induced by NIPS and salt leaching, stimulated Mo-DC to adopt a pro-inflammatory phenotype marked by a significant increase in the expression of IL1B and TNF genes, alongside decreased expression of anti-inflammatory markers, IL10 and TGF1B. Altogether, the results of the current study demonstrate the importance of tailoring porosity and stiffness of PCL scaffolds to direct their biological performance toward a more immune-mediated bone healing process.

Fibrosis signaling in endometrial cells and endometriosis development

In endometriosis, the tissues similar to the endometrial tissue attaches outside the uterine cavity, causing inflammation and fibrosis. The retrograde menstruation theory is the most plausible mechanism, though the detailed pathogenesis remains unclear. Our observations suggest that endometriosis-like lesions occur more often at sites of ovarian excision causing bleeding in mouse models. Additionally, prostaglandin E2 (PGE2) and thrombin, a protease-activated receptor (PAR) agonist in menstrual blood exacerbate inflammation in these lesions. Focusing on the hypoxic conditions of menstrual blood, we investigated the effects of PGE2/thrombin on inflammation and fibrosis using primary cultured endometrial stromal cells (ESCs) and glandular epithelial cells (EECs) under low oxygen conditions. Chemokine CXCL12 secreted by endometrial stromal cells under hypoxia acts on CXCR4 receptors on glandular epithelial cells, inducing epithelial-mesenchymal transition (EMT), suggesting a possible role in endometriosis progression. RNA-seq analysis of PGE2/thrombin effects on endometrial stromal cells revealed activation of the transforming growth factor (TGF)-β pathway, particularly increased production and secretion of activin A, a member of the TGFβ family. Activin A, via increased connective tissue growth factor (CTGF) expression, promotes differentiation of endometrial stromal cells from fibroblast-like to myofibroblast transdifferentiation (FMT) of ESCs. In conclusion, targeting the CXCL12/CXCR4 and activin A/CTGF signaling pathways holds promise for improving fibrosis in endometriosis lesions.

Comprehensive analysis of mesenchymal cells reveals a dysregulated TGF-β/WNT/HOXB7 axis in patients with myelofibrosis.

Despite the advances in the understanding and treatment of myeloproliferative neoplasm (MPN), the disease remains incurable with the risk of evolution to acute myeloid leukemia or myelofibrosis (MF). Unfortunately, the evolution of the disease to MF remains poorly understood, impeding preventive and therapeutic options. Recent studies in solid tumor microenvironment and organ fibrosis have shed instrumental insights on their respective pathogenesis and drug resistance, yet such precise data are lacking in MPN. In this study, through a patient sample-driven transcriptomic and epigenetic description of the MF microenvironment landscape and cell-based analyses, we identify homeobox B7 (HOXB7) overexpression and more precisely a potentially novel TGF-β/WNT/HOXB7 pathway as associated to a pro-fibrotic and pro-osteoblastic biased differentiation of mesenchymal stromal cells (MSCs). Using gene-based and chemical inhibition of this pathway, we reversed the abnormal phenotype of MSCs from patients with MF, providing the MPN field a potentially novel target to prevent and manage evolution to MF.

Effect of the polyphenol flavonoids fisetin and quercetin on the adipogenic differentiation of human mesenchymal stromal cells.

Fisetin and quercetin, polyphenol flavonoids, have been shown to have a wide range of beneficial pharmacological effects including anti-inflammatory, antioxidative, and anti-cancer. Our previous work shows that fisetin also affects the specification of the adipogenic-osteogenic lineage of human mesenchymal stem cells (hMSCs) by modulating the Hippo-YAP signaling pathway. Although quercetin has a structure similar to that of fisetin, its effects on the functional properties of hMSCs have not yet been investigated. The objective of the present study is to determine the effects of quercetin on the various properties of hMSCs, including proliferation, migration, and differentiation capacity toward adipogenic and osteogenic lineages. The results show that while fisetin increases hMSC adipogenic differentiation, quercetin inhibited adipogenic differentiation of hMSCs. The inhibition is mediated, at least in part, by the activation of hippo signaling and up-regulation of miR-27b, which inhibits the expression of genes involved in all critical steps of lipid droplet biogenesis, resulting in a decrease in the number of lipid droplets in hMSCs. It is possible that the lack of hydroxylation of the 5 position on the A ring of quercetin could be responsible for its different effect on the adipogenic-osteogenic lineage specification of hMSCs compared with fisetin. Molecular docking and molecular dynamics simulation suggested that fisetin and quercetin possibly bind to serine / threonine protein kinases 4 (STK4/MST1), which is an upstream kinase responsible for LATS phosphorylation. Taken together, our results demonstrate more insight into the mechanism underlying the role of flavonoid fisetin and quercetin in the regulation of adipogenesis.

Low-intensity pulsed ultrasound regulates bone marrow mesenchymal stromal cells differentiation and inhibits bone loss by activating the IL-11-Wnt/β-catenin signaling pathway

BackgroundOsteoporosis (OP) is a common metabolic bone disease. Low-intensity pulsed ultrasound (LIPUS) can effectively promote bone formation and fracture healing. The Wnt/β-catenin signaling pathway is crucial for regulating bone homeostasis and bone diseases, and its downregulation is one of the main mechanisms of osteoporosis pathogenesis. Interleukin-11 (IL-11), which is regulated by mechanical stress, is a key factor in bone remodeling. Here, we investigated the optimal intervention parameters for LIPUS, the relationships among LIPUS, IL-11, and the Wnt/β-catenin signaling pathway, and the effects of LIPUS on bone loss and potential molecular mechanisms in ovariectomized (OVX) mice. MethodsBone marrow mesenchymal stromal cells (BMSCs) were subjected to LIPUS intervention for 0, 10, or 20 min to determine the optimal intervention time. The mediating role of IL-11 in LIPUS intervention was explored through IL-11 knockdown and overexpression. Finally, animal experiments were conducted to investigate the in vivo therapeutic effects of LIPUS. ResultsThe optimal intervention time for LIPUS was 20 min. LIPUS promoted IL-11 expression and upregulated the Wnt/β-catenin signaling pathway, thereby promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. IL-11 mediates the regulation of the Wnt/β-catenin signaling pathway by LIPUS. Additionally, LIPUS effectively improved the bone microstructure in ovariectomized mice, inhibited bone loss, promoted IL-11 expression in bone tissue, and activated the Wnt/β-catenin signaling pathway in the femur. ConclusionLow-intensity pulsed ultrasound can regulate BMSCs differentiation and inhibit bone loss by promoting IL-11 expression and activating the Wnt/β-catenin signaling pathway.

Insights into Osteogenesis Induced by Crude Brassicaceae Seeds Extracts: A Role for Glucosinolates.

Background/Objectives: Crude extracts from the Brassica genus have recently emerged as promising phytochemicals for preventing bone loss. While the most documented evidence suggests that their general biological activity is due to glucosinolates' (GLSs') hydrolysis products, the direct activity of GLSs is beginning to be uncovered. However, the contribution of GLSs to the bone-sparing activity of crude Brassicaceae extracts has seldom been addressed. Here, we aimed to gain insights into this gap by studying in the same in vitro model of human osteogenesis the effect of two Brassica seed extracts (Eruca sativa and Lepidium sativum) obtained from defatted seed meals, comparing them to the isolated GLSs most represented in their composition, glucoerucin (GER) and glucotropaeolin (GTL), for Eruca sativa and Lepidium sativum, respectively. Methods: Osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was assessed by alizarin red staining assay and real-time PCR, respectively, evaluating mineral apposition and mRNA expression of specific osteogenic genes. Results: Both Brassica extracts and GLSs increased the osteogenic differentiation, indicating that the stimulating effect of Brassica extracts can be at least partially attributed to GLSs. Moreover, these data extend previous evidence of the effect of unhydrolyzed glucoraphanin (GRA) on osteogenesis to other types of GLSs: GER and GTL. Notably, E. sativa extract and GTL induced higher osteogenic stimulation than Lepidium sativum extract and GER, respectively. Conclusions: Overall, this study expands the knowledge on the possible application of Brassica-derived bioactive molecules as natural alternatives for the prevention and treatment of bone-loss pathologies.

A multifunctional self-reinforced injectable hydrogel for enhancing repair of infected bone defects by simultaneously targeting macrophages, bacteria, and bone marrow stromal cells

Injectable hydrogels (IHs) have demonstrated huge potential in promoting repair of infected bone defects (IBDs), but how to endow them with desired anti-bacterial, immunoregulatory, and osteo-inductive properties as well as avoid mechanical failure during their manipulation are challenging. In this regard, we developed a multifunctional AOHA-RA/Lap nanocomposite IH for IBDs repair, which was constructed mainly through two kinds of reversible cross-links: (i) the laponite (Lap) crystals mediated electrostatic interactions; (ii) the phenylboronic acid easter bonds between the 4-aminobenzeneboronic acid grafted oxidized hyaluronic acid (AOHA) and rosmarinic acid (RA). Due to the specific structural composition, the AOHA-RA/Lap IH demonstrated superior injectability, self-recoverability, spatial adaptation, and self-reinforced mechanical properties after being injected to the bone defect site. In addition, the RA molecules could be locally released from the hydrogel following a Weibull model for over 10 days. Systematic in vitro/vivo assays proved the strong anti-bacterial activity of the hydrogel against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Moreover, its capability of inducing M2 polarization of macrophages (Mφ) and osteogenic differentiation of bone marrow stromal cells (BMSCs) was verified either, and the mechanism of the former was identified to be related to the JAK1-STAT1 and PI3K-AKT signaling pathways and that of the latter was identified to be related to the calcium signaling pathway, extracellular matrix (ECM) receptor interaction and TGF-β signaling pathway. After being implanted to a S. aureus infected rat skull defect model, the AOHA-RA/Lap IH significantly accelerated repair of IBDs without causing significant systemic toxicity. Statement of significanceRosmarinic acid and laponite were utilized to develop an injectable hydrogel, promising for accelerating repair of infected bone defects in clinic. The gelation of the hydrogel was completely driven by two kinds of reversible cross-links, which endow the hydrogel superior spatial adaption, self-recoverability, and structural stability. The as-prepared hydrogel demonstrated superior anti-bacterial/anti-biofilm activity and could induce M2 polarization of macrophages and osteogenic differentiation of BMSCs. The mechanism behind macrophages polarization was identified to be related to the JAK1-STAT1 and PI3K-AKT signaling pathways. The mechanism behind osteogenic differentiation of BMSCs was identified to be related to the ECM receptor interaction and calcium signaling/TGF-β signaling pathways.

7954 Vitamin D Deficiency Underlies Defective Endometrial Decidualization In Vivo and In Vitro

Abstract Disclosure: M. Yi: None. T. Wang: None. A. Jukic: None. F.J. DeMayo: None. Several epidemiologic studies have reported associations between vitamin D and both menstrual cycles and fertility. Vitamin D signaling occurs most often through the interaction of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) with its cognate receptor, the transcription factor vitamin D receptor (VDR). This study aims to define the role of vitamin D and VDR on female reproduction and identify vitamin D-mediated signaling. In our studies, preliminary results demonstrate mice on a vitamin D-deficient diet showed a significantly impaired uterine decidualization reaction in response to exogenous hormones. To knockdown VDR expression, human endometrial stroma cells (THESCs) were transfected with either non-targeting control (siNT) or VDR siRNA (siVDR) for 48 h. And, to perform the in vitro decidualization, the cell culture media was replaced to differentiate the cells by treatment with 17β-estradiol (E), medroxyprogesterone acetate (P), and db-cAMP (C) for 72 h. At the 48 h exposure of EPC, the decidual media was replaced with containing 1,25(OH)2D3 or vehicle and cultured for 24 h additionally. Treatment of 1,25(OH)2D3 (ligand) and knockdown of VDR (receptor) in THESCs resulted in an increased expression of the decidualization markers, PRL and IGFBP1. Transcriptomic analysis was then conducted to identify the biological networks regulated by the ligand and receptor in THESCs. THESCs were transfected with either siNT or siVDR for 48 h, and the culture media was replaced with either treatment of 2 nM of 1,25(OH)2D3 or vehicle for an additional 24 h. Differentially expressed genes (DEGs) were affected by the ligand and receptor (N = 626 and 1499, respectively) based on a three-way ANOVA filtered with false discovery rate (FDR)-adjusted p-value<0.05 and fold-change (FC)≥|1.4|. Pathway analyses and predicted upstream regulators were analyzed by Ingenuity Pathways Analysis (IPA), and among the predicted upstream regulators of the DEGs, β-estradiol was predicted as an activator for both. Thus, to test the role of vitamin D in estrogen signaling, THESCs were treated with either EPC or PC for 72 h in total, and at the 48 h exposure, the decidual media was replaced with 1,25(OH)2D3 or vehicle and cultured for 24 h additionally. Removal of estrogen (PC) caused loss of the enhanced expression of decidual markers by 1,25(OH)2D3 that was observed in control EPC-treated cells. In conclusion, Vitamin D signaling modulates human and mouse uterine stromal cell differentiation by modulating the ability of estrogen to stimulate decidualization in endometrial stromal cells, a process critical to establishing pregnancy. Presentation: 6/2/2024