Proliferation Of Stromal Cells Research Articles

Persistent activation of signal transducer and activator of transcription 3 via interleukin-6 trans-signaling is involved in fibrosis of endometriosis.

Is activation of signal transducer and activator of transcription 3 (STAT3) via interleukin-6 (IL-6) trans-signaling involved in fibrosis of endometriosis? Persistent activation of STAT3 via IL-6 trans-signaling is involved in fibrosis of endometriosis. Our previous study showed that sustained low-grade inflammation promotes a fibrotic phenotype in endometriotic stromal cells. However, the underlying mechanisms of the establishment of non-resolving, low-grade inflammation in endometriosis remain to be clarified. Endometrial and/or endometriotic samples of 60 patients who had histological evidence of deep endometriosis and endometrial samples from 32 healthy fertile women were analyzed. The effects of priming with ligands of Toll-like receptors (TLRs) 2, 3 and 4 on secretion of inflammatory mediators (tumor necrosis factor-α, C-X-C motif chemokine ligand-10 [CXCL-10], IL6 and IL-10) after a second challenge with TLR ligands in endometrial and endometriotic stromal cells were investigated. Then, the effects of IL-6/soluble (s) IL-6 receptor (R)/STAT3 signaling, as well as inhibition of STAT3 activation by knockdown of STAT3 or pharmacological inhibition (S3I-201), on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Priming with TLR ligands for 4 h had no significant effects, whereas 24 h of priming significantly decreased secretion of IL-6, after a second challenge in endometrial stromal cells of healthy women. In endometriotic stromal cells, whereas 24 h of priming had no significant effects, priming with TLR ligands for 4 h significantly increased secretion of IL-6 after a second challenge. IL-6/soluble IL-6 receptor (sIL-6R) induced a pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers, cell migration and collagen gel contraction) as well as nuclear factor-kappa B (NF-κB) activation of endometriotic stromal cells. In contrast, IL-6/sIL-6R had no significant effects on either a pro-fibrotic phenotype or NF-κB activation of endometrial stromal cells of healthy women. Stimulation with transforming growth factor (TGF)-β1 and/or IL-6/sIL-6R for 1 h and 48 h activated STAT3, but induced very low or no suppressor of cytokine signaling (SOCS) 1 and 3 protein expression in endometriotic stromal cells. In endometrial stromal cells of healthy women, IL-6/sIL-6R-induced STAT3 and SOCS1/3 expression at 1 h, whereas no STAT3 activation was detected at 48 h. Knockdown of STAT3 gene or S3I-201 (a STAT3 inhibitor) decreased the IL-6/sIL-6R-induced pro-fibrotic phenotype as well as NF-κB activation and TGF-β1-induced cell proliferation of endometriotic stromal cells. N/A. In vivo studies are required to confirm the present in vitro results. However, it remains challenging to mimic non-resolving chronic inflammation in animal models, as active inflammation can resolve spontaneously. Dysfunction of negative regulators of IL-6/sIL-6R/STAT3 signaling may cause persistent activation of STAT3 in endometriosis. Since STAT3 activation in the endometrium is essential for successful embryo implantation, treatment with STAT3 inhibitors would not be appropriate for women wishing to conceive. However, targeting impaired negative regulation of IL-6/sIL-6R/STAT3 signaling may still represent a promising avenue for the treatment of endometriosis. This study was supported in part by the KARL STORZ SE & Co. KG (Tuttlingen, Germany). There are no conflicts of interest.

Exosomal AFAP1-AS1 binds to microRNA-15a-5p to promote the proliferation, migration, and invasion of ectopic endometrial stromal cells in endometriosis.

BackgroundEndometriosis (EMS) remains a major challenge to reproductive health due to multifactorial etiology, disease heterogeneity, and the lack of appropriate diagnostic markers and treatment. Eexosome (Exo) has become a major factor in progression of a variety of diseases. However, the mechanisms directing their role in the pathophysiology of EMS are ill-defined. Here, we aimed to investigate the clinical implications of actin filament associated protein 1-Antisense RNA 1 (AFAP1-AS1) in EMS.MethodsBioinformatics analysis was used to predict the expression and interaction of AFAP1-AS1, miR-15a-5p and BCL9 in EMS, and dual luciferase reporter assay was used to verify the targeted relationship of AFAP1-AS1, miR-15a-5p, and BCL9. The Exo from endometrial stromal cells (ESCs) was isolated and characterized by transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). Exosome uptake studies were performed. For in vitro assay, ectopic ESCs (EcESCs) proliferation, migration, and invasion were assessed by CCK-8 and Transwell assays. In vivo assay was performed by establishment of EMS mice to validate the result derived from in vitro assay.ResultsThe Exo was successfully isolated from ESCs and we observed high expression of AFAP1-AS1 and BCL9 but low expression of miR-15a-5p in EMS. Moreover, Exo derived from EcESCs could deliver AFAP1-AS1 to EcESCs and thus promoting proliferation, migration, and invasion of ESCs. AFAP1-AS1 bound to BCL9, which was targeted by miR-15a-5p in EMS. In vivo experiments in nude mice revealed that inhibition of Exosomal AFAP1-AS1 suppressed migration and invasion of EcESCs through miR-15a-5p/BCL9.ConclusionsCollectively, these findings suggested that ESCs-derived Exo carrying AFAP1-AS1 contributed to EMS pathogenesis. This study might help us realize the etiology of EMS and improve the treatment of the related complications.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12958-022-00942-1.

Degradation-Kinetics-Controllable and Tissue-Regeneration-Matchable Photocross-linked Alginate Hydrogels for Bone Repair.

Photocross-linked alginate hydrogels, due to their biodegradability, biocompatibility, strong control for gelling kinetics in space and time, and admirable adaptability for in situ polymerization with a minimally invasive approach in surgical procedures, have created great expectations in bone regeneration. However, hydrogels with suitable degradation kinetics that can match the tissue regeneration process have not been designed, which limits their further application in bone tissue engineering. Herein, we finely developed an oxidation strategy for alginate to obtain hydrogels with more suitable degradation rates and comprehensively explored their physical and biological performances in vitro and in vivo to further advance the clinical application for the hydrogels in bone repair. The physical properties of the gels can be tuned via tailoring the degree of alginate oxidation. In particular, in vivo degradation studies showed that the degradation rates of the gels were significantly increased by oxidizing alginate. The activity, proliferation, initial adhesion, and osteogenic differentiation of rat and rabbit bone marrow stromal cells (BMSCs) cultured with/in the hydrogels were explored, and the results demonstrated that the gels possessed excellent biocompatibility and that the encapsulated BMSCs were capable of osteogenic differentiation. Furthermore, in vivo implantation of rabbit BMSC-loaded gels into tibial plateau defects of rabbits demonstrated the feasibility of hydrogels with appropriate degradation rates for bone repair. This study indicated that hydrogels with increasingly controllable and matchable degradation kinetics and satisfactory bioproperties demonstrate great clinical potential in bone tissue engineering and regenerative medicine and could also provide references for drug/growth-factor delivery therapeutic strategies for diseases requiring specific drug/growth-factor durations of action.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

TCF3 regulates human endometrial stromal cell proliferation and migration in RPL.

Recurrent pregnancy loss (RPL) is a multifactorial condition with no explanation of miscarriage in approximately half of the RPL patients, consequently leaving deep physical and emotional sequels. Transcription factor 3 (TCF3 or E2A), is a unique member of the LEF/TCF family and plays an important role in embryogenesis. However, its function in RPL is poorly understood. Using real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry, we demonstrated that TCF3 was downregulated in decidual tissues from RPL patients compared with healthy control (HC). Further, TCF3 knockdown inhibited proliferation, induced G0/G1 phase arrest, and promoted migration in human endometrial stromal cells (HESCs), while overexpression of TCF3 exhibited the opposite effects. RNA-sequencing analysis combined with gene-set enrichment analysis results showed that the mitogen-activated protein kinase pathway is potentially downstream of TCF3. Knockdown of TCF3 confirmed increased p38 phosphorylation, while overexpression of TCF3 inhibited p38 phosphorylation. Furthermore, we found that TCF3 protein level was decreased in HESCs under hypoxic incubation, while hypoxia-inducible factor-1α (HIF1A) knockdown increased the expression of TCF3. TCF3 overexpression recovered the proliferation ability of HESCs inhibited by hypoxia and reversed hypoxia-induced migration. Consistently, we found that RPL patients had a significantly higher level of HIF1A in the decidual tissue than HC. Overall, this study clarifies that increased HIF1A in the decidua contributes to the occurrence of RPL through the TCF3/p38 signaling pathway.

C-X-C motif chemokine ligand 1 induced by Hedgehog signaling promotes mouse extrahepatic bile duct repair after acute injury.

In extrahepatic bile duct (EHBD) cholangiopathies, including primary sclerosing cholangitis, a reactive cholangiocyte phenotype is associated with inflammation and epithelial hyperproliferation. The signaling pathways involved in EHBD injury response are poorly understood. In this study, we investigated the role of Hedgehog (HH) signaling and its downstream effectors in controlling biliary proliferation and inflammation after EHBD injury. Using mouse bile duct ligation as an acute EHBD injury model, we used inhibitory paradigms to uncover mechanisms promoting the proliferative response. HH signaling was inhibited genetically in Gli1-/- mice or by treating wild-type mice with LDE225. The role of neutrophils was tested using chemical (SB225002) and biological (lymphocyte antigen 6 complex locus G6D [Ly6G] antibodies) inhibitors of neutrophil recruitment. The cellular response was defined through morphometric quantification of proliferating cells and CD45+ and Ly6G+ immune cell populations. Key signaling component expression was measured and localized to specific EHBD cellular compartments by in situ hybridization, reporter strain analysis, and immunohistochemistry. Epithelial cell proliferation peaked 24 h after EHBD injury, preceded stromal cell proliferation, and was associated with neutrophil influx. Indian HH ligand expression in the biliary epithelium rapidly increased after injury. HH-responding cells and neutrophil chemoattractant C-X-C motif chemokine ligand 1 (CXCL1) expression mapped to EHBD stromal cells. Inhibition of HH signaling blocked CXCL1 induction, diminishing neutrophil recruitment and the biliary proliferative response to injury. Directly targeting neutrophils by inhibition of the CXCL1/C-X-C motif chemokine receptor 2/Ly6G signaling axis also decreased biliary proliferation. HH-regulated CXCL1 orchestrates the early inflammatory response and biliary proliferation after EHBD injury through complex cellular crosstalk.

Downregulating HK2 inhibits proliferation of endometrial stromal cells through a noncanonical pathway involving phosphorylation of signal transducer and activator of transcription 1 in endometriosis.

Endometriosis is a benign gynecologic disease that causes chronic pelvic pain, dysmenorrhea and infertility and shares several characteristics with malignant tumors, afflicting women of reproductive age. Hexokinase 2 plays an essential role as the first rate-limiting enzyme in the metabolic glycolysis pathway, and its abnormal elevation in tumors is associated with tumor genesis and metastasis. However, the expression and role of hexokinase 2 in endometriosis remain unclear. We sequenced the primary endometrial stromal cells from patients with endometrioma and utilized immunohistochemistry, quantitative real-time PCR, and western blot to determine the expression of hexokinase 2. Then wound healing assays, cell invasion assays, and cell proliferation assays were performed to explore the functions of hexokinase 2 in endometrial stromal cells. Furthermore, mice models of endometriosis were used to observe the effects of hexokinase 2 inhibitors in vivo. Lastly, glycolysis metabolism detection and transcriptome sequencing were carried out in hexokinase 2-knockdown endometrial stromal cells to analyze the mechanism of hexokinase 2 affecting cell function. Endometrial stromal cells of endometrioma displayed active glycolysis metabolism and elevated expression of hexokinase 2. Downregulating hexokinase 2 reduced the migration, invasion, and proliferation capacity of endometrial stromal cells. Knockdown of hexokinase 2 induced upregulation of signal transducer and activator of transcription 1 and their phosphorylation to attenuate the proliferation of endometrial stromal cells. Hexokinase 2 is associated with the migration, invasion, and proliferation of endometrial stromal cells, which might provide new insights into the pathogenesis and treatment of endometriosis. HK2 is upregulated in ovarian endometrioma and knockdown of HK2 induced upregulation of signal transducer and activator of transcription 1 (STAT1) and their phosphorylation to attenuate the proliferation of endometrial stromal cells.

P281 In vitro evaluation of the response of human tendon-derived stromal cells to a novel electrospun suture

Abstract Background/Aims Recurrent tears after surgical tendon repair remain common, with 40% of rotator cuff repairs failing within one year. Repair failures can be partly attributed to the use of sutures not designed for the tendon cellular niche nor designed for the promotion of repair processes. Clinically-used sutures have high tensile strength to mechanically support injured tendon. However, these sutures do not support cell integration into the material, but instead can stimulate a chronic inflammatory response. Synthetic electrospun materials can mechanically support the tendon while providing topographical cues that modulate the immune response to promote wound healing. Electrospun material has an increased surface area and porosity which can mimic that of tendon tissue, thus promoting tendon cell infiltration and proliferation. Here, a novel electrospun suture made from twisted polydioxanone (PDO) polymer filaments is compared to PDS II, a clinically used PDO suture currently utilised in tendon repair. Methods We evaluated the ability of electrospun suture and PDS II to support the attachment and proliferation of human tendon-derived stromal cells using PrestoBlue cell viability assays and scanning electron microscopy. Suture surface chemistry was analysed using X-ray photoelectron spectroscopy. Bulk RNA-Seq interrogated the transcriptional response of primary tendon-derived stromal cells to sutures after 14 days. Results The electrospun suture showed increased initial cell attachment compared to PDS II. X-ray photoelectron spectroscopy revealed that the local surface chemistry of electrospun suture and PDS II is similar, indicating that the tendon-like architecture of electrospun suture was responsible for the greater cell attachment. Furthermore, electrospun suture elicited a stronger transcriptional response compared with PDS II, with relative enrichment of pathways including mTorc1 signalling and depletion of epithelial-to-mesenchymal transition and extracellular matrix gene sets. When compared to baseline, neither suture induced transcriptional upregulation of inflammatory pathways such as IL6-JAK-STAT3 signalling and TNF-α signalling via NF-κB. Conclusion Twisted electrospun sutures demonstrated a tendon-derived stromal cell response indicative of cell proliferation and wound healing, without significant fibrosis, when compared to clinically-used and safe PDS II suture. Twisted electrospun sutures therefore show promise in improving outcomes in surgical tendon repair by allowing increased cell attachment while maintaining an appropriate tissue response. Disclosure A. Nezhentsev: None. R.E. Abhari: None. M.J. Baldwin: None. J.Y. Mimpen: None. E. Augustyniak: None. M. Isaacs: None. P. Mouthuy: None. A.J. Carr: None. S.J.B. Snelling: None.

Comparing the Characteristics of Amniotic Membrane-, Endometrium-, and Urinary-Derived ECMs and Their Effects on Endometrial Regeneration in a Rat Uterine Injury Model

The decellularized extracellular matrices (d-ECMs) currently utilized to repair endometrial injuries are derived from three tissue sources, the endometrium (dE-ECM), placental amniotic membrane (dA-ECM), and urinary (dU-ECM). Notably, the structures of dU-ECM and dE-ECM are similar. These d-ECMs are derived from different tissues, and their specific roles in endometrial injury repair remain unclear. This study aimed to analyse the characteristics of the tissue microstructures and compositions to confirm specific differences among the three ECM types. And using a rat model of endometrial injury, the effects of all the matrices after implantation in vivo on the promotion of endometrial regeneration were analysed. After decellularization, dE-ECM had more residual active factors than the other two ECM types, while dA-ECM had significantly less DNA, α-Gal antigen components and extracellular matrix components than the other two groups. Although the three ECMs had no effect on the proliferation of stromal cells in vitro, dA-ECM may have increased the sensitivity of stromal cells to oestradiol (E2) responses. In vivo experiments confirmed the promotional effect of dA-ECM on endometrial regeneration. For example, the endometrial thickness, collagen deposition, endometrial tissue regeneration, vascular regeneration and pregnancy outcomes were significantly better in this group than in the other two groups. These findings might be associated with the excellent immune tolerance of dA-ECM. Therefore, when selecting a d-ECM for the treatment of endometrial injury, dE-ECM, which has the strongest tissue specificity, is not the preferred choice. Controlling the inflammatory responses in local lesions at the early stage may be a prerequisite for ECMs to exert their functions.

Elevated latent transforming growth factor beta binding protein 2 in endometriosis promotes endometrial stromal cell invasion and proliferation via the NF-kB signaling pathway

Endometriosis, defined as the abnormal growth of functional endometrium outside the uterus, is characterized by the abnormal phenotype of endometrial cells. This study aimed to investigate the role of latent transforming growth factor beta binding protein 2 (LTBP2), an extracellular matrix protein, in the occurrence and development of endometriosis. Elevated LTBP2 expression levels were observed in endometrial tissues and serum of endometriosis patients and their area under the ROC curve (AUC) values for distinguishing endometriosis were 0.9044 and 0.9534, respectively. Overexpressing-LTBP2 could promote proliferation, migration, and invasion, whereas suppressing apoptosis of endometrial stromal cells (ESCs). Moreover, LTBP2 downregulation induced the opposite effect. The supernatant from ESCs overexpressing LTBP2 promoted the tube formation of human umbilical vein endothelial cells (HUVECs), thus indicating an angiogenic effect. Furthermore, overexpression of LTBP2 facilitated the inflammation and might promote endometriosis progression through the NF-kB signaling pathway. Conclusively, LTBP2 might be a potential target in the diagnosis and treatment of endometriosis.

Ageing, a modulator of human endometrial stromal cell proliferation and decidualization: a role for implantation?

Research questionDoes ageing affect endometrial stromal cell function and gene expression involved in decidualization? DesignStromal cells were isolated and cultured (98% purity) from pipelle endometrial biopsies obtained from female donors, at the proliferative phase of the menstrual cycle. Initially, 28 samples were collected (age range 25–46 years). These samples were divided into two groups: 25–35 years and 36–46 years. Cell proliferation assays were carried out to determine changes in stromal cell proliferation, in vitro, between the two groups. In addition, mRNA and protein expression of decidualization factors, BMP2 and STAT3, were analysed for the same age groups and samples using real-time polymerase chain reaction and western blot analysis, respectively. Finally, another 12 pipelle endometrial biopsies (age range 25–46 years) were used in separate in-vitro decidualization experiments. The mRNA expression of decidualization markers, prolactin and IGFBP-1 were analysed in cultured stromal cells after adding 8-bromo-cAMP. ResultsA statistically significant decrease was observed in stromal cell proliferation with increasing age (P = 0.0006). Messenger RNA expression of BMP2 and STAT3 were found to decrease (P = 0.0167; P = 0.0037, respectively) in the older age group. In addition, BMP2 and STAT3 protein expression between the samples analysed changed significantly (P = 0.0085; P = 0.0463, respectively) with increasing age. In-vitro induced decidualization experiments showed significant changes in stromal cell mRNA expression of decidualization markers (IGFBP1 and prolactin) between different age groups. ConclusionAgeing affected endometrial cell function and gene expression. Changes in cell function and expression of associated molecules that differ in the ageing endometrium can help understand the causes of infertility.

Preparation of BMP-2/PDA-BCP Bioceramic Scaffold by DLP 3D Printing and its Ability for Inducing Continuous Bone Formation.

Digital light processing (DLP)-based 3D printing is suitable to fabricate bone scaffolds with small size and high precision. However, the published literature mainly deals with the fabrication procedure and parameters of DLP printed bioceramic scaffold, but lacks the subsequent systematic biological evaluations for bone regeneration application. In this work, a biphasic calcium phosphate (BCP) macroporous scaffold was constructed by DLP-based 3D printing technique. Furthermore, bone morphogenetic protein-2 (BMP-2) was facilely incorporated into this scaffold through a facile polydopamine (PDA) modification process. The resultant scaffold presents an interconnected porous structure with pore size of ∼570 μm, compressive strength (∼3.6 MPa), and the self-assembly Ca-P/PDA nanocoating exhibited excellent sustained-release property for BMP-2. Notably, this BMP-2/PDA-BCP scaffold presents favorable effects on the adhesion, proliferation, osteogenic differentiation, and mineralization of bone marrow stromal cells (BMSCs). Furthermore, in vivo experiments conducted on rats demonstrated that the scaffolds could induce cell layer aggregation adjacent to the scaffolds and continuous new bone generation within the scaffold. Collectively, this work demonstrated that the BMP-2/PDA-BCP scaffold is of immense potential to treat small craniofacial bone defects in demand of high accuracy.

Effect of Acute Myeloid Leukemia Cells on the Proliferation and Apoptosis of Bone Marrow-Derived Mesenchymal Stromal Cells

To investigate the effect of acute myeloid leukemia cells in leukemia-microenvironment on proliferation and apoptosis of bone marrow-derived mesenchymal stromal cells (BM-MSC). Acute myeloid leukemia (AML) murine models overexpressing MLL-AF9 were established. The number of BM-MSC of wild type (WT) and AML-derived mice were analyzed by flow cytometry. Morphology and growth differences between WT and AML-derived BM-MSC were analyzed by inverted fluorescence microscope. Proliferation and apoptosis of BM-MSC between these two groups were detected by Brdu and Annexin V/PI. Compared with WT-derived BM-MSC, the number and proliferation rate of AML-derived BM-MSC significantly increased (P<0.01, P<0.001), while apoptosis rate decreased (P<0.05). When cultured in vitro, BM-MSC grew faster under conditional medium. AML cells can promote proliferation and inhibit apoptosis of BM-MSC.

Scaphechinus mirabilis Extract Effectively Inhibits the Proliferation of BPH-1 and LNCaP Prostate Epithelial Cells

Background: Benign Prostatic Hyperplasia (BPH) is an age-related disease characterized by progressive proliferation of prostate stromal and epithelial cells. While the precise etiology of BPH is still not clear, the proliferation of epithelial cells has been implicated in the development of the disease. Scaphechinus mirabilis (S. mirabilis) is a marine species belonging to the order Clypeasteroida, which contains flat, burrowing sea urchins. Objective: This study examined the effects of S. mirabilis extract on the proliferation of BPH-1 and LNCaP prostate epithelial cells. Methods: BPH-1 and LNCaP cells were cultured and treated with S. mirabilis extract (50, 100, and 200 μg/ml). The viability of cells treated with S. mirabilis extract was determined by the MTT assay. Results: Proliferation of BPH-1 and testosterone-induced LNCaP cells was inhibited by treatment with S. mirabilis extract. S. mirabilis extract suppressed the expression of androgen-related proteins, such as androgen receptor, prostate-specific antigen, and 5α-reductase 2. S. mirabilis extract inhibited testosterone-induced proliferation. Moreover, S. mirabilis extract induced apoptotic responses by regulating the expression of caspase-9, Bcl-2, and Bax. Conclusion: These findings suggest that S. mirabilis extract abrogated the expression of androgen- related proteins by inducing apoptotic responses, which could be valuable for the design of new therapeutic agents for the treatment of BPH.

An ECM-Mimetic Hydrogel to Promote the Therapeutic Efficacy of Osteoblast-Derived Extracellular Vesicles for Bone Regeneration.

The use of extracellular vesicles (EVs) is emerging as a promising acellular approach for bone regeneration, overcoming translational hurdles associated with cell-based therapies. Despite their potential, EVs short half-life following systemic administration hinders their therapeutic efficacy. EVs have been reported to bind to extracellular matrix (ECM) proteins and play an essential role in matrix mineralisation. Chitosan and collagen type I are naturally-derived pro-osteogenic biomaterials, which have been demonstrated to control EV release kinetics. Therefore, this study aimed to develop an injectable ECM-mimetic hydrogel capable of controlling the release of osteoblast-derived EVs to promote bone repair. Pure chitosan hydrogels significantly enhanced compressive modulus (2.48-fold) and osteogenic differentiation (3.07-fold), whilst reducing gelation times (2.09-fold) and proliferation (2.7-fold) compared to pure collagen gels (p ≤ 0.001). EV release was strongly associated with collagen concentration (R2 > 0.94), where a significantly increased EV release profile was observed from chitosan containing gels using the CD63 ELISA (p ≤ 0.001). Hydrogel-released EVs enhanced human bone marrow stromal cells (hBMSCs) proliferation (1.12-fold), migration (2.55-fold), and mineralisation (3.25-fold) compared to untreated cells (p ≤ 0.001). Importantly, EV-functionalised chitosan-collagen composites significantly promoted hBMSCs extracellular matrix mineralisation when compared to the EV-free gels in a dose-dependent manner (p ≤ 0.001). Taken together, these findings demonstrate the development of a pro-osteogenic thermosensitive chitosan-collagen hydrogel capable of enhancing the therapeutic efficacy of osteoblast-derived EVs as a novel acellular tool for bone augmentation strategy.

Fibroblast growth factor receptor 2 promotes the proliferation, migration, and invasion of ectopic stromal cells via activation of extracellular-signal-regulated kinase signaling pathway in endometriosis

ABSTRACT Endometriosis is defined as the presence of endometrial tissues with cancer-like features in extrauterine locations. Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase that is involved in cancer pathogenesis. This study aimed to determine the role of FGFR2 in endometriosis. A total of 29 pairs of ectopic and eutopic endometrial tissues were collected from women with endometriosis. Endometrial tissues from women with hysteromyomas were considered as normal controls. Primary ectopic stromal cells (ESCs) were isolated from the ectopic endometrium. The role of FGFR2 in ESCs was assessed using immunohistochemistry, polymerase chain reaction, cell counting kit-8 assay, EdU staining, flow cytometry, transwell assay, and western blotting. The following signaling pathways were detected using bioinformatic analysis and confirmed in vitro. By searching the GSE171154, GSE86543, and GSE77182 datasets, FGFR2 was identified as an upregulated overlapping gene in endometriosis. Compared to eutopic and normal endometria, FGFR2 was highly expressed in ectopic tissues. Transfection of primary ESCs with FGFR2 small interfering RNA (siRNA) repressed the viability and proliferation of cells and induced apoptosis. FGFR2 siRNA inhibited the migration, invasion, and transforming growth factor-β1-triggered epithelial-mesenchymal transition (EMT). Extracellular signal-regulated kinase (ERK) signaling was found to be a downstream signaling pathway for FGFR2. The ERK1/2 inhibitor PD98059 was found to reverse the promoting effects of FGFR2 on ESC proliferation and invasion. FGFR2 silencing effectively inhibited the growth, migration, invasion, and EMT of ESCs. The effects of FGFR2 on endometriosis might be mediated via the activation of ERK signaling.

Improvement of Mesenchymal Stromal Cell Proliferation and Differentiation via Decellularized Extracellular Matrix on Substrates With a Range of Surface Chemistries.

Decellularized extracellular matrix (dECM) deposited by mesenchymal stromal cells (MSCs) has emerged as a promising substrate for improved expansion of MSCs. To date, essentially all studies that have produced dECM for MSC expansion have done so on tissue culture plastic or glass. However, substrate surface chemistry has a profound impact on the adsorption of proteins that mediate cell-material interactions, and different surface chemistries can cause changes in cell behavior, ECM deposition, and the in vivo response to a material. This study tested the hypothesis that substrate surface chemistry impacts the deposition of ECM and its subsequent bioactivity. This hypothesis was tested by producing glass surfaces with various surface chemistries (amine, carboxylic acid, propyl, and octyl groups) using silane chemistry. ECM was deposited by an immortalized MSC line, decellularized, and characterized through SDS-PAGE and immunofluorescence microscopy. No significant difference was observed in dECM composition or microarchitecture on the different surfaces. The decellularized surfaces were seeded with primary MSCs and their proliferation and differentiation were assessed. The presence of dECM improved the proliferation of primary MSCs by ~100% in comparison to surface chemistry controls. Additionally, the adipogenesis increased by 50–90% on all dECM surfaces in comparison to surface chemistry controls, and the osteogenesis increased by ~50% on the octyl-modified surfaces when dECM was present. However, no statistically significant differences were observed within the set of dECM surfaces or control surfaces. These results support the null hypothesis, meaning surface chemistry (over the range tested in this work) is not a key regulator of the composition or bioactivity of MSC-derived dECM. These results are significant because they provide an important insight into regenerative engineering technologies. Specifically, the utilization of dECM in stem cell manufacturing and tissue engineering applications would require the dECM to be produced on a wide variety of substrates. This work indicates that it can be produced on materials with a range of surface chemistries without undesired changes in the bioactivity of the dECM.

Magnetically responsive nanofibrous ceramic scaffolds for on-demand motion and drug delivery.

Smart biomaterials, featuring not only bioactivity, but also dynamic responsiveness to external stimuli, are desired for biomedical applications, such as regenerative medicine, and hold great potential to expand the boundaries of the modern clinical practice. Herein, a magnetically responsive three-dimensional scaffold with sandwich structure is developed by using hydroxyapatite (HA) nanowires and ferrosoferric oxide (Fe3O4) nanoparticles as building blocks. The magnetic HA/Fe3O4 scaffold is fully inorganic in nature, but shows polymeric hydrogel-like characteristics including a 3D fibrous network that is highly porous (>99.7% free volume), deformable (50% deformation) and elastic, and tunable stiffness. The magnetic HA/Fe3O4 scaffold has been shown to execute multimodal motion upon exposure to an external magnetic field including shape transformation, rolling and somersault. In addition, we have demonstrated that the magnetic scaffold can serve as a smart carrier for remotely controlled, on-demand delivery of compounds including an organic dye and a protein. Finally, the magnetic scaffold has exhibited good biocompatibility, supporting the attachment and proliferation of human mesenchymal stromal cells, thereby showing great potential as smart biomaterials for a variety of biomedical applications.

Magnesium oxide regulates the degradation behaviors and improves the osteogenesis of poly(lactide-co-glycolide) composite scaffolds

Poly (lactic-co-glycolic acid) (PLGA) is a star biodegradable polymer widely studied and applied in the biomedical field. Improving the acidic microenvironment caused by its degradation products and regulating its degradation behavior are still urgent scientific and technological problems to be solved. In this study, to regulate the degradation behaviors of PLGA and improve its bioactivity, hydroxyapatite (HA) and magnesium oxide (MgO) were incorporated into PLGA substrate in different proportions and a series of 3D-printing PLGA/HA/MgO (PHM) composite porous scaffolds were prepared. Then the physicochemical properties, degradation behaviors, in vitro and in vivo biological performance of fabricated scaffolds were systematically studied. The in vitro experimental results showed that PHM composite scaffolds exhibited hydrophilic and mechanical properties similar to natural bone, the presence of MgO could effectively neutralize the acidic environment, and promote bone marrow stromal cells (BMSCs) adhesion, proliferation, and differentiation. The in vivo osteogenic experiments demonstrated that the PHM5 (25 wt% HA, 5 wt% MgO) scaffold could reduce the inflammatory response and promote new bone formation. This study provides a new idea for the regulation of the degradation behavior and the improvement of osteogenic properties of PLGA-based biomaterials. The prepared 3D-printed PHM5 scaffold shows great application potential in the bone repair field.

Long Intergenic Non-Protein Coding RNA 02381 Promotes the Proliferation and Invasion of Ovarian Endometrial Stromal Cells through the miR-27b-3p/CTNNB1 Axis.

Purpose: Catenin Beta 1 (CTNNB1) is a key regulator of cell proliferation and invasion in endometriosis; however, its upstream factor is not clear. Long noncoding RNAs may participate in endometriosis. The aim of this study was to investigate the mechanism of interaction between LINC02381 and CTNNB1 in endometriosis. Method: Screening and validation of RNAs were completed by whole transcriptional sequencing and qRT-PCR. The subcellular localization of LINC02381 was determined by RNA in situ hybridization and nucleo-cytoplasmic separation. Plasmids were transfected for functional experiments. Luciferase assay was used to verify the binding relationship. Results: The expression of LINC02381 and CTNNB1 was significantly increased in ovarian ectopic endometrial tissues (OSAs) and ectopic endometrial stromal cells (ESCs). When LINC02381 was downregulated in ESCs, the expression of CTNNB1, metallopeptidase 9 (MMP9) and cyclinD1, as well as ESCs invasion and proliferation, decreased. LINC02381 was mainly present in the cytoplasm of ESCs, indicating that it may act as a competitive endogenous RNA. Bioinformatic analysis revealed that microRNA-27b-3p (miR-27b-3p) is a downstream target of LINC02381. miR-27b-3p decreased in OSAs and ESCs. Moreover, when miR-27b-3p was upregulated in ESCs, the expression of CTNNB1, MMP9 and cyclinD1, as well as the invasion and proliferation ability of ESCs, were reduced. Additionally, rescue experiments demonstrated that the expression of CTNNB1, MMP9 and cyclinD1, as well as the invasion and proliferation ability, were significantly increased in the group transfected with both sh-LINC02381 and a miR-27b-3p inhibitor. Conclusion: LINC02381 upregulated CTNNB1 by adsorbing miR-27b-3p, causing increased proliferation and invasion of ESCs.