MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.
The development of the field of biomaterials engineering is rapid. Various bioactive coatings are created to improve the biocompatibility of substrates used for bone regeneration, which includes formulation of thin zirconia coatings with pro-osteogenic properties. The aim of this study was to assess the biological properties of ZrO 2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO 2 ALD). Methodology: The cytocompatibility of the obtained layers was analysed using the mice preosteoblastic cell line (MC3T3) characterized by decreased expression of microRNA 21-5p (miR-21-5p) in order to evaluate the potential pro-osteogenic properties of the coatings. The in vitro experiments were designed to determine the effect of ZrO 2 ALD coatings on cell morphology (confocal microscope), proliferative activity (cell cycle analysis) and metabolism, reflected by mitochondrial membrane potential (cytometric-based measurement). Additionally, the influence of layers on the expression of genes associated with cell survival and osteogenesis was studied using RT-qPCR. The following genes were investigated: B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), p53 and p21, as well as osteogenic markers, i.e. collagen type 1 (Coll-1), osteopontin (Opn), osteocalcin (Ocl) and runt-related transcription factor 2 (Runx-2). The levels of microRNA (miRNA/miR) involved in the regulation of osteogenic genes were determined, including miR-7, miR-21, miR-124 and miR-223. Results: The analysis revealed that the obtained coatings are cytocompatible and may increase the metabolism of pre-osteoblast, which was correlated with increased mitochondrial membrane potential and extensive development of the mitochondrial network. The obtained coatings affected the viability and proliferative status of cells, reducing the population of actively dividing cells. However, in cultures propagated on ZrO 2 ALD coatings, the up-regulation of genes essential for bone metabolism was noted. Discussion: The data obtained indicate that ZrO 2 coatings created using the ALD method may have pro-osteogenic properties and may improve the metabolism of bone precursor cells. Given the above, further development of ZrO 2 ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.
Osteosarcoma (OSA) is malignant bone tumor, occurring in children and adults, characterized by poor prognosis. Despite advances in chemotherapy and surgical techniques, the survival of osteosarcoma patients is not improving significantly. Currently, great efforts are taken to identify novel selective strategies, distinguishing between cancer and normal cells. This includes development of biomimetic scaffolds with anticancer properties that can simultaneously support and modulate proper regeneration of bone tissue. In this study cytotoxicity of scaffolds composed from poly (L-lactic acid) functionalized with nanohydroxyapatite (nHAp) and doped with europium (III) ions—10 wt % 3 mol % Eu3+: nHAp@PLLA was tested using human osteosarcoma cells: U-2 OS, Saos-2 and MG-63. Human adipose tissue-derived stromal cells (HuASCs) were used as non-transformed cells to determine the selective cytotoxicity of the carrier. Analysis included evaluation of cells morphology (confocal/scanning electron microscopy (SEM)), metabolic activity and apoptosis profile in cultures on the scaffolds. Results obtained indicated on high cytotoxicity of scaffolds toward all OSA cell lines, associated with a decrease of cells’ viability, deterioration of metabolic activity and activation of apoptotic factors determined at mRNA and miRNA levels. Simultaneously, the biomaterials did not affect HuASCs’ viability and proliferation rate. Obtained scaffolds showed a bioimaging function, due to functionalization with luminescent europium ions, and thus may find application in theranostics treatment of OSA.
Purpose: Osteoporosis results in a severe decrease in the life quality of many people worldwide. The latest data shows that the number of osteoporotic fractures is becoming an increasing international health service problem. Therefore, a new kind of controllable treatment methods for osteoporotic fractures is extensively desired. For that reason, we have manufactured and evaluated nanohydroxyapatite (nHAp)-based composite co-doped with iron oxide (IO) nanoparticles. The biomaterial was used as a matrix for the controlled delivery of miR-21-5p and miR-124-3p, which have a proven impact on bone cell metabolism. Methods: The nanocomposite Ca 5 (PO 4 ) 3 OH/Fe 3 O 4 (later called nHAp/IO) was obtained by the wet chemistry method and functionalised with microRNAs (nHAp/IO@miR-21/124). Its physicochemical characterization was performed using XRPD, FT-IR, SEM-EDS and HRTEM and SAED methods. The modulatory effect of the composite was tested in vitro using murine pre-osteoblasts MC3T3-E1 and pre-osteoclasts 4B12. Moreover, the anti-inflammatory effects of biomaterial were analysed using a model of LPS-treated murine macrophages RAW 264.7. We have analysed the cells' viability, mitochondria membrane potential and oxidative stress under magnetic field (MF+) and without (MF-). Moreover, the results were supplemented with RT-qPCR and Western blot assays to evaluate the expression profile for master regulators of bone metabolism. Results: The results indicated pro-osteogenic effects of nHAp/IO@miR-21/124 composite enhanced by exposure to MF. The enhanced osteogenesis guided by nHAp/IO@miR-21/124 presence was associated with increased metabolism of progenitor cells and activation of osteogenic markers (Runx-2, Opn, Coll-1). Simultaneously, nanocomposite decreased metabolism and differentiation of pre-osteoclastic 4B12 cells accompanied by reduced expression of CaII and Ctsk. Obtained composite regulated viability of bone progenitor cells and showed immunomodulatory properties inhibiting the expression of inflammatory markers, ie, TNF-α, iNOs or IL-1β, in LPS-stimulated RAW 264.7 cells. Conclusion:We have described for the first time a new concept of osteoporosis treatment based on nHAp/IO@miR-21/124 application. Obtained results indicated that fabricated nanocomposite might impact proper regeneration of osteoporotic bone, restoring the balance between osteoblasts and osteoclast.
The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism, and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K, and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predicts lower regenerative capacity if used as autologous transplants.
Atomic layer deposition (ALD) technology has started to attract attention as an efficient method for obtaining bioactive, ultrathin oxide coatings. In this study, using ALD, we have created titanium dioxide (TiO2) layers. The coatings were characterised in terms of physicochemical and biological properties. The chemical composition of coatings, as well as thickness, roughness, wettability, was determined using XPS, XRD, XRR. Cytocompatibillity of ALD TiO2 coatings was accessed applying model of mouse pre-osteoblast cell line MC3T3-E1. The accumulation of transcripts essential for bone metabolism (both mRNA and miRNA) was determined using RT-qPCR. Obtained ALD TiO2 coatings were characterised as amorphous and homogeneous. Cytocompatibility of the layers was expressed by proper morphology and growth pattern of the osteoblasts, as well as their increased viability, proliferative and metabolic activity. Simultaneously, we observed decreased activity of osteoclasts. Obtained coatings promoted expression of Opn, Coll-1, miR-17 and miR-21 in MC3T3-E1 cells. The results are promising in terms of the potential application of TiO2 coatings obtained by ALD in the field of orthopaedics, especially in terms of metabolic- and age-related bone diseases, including osteoporosis.
In chronic upper respiratory tract diseases, increased cell proliferative activity is observed, which is coordinated by BCL-2 proteins and small non-coding RNAs. This study aimed to determine the expression of critical apoptosis markers at the mRNA and miRNA levels in patients with chronic rhinosinusitis with nasal polyps (CSRwNP). The study group consisted of ten patients with CSRwNP and ten healthy subjects. To detect in situ apoptosis in the maxillary sinus mucosa, TUNEL staining was performed. The expression of transcripts was determined by RT-qPCR and included the detection of markers associated with cell survival and apoptosis, i.e., BAX, p53, p21, CASP3, CASP9, c-MYC, CCND1, BRIC5, and APAF1. Levels of miR-17-5p, miR-145-5p, miR-146a-5p, and miR-203a-3p were also measured by RT-qPCR. The obtained results indicated increased apoptosis determined by a TUNEL assay in CSRwNP patients and accompanied by an increased expression of BAX, P21, P53, CASP3, CASP9, c-MYC, and APAF-1 transcripts and decreased mRNA levels of BCL-2 and BIRC5. Furthermore, the nasal sinus epithelium of patients with CSRwNP showed increased levels of miR-203a-3p while also showing a decreased expression of miR-17-5p and miR-145-5p. Our results showed that pro-apoptotic transcripts detected at mRNA and miRNA levels might be involved in the pathogenesis of chronic sinusitis with polyps. The identification of those key molecular mediators may be applicable for the specific diagnostic and/or development of targeted therapies for chronic sinusitis with polyps.
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