Identification and characterization of stromal-like cells with CD207+/low CD1a+/low phenotype derived from histiocytic lesions - a perspective in vitro model for drug testing.

BACKGROUND: Histiocytoses are rare disorders manifested by increased proliferation of pathogenic myeloid cells sharing histological features with macrophages or dendritic cells and accumulating in various organs, i.a., bone and skin. Pre-clinical in vitro models that could be used to determine molecular pathways of the disease are limited, hence research on histiocytoses is challenging. The current study compares cytophysiological features of progenitor, stromal-like cells derived from histiocytic lesions (sl-pHCs) of three pediatric patients with different histiocytoses types and outcomes. The characterized cells may find potential applications in drug testing. METHODS: Molecular phenotype of the cells, i.e. expression of CD1a and CD207 (langerin), was determined using flow cytometry. Cytogenetic analysis included GTG-banded metaphases and microarray (aCGH) evaluation. Furthermore, the morphology and ultrastructure of cells were evaluated using a confocal and scanning electron microscope. The microphotographs from the confocal imaging were used to reconstruct the mitochondrial network and its morphology. Basic cytophysiological parameters, such as viability, mitochondrial activity, and proliferation, were analyzed using multiple cellular assays, including Annexin V/7-AAD staining, mitopotential analysis, BrdU test, clonogenicity analysis, and distribution of cells within the cell cycle. Biomarkers potentially associated with histiocytoses progression were determined using RT-qPCR at mRNA, miRNA and lncRNA levels. Intracellular accumulation of histiocytosis-specific proteins was detected with Western blot. Cytotoxicyty and IC50 of vemurafenib and trametinib were determined with MTS assay. RESULTS: Obtained cellular models, i.e. RAB-1, HAN-1, and CHR-1, are heterogenic in terms of molecular phenotype and morphology. The cells express CD1a/CD207 markers characteristic for dendritic cells, but also show intracellular accumulation of markers characteristic for cells of mesenchymal origin, i.e. vimentin (VIM) and osteopontin (OPN). In subsequent cultures, cells remain viable and metabolically active, and the mitochondrial network is well developed, with some distinctive morphotypes noted in each cell line. Cell-specific transcriptome profile was noted, providing information on potential new biomarkers (non-coding RNAs) with diagnostic and prognostic features. The cells showed different sensitivity to vemurafenib and trametinib. CONCLUSION: Obtained and characterized cellular models of stromal-like cells derived from histiocytic lesions can be used for studies on histiocytosis biology and drug testing.

ß-Lactoglobulin affects the oxidative status and viability of equine endometrial progenitor cells via lncRNA-mRNA-miRNA regulatory associations.

The ß-lactoglobulin (ß-LG) was previously characterized as a mild antioxidant modulating cell viability. However, its biological action regarding endometrial stromal cell cytophysiology and function has never been considered. In this study, we investigated the influence of ß-LG on the cellular status of equine endometrial progenitor cells under oxidative stress. The study showed that ß-LG decreased the intracellular accumulation of reactive oxygen species, simultaneously ameliorating cell viability and exerting an anti-apoptotic effect. However, at the transcriptional level, the reduced mRNA expression of pro-apoptotic factors (i.e. BAX and BAD) was accompanied by decreased expression of mRNA for anti-apoptotic BCL-2 and genes coding antioxidant enzymes (CAT, SOD-1, GPx). Still, we have also noted the positive effect of ß-LG on the expression profile of transcripts involved in endometrial viability and receptivity, including ITGB1, ENPP3, TUNAR and miR-19b-3p. Finally, the expression of master factors of endometrial decidualization, namely prolactin and IGFBP1, was increased in response to ß-LG, while non-coding RNAs (ncRNAs), that is lncRNA MALAT1 and miR-200b-3p, were upregulated. Our findings indicate a novel potential role of ß-LG as a molecule regulating endometrial tissue functionality, promoting viability and normalizing the oxidative status of endometrial progenitor cells. The possible mechanism of ß-LG action includes the activation of ncRNAs essential for tissue regeneration, such as lncRNA MALAT-1/TUNAR and miR-19b-3p/miR-200b-3p.

MiR-21-5p regulates the dynamic of mitochondria network and rejuvenates the senile phenotype of bone marrow stromal cells (BMSCs) isolated from osteoporotic SAM/P6 mice.

BACKGROUND: Progression of senile osteoporosis is associated with deteriorated regenerative potential of bone marrow-derived mesenchymal stem/stromal cells (BMSCs). According to the recent results, the senescent phenotype of osteoporotic cells strongly correlates with impaired regulation of mitochondria dynamics. Moreover, due to the ageing of population and growing osteoporosis incidence, more efficient methods concerning BMSCs rejuvenation are intensely investigated. Recently, miR-21-5p was reported to play a vital role in bone turnover, but its therapeutic mechanisms in progenitor cells delivered from senile osteoporotic patients remain unclear. Therefore, the goal of this paper was to investigate for the first time the regenerative potential of miR-21-5p in the process of mitochondrial network regulation and stemness restoration using the unique model of BMSCs isolated from senile osteoporotic SAM/P6 mice model. METHODS: BMSCs were isolated from healthy BALB/c and osteoporotic SAM/P6 mice. We analysed the impact of miR-21-5p on the expression of crucial markers related to cells' viability, mitochondria reconstruction and autophagy progression. Further, we established the expression of markers vital for bone homeostasis, as well as defined the composition of extracellular matrix in osteogenic cultures. The regenerative potential of miR-21 in vivo was also investigated using a critical-size cranial defect model by computed microtomography and SEM-EDX imaging. RESULTS: MiR-21 upregulation improved cells' viability and drove mitochondria dynamics in osteoporotic BMSCs evidenced by the intensification of fission processes. Simultaneously, miR-21 enhanced the osteogenic differentiation of BMSCs evidenced by increased expression of Runx-2 but downregulated Trap, as well as improved calcification of extracellular matrix. Importantly, the analyses using the critical-size cranial defect model indicated on a greater ratio of newly formed tissue after miR-21 application, as well as upregulated content of calcium and phosphorus within the defect site. CONCLUSIONS: Our results demonstrate that miR-21-5p regulates the fission and fusion processes of mitochondria and facilitates the stemness restoration of senile osteoporotic BMSCs. At the same time, it enhances the expression of RUNX-2, while reduces TRAP accumulation in the cells with deteriorated phenotype. Therefore, miR-21-5p may bring a novel molecular strategy for senile osteoporosis diagnostics and treatment.

Comparison of Selected Non-Coding RNAs and Gene Expression Profiles between Common Osteosarcoma Cell Lines.

Osteosarcoma (OS) is a bone tumour affecting adolescents and elderly people. Unfortunately, basic treatment methods are still underdeveloped, which has a high impact on the poor survivability of the patients. Studies designed to understand the underlying mechanisms of osteosarcoma development, as well as preclinical investigations aimed at establishing novel therapeutic strategies, rely significantly upon in vitro models, which apply well-established cell lines such as U-2 OS, Saos-2 and MG-63. In this study, the expression of chosen markers associated with tumour progression, metastasis and survival were identified using RT-qPCR. Levels of several onco-miRs (miR-21-5p, miR-124-3p, miR-223-3p and miR-320a-3p) and long non-coding RNA MEG3 were established. The mRNA expression of bone morphogenetic proteins (BMPs), including BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, as well as their receptors: BMPR-IA, BMPR-IB and BMPR-II was also determined. Other tested markers included metalloproteinases, i.e., MMP-7 and MMP-14 and survivin (BIRC5), C-MYC, as well as CYCLIN D (CCND1). The analysis included comparing obtained profiles with transcript levels established for the osteogenic HeLa cell line and human adipose-derived stromal cells (hASCs). The tested OS cell lines were characterised by a cancer-related phenotype, such as increased expression of mRNA for BMP-7, as well as MMP-7 and MMP-14. Osteosarcoma cells differ considerably in miR-21-5p and miR-124-3p levels, which can be related to uncontrolled tumour growth. The comprehensive examination of osteosarcoma transcriptome profiles may facilitate the selection of appropriate cell models for preclinical investigations aimed at the development of new strategies for OS treatment.

Sex hormone binding globulin as a potential drug candidate for liver-related metabolic disorders treatment.

Sex hormone binding globulin (SHBG) is a hepatokine that binds to circulating steroid hormones (testosterone, oestradiol) to regulate their concentration in the bloodstream. Recently SHBG was recognized as an essential biomarker for metabolic syndrome (MetS) and hepatic steatosis development. At the hepatic level, the production of SHBG is mainly regulated by sex steroids and thyroxine. Studies of various research groups, including ours, showed that SHBG could be considered a reliable marker of insulin resistance and, therefore, can serve as a predictor of type 2 diabetes. Moreover, increased levels of circulating pro-inflammatory mediators strongly correlate with lowered serum levels of SHBG. This review paper emphasizes the role of SHBG as a potential drug candidate in the course of various metabolic dysfunctions, including non-alcoholic fatty liver disease (NAFLD), obesity, diabetes mellitus and insulin resistance. The studies related to SHBG and its role in the course of metabolic disorders are very limited. Here, we have summarized the most current knowledge about SHBG and its mechanism of action, indicating a novel concept for its possible therapeutic application in the management framework of commonly occurring metabolic dysfunctions.

Expression of Apoptosis-Related Biomarkers in Inflamed Nasal Sinus Epithelium of Patients with Chronic Rhinosinusitis with Nasal Polyps (CRSwNP)-Evaluation at mRNA and miRNA Levels.

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.

Obesity Affects the Proliferative Potential of Equine Endometrial Progenitor Cells and Modulates Their Molecular Phenotype Associated with Mitochondrial Metabolism.

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.

Novel Nanohydroxyapatite (nHAp)-Based Scaffold Doped with Iron Oxide Nanoparticles (IO), Functionalized with Small Non-Coding RNA (miR-21/124) Modulates Expression of Runt-Related Transcriptional Factor 2 and Osteopontin, Promoting Regeneration of Osteoporotic Bone in Bilateral Cranial Defects in a Senescence-Accelerated Mouse Model (SAM/P6). PART 2.

PURPOSE: Healing of osteoporotic defects is challenging and requires innovative approaches to elicit molecular mechanisms promoting osteoblasts-osteoclasts coupling and bone homeostasis. METHODS: Cytocompatibility and biocompatibility of previously characterised nanocomposites, i.e Ca5(PO4)3OH/Fe3O4 (later called nHAp/IO) functionalised with microRNAs (nHAp/IO@miR-21/124) was tested. In vitro studies were performed using a direct co-culture system of MC3T3-E1 pre-osteoblast and 4B12 pre-osteoclasts. The analysis included determination of nanocomposite influence on cultures morphology (confocal imaging), viability and metabolic activity (Alamar Blue assay). Pro-osteogenic signals were identified at mRNA, miRNA and protein level with RT-qPCR, Western blotting and immunocytochemistry. Biocompatibility of biomaterials was tested using bilateral cranial defect performed on a senescence-accelerated mouse model, ie SAM/P6 and Balb/c. The effect of biomaterial on the process of bone healing was monitored using microcomputed tomography. RESULTS: The nanocomposites promoted survival and metabolism of bone cells, as well as enhanced functional differentiation of pre-osteoblasts MC3T3-E1 in co-cultures with pre-osteoclasts. Differentiation of MC3T3-E1 driven by nHAp/IO@miR-21/124 nanocomposite was manifested by improved extracellular matrix differentiation and up-regulation of pro-osteogenic transcripts, ie late osteogenesis markers. The nanocomposite triggered bone healing in a cranial defect model in SAM/P6 mice and was replaced by functional bone in Balb/c mice. CONCLUSION: This study demonstrates that the novel nanocomposite nHAp/IO can serve as a platform for therapeutic miRNA delivery. Obtained nanocomposite elicit pro-osteogenic signals, decreasing osteoclasts differentiation, simultaneously improving osteoblasts metabolism and their transition toward pre-osteocytes and bone mineralisation. The proposed scaffold can be an effective interface for in situ regeneration of osteoporotic bone, especially in elderly patients.

Bone marrow stromal cells (BMSCs CD45- /CD44+ /CD73+ /CD90+ ) isolated from osteoporotic mice SAM/P6 as a novel model for osteoporosis investigation.

Available therapies aimed at treating age-related osteoporosis are still insufficient. Therefore, designing reliable in vitro model for the analysis of molecular mechanisms underlying senile osteoporosis is highly required. We have isolated and characterized progenitor cells isolated from bone marrow (BMSCs) of osteoporotic mice strain SAM/P6 (BMSCSAM/P6 ). The cytophysiology of BMSCSAM/P6 was for the first time compared with BMSCs isolated from healthy BALB/c mice (BMSCBALB/c ). Characterization of the cells included evaluation of their multipotency, morphology and determination of specific phenotype. Viability of BMSCs cultures was determined in reference to apoptosis profile, metabolic activity, oxidative stress, mitochondrial membrane potential and caspase activation. Additionally, expression of relevant biomarkers was determined with RT-qPCR. Obtained results indicated that BMSCSAM/P6 and BMSCBALB/c show the typical phenotype of mesenchymal stromal cells (CD44+, CD73+, CD90+) and do not express CD45. Further, BMSCSAM/P6 were characterized by deteriorated multipotency, decreased metabolic activity and increased apoptosis occurrence, accompanied by elevated oxidative stress and mitochondria depolarisation. The transcriptome analyses showed that BMSCSAM/P6 are distinguished by lowered expression of molecules crucial for proper osteogenesis, including Coll-1, Opg and Opn. However, the expression of Trap, DANCR1 and miR-124-3p was significantly up-regulated. Obtained results show that BMSCSAM/P6 present features of progenitor cells with disturbed metabolism and could serve as appropriate model for in vitro investigation of age-dependent osteoporosis.

Nanohydroxyapatite (nHAp) Doped with Iron Oxide Nanoparticles (IO), miR-21 and miR-124 Under Magnetic Field Conditions Modulates Osteoblast Viability, Reduces Inflammation and Inhibits the Growth of Osteoclast - A Novel Concept for Osteoporosis Treatment: Part 1.

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 Ca5(PO4)3OH/Fe3O4 (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-1ß, 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.

Titanium Dioxide Thin Films Obtained by Atomic Layer Deposition Promotes Osteoblasts' Viability and Differentiation Potential While Inhibiting Osteoclast Activity-Potential Application for Osteoporotic Bone Regeneration.

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.

Small and Long Non-coding RNAs as Functional Regulators of Bone Homeostasis, Acting Alone or Cooperatively.

Emerging knowledge indicates that non-coding RNAs, including microRNAs (miRNAs) and long-noncoding RNAs (lncRNAs), have a pivotal role in bone development and the pathogenesis of bone-related disorders. Most recently, miRNAs have started to be regarded as potential biomarkers or targets for various sets of diseases, while lncRNAs have gained attention as a new layer of gene expression control acting through versatile interactions, also with miRNAs. The rapid development of RNA sequencing techniques based on next-generation sequencing (NGS) gives us better insight into molecular pathways regulated by the miRNA-lncRNA network. In this review, we summarize the current knowledge related to the function of miRNAs and lncRNAs as regulators of genes that are crucial for proper bone metabolism and homeostasis. We have characterized important non-coding RNAs and their expression signatures, in relationship to bone. Analysis of the biological function of miRNAs and lncRNAs, as well as their network, will pave the way for a better understanding of the pathogenesis of various bone disorders. We also think that this knowledge may lead to the development of innovative diagnostic tools and therapeutic approaches for bone-related disorders.

Zirconium Oxide Thin Films Obtained by Atomic Layer Deposition Technology Abolish the Anti-Osteogenic Effect Resulting from miR-21 Inhibition in the Pre-Osteoblastic MC3T3 Cell Line.

INTRODUCTION: 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 ZrO2 thin films grown by Atomic Layer Deposition (ALD) technology (ZrO2 ALD). METHODOLOGY: The cytocompatibility of the obtained layers was analysed using the mice pre-osteoblastic 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 ZrO2 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 ZrO2 ALD coatings, the up-regulation of genes essential for bone metabolism was noted. DISCUSSION: The data obtained indicate that ZrO2 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 ZrO2 ALD layers is essential in terms of their potential clinical application in bone regenerative medicine.

Three dimensional (3D) printed polylactic acid with nano-hydroxyapatite doped with europium(III) ions (nHAp/PLLA@Eu3+) composite for osteochondral defect regeneration and theranostics.

In the current research previously developed composites composed from poly (l-lactide) (PLLA) and nano-hydroxyapatite (10 wt% nHAp/PLLA) were functionalized with different concentrations of europium (III) (Eu3+). The aim of this study was to determine whether Eu3+ ions doped within the 10 wt% nHAp/PLLA scaffolds will improve the bioactivity of composites. Therefore, first set of experiments was designed to evaluate the effect of Eu3+ ions on morphology, viability, proliferation and metabolism of progenitor cells isolated from adipose tissue (hASC). Three different concentration were tested i.e. 1 mol%, 3 mol% and 5%mol. We identified the 10 wt% nHAp/PLLA@3 mol% Eu3+ scaffolds as the most cytocompatible. Further, we investigated the influence of the composites doped with 3 mol% Eu3+ ions on differentiation of hASC toward bone and cartilage forming cells. Our results showed that 10 wt% nHAp/PLLA@3 mol% Eu3+ scaffolds promotes osteogenesis and chondrogenesis of hASCs what was associated with improved synthesis and secretion of extracellular matrix proteins specific for bone and articular cartilage tissue. We also proved that obtained biomaterials have bio-imaging function and their integration with bone can be monitored using micro computed tomography (µCT).

The Role of miR-21 in Osteoblasts-Osteoclasts Coupling In Vitro.

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.

Theranostic Applications of Nanostructured Silicate-Substituted Hydroxyapatite Codoped with Eu3+ and Bi3+ Ions-A Novel Strategy for Bone Regeneration.

In this paper, nanocrystalline silicate-substituted hydroxyapatites (nSi-HAps) codoped with Eu3+ were functionalized with Bi3+ ions. Biomaterials were synthesized using a microwave-assisted hydrothermal method and heat-treated at 700 °C. The concentration of Eu3+ ions was established at 1 mol %, and the concentration of Bi3+ was in the range of 0.5-2 mol %. The physicochemical properties of the obtained biomaterials were determined using previously established methods, including X-ray powder diffraction, scanning electron microscopy techniques, and IR spectroscopy. Particle sizes obtained in this study were in the range of 22-65 nm, which was established by the Rietveld method. The luminescence properties of the Eu3+ ion-doped silicate-substituted apatite were recorded depending on the bismuth(III) concentration. The cytocompatibility of obtained biomaterials was tested using the model of mouse pre-osteoblasts cell line, that is, MC3T3-E1. We showed that the obtained biomaterials exerted anti-apoptotic effect, reducing the number of early and late apoptotic cells and decreasing caspase activity and reactive oxygen species accumulation. The transcripts levels of genes associated with apoptosis confirmed the anti-apoptotic effect of the biomaterials. Increased metabolic activity of MC3T3-E1 in cultures with biomaterials functionalized with Bi3+ ions has been observed. Moreover, the determined profile of osteogenic markers indicates that the obtained matrices, that is, Eu3+:nSi-HAp functionalized with Bi3+ ions, exert pro-osteogenic properties. The biological features of Eu3+:nSi-HAp modified with Bi3+ ions are highly desired in terms of functional tissue restoration and further efficient osteointegration.

The Potential Selective Cytotoxicity of Poly (L- Lactic Acid)-Based Scaffolds Functionalized with Nanohydroxyapatite and Europium (III) Ions toward Osteosarcoma Cells.

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.

The Haematococcus pluvialis extract enriched by bioaccumulation process with Mg(II) ions improves insulin resistance in equine adipose-derived stromal cells (EqASCs).

Insulin resistance (IR) is one of the characteristic features of equine metabolic syndrome (EMS). Presently, the only therapies of choice are caloric restrictions combined with mineral supplementation, which might improve insulin sensitivity. In this study we investigated the effect of Haematococcus pluvialis algae water extract enriched in bioaccumulation process in magnesium ions (Hp_Mg(II)) on equine adipose derived mesenchymal stromal stem cells, in which insulin resistance was induced by palmitic acid (IR-EqASCs). For this purpose, chemical characterization of H. pluvialis was performed with special emphasis on the analysis of minerals composition, total phenolic and carotenoids contents, as well as scavenging activity. To examine the influence of H. pluvialis extract on IR-EqASCs, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, gene expression by RT-qPCR, proliferative and metabolic cells activity analysis) were applied to investigate in vitro viability, oxidative stress markers and apoptosis-related factor accumulation, along with insulin resistance-related genes expression. Obtained results show, that Hp_Mg(II) significantly improves proliferative and metabolic activity of IR-EqASCs, shortens their population doubling time, improves their clonogenic potential and reduces expression of apoptosis related genes. Moreover, anti-oxidative effect of extract was presented.

Nanocrystalline Hydroxyapatite Loaded with Resveratrol in Colloidal Suspension Improves Viability, Metabolic Activity and Mitochondrial Potential in Human Adipose-Derived Mesenchymal Stromal Stem Cells (hASCs).

In response to the demand for new multifunctional materials characterized by high biocompatibility, hydrogel (HG) nanocomposites as a platform for bioactive compound delivery have been developed and fabricated. A specific crosslinking/copolymerization chemistry was used to construct hydrogels with a controlled network organization. The hydrogels were prepared using 3,6-anhydro-α-l-galacto-ß-d-galactan (galactose hydrogel) together with resveratrol (trans-3,5,4'-trihydroxystilbene) and calcium hydroxyapatite nanoparticles. The resveratrol was introduced in three different concentrations of 0.1, 0.5, and 1 mM. Nanosized calcium hydroxyapatite was synthesized by a microwave-assisted hydrothermal technique, annealed at 500 °C for 3 h, and introduced at a concentration 10% (m/v). The morphology and structural properties of Ca10(PO4)6(OH)2 and its composite were determined by using XRPD (X-ray powder diffraction) techniques, as well as the absorption and IR (infrared) spectroscopy. The average nanoparticle size was 35 nm. The water affinity, morphology, organic compound release profile, and cytocompatibility of the obtained materials were studied in detail. The designed hydrogels were shown to be materials of biological relevance and of great pharmacological potential as carriers for bioactive compound delivery. Their cytocompatibility was tested using a model of human multipotent stromal cells isolated from adipose tissue (hASCs). The biomaterials increased the proliferative activity and viability of hASCs, as well as reduced markers of oxidative stress. In light of the obtained results, it has been thought that the designed materials meet the requirements of the tissue engineering triad, and may find application in regenerative medicine, especially for personalized therapies.

Intra-Vitreal Administration of Microvesicles Derived from Human Adipose-Derived Multipotent Stromal Cells Improves Retinal Functionality in Dogs with Retinal Degeneration.

This study was designed to determine the influence of microvesicles (MVs) derived from multipotent stromal cells isolated from human adipose tissue (hASCs) on retinal functionality in dogs with various types of retinal degeneration. The biological properties of hASC-MVs were first determined using an in vitro model of retinal Muller-like cells (CaMLCs). The in vitro assays included analysis of hASC-MVs influence on cell viability and metabolism. Brain-derived neurotrophic factor (BDNF) expression was also determined. Evaluation of the hASC-MVs was performed under normal and oxidative stress conditions. Preliminary clinical studies were performed on ten dogs with retinal degeneration. The clinical studies included behavioral tests, fundoscopy and electroretinography before and after hASC-MVs intra-vitreal injection. The in vitro study showed that CaMLCs treated with hASC-MVs were characterized by improved viability and mitochondrial potential, both under normal and oxidative stress conditions. Additionally, hASC-MVs under oxidative stress conditions reduced the number of senescence-associated markers, correlating with the increased expression of BDNF. The preliminary clinical study showed that the intra-vitreal administration of hASC-MVs significantly improved the dogs' general behavior and tracking ability. Furthermore, fundoscopy demonstrated that the retinal blood vessels appeared to be less attenuated, and electroretinography using HMsERG demonstrated an increase in a- and b-wave amplitude after treatment. These results shed promising light on the application of cell-free therapies in veterinary medicine for retinal degenerative disorders treatment.