Extracellular vesicles (EVs) derived from stem cells have emerged as promising mediators of osteogenesis, suggesting cell-free alternatives for bone tissue engineering and regenerative medicine. This review provides a comprehensive analysis of the main stem cell sources used for EV production, including bone marrow mesenchymal stem cells (BM-MSCs), adipose-derived stem cells (ADSCs), umbilical cord MSCs (UC-MSCs), induced pluripotent stem cells (iPSCs), and alternative stromal populations. Particular attention is given to the ways in which different conditioning and differentiation strategies, such as osteogenic induction, hypoxia, and mechanical stimulation, modulate EV cargo composition and enhance their therapeutic potential. We further discuss the in vitro models employed to evaluate EV-mediated bone regeneration, ranging from 2D cultures to complex 3D spheroids, scaffold-based systems, and bone organoids. Overall, this review emphasizes the current challenges related to standardization, scalable production, and clinical translation. It also outlines future directions, including bioengineering approaches, advanced preclinical models, and the integration of multi-omics approaches and artificial intelligence to optimize EV-based therapies. By integrating current knowledge, this work aims to guide researchers toward more consistent and physiologically relevant strategies to harness EVs for effective bone regeneration. Finally, this work uniquely integrates a comparative analysis of EVs from multiple stem cell sources with engineering strategies and emerging clinical perspectives, thereby providing an updated and translational framework for their application in bone regeneration.

Runt-related transcription factor 2(RUNX2),also called core-binding factor subunit alpha-1(CBFA1),is the bone-specific transcription factor considered the master gene in osteogenesis,contains a crucial RUNT domain for DNA binding,and is regulated by multiple mechanisms.During the initial stages of osteogenesis,the expression levels of RUNX2 are primarily elevated and then gradually decrease during the formation of osteoblasts and osteocytes.

Overexpression of the Runt-related transcription factor 2 (RUNX2) has been reported in several cancer types, and the C-X-C motif chemokine receptor 4 (CXCR4) has an important role in tumour progression. However, the interplay between CXCR4 and RUNX2 in melanoma cells remains poorly understood. In the present study, we used melanoma cells and a RUNX2 knockout (RUNX2-KO) in vitro model to assess the influence of RUNX2 on CXCR4 protein levels along with its effects on markers associated with cell invasion and autophagy. Osteotropism was assessed using a 3D microfluidic model. Moreover, we assessed the impact of CXCR4 on the cellular levels of key cellular signalling proteins involved in autophagy. We observed that melanoma cells express both RUNX2 and CXCR4. Restored RUNX2 expression in RUNX2 KO cells increased the expression levels of CXCR4 and proteins associated with the metastatic process. The protein markers of autophagy LC3 and beclin were upregulated in response to increased CXCR4 levels. The CXCR4 inhibitor WZ811 reduced osteotropism and activated the mTOR and p70-S6 cell signalling proteins. Our data indicate that the RUNX2 transcription factor promotes the expression of the CXCR4 chemokine receptor on melanoma cells, which in turn promotes autophagy, cell invasiveness, and osteotropism, through the inhibition of the mTOR signalling pathway. Our data suggest that RUNX2 promotes melanoma progression by upregulating CXCR4, and we identify the latter as a key player in melanoma-related osteotropism.

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells.

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 µM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation.

Cleidocranial dysplasia (CCD), a dominantly inherited skeletal disease, is characterized by a variable phenotype ranging from dental alterations to severe skeletal defects. Either de novo or inherited mutations in the RUNX2 gene have been identified in most CCD patients. Transcription factor RUNX2, the osteogenic master gene, plays a central role in the commitment of mesenchymal stem cells to osteoblast lineage. With the aim to analyse the effects of RUNX2 mutations in CCD patients, we investigated RUNX2 gene expression and the osteogenic potential of two CCD patients' cells. In addition, with the aim to better understand how RUNX2 mutations interfere with osteogenic differentiation, we performed string analyses to identify proteins interacting with RUNX2 and analysed p53 expression levels. Our findings demonstrated for the first time that, in addition to the alteration of downstream gene expression, RUNX2 mutations impair p53 expression affecting osteogenic maturation. In conclusion, the present work provides new insights into the role of RUNX2 mutations in CCD patients and suggests that an in-depth analysis of the RUNX2-associated gene network may contribute to better understand the complex molecular and phenotypic alterations in mutant subjects.

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy.

Tissue non-specific alkaline phosphatase (TNSALP) is an ubiquitary enzyme fundamental for bone tissue development and growth. Adult Hypophosphatasia (HPP) is a rare genetic disease caused by a defective TNSALP activity. HPP is frequently misdiagnosed due to the lack of clinical manifestation. We report an asymptomatic woman with HPP that during 5 years follow-up suffered of a bone fracture. Sequencing of genomic DNA revealed a previously unreported mutation at nucleotide 1252G > A (Gly418Arg) leading to a defective enzyme product in the subject. HPP was suspected because of the lack of the expected increase of serum TNSALP at menopause. Our case report underlines the importance of early diagnosis and that the lack of increase of TNSALP at meno pause must be a wake-up call for clinicians.

BACKGROUND:Several studies evidenced a possible role of the d3-Growth Hormone Receptor (GHR) polymorphism in fetal growth. The GHR genotype distribution was studied in small (SGA) and appropriate (AGA) for gestational age newborns but never in the large (LGA) for gestational age babies. The aim of this study was to evaluate the frequencies of this polymorphism in a large cohort of SGA, AGA and LGA newborns.METHODS:A total of 536 healthy newborns, randomly selected among the infants referred to the Italian North-Eastern centre for endocrinological and metabolic newborn screening, were enrolled: 192 SGA, 200 LGA and 144 AGA. Weight was recorded at birth. Isoforms of d3-GHR gene (fl/fl, d3/fl, and d3/d3) were analysed.RESULTS:The analysis of the GHR genotype evidenced a lower frequency of the d3/d3 genotype in SGA cohort compared to the AGA population (P=0.005), or to the total population (P=0.035). No differences were found in the genotypic distribution between LGA and AGA population (P=0.373), or between LGA and the whole population (P=0.292).CONCLUSIONS:d3/d3 GHR genotype was found twice as frequent in AGA and LGA cohorts compared to SGA subjects, whereas no significant differences in the frequency distribution of the GHR genotypes between LGA and AGA newborns were detected. The data leads to the exclusion of the GHR exon 3 deletion polymorphism as a possible genetic factor leading to LGA pregnancies.

Recent studies have identified the molecular defect underlying autosomal dominant osteogenesis imperfecta (OI) type V. Unlike all other OI types, which are characterized by high genetic heterogeneity, OI type V appears consistently associated to a unique de novo C>T transition within the 5' UTR of the IFITM5 gene. Although the precise frequency of OI type V is not known, this recurrent base substitution may well represent a mutational hotspot in the human genome. We show that it occurs at a CpG dinucleotide that is highly methylated in several tissues and particularly in the sperm DNA, suggesting a mutational mechanism common to other de novo recurrent dominant mutations.

CONTEXT Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disease resulting from mutations within the hydroxysteroid (11β-dehydrogenase2 [HSD11B2]) gene causing a prominent mineralocorticoid receptor activation by cortisol and hypokalemic low renin hypertension as the main clinical feature. OBJECTIVE The objective of the study was to characterize AME for possible novel HSD11B2 mutations and to define the role of HSD11B2 promoter methylation in the phenotypic expression of the disease. SUBJECTS Two proband brothers and 10 relatives participated in the study. METHODS Peripheral blood mononuclear cell DNA was used for HSD11B2 exon sequencing, and a new predicted structure of 11β-hydroxysteroid dehydrogenase type 2 was generated by an in silico three-dimensional modeling. Promoter methylation was determined by bisulfite pyrosequencing. Urinary tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone ratio, a surrogate marker of 11β-hydroxysteroid dehydrogenase type 2 activity, was measured by gas chromatography-mass spectrometry. RESULTS A novel homozygous variant at HSD11B2 exon 3 site (c.C662G) resulting in an alanine-to-glycine change at position 221 was discovered by sequencing the DNA of the probands. A monoallelic mutation was found in the DNA of the parents and other four relatives. In silico three-dimensional modeling showed that the Ala221Gly substitution could perturb a hydrophobic interaction by reducing the enzymatic affinity for the substrate. The HSD11B2 promoter methylation of normotensive heterozygous relatives was similar to that of wild types, whereas the hypertensive heterozygous subjects showed higher methylation than wild types, consistently with a transcriptional repressive effect of promoter hypermethylation. CONCLUSIONS A novel HSD11B2 functional mutation accounting for an Ala221Gly substitution causes AME. The hypertension phenotype is also epigenetically modulated by HSD11B2 methylation in subjects heterozygous for the mutation.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)