FGF23-related hypophosphatemia in patients with low bone mineral density and fragility fractures: challenges in diagnosis and management.

PURPOSE: Hypophosphatemia (HP) can be observed in patients evaluated for skeletal fragility. We investigated prevalence of HP among outpatients referred for low bone density or fragility fractures, HP-associated clinical and biochemical features and outcomes of recommended diagnostic algorithm in our cohort. METHODS: Chronic HP (phosphate ≤ 2.7 mg/dL over 6 months or longer) was retrospectively investigated among 2319 patients. In renal wasting-related HP, intact FGF23 was assessed; non-suppressed FGF23 prompted the performance of 68Ga-DOTATOC PET/CT in the suspicion of tumor-induced steomalacia (TIO). RESULTS: Renal wasting-related HP (median 2.2, range 1.6-2.6 mg/dL) was observed in 19 patients (0.82%). FGF23 levels were suppressed in two patients diagnosed with renal tubular disease, increased in one and within normal range in most patients. X-linked hypophosphatemic rickets was diagnosed in one woman. In the remaining 16 patients, highly prevalent fragility fractures (50%) and severely reduced bone mineral density were detected, though diagnostic criteria for osteomalacia were not fulfilled. 68Ga-PET was performed in nine patients and was positive in four. While intact FGF23 levels alone failed to differentiate PET's outcomes (positive: FGF23 median 70.5 pg/mL; negative: 52 pg/mL, P = 0.462), the coexistence of multiple biochemical and radiologic alterations performed better in prediction of PET's positivity. CONCLUSION: Mild, apparently unexplained HP is observed in 0.82% of patients with low bone density or fragility fractures. In asymptomatic patients with isolated mild hypophosphatemia, the probability of finding an underlying tumor disease is very low, and utility of extensive and expensive diagnostic workup should be carefully considered in this setting.

Genome-wide DNA methylation profiling of recurrent and non-recurrent chordomas.

Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1-4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2'-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (>0.2 average ß-value difference) between chordomas and nucleus pulposus samples (adjusted P < 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.

Stemness and osteogenic and adipogenic potential are differently impaired in subcutaneous and visceral adipose derived stem cells (ASCs) isolated from obese donors.

Today adipose tissue is not just considered as the primary energy storage organ, but it is also recognized as an important endocrine tissue and an abundant source of mesenchymal stem cells (adipose-derived stem cells, ASCs). During the last decade, several studies have provided preclinical data on the safety and efficacy of ASCs, supporting their use in cell-based therapy for regenerative medicine purposes. Little is known about the effect of obesity on ASCs properties. Since ASCs differentiation and proliferation are determined by their niche, the differences in body fat distribution and the obesity-related co-morbidities may have several consequences. In this study we compared ASCs of subcutaneous adipose tissue from obese (obS-ASCs) and non-obese (nS-ASCs) donors in order to compare their immunophenotype and osteogenic and adipogenic potential. Moreover, in order to evaluate the possible difference between subcutaneous and visceral fat, obS-ASCs were also compared to ASCs derived from visceral adipose tissue of the same obese donors (obV-ASCs). Our results show that subcutaneous and visceral ASCs derived from obese donors have an impaired cell proliferation, clonogenic ability and immunophenotype. Nevertheless, obS-ASCs are able to differentiate toward osteogenic and adipogenic lineages, although to a small extent with respect to non-obese donors, whereas obV-ASCs lose most of their stem cell characteristics, including multi-differentiation potential. Taken together our findings confirm that not all ASCs present the same behavior, most likely due to their biological microenvironment in vivo. The specific stimuli which can play a key role in ASCs impairment, including the effects of the obesity-related inflammation, should be further investigated to have a complete picture of the phenomenon.

Two bone substitutes analyzed in vitro by porcine and human adipose-derived stromal cells.

Nowadays, the repair of large bone defects is an important goal in orthopaedic and dental fields. Tissue engineering, applied to increase the bone regeneration process, combines suitable scaffolds with either terminally differentiated cells or Mesenchymal Stromal Cells. In vitro studies with Adipose-derived Stromal Cells (ASCs) may identify new bioactive supports, to be tested in preclinical model. In this study, we evaluated the biocompatibility and the osteoinductive properties of two bone substitutes, RegenOSS (RO-1) and a new generation scaffold (RO-2), on both porcine and human ASCs. Porcine ASCs need a prolonged initial phase to adapt to both substitutes; indeed, their growth was initially reduced respect to cells cultured in their absence. In contrast, human ASCs were not negatively affected. However, no toxicity of RO-1 and -2 was observed on both ASC populations which are able to stick to both biomaterials. RO-1 and -2 supported osteogenic differentiation of porcine and human ASCs in a different manner: the presence of RO-1 up-regulated both alkaline phosphatase (ALP) activity and collagen production of human ASCs, whereas in porcine ASCs, RO-2 seemed to up-regulate ALP activity, while the production of collagen is mainly stimulated by the presence of RO-1. We suggest to use not just human ASCs, but also animal ones to select suitable scaffolds to generate bio-constructs in vitro, which then need to be tested in animal model before reaching the market.