PDF ) Rev Osteoporos Metab Miner. 2019; 11 (3): 15-53


1. Serum 25 hydroxyvitamin D levels, trabecular bone score and bone mineral density in patients with differentiated thyroid cancer and tsh suppression therapy
Allo Miguel G1, Guadalix Iglesias S1, Martín-Arriscado Arroba C2, Mola Reyes L1, Martínez Díaz-Guerra G1, Hawkins F2
1 Endocrinology Service. 12 de Octubre University Hospital; 2 Research Institute i+12, University Hospital 12 de Octubre. Complutense University

Background: It has been reported an increased rate of 25OHD insufficiency and deficiency in patients with differentiated thyroid cancer (DTC). This study evaluates the association between different levels of serum 25OHD, and its relation with trabecular bone score (TBS) and BMD in patients with DTC and long-term TSH suppression therapy.
Material and methods: 134 caucasian women with total thyroidectomy due to DTC were evaluated in this cross-sectional study. Patients had undergone total thyroidectomy with 1,31I ablation when indicated, and were receiving long-term TSH with LT4 (median 10 years; range 2 to 22 years). BMD was assessed by DXA, (densitometer QDR 4500, Hologic), and TBS applying iNsight2.0 software (Med-Imaps, Swiss). Serum 25OHD was measured by enzyme immunoassay (Roche Diagnostics). Statistics: pearson test was performed to evaluate the correlations between serum 25OHD, laboratory parameters, DXA and TBS.A level of α=0.05 was considered significant in all statistical procedures.
Results: 107 patients with 25OHD <30 ng/ml and 27 >30 ng/ml were included in the study. Mean age: 64±10.8 years. Mean serum 25OHD was 23.09±7.9 ng/ml. Mean serum 25OHD according to the categorization of deficiency, insufficiency and sufficiency groups were: 15.64±2.9, 25.27±2.7 and 34.7ng/ml, respectively. Serum PTH was higher in deficient and insufficient patients (57.65±22.6; 45.88±19.8 ng/ml) compared with sufficient patients (47.13±16; p=n.s.). BAP was significantly lower in the 25OHD sufficiency group (22.14±9.7 ng/ml) compared with the deficiency and insufficiency groups (29.5±14 ng/ml) (p=0.0440). Patients with 25OHD <30 ng/ml had significantly lower L-T, FN-T and TH-T scores compared with patients with 25OHD >30 (p<0.01; p=0.01 and p<0.01, respectively). TBS was lower in patients with 25OHD deficiency (1.24±0.13) compared with patients with insufficiency (1.27±0.13; p<0.05) and sufficiency (1.31±0.11; p<0.01). 25OHD levels were not associated with BMI, cancer stage or accumulative iodine radioactive dose.
Conclusion: DTC patients with long-term TSH suppression are at high risk of 25OHD deficiency and insufficiency. In our study, patients with serum 25OHD >30 ng/ml show better TBS and BMD, compared with patients with lower values. Based on these, we recommend 25(OH)D screening to avoid its deficiency with proper supplementation.


2. Primary cilia mediates PTHrP-dependent prosurvival actions on bone cells via Hedgehog signaling pathway
Martín Guerrero E, Tirado Cabrera I, Buendía I, Heredero Jiménez S, Rodríguez de Gortázar A, Ardura JA
Universidad San Pablo CEU. Madrid

Activation of primary cilia in osteocytes and osteoblasts has been proposed as a mechanism that participates in cell survival and bone remodeling. Among different signaling pathways stimulated by the primary cilia, Hedgehog signaling has been associated with regulation of bone development. Parathyroid hormone (PTH)-related protein (PTHrP) signaling through PTH 1 receptor (PTH1R) also regulates bone cell survival and remodeling and modulates Hedgehog pathway during skeletal development. We hypothesize that PTH1R and primary cilia concomitantly regulate bone remodeling and cell survival and aim to describe the mechanisms that mediate these effects in osteocytes and osteoblasts. MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells were stimulated or not with PTHrP (1-37). siRNAs for the primary cilia protein IFT88, a primary cilia specific inhibitor [chloral hydrate] and a Hedgehog signaling inhibitor [Gli-1-Antagonist 61: GANT61] were used to inhibit primary cilia formation and primary cilia and Hedgehog signaling, respectively. Cell survival in serum-depleted conditions and gene expression of bone remodeling and formation markers including OPG, RANKL, Runx2 and osteocalcin were studied. Protein levels of Gli-1 (a transcription factor that mediates gene expression in Hedgehog pathway) were also evaluated in these cells. In MLO-Y4 cells, PTHrP increased OPG, osteocalcin and RANK-L without modifying the OPG/RANK-L ratio whereas in MC3T3-E1 cells PTHrP overexpressed OPG, Runx2 and osteocalcin increasing the OPG/RANK-L ratio without modifying RANK-L expression. In addition, PTHrP increased cell survival and Gli-1 protein levels in both cell lines. Primary cilia siRNAs and chloral hydrate reverted osteocalcin stimulation in osteocytes and osteoblasts but decreased the OPG/RANKL ratio only in osteoblasts. Preliminary findings show that the Hedgehog inhibitor GANT61 did not reproduce the aforementioned inhibitory actions. In contrast, primary cilia siRNAs, chloral hydrate and GANT61 similarly inhibited PTHrP-dependent pro-survival effects on MLO-Y4 osteocytes and MC3T3-E1 osteoblasts. PTHrP increased GLI-1 protein levels in MLO-Y4 and MC3T3-E1 cell and these effects decreased by pre-incubation with the primary cilia inhibitor chloral hydrate. We conclude that PTH1R promotes osteocyte and osteoblast cell survival via Hedgehog- and primary cilia-dependent mechanisms and overexpress bone formation genes via primary cilia-dependent and hedhehog-independent mechanisms.


3. Bone mineral density and 3D-DXA assessment in adult patients with a positive and negative genetical testing for hypophosphatasia compared with a healthy control group
Tornero C1, Coronado M2, Humbert L3, García S1, Lanchas C, Monachello D2, Mateo C4, Montero A4, Domínguez L2, Balsa A1, Aguado P1
1 Rheumatology Department. La Paz Hospital. Madrid; 2 Nuclear Medicine Department. La Paz Hospital. Madrid; 3 Galgo Medical. Spain; 4 Primary Health Care Center Fuencarral. Madrid (Spain)

Introduction: Scarce evidence exists on the assessment of bone mineral density (BMD) in adult hypophosphatasia (HPP). Recent studies suggest that dual-energy X-ray absorptiometry (DXA) could not appropriately predict their fracture risk and note the importance on further explore bone microstructure.
Objectives: To evaluate BMD at proximal femur (PF) assessed by DXA in patients with persistent low alkaline phosphatase levels (ALP) and HPP genetically confirmed (HPP TG +) compared with a group of subjects with the same biochemical abnormality and a negative HPP genetic test (HPP TG-) and a healthy control group (3:1). To assess the cortical and trabecular bone at the PF using DXA-based 3D modelling.
Methods: Fifty-two subjects with persistent low ALP levels –at least two values <35 IU/L and none >45 IU/L– and a genetic test for HPP performed and 78 healthy subjects matched by age, gender and body mass index were included. Individuals with hypophosphatasaemia were distributed into 2 groups according to their genetic status and the other group was formed by the healthy controls. Secondary causes of hypophosphatasaemia were excluded by reviewing patients’ medical histories. BMD was measured using GE-LUNAR iDX and the 3D-SHAPER software (Galgo Medical, Barcelona) was also employed.
Results: Demographic, densitometric, and 3D-DXA parameters are shown in Table. Of the 52 subjects with low ALP levels included, half of them had pathogenic heterocygous mutations in ALPL. When subjects with low ALPL levels were compared, BMD at femoral neck was significantly lower in the HPP + GT group compared to the HPP- GT group (0.886±0.11 vs. 0.972±0.12 (p<0.05), and no differences were observed in the other regions. The 3D-SHAPER evaluation showed that integral and cortical vBMD at the neck was also lower in the HPP + GT (p<0.05) and a trend was observed in trabecular vBMD at this region (p=0.055). Statistical significance was also observed in the total cortical vBMD (829.35±95 vs. 867±84, p<0.05). On the other hand, in the HPP + GT group, there were no differences in DXA and 3D-SHAPER measurements when compared with the healthy control group. Additionally, BMD at the neck was higher in the HPP – GT group (0.972±0.11) in comparison with the healthy controls (0.914±0.1, p<0.05) and, in the 3D-SHAPER assessment, higher levels of vBMD at the neck were also observed, in contrast to parameters of total vBMD, that showed no differences.
Conclusion: The HPP + GT group did not show differences in DXA and 3D-SHAPER parameters when compared with the control group, probably reflecting mild or moderate forms of adult HPP. Besides that, differences in BMD and vBMD at the neck in the HPP – GT group suggest that an effort to explore possible genetic mechanisms regulating the alkaline phosphatase enzyme different to the ALPL gene is mandatory in these subjects.


4. The missense mutations p.Met16Leu, p.Ala41Thr, p.Tyr74Phe and p.Arg120Leu in DKK1 affect its inhibitory capacity
Martínez Gil N1, Roca Ayats N1, García Giralt N2, Van Hul W, Nogués X2, Mellibovsky L2, Díez Pérez A2, Grinberg D1, Balcells S1
1 Departamento Genética, Microbiología i Estadistica. Universitat de Barcelona. IBUB. CIBERER. IRSJD. Barcelona; 2 URFOA. IMIM. Parc de Salut Mar. CIBERFES. Barcelona

DKK1 encodes a protein of the same name, which acts in the extracellular space as an inhibitor of the Wnt signaling pathway. Numerous studies have associated this pathway with bone formation and risk of fracture. Our group has identified two missense variants in the DKK1 gene in two women with high bone mass (HBM) phenotype. These two variants are p.Tyr74Phe and p.Arg120Leu. The first cosegregates with HBM phenotype in a family (Sarrión et al., PLoS One 2014 Apr 15;9(4):e94607) and the second was found in an unrelated case of HBM (Martinez-Gil et al., Sci Rep 2018 19;8(1):10951). On the other hand, in the general population there are additional DKK1 missense variants with unknown effects in terms of bone mass, as can be found in the ExAC database. In fact, in ExAC, p.Arg120Leu appears as the most frequent amino acid change of DKK1 [MAF (minor allele frequency)=0.003]. Other relatively frequent missense variants (MAF between 0.0024 and 0.00012) are p.Ala106Thr, p.Met16Leu, p.Ser157Ile, p.Pro84Leu and p.Ala41Thr. Our hypothesis is that variants found in individuals with HBM could cause a partial loss of function of the DKK1 protein, leading to a greater activity of the canonical Wnt pathway, due to lack of inhibition. They would, therefore, contribute to the HBM phenotype and not mere accidental findings. Similarly, some of the variants found in the general population could also affect the functionality of the protein and contribute to the population variability of bone mineral density. To test the functionality of DKK1 proteins with these missense mutations, we performed reporter gene (luciferase) and Western blot assays. The former has been designed to measure the activity of the Wnt pathway in the presence or absence of wild-type or mutated DKK1 protein. Our results show that there are significant differences in the inhibitor capacity of DKK1 with the mutations p.Met16Leu, p.Ala41Thr, p.Tyr74Phe and p.Arg120Leu. Additionally, Western blot assays show that the amount of protein in the extracellular space is the same in all cases suggesting, that the loss of activity is not determined by a lack of protein but by a loss of intrinsic activity. In conclusion, these studies show that missense variants in the general population may produce an increase in BMD.


5. A sensitive method to monitor the migration of human bone marrow mesenchymal stem cells in mice models
Real A del, López Delgado L, Sañudo C, Pérez Nuñez MI, Laguna E, Menéndez G, Alfonso A, González Montesinos B, Ferreño D, Sainz Aja J, Valero C, Santurtun A, Riancho JA
Servicios de Medicina Interna y Traumatología. Hospital UM Valdecilla. Universidad de Cantabria. IDIVAL. Santander

It is critical to monitor the distribution and homing of the infused cells into the host tissues of animal models by sensitive, specific and efficient procedures. The aim of this study was to determine the performance of PCR-based detection of human Alu sequences to detect human DNA after the infusion of human bone marrow stem cells (hBMSCs) into immunodeficient mice.
hBMSCs were obtained from the femoral head of patients undergoing hip replacement surgery. After grown in culture,106 hBMSCs were infused intravenously by injection in the retro-orbital plexus of NOD/SCID mice. Several mice groups were used, including mice with mid-diaphysis femoral fractures with an intramedullary needle. After tissue extraction by standard procedures, the presence of human DNA was evaluated in lung, liver and bone by using the primers and protocol proposed by Funakoshi (Sci Rep. 2017;7(1):13202). Besides no template controls (NTC), we included control experiments with DNA extracted from tissues of mice without hBMSCs, as well as artificial mixtures of human and mouse cells.
In several independent experiments, the threshold cycle (Ct) for NTC was 34.6±1.8. No signal was detected when up to 100 ng of mouse DNA was tested (Ct was 34.7±1.6). However, human DNA was readily detected, down to 0.1 pg (Ct=31.4), with a good log-linear relationship (Ct was 11.5, 14.6, 17.2, 21.0, 23.8, 26.8, 30, and 32.5; for 100 ng, 10 ng, 1ng, 0.1 ng, 0.01 ng, 1 pg, 0.1 pg, and 0.01 pg of human DNA, respectively; r2=0.992; p<0.0001). Similarly, when we mixed human and mouse osteoblasts, we easily detected 10 or even 1 human cells within 105 mouse cells (Ct 25.6 and 30.8, respectively).
Then, we analyzed tissues from non-fractured NOD/SCID mice treated with hBMSCs intravenously. We readily detected human DNA in the lungs 1 and 7 days after cell infusions (Ct 22.6±0.7 and 30.6±3.7, respectively). However, human DNA was inconsistently detected in liver
and bone. In fractured mice models, tissues were analyzed 4 weeks after fracture. Very low level of human DNA was detected in 20% fractured femurs and a 33% of lung samples.
In conclusion, the Alu-quantification method used in this work able to detect down to 1-10 human cells among 100,000 mouse cells. Our results confirm that most hBMSCs injected intravenously into NOD/SCID mice are trapped into the lungs. Therefore, procedures to increase cell homing into bone are needed if hBMSCs are to be used in skeletal regenerative procedures.