Isabella Villa

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [alpha1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases.

Effects of calcitonin gene-related peptide and amylin on human osteoblast-like cells proliferation.

Expression of mRNA for calcitonin gene-related peptide (CGRP) and CGRP receptor has been detected in osteoblasts indicating that CGRP could play a role in bone metabolism. In the present study, we evaluated the effect of CGRP on primary culture of human osteoblast-like cells proliferation. The peptide was able to stimulate [3H]thymidine incorporation in human osteoblast-like cells with a maximal effect at 10(-8) M. The proliferating activity of CGRP was not inhibited by the two antagonists, CGRP-(8-37) or amylin-(8-37), whereas amylin fragment antagonized the proliferating activity of amylin. In human osteoblast-like cells CGRP, but not amylin, was able to stimulate adenylyl cyclase activity and this effect was completely antagonized only by CGRP-(8-37) and not by amylin-(8-37). These data suggest that the CGRP induced stimulation of cAMP is not involved in the peptide proliferating effect in human osteoblast-like cells and that in this cell population there are receptor subtypes for CGRP, distinct from that of amylin.

In vivo leptin expression in cartilage and bone cells of growing rats and adult humans

The present investigation was carried out to analyse, immunohistochemically, in vivo leptin expression in cartilage and bone cells, the latter restricted to the elements of the osteogenic system (stromal cells, osteoblasts, osteocytes, bone lining cells). Observations were performed on the first lumbar vertebra, tibia and femur of four rats and on the humerus, femur and acromion of four patients. Histological sections of paraffin‐embedded bone samples were immunostained using antibody to leptin. The results showed that, in growing rat bone, leptin is expressed in chondrocytes and stromal cells, but not in osteoblasts; bone lining cells were not found in the microscopic fields examined. In adult human bone, leptin is expressed in chondrocytes, stromal cells and bone lining cells; osteoblasts were not found in the microscopic fields examined. Osteocytes were found to be leptin positive only occasionally and focally in both rat and human bone. The in vivo findings reported show, for the first time, that leptin appears to be expressed only in the cells of the osteogenic lineage (stromal cells, bone lining cells, osteocytes) that, with respect to osteoblasts, are permanent and inactive, i.e. in those cells that according to our terminology constitute the bone basic cellular system (BBCS). Because the BBCS seems to be primarily involved in sensing and integrating mechanical strains and biochemical factors and then in triggering and driving bone formation and/or bone resorption, it appears that leptin seems to be mainly involved in modulating the initial phases of bone modelling and remodelling processes.

Impaired osteoblastogenesis in a murine model of dominant osteogenesis imperfecta: A new target for osteogenesis imperfecta pharmacological therapy

The molecular basis underlying the clinical phenotype in bone diseases is customarily associated with abnormal extracellular matrix structure and/or properties. More recently, cellular malfunction has been identified as a concomitant causative factor and increased attention has focused on stem cells differentiation. Classic osteogenesis imperfecta (OI) is a prototype for heritable bone dysplasias: it has dominant genetic transmission and is caused by mutations in the genes coding for collagen I, the most abundant protein in bone. Using the Brtl mouse, a well‐characterized knockin model for moderately severe dominant OI, we demonstrated an impairment in the differentiation of bone marrow progenitor cells toward osteoblasts. In mutant mesenchymal stem cells (MSCs), the expression of early (Runx2 and Sp7) and late (Col1a1 and Ibsp) osteoblastic markers was significantly reduced with respect to wild type (WT). Conversely, mutant MSCs generated more colony‐forming unit‐adipocytes compared to WT, with more adipocytes per colony, and increased number and size of triglyceride drops per cell. Autophagy upregulation was also demonstrated in mutant adult MSCs differentiating toward osteogenic lineage as consequence of endoplasmic reticulum stress due to mutant collagen retention. Treatment of the Brtl mice with the proteasome inhibitor Bortezomib ameliorated both osteoblast differentiation in vitro and bone properties in vivo as demonstrated by colony‐forming unit‐osteoblasts assay and peripheral quantitative computed tomography analysis on long bones, respectively. This is the first report of impaired MSC differentiation to osteoblasts in OI, and it identifies a new potential target for the pharmacological treatment of the disorder. STEM CELLS2012;30:1465–1476

Calcitonin gene-related peptide (CGRP) inhibits apoptosis in human osteoblasts by β-catenin stabilization.

Transgenic mice over‐expressing calcitonin gene‐related peptide (CGRP) in osteoblasts have increased bone density due to increased bone formation, thus suggesting that CGRP plays a role in bone metabolism. In this study we determined the relationship between CGRP, the canonical Wnt signaling and apoptosis in human osteoblasts (hOBs) in consideration of the well‐documented involvement of this pathway in bone cells. Primary cultures of hOBs were treated with CGRP 10−8 M. Levels of β‐catenin, which is the cytoplasmic protein mediator of canonical Wnt signaling, and mRNA were determined. CGRP increases both the expression and the levels of cytoplasmic β‐catenin by binding to its receptor, as this effect is blocked by the antagonist CGRP8–37. This facilitatory action on β‐catenin appears to be mediated by the inhibition of the enzyme GSK‐3β via protein kinase A (PKA) activation. GSK‐3β is a glycogen synthase kinase that, by phosphorylating β‐catenin, promotes its degradation by the proteosomal machinery. Moreover, the peptide is able to inhibit hOBs apoptosis stimulated by dexamethasone or by serum deprivation, possibly through the accumulation of β‐catenin, since the inhibitor of PKA activity H89 partially prevents the antiapoptotic effect of the peptide. In conclusion CGRP, released by nerve fibers, exerts its anabolic action on bone cells by stimulating canonical Wnt signaling and by inhibiting hOBs apoptosis, thus favoring local bone regeneration. J. Cell. Physiol. 225: 701–708, 2010.

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

Effects of amylin on human osteoblast-like cells

Amylin has been reported to have bone-conserving effects. In the present study we evaluated the possible activity of the peptide on human osteoblast-like (hOB) cells in primary culture. Amylin between 10(-9) and 10(-6) M, dose-dependently stimulated cell proliferation with a maximal effect (200%) at 10(-6) M. In addition, amylin increased osteocalcin production when hOB cells were exposed to 1,25(OH)2D3 (10(-8)M) but there was a nonsignificant upward trend on alkaline phosphatase activity. The present results suggest that amylin could be included among the group of peptides endowed with osteogenic activity.

Osteocyte-Bone Lining Cell System at the Origin of Steady Ionic Current in Damaged Amphibian Bone

Abstract. A wound-generated steady electric current was measured by a two-dimensional vibrating probe system in the metatarsal bones of 22 adult frogs (Xenopus laevis) placed in amphibian Ringer. Inward currents were recorded entering a micrometric hole drilled through the cortex at middiaphyseal level. These steady state currents (mean ± SD 8.50 ± 2.77 μA/cm2) last approximately 2 hours, were dependent on the presence of sodium in the incubation medium, were no more detectable after fixation, and were reduced to background level when the cell membranes were solubilized. These results agree with previous recordings of metatarsal bones of weanling mice, under identical conditions. Both results suggest that the measured ionic currents have a cellular origin. Metatarsal bones of adult amphibian were purposely selected for this study because, unlike mammalian bones, their shafts are avascular and only contain an osteocyte-bone lining cell system, as documented by scanning and transmission electron observations. Thus, unlike the data from previous investigations on mammals, the results succeeded in giving the first convincing evidence that the osteocyte-bone lining cell system is the origin of damage-generated ionic currents. As damage exposes bone ionic compartment to plasma, damage-generated ionic currents are representative of ion fluxes at bone plasma interface, and cells at the origin of the current generate the driving force of such fluxes. By demonstrating that osteocytes and bone lining cells are at the origin of the current, this study suggests that the osteocyte-bone lining cell system, though operating as a cellular membrane partition, regulates ionic flow between bone and plasma. Since strain-related adaptive remodeling could also depend on ionic characteristics and flow of the bone fluid through the osteocyte lacuno-canalicular network, the results reported here support the view that osteocyte and bone lining cells may constitute a functional syncytium involved in mineral homeostasis as well as in bone adaptation to mechanical loading.

Microarray analysis of 1,25(OH)2D3 regulated gene expression in human primary osteoblasts

Though extensive studies have been conducted, questions regarding the molecular effectors and pathways underlying the regulatory role of 1,25(OH)2D3 in human osteoblasts other than cell differentiation and matrix protein production remain unanswered. This study aims to identify genes and pathways that are modulated by 1,25(OH)2D3 treatment in human osteoblasts. Primary osteoblast cultures obtained from human bone tissue samples were treated with 1,25(OH)2D3 (10−7 M) for 24 h and their transcritptomes were profiled by microarray analysis using the Affymetrix GeneChip®. Statistical analysis was conducted to identify genes whose expression is significantly modulated following 1,25(OH)2D3 treatment. One hundred and fifty‐eight genes were found to be differentially expressed. Of these, 136 were upregulated, indicating clear transcriptional activation by 1,25(OH)2D3. Biostatistical evaluation of microarray data by Ingenuity Pathways Analysis (IPA) revealed a relevant modulation of genes involved in vitamin D metabolism (CYP24), immune functions (CD14), neurotransmitter transporters (SLC1A1, SLC22A3), and coagulation [thrombomodulin (THBD), tissue plasminogen activator (PLAT), endothelial protein C receptor (PROCR), thrombin receptor (F2R)]. We identified a restricted number of highly regulated genes and confirmed their differential expression by real‐time quantitative PCR (RT qPCR). The present genome‐wide microarray analysis on 1,25(OH)2D3‐treated human osteoblasts reveals an interplay of critical regulatory and metabolic pathways and supports the hypothesis that 1,25(OH)2D3 can modulate the coagulation process through osteoblasts, activates osteoclastogenesis through inflammation signaling, modulates the effects of monoamines by affecting their reuptake. J. Cell. Biochem. 113: 640–649, 2012.

Different skeletal regional response to continuous brain infusion of leptin in the rat

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.