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Biomedical subjects

Paolo Bianco

Publications and source records attributed to Paolo Bianco.

At least 19 recordsLinked to original sources
PubMed2006

Postnatal skeletal stem cells.

Postnatal skeletal stem cells are a subpopulation of the bone marrow stromal cell network. To date, the most straightforward way of assessing the activity of skeletal stem cells within the bone marrow stromal cell (BMSC) population is via analysis of the rapidly adherent, colony-forming unit-fibroblast (CFU-F), and their progeny, BMSCs. Several in vitro methods are employed to determine the differentiation capacity of BMSCs, using osteogenic and adipogenic "cocktails" and staining protocols, and pellet cell culture for chondrogenic differentiation. However, true differentiation potential is best determined by in vivo transplantation in either closed or open systems. By in vivo transplantation, approximately 10% of the clonal strains are able to form bone, stroma, and marrow adipocytes, and are true skeletal stem cells. Furthermore, when derived from patients or animal models with abnormalities in gene expression, they recapitulate the disease phenotype on in vivo transplantation. Although ex vivo expansion of BMSCs inevitably dilutes the skeletal stem cells, when used en masse, they are attractive candidates for reconstruction of segmental bone defects, and as targets for gene therapy.

Adult Stem Cells↗
PubMed2006

Reconstruction of extensive long bone defects in sheep using resorbable bioceramics based on silicon stabilized tricalcium phosphate.

In this study we evaluated the performance of Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (Si-TCP), in promoting the repair of a large-sized, experimentally induced defect in a weight-bearing long bone sheep model. Eighteen 2-year-old ewes were used in this study. Animals were sacrificed at 3, 6, and 12 months. One animal entered a very prolonged followup and was sacrificed 2 years after surgery. Bone formation and scaffold resorption were evaluated by sequential x-ray studies, CT scans, histology, immunohistology, microradiography, and quantitative analysis of x-ray studies (optical density) and microradiographs (percentage of bone and scaffold area). Our data show an excellent implant integration and significant bone regeneration within the bone substitute over the course of the experiment. Progressive osteoclastic resorption of the biomaterial was also evident. At 1 year from surgery, the remaining scaffold was approximately 10-20% of the scaffold initially implanted, while after 2 years it was essentially completely resorbed. At the end of the observation period, the segmental defect was filled with newly formed, highly mineralized, lamellar bone.

Absorbable Implants↗
PubMed2006

Monostotic fibrous dysplasia of the proximal femur and liposclerosing myxofibrous tumor: which one is which?

Clinical, histological, and genetic studies of two cases of isolated fibro-osseous lesions of the femur in adults show the overlap between monostotic fibrous dysplasia (MFD) of the proximal femur and the so-called liposclerosing myxofibrous tumor. The two cases highlight how the incomplete understanding of the natural history of MFD may result in diagnostic pitfalls or incorrect classification of individual lesions.

Adult↗
PubMed2006

Fibrous dysplasia as a stem cell disease.

At a time when significant attention is devoted worldwide to stem cells as a potential tool for curing incurable diseases, fibrous dysplasia of bone (FD) provides a paradigm for stem cell diseases. Consideration of the time and mechanism of the causative mutations and of nature of the pluripotent cells that mutate in early embryonic development indicates that, as a disease of the entire organism, FD can be seen as a disease of pluripotent embryonic cells. As a disease of bone as an organ, in turn, FD can be seen as a disease of postnatal skeletal stem cells, which give rise to dysfunctional osteoblasts. Recognizing FD as a stem cell disease provides a novel conceptual angle and a way to generate appropriate models of the disease, which will continue to provide further insight into its natural history and pathogenesis. In addition, skeletal stem cells may represent a tool for innovative treatments. These can be conceived as directed to alter the in vivo behavior of mutated stem cells, to replace mutated cells through local transplantation, or to correct the genetic defect in the stem cells themselves. In vitro and in vivo models are currently being generated that will permit exploration of these avenues in depth.

Bone and Bones↗
PubMed2006

The use of adult stem cells in rebuilding the human face.

BACKGROUND: Stem cells have been isolated from a variety of embryonic and postnatal (adult) tissues, including bone marrow. Bone marrow stromal cells (BMSCs), which are non-blood-forming cells in marrow, contain a subset of skeletal stem cells (SSCs) that are able to regenerate all types of skeletal tissue: bone, cartilage, blood-supportive stromal cells and marrow fat cells. METHODS: Bone marrow suspensions are placed into culture for analysis of their biological character and for expansion of their number. The resulting populations of cells are used in a variety of assays to establish the existence of an adult SSC, and the ability of BMSC populations to regenerate hard tissues in the craniofacial region, in conjunction with appropriate scaffolds. RESULTS: Single-cell analysis established the existence of a true adult SSC in bone marrow. Populations of ex vivo expanded BMSCs (a subset of which are SSCs) are able to regenerate a bone/marrow organ. In conjunction with appropriate scaffolds, these cells can be used to regenerate bone in a variety of applications. CONCLUSIONS: BMSCs have the potential to re-create tissues of the craniofacial region to restore normal structure and function in reconstructing the hard tissues of a face. Ex vivo expanded BMSCs with scaffolds have been used in a limited number of patients to date, but likely will be used more extensively in the near future.

Adult↗
PubMed2004

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow.

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.

Animals↗
PubMed2004

The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Animals↗
PubMed2004

An instrument to measure skeletal burden and predict functional outcome in fibrous dysplasia of bone.

UNLABELLED: An instrument to measure skeletal burden in fibrous dysplasia was developed. Biological and clinical relevance was shown by correlating skeletal burden scores with bone markers, quality of life, and ambulatory status. Childhood scores predict adult ambulatory status, and scores were unaffected when bone markers decreased with bisphosphonate treatment or aging. INTRODUCTION: Fibrous dysplasia (FD) is a skeletal disease with a broad clinical expression. There is no objective method to assess the extent of skeletal involvement or predict outcome. We developed an instrument to measure skeletal burden that correlates with physical function, health-related quality of life (HRQL), and ambulatory status. MATERIALS AND METHODS: Seventy-nine patients with FD underwent bone scintigraphy. The skeletal burden score was derived from a weighted score based on the regional measurement using bone scintigraphy to estimate the amount of FD in anatomical segments. Six readers scored 20 scans twice to determine the inter- and intrareader agreement. To assess biological significance, scores were correlated with bone markers. To assess functional outcome, scores on the SF-36 (adults) or CHQ-PF50 (children) were correlated with skeletal burden scores. In a group of patients who had bone scans as children and adults (n = 6), the ability to predict ambulatory status was tested. Skeletal burden scores were assessed in patients before and after treatment with pamidronate (n = 5). RESULTS: The inter- and intrareader agreement of burden scores were r = 0.96, and 0.98, respectively (p < 0.001 for both). The scores correlated with markers of bone metabolism and HRQL (Spearman rho, 0.54-0.67 p < 0.001 and -0.43, p = 0.001, respectively). The mean score of patients who ambulated unassisted was significantly lower than those requiring assistance (p < 0.001 unassisted versus crutch and/or wheelchair). In unassisted ambulators, younger patients had higher scores, suggesting high childhood scores may predict adulthood impairment. In six patients with childhood and adulthood scans, childhood scores >30 predicted assisted ambulation in adulthood. There was a negative correlation between bone markers and age (Spearman rho, -0.42 to -0.70; p < 0.001), but not age and skeletal burden score. Pamidronate treatment decreased serum alkaline phosphatase but had no effect on the skeletal burden score. CONCLUSIONS: This is a validated and reliable instrument for the measurement of skeletal burden of FD and is able to predict functional outcome.

Adolescent↗
PubMed2004

TGFbeta/BMP activate the smooth muscle/bone differentiation programs in mesoangioblasts.

Mesoangioblasts are vessel-derived stem cells that can be induced to differentiate into different cell types of the mesoderm such as muscle and bone. The gene expression profile of four clonal derived lines of mesoangioblasts was determined by DNA micro-array analysis: it was similar in the four lines but different from 10T1/2 embryonic fibroblasts, used as comparison. Many known genes expressed by mesoangioblasts belong to response pathways to developmental signalling molecules, such as Wnt or TGFbeta/BMP. Interestingly, mesoangioblasts express receptors of the TGFbeta/BMP family and several Smads and, accordingly, differentiate very efficiently into smooth muscle cells in response to TGFbeta and into osteoblasts in response to BMP. In addition, insulin signalling promotes adipogenic differentiation, possibly through the activation of IGF-R. Several Wnts and Frizzled, Dishevelled and Tcfs are expressed, suggesting the existence of an autocrine loop for proliferation and indeed, forced expression of Frzb-1 inhibits cell division. Mesoangioblasts also express many neuro-ectodermal genes and yet undergo only abortive neurogenesis, even after forced expression of neurogenin 1 or 2, MASH or NeuroD. Finally, mesoangioblasts express several pro-inflammatory genes, cytokines and cytokine receptors, which may explain their ability to be recruited by tissue inflammation. Our data define a unique phenotype for mesoangioblasts, explain several of their biological features and set the basis for future functional studies on the role of these cells in tissue histogenesis and repair.

Animals↗
PubMed2004

A novel technique based on a PNA hybridization probe and FRET principle for quantification of mutant genotype in fibrous dysplasia/McCune-Albright syndrome.

Somatic mutations are present in various proportions in numerous developmental pathologies. Somatic activating missense mutations of the GNAS gene encoding the Gs(alpha) protein have previously been shown to be the cause of fibrous dysplasia of bone (FD)/McCune-Albright syndrome (MAS). Because in MAS patients, tissues as diverse as melanocytes, gonads and bone are affected, it is generally accepted that the GNAS mutation in this disease must have occurred early in development. Interestingly, it has been shown that the development of an active FD lesion may require both normal and mutant cells. Studies of the somatic mosaic states of FD/MAS and many other somatic diseases need an accurate method to determine the ratio of mutant to normal cells in a given tissue. A new method for quantification of the mutant:normal ratio of cells using a PNA hybridization probe-based FRET technique was developed. This novel technique, with a linear sensitivity of 2.5% mutant alleles, was used to detect the percentage mutant cells in a number of tissue and cell culture samples derived from FD/MAS lesions and could easily be adapted for the quantification of mutations in a large spectrum of diseases including cancer.

Alleles↗
PubMed2004

MT1-MMP: a tethered collagenase.

Gene ablation in mice offers a powerful tool to assay in vivo the role of selected molecules. Numerous new mouse models of matrix metalloproteinases (MMP) deficiency have been developed in the past 5 years and have yielded a new understanding of the role of MMPs while also putting to rest assumptions based on data predating the days of mouse models. The phenotype of the MT1-MMP deficient mouse is one example which illustrates the sometimes rather surprising insights into extracellular matrix remodeling in development and growth that can be gained with mouse genetics. While MT1-MMP appears to play little or no role in embryonic development, loss of this enzyme results in progressive impairment of postnatal growth and development affecting both the skeleton and the soft connective tissues. The underlying pathologic mechanism is loss of an indispensable collagenolytic activity, which remains essentially uncompensated. Our findings demonstrate that growth and maintenance of the skeleton requires coordinated and simultaneous MT1-MMP-dependent remodeling of all soft tissue attachments (ligaments, tendons, joint capsules). We note that the phenotype of the MT1-MMP deficient mouse bears no resemblance to those of mice deficient in MMP-2 and tissue inhibitors of metallo-proteinase (TIMP)-2 all but dispelling the view that activation of MMP-2 by the MT1-MMP/TIMP-2/proMMP-2 axis plays a significant role in growth and development throughout life. It is of interest to note that loss of a single catabolic function such as selective collagen degradation mediated by MT1-MMP gives rise to profound impairment of a number of both anabolic and catabolic functions.

Animals↗
PubMed2003

Fracture incidence in polyostotic fibrous dysplasia and the McCune-Albright syndrome.

UNLABELLED: In patients with polyostotic fibrous dysplasia of bone, the peak incidence of fractures is during the first decade of life, followed by a decrease thereafter. Phosphaturia is associated with an earlier incidence and increased frequency of fractures. INTRODUCTION: Fibrous dysplasia (FD) is a disorder involving either one (monostotic) or several bones (polyostotic FD [PFD] and sometimes is associated with cafe-au-lait hyperpigmentation of the skin and one or more hyperfunctioning endocrinopathies (McCune-Albright syndrome [MAS]). Both PFD and MAS are often associated with phosphaturia. Although fractures occur frequently in PFD/MAS, fracture incidence and the effect of age and co-existing metabolic abnormalities (endocrinopathy and/or phosphaturia) on fractures are ill defined. MATERIALS AND METHODS: We reviewed the medical records and examined the endocrine and phosphorus metabolism of 35 patients with PFD/MAS. We report on the age at which extremity fractures occurred and their location and treatment. The results of endocrine and phosphorus metabolism testing and associations between age of first fractures, number of fractures, fracture rate, and metabolic abnormalities were noted. RESULTS: The average follow-up was 14.2 years (range, 2-39 years), during which 172 fractures occurred. The number and sites of fractures were 103 femoral, 25 tibial, 33 humeral, and 11 forearm. Twenty-seven patients had PFD with one or more endocrinopathies and/or phosphaturia, and eight had PFD alone. The endocrinopathies included precocious puberty (n = 19), hyperthyroidism (n = 9), growth hormone excess (n = 6), and one patient each with Cushing syndrome and primary hyperparathyroidism. Twelve patients had phosphaturia. The peak rate of fractures occurred between 6 and 10 years of age and decreased thereafter. Patients with metabolic abnormalities sustained their first fracture at an earlier age (6.9 versus 16.6 years, p < 0.005) and had a higher lifetime rate of fractures (0.29 versus 0.08 fractures/year), relative to patients with PFD alone. Phosphaturia was the single metabolic dysfunction associated with both an earlier age of first fracture (5.1 versus 16.6 years, p < 0.05) and a greater lifetime fracture rate (0.35 versus 0.08 fractures/year, p < 0.05). CONCLUSIONS: The occurrence of extremity fractures in FD peaks between 6 and 10 years of age and declines thereafter. Fractures occur earlier and more frequently in the presence of phosphaturia. These data have implications for long-term prognosis, clinical management, and interpretation of therapeutic interventions.

Adolescent↗
PubMed2003

MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth.

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)-dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of "ghost" cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.

Animals↗
PubMed2003

The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility.

Using the Cre/loxP system we conditionally inactivated beta-catenin in endothelial cells. We found that early phases of vasculogenesis and angiogenesis were not affected in mutant embryos; however, vascular patterning in the head, vitelline, umbilical vessels, and the placenta was altered. In addition, in many regions, the vascular lumen was irregular with the formation of lacunae at bifurcations, vessels were frequently hemorrhagic, and fluid extravasation in the pericardial cavity was observed. Cultured beta-catenin -/- endothelial cells showed a different organization of intercellular junctions with a decrease in alpha-catenin in favor of desmoplakin and marked changes in actin cytoskeleton. These changes paralleled a decrease in cell-cell adhesion strength and an increase in paracellular permeability. We conclude that in vivo, the absence of beta-catenin significantly reduces the capacity of endothelial cells to maintain intercellular contacts. This may become more marked when the vessels are exposed to high or turbulent flow, such as at bifurcations or in the beating heart, leading to fluid leakage or hemorrhages.

Actins↗
PubMed2003

Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation.

We have generated transgenic mice harboring the deletion of exon 48 in the mouse alpha1(II) procollagen gene (Col2a1). This was the first dominant negative mutation identified in the human alpha1(II) procollagen gene (COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.

Animals↗
PubMed2003

Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells.

Bone-marrow stromal cells can differentiate into multiple mesenchymal lineages including cartilage and bone. When these cells are seeded in high-density 'pellet culture', they undergo chondrogenesis and form a tissue that is morphologically and biochemically defined as cartilage. Here, we show that dual chondro-osteogenic differentiation can be obtained in the same micromass culture of human bone-marrow stromal cells. Human bone-marrow stromal cells were pellet cultured for 4 weeks in chondro-inductive medium. Cartilage 'beads' resulting from the micromass culture were then subcultured for further 1-3 weeks in osteo-inductive medium. This resulted in the formation of a distinct mineralized bony collar around hyaline cartilage. During the chondrogenesis phase, type I collagen and bone sialoprotein were produced in the outer portion of the cartilage bead, which, upon subsequent exposure to beta-glycerophosphate, mineralized and accumulated extracellular bone sialoprotein and osteocalcin. Our modification of the pellet culture system results in the formation of a chondro-osseous 'organoid' structurally reminiscent of pre-invasion endochondral rudiments, in which a bony collar forms around hyaline cartilage. The transition from a cell culture to an organ culture dimension featured by our system provides a suitable model for the dissection of molecular determinants of endochondral bone formation, which unfolds in a precisely defined spatial and temporal frame

Adolescent↗
PubMed2003

Mesoangioblasts--vascular progenitors for extravascular mesodermal tissues.

Mesoangioblasts are multipotent progenitors of mesodermal tissues that express the key marker of angiopoietic progenitors, Flk1 (VEGF-receptor 2), and are physically associated with the embryonic dorsal aorta in avian and mammalian species. When transplanted in vivo, they give rise to multiple differentiated mesodermal phenotypes. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Mesoangioblasts disclose not only an unexpected source of progenitors for skeletal muscle and a variety of other mesoderm-derived tissues, but also establish a lineage kinship between progenitors of vascular and extravascular mesodermal tissues, with important basic and applicative implications.

Animals↗