Paolo Bianco

Targeted disruption of the biglycan gene leads to an osteoporosis-like phenotype in mice

The resilience and strength of bone is due to the orderly mineralization of a specialized extracellular matrix (ECM) composed of type I collagen (90%) and a host of non-collagenous proteins that are, in general, also found in other tissues. Biglycan (encoded by the gene Bgn) is an ECM proteoglycan that is enriched in bone and other non-skeletal connective tissues. In vitro studies indicate that Bgn may function in connective tissue metabolism by binding to collagen fibrils and TGF-ß (Refs 5,6), and may promote neuronal survival. To study the role of Bgn in vivo, we generated Bgn-deficient mice. Although apparently normal at birth, these mice display a phenotype characterized by a reduced growth rate and decreased bone mass due to the absence of Bgn. To our knowledge, this is the first report in which deficiency of a non-collagenous ECM protein leads to a skeletal phenotype that is marked by low bone mass that becomes more obvious with age. These mice may serve as an animal model to study the role of ECM proteins in osteoporosis.

FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting

FGF-23, a novel member of the FGF family, is the product of the gene mutated in autosomal dominant hypophosphatemic rickets (ADHR). FGF-23 has been proposed as a circulating factor causing renal phosphate wasting not only in ADHR (as a result of inadequate degradation), but also in tumor-induced osteomalacia (as a result of excess synthesis by tumor cells). Renal phosphate wasting occurs in approximately 50% of patients with McCune-Albright syndrome (MAS) and fibrous dysplasia of bone (FD), which result from postzygotic mutations of the GNAS1 gene. We found that FGF-23 is produced by normal and FD osteoprogenitors and bone-forming cells in vivo and in vitro. In situ hybridization analysis of FGF-23 mRNA expression identified fibrous cells, osteogenic cells, and cells associated with microvascular walls as specific cellular sources of FGF-23 in FD. Serum levels of FGF-23 were increased in FD/MAS patients compared with normal age-matched controls and significantly higher in FD/MAS patients with renal phosphate wasting compared with those without, and correlated with disease burden bone turnover markers commonly used to assess disease activity. Production of FGF-23 by FD tissue may play an important role in the renal phosphate-wasting syndrome associated with FD/MAS.

Expression of bone sialoprotein (BSP) in developing human tissues

SummaryBone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry andin situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains byin situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone.

Phenotypic Effects of Biglycan Deficiency Are Linked to Collagen Fibril Abnormalities, Are Synergized by Decorin Deficiency, and Mimic Ehlers-Danlos-Like Changes in Bone and Other Connective Tissues†

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine‐rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a “subclinical” cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn‐ and dcn‐deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double‐knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double‐KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double‐KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked “serrated fibril” morphology. The phenotype of the double‐KO animal mimics directly the rare progeroid variant of human Ehlers‐Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and dcn deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

Osteoconduction in large macroporous hydroxyapatite ceramic implants: evidence for a complementary integration and disintegration mechanism

Large, cylindrical implants of a porous calcium phosphate ceramic (hydroxyapatite starting material, HAC) were used to replace far greater than critical-sized sections of the midshaft of sheep tibiae and retrieved at 2 and 9 months; external fixation was used in the first 5 months. Excellent clinical function of these implants was reported in a previous study. The material retrieved was embedded in PMMA, and blocks were sectioned and surfaces were polished and carbon coated prior to study using digital backscattered electron (BSE) imaging. Detailed scanning electron microscopy study of the pattern of osseointegration of the implanted material at early (2 months) and late (9 months) timepoints revealed a previously unrecognized pattern of integration/disintegration of this implant material in tandem with bone growth. We conclude that bone adaptation to the HAC leads to its fracture and that the newly generated surfaces are equally osteoconductive. This leads to a self-propagating, self-annealing system in which defects in the HAC are mended by intercalation of bone.

Fracture Incidence in Polyostotic Fibrous Dysplasia and the McCune‐Albright Syndrome

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.

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.

An Instrument to Measure Skeletal Burden and Predict Functional Outcome in Fibrous Dysplasia of Bone

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.

Osteoclastogenesis in fibrous dysplasia of bone: in situ and in vitro analysis of IL-6 expression

Fibrous dysplasia of bone (FD) is caused by somatic mutations of the GNAS1 gene, which lead to constitutive activation of adenylyl cyclase and overproduction of cAMP in osteogenic cells. Previous in vitro studies using nonclonal, heterogeneous strains of FD-derived cells suggested that IL-6 might play a critical role in promoting excess osteoclastogenesis in FD. In this study, we investigated IL-6 expression in FD in situ and its relationship to the actual patterns of osteoclastogenesis within the abnormal tissue. We found that osteoclastogenesis is not spatially restricted to bone surfaces in FD but occurs to a large extent ectopicly in the fibrous tissue, where stromal cells diffusely express IL-6 mRNA and exhibit a characteristic cell morphology. We also observed specific expression of IL-6 mRNA in a proportion of osteoclasts, suggesting that an autocrine/paracrine loop may contribute to osteoclastogenesis in vivo in FD, as in some other bone diseases, including Pagets disease. We also generated homogeneous, clonally derived strains of wild-type and GNAS1-mutated stromal cells from the same individual, parent FD lesions. In this way, we could show that mutated stromal cells produce IL-6 at a basal magnitude and rate that are significantly higher than in the cognate wild-type cells. Conversely, wild-type cells respond to db-cAMP with a severalfold increase in magnitude and rate of IL-6 production, whereas mutant strains remain essentially unresponsive. Our data establish a direct link between GNAS1 mutations in stromal cells and IL-6 production but also define the complexity of the role of IL-6 in regulating osteoclastogenesis in FD in vivo. Here, patterns of osteoclastogenesis and bone resorption reflect not only the cell-autonomous effects of GNAS1 mutations in osteogenic cells (including IL-6 production) but also the local and systemic context to which non-osteogenic cells, local proportions of wild-type vs mutated cells, and systemic hormones contribute.

Gnathodiaphyseal Dysplasia: A Syndrome of Fibro‐Osseous Lesions of Jawbones, Bone Fragility, and Long Bone Bowing

We report an unusual generalized skeletal syndrome characterized by fibro‐osseous lesions of the jawbones with a prominent psammomatoid body component, bone fragility, and bowing/sclerosis of tubular bones. The case fits with the emerging profile of a distinct syndrome with similarities to previously reported cases, some with an autosomal dominant inheritance and others sporadic. We suggest that the syndrome be named gnathodiaphyseal dysplasia. The patient had been diagnosed previously with polyostotic fibrous dysplasia (PFD) elsewhere, but further clinical evaluation, histopathological study, and mutation analysis excluded this diagnosis. In addition to providing a novel observation of an as yet poorly characterized syndrome, the case illustrates the need for stringent diagnostic criteria for FD. The jaw lesions showed fibro‐osseous features with the histopathological characteristics of cemento‐ossifying fibroma, psammomatoid variant. This case emphasizes that the boundaries between genuine GNAS1 mutation‐positive FD and other fibro‐osseous lesions occurring in the jawbones should be kept sharply defined, contrary to a prevailing tendency in the literature. A detailed pathological study revealed previously unreported features of cemento‐ossifying fibroma, including the participation of myofibroblasts and the occurrence of psammomatoid bodies and aberrant mineralization, within the walls of blood vessels. Transplantation of stromal cells grown from the lesion into immunocompromised mice resulted in a close mimicry of the native lesion, including the sporadic formation of psammomatoid bodies, suggesting an intrinsic abnormality of bone‐forming cells.