Alessandro Corsi

AUTOLOGOUS BONE MARROW STROMAL CELLS LOADED ONTO POROUS HYDROXYAPATITE CERAMIC ACCELERATE BONE REPAIR IN CRITICAL-SIZE DEFECTS OF SHEEP LONG BONES

The ability of marrow-derived osteoprogenitor cells to promote repair of critical-size tibial gaps upon autologous transplantation on a hydroxyapatite ceramic (HAC) carrier was tested in a sheep model. Conditions for in vitro expansion of sheep bone marrow stromal cells (BMSC) were established and the osteogenic potential of the expanded cells was validated. Ectopic implantation of sheep BMSC in immunocompromised mice led to extensive bone formation. When used to repair tibial gaps in sheep, cell-loaded implants (n = 2) conducted a far more extensive bone formation than did cell-free HAC cylinders (n = 2) over a 2-month period. In cell-loaded implants, bone formation was found to occur both within the internal macropore space and around the HAC cylinder while in control cell-free implants, bone formation was limited mostly to the outer surface and was not observed in most of the inner pores. As tested in an indentation assay, the stiffness of the complex HAC-bone material was found to be higher in cell-loaded implants compared to controls. Our pilot study on a limited number of large-sized animals suggests that the use of autologous BMSC in conjunction with HAC-based carriers results in faster bone repair compared to HAC alone. Potentially this combination could be used clinically in the treatment of extensive long bone defects.

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

Using the Cre/loxP system we conditionally inactivated β-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 β-catenin −/− endothelial cells showed a different organization of intercellular junctions with a decrease in α-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 β-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.

Mutations of the GNAS1 Gene, Stromal Cell Dysfunction, and Osteomalacic Changes in Non–McCune–Albright Fibrous Dysplasia of Bone

Activating missense mutations of the GNAS1 gene, encoding the α subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune–Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non‐MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non‐MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS‐related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non‐MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism‐targeted therapeutic approaches to be developed, must also be the same in MAS and non‐MAS FD.

The histopathology of fibrous dysplasia of bone in patients with activating mutations of the Gsα gene : Site-specific patterns and recurrent histological hallmarks

Gsα mutations and histopathology have been analysed in a series of 13 patients with fibrous dysplasia (FD) of bone, including 12 patients with the McCune–Albright syndrome (MAS) and one patient with monostotic FD. Activating mutations (either R201C or R201H) of the gene encoding the α subunit of the stimulatory G protein, Gs, were detected in all cases, including the case of monostotic FD, using a variety of techniques [reverse transcription‐polymerase chain reaction (RT‐PCR) with allele‐specific primers, allele‐specific oligonucleotide hybridization, and DNA sequencing]. A spectrum of bone lesions associated with such mutations was identified and it was possible to recognize three primary, but distinct, histological patterns, defined here as Chinese writing type, sclerotic/Pagetoid type, and sclerotic/hypercellular type, which are characteristically associated with the axial/appendicular skeleton, cranial bones, or gnathic bones, respectively. Features of FD histopathology were characterized by confocal fluorescence microscopy, which allowed the definition of osteogenic cell shape changes and ‘Sharpey fibre bone’ as common denominators of all histological subtypes. Defining characteristics of the different subtypes, two of which diverge from standard descriptions of FD and have never been characterized before, were dependent on the amount and structure of bone tissue within the FD lesion. These data emphasize the non‐random (site‐specific) variability of FD histopathology in patients carrying activating mutations of the Gsα gene and provide additional evidence for the occurrence of Gsα mutations in cases of FD other than typical MAS. Copyright

Reconstruction of Extensive Long-Bone Defects in Sheep Using Porous Hydroxyapatite Sponges

Abstract. The capacity of hydroxyapatite (HA) implants to support large defect repair in weight-bearing long bones of large size animals was investigated. Diaphyseal resections 3.5 cm of the tibia were performed in five adult sheep. They were substituted with HA macroporous ceramic cylinders anatomically shaped, and an external fixator was assembled. The sheep were sacrificed at 20, 40, 60, 120, and 270 days after surgery, respectively. Histology and micro X-ray study of resected implants and adjacent tissues showed proper integration of ceramic with newly formed periosteal bone as early as 20 days after surgery. In one sheep, the external fixator was removed 5 months after surgery. The animal gained the ability to walk with no functional impairment until it was sacrificed 4 months later. At this time, extensive integration of ceramic with bone was detected radiographically and confirmed by a morphological study of the resected sample. Our data indicate that large defects in a weight-bearing long bone can be repaired to the extent necessary for full functional recovery in large animals. These data set the stage for further intervention on material properties as well as for preliminary attempts to use ceramic prostheses for reconstruction of large bone defects in humans.

Biglycan Deficiency Causes Spontaneous Aortic Dissection and Rupture in Mice

Background— For the majority of cases, the cause of spontaneous aortic dissection and rupture is unknown. An inherited risk is associated with Marfan syndrome, Ehlers-Danlos syndrome type IV, and loci mapped to diverse autosomal chromosomes. Analysis of pedigrees however has indicated that it may be also inherited as an X-linked trait. The biglycan gene, found on chromosome X in humans and mice, encodes a small leucine-rich proteoglycan involved in the integrity of the extracellular matrix. A vascular phenotype has never been described in mice deficient in the gene for small leucine-rich proteoglycans. In the breeding of BALB/cA mice homozygous for a null mutation of the biglycan gene, we observed that 50% of biglycan-deficient male mice died suddenly within the first 3 months of life. Methods and Results— Necropsies revealed a major hemorrhage in the thoracic or abdominal cavity, and histology showed aortic rupture that involved an intimal and medial tear as well as dissection between the media and adventitia. By transmission electron microscopy and biomechanical testing, the aortas of biglycan-deficient mice showed structural abnormalities of collagen fibrils and reduced tensile strength. Similar collagen fibril changes were observed in male as well as in female biglycan-deficient mice, which implies a role of additional determinants such as gender-related response to stress in the development of this vascular catastrophe only in male mice. Conclusions— The spontaneous death of biglycan-deficient male mice from aortic rupture implicates biglycan as essential for the structural and functional integrity of the aortic wall and suggests a potential role of biglycan gene defects in the pathogenesis of aortic dissection and rupture in humans.

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.

Osteomalacic and hyperparathyroid changes in fibrous dysplasia of bone: Core biopsy studies and clinical correlations

Deposition, mineralization, and resorption of FD bone compared with unaffected bone from FD patients was investigated in iliac crest biopsy specimens from 13 patients. Compared with unaffected bone, lesional FD bone seemed to be very sensitive to the effects of PTH and renal phosphate wasting, which respectively bring about hyperparathyroid or osteomalacic changes in the lesional bone.

Pathology of bone lesions associated with congenital pseudarthrosis of the leg.

&NA; Congenital pseudarthrosis of the leg remains one of the most controversial pediatric entities in terms of etiopathogenesis, pathology, treatment, and prognosis. The authors reviewed the pathologic material of 24 patients with congenital pseudarthrosis of the leg along with clinical and radiographic data. The tibia was affected in 22 patients; in two patients the disease was limited to the fibula. Fifteen patients were male and nine were female. Age at surgery ranged from 1 to 26 years. Nineteen patients were classified as having dysplastic type, one cystic, and four mixed. Clinical evidence of neurofibromatosis type I (NF‐I) was found in 17 patients. The main histopathologic change observed was the growth of a highly cellular, fibromatosis‐like tissue. In the dysplastic type, such tissue was associated with the periosteum. In the cystic type, a closely similar tissue occupied the lytic area. In cases classified as of mixed type, the coexistence of endosteal/medullary and periosteal involvement by the fibromatosis‐like tissue was observed. In the cystic lesion, evidence of de novo bone formation within the lesional tissue was obvious. Overall, the histologic features of the cystic lesion were similar to those of osteofibrous dysplasia. In the dysplastic type, the proliferation of the fibrovascular tissue was associated with active osteoclastic resorption of the cortex, which remodeled into a trabecular rather than a compact type of structure. Histologic comparison of the pathologic samples of patients with and without NF‐1 revealed no significant differences. The pseudarthrosis gap was continuous with periosteal soft tissues and filled by fibrous tissue, fibrocartilage, and hyaline cartilage with features of enchondral ossification. The authors suggest that the clinical diversity of congenital pseudarthrosis of the leg results from the diverse location of a single pathologic process—namely the growth of an abnormal, fibromatosis‐like tissue either within the periosteum or within the endosteal/marrow tissues. It is tempting to suggest that such an “osteofibromatosis” represent a skeletal expression of neurofibromatosis, either within the fully expressed syndrome (patients with known neurofibromatosis) or as isolated lesion (patients with unknown/cryptic neurofibromatosis).

Regeneration of large bone defects in sheep using bone marrow stromal cells

Bone repair was addressed in a critical‐sized defect model in sheep, combining a ceramic biomaterial and mesenchymal progenitor cells. The defects in the tibial mid‐diaphysis were treated with autologous bone or with a silicon‐stabilized tricalcium phosphate biomaterial, implemented or not by the addition of expanded bone marrow stromal cells. An internal locking compression plate and an external fixator were applied for stabilization. Radiographies were taken during the 8 months follow‐up: the pixel grey levels of the lesion areas were determined to evaluate the repair process radiologically. Microradiography, histology and vascular density tests were performed. The autologous bone‐treated group performed best, as assessed radiologically, within 20–24 weeks after surgery. Very limited healing was detected in the other experimental group: a partial bone deposition occurred at the periphery of the bony stumps only in the cell‐seeded scaffolds. Interestingly, this effect ended within 20–24 weeks, as for the autologous bone, suggesting similar kinetics of the repair processes involved. Moreover, bone deposition was located where a significant reduction of the ceramic scaffold was detected. Faxitron microradiography and histology data confirmed these results. Vascular density analysis evidenced that cell‐seeded scaffolds supported an increased vascular ingrowth. Thus, the interactions with the proper microenvironment and the oxygen and nutrient supply in the inner part of the constructs seem fundamental to initiate scaffold substitution and to improve cell performance in tissue‐engineered approaches to bone repair. Copyright