SH3BP2-related fibro-osseous disorders of the maxilla and mandible: A systematic review.

Cherubism is a disorder of bony overgrowth of the jaws that manifests in childhood. SH3BP2 gene variants have been associated with cherubism; this gene plays a major role in bone homeostasis. Due to its rare occurrence, there is as yet no comprehensive understanding of the natural history and clinical course of the disease. The aim of this review was to compile and analyze all cases of SH3BP2-related cherubism and cherubism-like disorders. Thirty publications were identified, including 92 individuals from 34 families, who were diagnosed with SH3BP2-related fibro-osseous lesions of the jaw. Only 15% of cases included in this review had no known family history of the disease. The distribution of cherubism was equal with respect to biological sex. Missing teeth were reported in 38% of cases. Lesions were restricted to the mandible in 36% of cases and involved both the maxilla and mandible in 54% of cases. The clinical phenotypes reported in the articles analyzed varied greatly in detail, making comparisons between studies and conclusive analysis difficult. Further work is necessary to describe the connection between SH3BP2 gene variants and cherubism in order to advance its diagnosis and treatment.

Evidence to support the hypothesis of tuberculosis as a cause of extreme osteonecrosis and osteomyelitis of the mandible in a West African population.

Maxillofacial tuberculosis (TB) is rare. The cases of 19 patients showing extreme bony destruction in the mandible, collected over a 3-month period in West Africa, are presented. Clinical, radiographic, and histological evidence indicated Mycobacterium tuberculosis as a possible cause. Further studies are in progress.

Regulation of adipogenesis and osteogenesis in mesenchymal stem cells by vascular endothelial growth factor A.

Understanding the mechanisms by which bone marrow mesenchymal stem cells (BMSCs) differentiate into bone-forming osteoblasts and marrow adipocytes is crucial to develop strategies for the treatment of several bone diseases. Age-related bone loss resulting in osteopenia and osteoporosis has been associated with reduced numbers of osteoblasts and increased numbers of adipocytes, likely originating from differentiation defects in BMSCs. Although many factors involved in the complex regulation of osteoblast and adipocyte cell lineages have previously been identified, their functional interactions in the context of BMSC differentiation and maintenance of bone homeostasis during ageing are unknown. Recent discoveries have provided important new insights into the mechanisms by which the nuclear envelope protein lamin A and vascular endothelial growth factor A (VEGF) mutually control BMSC fate. Particularly interesting is the finding that VEGF in this context functions as an intracellular protein, unaffected by neutralizing antibodies, and not as a secreted growth factor. These insights may not only facilitate the identification of new targets for treating bone diseases but also lead to improved design of tissue engineering approaches aimed at stimulating bone regeneration and repair.

The mouse palate and its cellular responses to midpalatal suture expansion forces.

OBJECTIVES: To investigate the anatomy of the mouse palate, the midpalatal suture, and the cellular characteristics in the sutures before and immediately after midpalatal suture expansion. MATERIALS AND METHODS: Wild-type C57BL/6 male mice, aged between 6 weeks and 12 months, were chosen for all the experiments. The complete palate of the non-operated group and the midpalatal suture-expanded group at different ages was used for histological, micro-CT, immunohistochemistry, and sutural cell analyses. RESULTS: This study documents precise morphological and histological characteristics of the mouse palatal sutures. In addition to the opening of the midpalatal suture caused by expansion, both transverse and interpalatine sutures were also seen to be affected. Cellular density was decreased in different types of sutures following the application of mechanical force. CONCLUSIONS: The detailed morphology and histology of the mouse palate and the cellular changes that occur following midpalatal suture expansion, as described here, will be helpful as a basis for further investigations of palatal suture tissue responses to mechanical force.

Development and tissue origins of the mammalian cranial base.

The vertebrate cranial base is a complex structure composed of bone, cartilage and other connective tissues underlying the brain; it is intimately connected with development of the face and cranial vault. Despite its central importance in craniofacial development, morphogenesis and tissue origins of the cranial base have not been studied in detail in the mouse, an important model organism. We describe here the location and time of appearance of the cartilages of the chondrocranium. We also examine the tissue origins of the mouse cranial base using a neural crest cell lineage cell marker, Wnt1-Cre/R26R, and a mesoderm lineage cell marker, Mesp1-Cre/R26R. The chondrocranium develops between E11 and E16 in the mouse, beginning with development of the caudal (occipital) chondrocranium, followed by chondrogenesis rostrally to form the nasal capsule, and finally fusion of these two parts via the midline central stem and the lateral struts of the vault cartilages. X-Gal staining of transgenic mice from E8.0 to 10 days post-natal showed that neural crest cells contribute to all of the cartilages that form the ethmoid, presphenoid, and basisphenoid bones with the exception of the hypochiasmatic cartilages. The basioccipital bone and non-squamous parts of the temporal bones are mesoderm derived. Therefore the prechordal head is mostly composed of neural crest-derived tissues, as predicted by the New Head Hypothesis. However, the anterior location of the mesoderm-derived hypochiasmatic cartilages, which are closely linked with the extra-ocular muscles, suggests that some tissues associated with the visual apparatus may have evolved independently of the rest of the "New Head".

Age-dependent increase of discoidin domain receptor 2 and matrix metalloproteinase 13 expression in temporomandibular joint cartilage of type IX and type XI collagen-deficient mice.

Our previous studies demonstrated that mutations in type IX and type XI collagens in mice caused osteoarthritis (OA)-like changes in knee and temporomandibular (TM) joints. We also found that the overexpression of matrix metalloproteinase 13 (Mmp-13) was probably due to the up-regulation of a collagen receptor, discoidin domain receptor 2 (Ddr2), which was responsible for knee cartilage degeneration in mutant mice. The objective of our study was to determine whether the expression of Mmp-3, Mmp-13 and Ddr2 was increased in OA-like TM joints in mutant mice using immunohistochemistry. We found that the staining for Ddr2, Mmp-13 and Mmp-derived type II collagen fragments in tissue sections from 6-month-old mice was increased in TM joints of the mutant mice. In contrast, we found no difference in the staining for Mmp-3 amongst the two mutant mice and their wild-type littermates. We conclude that, similar to previous observations in knee joints, the overexpression of Ddr2 and Mmp-13 may be responsible for the OA-like change in TM joints in mutant mice.

Osteoarthritis-like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho).

OBJECTIVE: To investigate whether heterozygosity for a loss-of-function mutation in the gene encoding the alpha1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints. METHODS: Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild-type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP-3) and MMP-13 in knee joints. In 6-month-old animals, fixed-charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique. RESULTS: The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA-like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP-3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP-13 was higher in knee joints from cho/+ mice than in joints from their wild-type littermates, and negative fixed-charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration. CONCLUSION: Heterozygosity for a loss-of-function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

Delivery of plasmid DNA to articular chondrocytes via novel collagen-glycosaminoglycan matrices.

Our primary objective was to fabricate a porous gene-supplemented collagen-glycosaminoglycan (GSCG) matrix for sustained delivery (over a period of several weeks) of plasmid DNA to articular chondrocytes when implanted into cartilage lesions. The specific aims of this in vitro study were to determine the release kinetics profiles of plasmid DNA from the GSCG matrices, and to determine the ability of the released plasmid DNA to transfect adult canine articular chondrocytes. In particular, we evaluated the effects of two variables, cross-linking treatment and the pH at which the DNA was incorporated into the matrices, on the amount of the plasmid DNA that remained bound to the GSCG matrices after passive (nonenzymatic) leaching and on the expression of a reporter gene in articular chondrocytes grown in the GSCG matrices. Collagen-glycosaminoglycan matrices were synthesized without cross-linking, and by three cross-linking treatments: dehydrothermal (DHT) treatment, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) treatment, and exposure to ultraviolet (UV) radiation. The plasmid DNA was incorporated into the collagen-glycosaminoglycan matrices in solutions at pH 2.5 or 7.5. Transmission electron microscopy studies revealed plasmid DNA bound to the walls of the porous GSCG matrices. In general, the GSCG matrices fabricated at pH 2.5 retained a larger fraction of the initial DNA load after 28 days of incubation in Tris-EDTA buffer. The passive, solvent-mediated release of the plasmid DNA from the GSCG matrices showed a biphasic pattern consisting of a faster, early release rate over the initial 8 hr of leaching followed by a slower, late release rate that was relatively constant over the subsequent 28 days of leaching. Electrophoretic analyses revealed that the plasmid DNA released from the GSCG matrices fabricated at pH 2.5 had been linearized and/or degraded; whereas the plasmid DNA leached from the GSCG matrices prepared with a DNA solution at pH 7.5 was primarily supercoiled and linear. Plasmid DNA released from all GSCG matrix formulations was able to generate luciferase reporter gene expression in monolayer-cultured chondrocytes transfected with the aid of a commercial lipid reagent, and in chondrocytes cultured in the GSCG matrices without the aid of a supplemental transfection reagent. Luciferase expression in chondrocyte-seeded GSCG constructs was evident throughout the culture period (28 days), with the EDC and UV cross-linked matrices prepared at pH 7.5 providing the highest transgene expression levels. We conclude that released plasmid DNA continually transfected canine articular chondrocytes seeded into GSCG matrices in vitro for a 4-week period as evidenced by luciferase reporter gene expression. Thus, GSCG matrices can be fabricated to provide sustained release of plasmid DNA carrying a potential therapeutic gene.

Mouse models of abnormal skeletal development and homeostasis.

Studies of a number of mouse mutations with skeletal defects have contributed significantly to the understanding of bone development and homeostasis. In many cases, such mutants are also genetic models of disorders in humans, characterized by reduced bone mass (osteoporosis), increased bone mass (osteopetrosis), or abnormalities in endochondral ossification (chondrodysplasias).

The role of collagen-derived proteolytic fragments in angiogenesis.

Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous (NC1) domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIII/NC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth.

Intravital microscopy reveals novel antivascular and antitumor effects of endostatin delivered locally by alginate-encapsulated cells.

The current study describes new, antivascular, and antitumor effects of human endostatin. A novel system for continuous, localized delivery of antiangiogenic compounds to brain tumors was used. The delivery system was composed of endostatin-producing 293 cells encapsulated into immuno-isolating sodium alginate. Intravital multifluorescence microscopy was used to assess vascular and antitumor effects of endostatin in C6 glioma spheroids implanted into an ectopic as well as an orthotopic setting. Analysis of total and functional vascular density, microvascular diameters, vessel perfusion, tumor growth, and tumor cell migration were performed repetitively. Tumor growth was reduced by 35% in treated animals. It was of interest that tumor cell invasion into the surrounding tissue was also inhibited. The total vascular density was reduced by 67.6%, perfusion by 67%, and vessel diameters by 37%. This resulted in a significant reduction in tumor perfusion, although the vessel permeability was not influenced. We have demonstrated that human endostatin not only reduces total vascular density, as shown previously, but also greatly reduces the functionality and the diameters of the vessels. Furthermore, we show that this therapeutic approach also inhibits tumor cell invasion, thus supporting the hypothesis that tumor angiogenesis and invasion represent two interrelated processes. Finally, this work further confirms the new therapeutic concept using alginate cell-encapsulation technology for the localized delivery of therapeutic compounds to central nervous system malignancies.

Gene for the human transmembrane-type protein tyrosine phosphatase H (PTPRH): genomic structure, fine-mapping and its exclusion as a candidate for Peutz-Jeghers syndrome.

Mutations in the serine/threonine kinase STK11 lead to Peutz-Jeghers syndrome (PJS) in a subset of affected individuals. Significant evidence for linkage to a second potential PJS disease locus on 19q13.4 has previously been described in one PJS family (PJS07). In the current study, we investigated this second locus for PJS gene candidates. We mapped the main candidate gene in this region, the gene for the transmembrane-type protein tyrosine phosphatase H (PTPRH), within 15 kb telomeric to the marker D19S880. We determined its genomic structure, and performed mutation analysis of all exons and the exon-intron junctions of the PTPRH gene in the PJS07 family. No disease causing mutation was identified in PTPRH in affected individuals, suggesting the existence of an as yet not identified gene on 19q13.4 as a second PJS gene.

Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. We provide the first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation.

A nonsense mutation in MSX1 causes Witkop syndrome.

Witkop syndrome, also known as tooth and nail syndrome (TNS), is a rare autosomal dominant disorder. Affected individuals have nail dysplasia and several congenitally missing teeth. To identify the gene responsible for TNS, we used candidate-gene linkage analysis in a three-generation family affected by the disorder. We found linkage between TNS and polymorphic markers surrounding the MSX1 locus. Direct sequencing and restriction-enzyme analysis revealed that a heterozygous stop mutation in the homeodomain of MSX1 cosegregated with the phenotype. In addition, histological analysis of Msx1-knockout mice, combined with a finding of Msx1 expression in mesenchyme of developing nail beds, revealed that not only was tooth development disrupted in these mice, but nail development was affected as well. Nail plates in Msx1-null mice were defective and were thinner than those of their wild-type littermates. The resemblance between the tooth and nail phenotype in the human family and that of Msx1-knockout mice strongly supports the conclusions that a nonsense mutation in MSX1 causes TNS and that Msx1 is critical for both tooth and nail development.

Expression of frizzled genes in mouse costochondral chondrocytes.

Protein products of frizzled genes are cell membrane receptors for Wnt proteins that play multiple roles during development. We examined the expression of nine frizzled genes in mouse chondrocytes, and detected transcripts of six of the nine genes. We also cloned the entire cDNA of mouse frizzled-1 and compared its cDNA sequence and the cysteine-rich and transmembrane domains of its translated product to sequences of frizzled-1 from C. elegans, Drosophila, chicken and human. We used the T31 Mouse/Hamster radiation hybrid panel to map the mouse frizzled-1 to mouse chromosome 5 (5 cM from the centromere), and frizzled-9 to mouse chromosome 5 (74 cM from the centromere).

Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism.

Cherubism (MIM 118400) is an autosomal dominant inherited syndrome characterized by excessive bone degradation of the upper and lower jaws followed by development of fibrous tissue masses, which causes a characteristic facial swelling. Here we describe seven mutations in the SH3-binding protein SH3BP2 (MIM 602104) on chromosome 4p16.3 that cause cherubism.

Autosomal dominant craniometaphyseal dysplasia is caused by mutations in the transmembrane protein ANK.

Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an approximately 5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.

Clonality and altered behavior of endothelial cells from hemangiomas.

Hemangioma, the most common tumor of infancy, is a benign vascular neoplasm of unknown etiology. We show, for the first time to our knowledge, that endothelial cells from proliferating hemangioma are clonal, and we demonstrate that these hemangioma-derived cells differ from normal endothelial cells in their rates of proliferation and migration in vitro. Furthermore, migration of hemangioma endothelial cells is stimulated by the angiogenesis inhibitor endostatin, unlike the inhibition seen with normal endothelial cells. We conclude that hemangiomas constitute clonal expansions of endothelial cells. This is consistent with the possibility that these tumors are caused by somatic mutations in one or more genes regulating endothelial cell proliferation.

Linkage disequilibrium narrows locus for venous malformation with glomus cells (VMGLOM) to a single 1.48 Mbp YAC.

Venous malformations with glomus cells are localised cutaneous lesions of vascular dysmorphogenesis. They are usually sporadic, but sometimes familial. Using five families, we mapped the locus, VMGLOM, to chromosome 1p21-p22. In order to refine this locus, spanning 4-6 Mbp, we then studied seven additional families. They exhibited linkage to VMGLOM and the combined lod score for all 12 families was 18.41 at theta = 0.0 for marker D1S188. We found a distinct haplotype shared by seven families, comprising seven alleles which are rare in the general population (P < 0.01). This indicates that the haplotype is identical by descent in all seven families, and hence the locus can be refined by inferring ancestral crossovers. Using this approach, we position the causative gene between two markers on the same non-chimeric YAC of 1.48 Mbp, a feasible size for positional cloning. As there is no known gene involved in vasculogenesis and/or angiogenesis in this YAC, the identification of the causative gene is likely to reveal a novel regulator or vascular development.

Local endostatin treatment of gliomas administered by microencapsulated producer cells.

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.