Abnormal Skeletal Development Research Articles

Evaluating the abnormal ossification in tibiotarsi of developing chick embryos exposed to 1.0 ppm doses of platinum group metals by spectroscopic techniques

Platinum group metals (PGMs), i.e., palladium (Pd), platinum (Pt) and rhodium (Rh), are found at pollutant levels in the environment and are known to accumulate in plant and animal tissues. However, little is known about PGM toxicity. Our previous studies showed that chick embryos exposed to PGM concentrations of 1mL of 5.0ppm (LD50) and higher exhibited severe skeletal deformities. This work hypothesized that 1.0ppm doses of PGMs will negatively impact the mineralization process in tibiotarsi. One milliliter of 1.0ppm of Pd(II), Pt(IV), Rh(III) aqueous salt solutions and a PGM-mixture were injected into the air sac on the 7th and 14th day of incubation. Control groups with no-injection and vehicle injections were included. On the 20th day, embryos were sacrificed to analyze the PGM effects on tibiotarsi using four spectroscopic techniques. 1) Micro-Raman imaging: Hyperspectral Raman data were collected on paraffin embedded cross-sections of tibiotarsi, and processed using in-house-written MATLAB codes. Micro-Raman univariate images that were created from the ν1(PO43−) integrated areas revealed anomalous mineral inclusions within the bone marrow for the PGM-mixture treatment. The age of the mineral crystals (ν(CO32−)/ν1(PO43−)) was statistically lower for all treatments when compared to controls (p≤0.05). 2) FAAS: The percent calcium content of the chemically digested tibiotarsi in the Pd and Pt groups changed by ~45% with respect to the no-injection control (16.1±0.2%). 3) Micro-XRF imaging: Abnormal calcium and phosphorus inclusions were found within the inner longitudinal sections of tibiotarsi for the PGM-mixture treatment. A clear increase in the mineral content was observed for the outer sections of the Pd treatment. 4) ICP-OES: PGM concentrations in tibiotarsi were undetectable (<5ppb). The spectroscopic techniques gave corroborating results, confirmed the hypothesis, and explained the observed pathological (skeletal developmental abnormalities) and histological changes (tibiotarsus ischemia and nuclear fragmentation in chondrocytes).

The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia, which play important roles in development and homeostasis. IFT80 is a newly defined IFT protein. Partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development. However, the role and mechanism of IFT80 in osteogenesis is unknown. Here, we first detected IFT80 expression pattern and found that IFT80 was highly expressed in mouse long bone, skull, and during osteoblast differentiation. By using lentivirus-mediated RNA interference (RNAi) technology to silence IFT80 in murine mesenchymal progenitor cell line-C3H10T1/2 and bone marrow derived stromal cells, we found that silencing IFT80 led to either shortening or loss of cilia and the decrease of Arl13b expression — a small GTPase that is localized in cilia. Additionally, silencing IFT80 blocked the expression of osteoblast markers and significantly inhibited ALP activity and cell mineralization. We further found that IFT80 silencing inhibited the expression of Gli2, a critical transcriptional factor in the hedgehog signaling pathway. Overexpression of Gli2 rescued the deficiency of osteoblast differentiation from IFT80-silenced cells, and dramatically promoted osteoblast differentiation. Moreover, introduction of Smo agonist (SAG) promotes osteoblast differentiation, which was partially inhibited by IFT80 silencing. Thus, these results suggested that IFT80 plays an important role in osteogenesis through regulating Hedgehog/Gli signal pathways.

NF-κB signaling and bone resorption

The transcription factor NF-κB is a family of proteins involved in signaling pathways essential for normal cellular functions and development. Deletion of various components of this pathway resulted with abnormal skeletal development. Research in the last decade has established that NF-κB signaling mediates RANK ligand-induced osteoclastogenesis. Consistently, it was shown that inhibition of NF-κB was an effective approach to inhibit osteoclast formation and bone resorptive activity. Identification of the molecular machinery underlying NF-κB activation permitted osteoclast-specific deletion of the major components of this pathway. As a result, it was clear that deletion of members of the proximal IKK kinase complex and the distal NF-κB subunits and downstream regulators affected skeletal development. These studies provided several targets of therapeutic intervention in osteolytic diseases. NF-κB activity has been also described as the centerpiece of inflammatory responses and is considered a potent mediator of inflammatory osteolysis. Indeed, inflammatory insults exacerbate physiologic RANKL-induced NF-κB signals leading to exaggerated responses and to inflammatory osteolysis. These superimposed NF-κB activities appear to underlie several bone pathologies. This review will describe the individual roles of NF-κB molecules in bone resorption and inflammatory osteolysis.

Constitutively Activated NLRP3 Inflammasome Causes Inflammation and Abnormal Skeletal Development in Mice

The NLRP3 inflammasome complex is responsible for maturation of the pro-inflammatory cytokine, IL-1β. Mutations in NLRP3 are responsible for the cryopyrinopathies, a spectrum of conditions including neonatal-onset multisystem inflammatory disease (NOMID). While excessive production of IL-1β and systemic inflammation are common to all cryopyrinopathy disorders, skeletal abnormalities, prominently in the knees, and low bone mass are unique features of patients with NOMID. To gain insights into the mechanisms underlying skeletal abnormalities in NOMID, we generated knock-in mice globally expressing the D301N NLRP3 mutation (ortholog of D303N in human NLRP3). NOMID mice exhibit neutrophilia in blood and many tissues, including knee joints, and high levels of serum inflammatory mediators. They also exhibit growth retardation and severe postnatal osteopenia stemming at least in part from abnormally accelerated bone resorption, attended by increased osteoclastogenesis. Histologic analysis of knee joints revealed abnormal growth plates, with loss of chondrocytes and growth arrest in the central region of the epiphyses. Most strikingly, a tissue “spike" was observed in the mid-region of the growth plate in the long bones of all NOMID mice that may be the precursor to more severe deformations analogous to those observed in NOMID patients. These findings provide direct evidence linking a NOMID-associated NLRP3-activating mutation to abnormalities of postnatal skeletal growth and bone remodeling.

DLK1 Is a Somato-Dendritic Protein Expressed in Hypothalamic Arginine-Vasopressin and Oxytocin Neurons

Delta-Like 1 Homolog, Dlk1, is a paternally imprinted gene encoding a transmembrane protein involved in the differentiation of several cell types. After birth, Dlk1 expression decreases substantially in all tissues except endocrine glands. Dlk1 deletion in mice results in pre-natal and post-natal growth deficiency, mild obesity, facial abnormalities, and abnormal skeletal development, suggesting involvement of Dlk1 in perinatal survival, normal growth and homeostasis of fat deposition. A neuroendocrine function has also been suggested for DLK1 but never characterised. To evaluate the neuroendocrine function of DLK1, we first characterised Dlk1 expression in mouse hypothalamus and then studied post-natal variations of the hypothalamic expression. Western Blot analysis of adult mouse hypothalamus protein extracts showed that Dlk1 was expressed almost exclusively as a soluble protein produced by cleavage of the extracellular domain. Immunohistochemistry showed neuronal DLK1 expression in the suprachiasmatic (SCN), supraoptic (SON), paraventricular (PVN), arcuate (ARC), dorsomedial (DMN) and lateral hypothalamic (LH) nuclei. DLK1 was expressed in the dendrites and perikarya of arginine-vasopressin neurons in PVN, SCN and SON and in oxytocin neurons in PVN and SON. These findings suggest a role for DLK1 in the post-natal development of hypothalamic functions, most notably those regulated by the arginine-vasopressin and oxytocin systems.

Autophagy: A New Therapeutic Target in Cartilage Injury and Osteoarthritis

Autophagy is a cellular homeostasis mechanism that plays an essential role in energy and nutrient regulation as well as in the removal of damaged and dysfunctional macromolecules and organelles.1 Failure of autophagy results in the increased production of reactive oxygen species and to abnormal gene expression, and it can lead to cell death. Autophagy failure as observed in aging tissues and cells leads to neurodegeneration, cardiomyopathies, abnormal skeletal development, and premature death.2 In human osteoarthritis (OA) and in a mouse model, autophagy proteins were expressed at reduced levels; this measure was associated with increased cell death and degradation in articular cartilage.3 An important suppressor of autophagy is the protein mammalian target of rapamycin (mTOR) as part of the complex mTORC1. Rapamycin is a lipophilic macrolide antibiotic that is used as an immunosuppressive drug in solidorgan transplantation for the management of renal cell carcinoma, and it is under evaluation for the treatment of certain types of cancer. It can induce autophagy in a variety of cell types.4 Rapamycin was tested for chondroprotective activities in an in vitro model of cartilage injury and in a mouse model of OA. Cartilage injury was induced by subjecting bovine tissue explants to singleimpact loading, which results in cell death and extracellular matrix damage; single-impact loading was also associated with a suppression of autophagy regulators. Treatment of the explants with rapamycin improved cell viability and attenuated tissue damage.5 In the mouse model, OA was induced by transection of the medial meniscotibial ligament and the medial collateral ligament. Intraperitoneal administration of rapamycin affected the mTOR signaling pathway, as indicated by inhibition of phosphorylation of ribosomal protein S6, a target of mTORC1, and by activation of light chain 3 (LC3), a main marker of autophagy.6 The severity of cartilage degradation was reduced in the rapamycin-treated group compared with the control group, and this reduction was associated with a decrease in synovitis.6 Rapamycin treatment also maintained cartilage cellularity and decreased the expression of ADAMTS-5, an important enzyme in cartilage extracellular matrix degradation,7 and of interleukin-1β, a potent inflammatory and catabolic stimulus in OA.8 These results suggest that rapamycin reduces the severity of traumatic cartilage injury and of experimental OA at least in part by autophagy activation. Drugs that inhibit one of a large number of extracellular matrix-degrading enzymes may not provide sufficient impact to change the overall course of the disease and failed in previous clinical trials.9,10 By contrast, inhibiting the failure of a critical cellular homeostasis mechanism, such as autophagy, leads to global changes in gene expression and can ultimately cause cell death and extracellular matrix destruction,2 two main features of OA-affected cartilage.11 The in vitro and in vivo studies of rapamycin establish proof of principle for chondroprotective effects of rapamycin. The two models are associated with the rapid development of cartilage lesions; it remains to be determined whether rapamycin is also effective in models of spontaneous or aging-associated OA. However, short-term use of rapamycin may be effective and feasible in clinical conditions associated with joint injury.12 One important limitation of rapamycin for long-term administration in humans is its immunosuppressive effect. This will certainly be a concern if the drug were to be used to slow progression of established disease. However, the clinical feasibility of using mTOR inhibitors for the management of OA is supported by recent advances in the development of new and safer rapamycin analogs. It can also be anticipated that, with more precise resolution of the different effector mechanisms that are regulated by mTOR, there will be new drugs with greater specificity.13 Recent observations on suppression of autophagy in aging and injured cartilage and chondroprotective activities of rapamycin suggest that pharmacologic enhancement of autophagy may be a promising new approach to prevent or manage OA.

Status of muscle signaling pathways in the early pathogenesis of dystroglycanopathy‐type muscular dystrophy

A subset of muscular dystrophies are caused by abnormal glycosylation of α‐dystroglycan. Without its proper sugar structure, α‐dystroglycan binding to extracellular matrix proteins (e.g. laminin, perlecan) is compromised, disrupting the structural integrity of muscle and other tissues. Mutations in the FCMD gene, encoding fukutin, cause severe and milder dystroglycanopathies, Fukuyama congenital muscular dystrophy (FCMD) and limb‐girdle muscular dystrophy type 2M (LGMD 2M), respectively. Using fukutin conditional knockout mice, we have evidence that severe congenital muscular dystrophy phenotypes involve abnormal skeletal muscle development and/or differentiation. We will present a molecular, biochemical and histological approach, screening select muscle signaling pathways in young knockout mice, to identify abnormalities in skeletal muscle development/differentiation. These findings bring new insight to potential mechanisms underlying early disease pathogenesis and severe phenotypic outcomes in dystroglycanopathy‐type congenital muscular dystrophies.This work has been supported by the Muscular Dystrophy Association (Development Grant) and the University of Georgia, College of Pharmacy.

Hyperactivation of mTOR critically regulates abnormal osteoclastogenesis in neurofibromatosis type 1

Individuals with nerofibromatosis Type 1 (NF1) frequently suffer a spectrum of bone pathologies, such as abnormal skeletal development (scoliosis, congenital bowing, and congenital pseudoarthroses, etc), lower bone mineral density with increased fracture risk. These skeletal problems may result, in part, from abnormal osteoclastogenesis. Enhanced RAS/PI3K activity has been reported to contribute to abnormal osteoclastogenesis in Nf1 heterozygous (Nf1+/-) mice. However, the specific downstream pathways linked to NF1 abnormal osteoclastogenesis have not been defined. Our aim was to determine whether mammalian target of rapamycin (mTOR) was a key effector responsible for abnormal osteoclastogenesis in NF1. Primary osteoclast-like cells (OCLs) were cultured from Nf1 wild-type (Nf1+/+) and Nf1+/- mice. Compared to Nf1+/+ controls, there were 20% more OCLs induced from Nf1+/- mice. Nf1+/- OCLs were larger and contained more nuclei. Hyperactive mTOR signaling was detected in Nf1+/- OCLs. Inhibition of mTOR signaling by rapamycin in Nf1+/- OCLs abrogated abnormalities in cellular size and number. Moreover, we found that hyperactive mTOR signaling induced abnormal osteoclastogenesis major through hyper-proliferation. Our research suggests that neurofibromin directly regulates osteoclastogenesis through mTOR signaling pathway. Inhibiting mTOR may represent a viable strategy to treat NF1 bone diseases.

Congenital hypothyroidism of dogs and cats: A review

Congenital hypothyroidism is a rare and underdiagnosed congenital endocrine disorder in dogs and cats and the true incidence is unknown. The disorder may cause a range of clinical signs depending on the primary defect, which affect production of thyroid hormones; some cases present when adult. Hallmark clinical signs of congenital hypothyroidism are mental impairment and skeletal developmental abnormalities, resulting in disproportionate dwarfism; goitre may or may not be present. Documented causes of congenital hypothyroidism in dogs include deficiency of, or unresponsiveness to, thyrotropin-releasing hormone (TRH) or thyroid-stimulating hormone (TSH), thyroid dysgenesis, dyshormonogenesis and iodine deficiency. In cats, TSH unresponsiveness, thyroid dysgenesis, dyshormonogenesis and iodine deficiency have been confirmed. Adequate replacement therapy results in a successful outcome in the majority of cases, especially when started early in life, as permanent developmental abnormalities can be prevented. This review describes reported cases in dogs and cats, diagnostic investigation, and recommendations for treatment.

Biophysical stimuli induced by passive movements compensate for lack of skeletal muscle during embryonic skeletogenesis

In genetically modified mice with abnormal skeletal muscle development, bones and joints are differentially affected by the lack of skeletal muscle. We hypothesise that unequal levels of biophysical stimuli in the developing humerus and femur can explain the differential effects on these rudiments when muscle is absent. We find that the expression patterns of four mechanosensitive genes important for endochondral ossification are differentially affected in muscleless limb mutants, with more extreme changes in the expression in the humerus than in the femur. Using finite element analysis, we show that the biophysical stimuli induced by muscle forces are similar in the humerus and femur, implying that the removal of muscle contractile forces should, in theory, affect the rudiments equally. However, simulations in which a displacement was applied to the end of the limb, such as could be caused in muscleless mice by movements of the mother or normal littermates, predicted higher biophysical stimuli in the femur than in the humerus. Stimuli induced by limb movement were much higher than those induced by the direct application of muscle forces, and we propose that movements of limbs caused by muscle contractions, rather than the direct application of muscle forces, provide the main mechanical stimuli for normal skeletal development. In muscleless mice, passive movement induces unequal biophysical stimuli in the humerus and femur, providing an explanation for the differential effects seen in these mice. The significance of these results is that forces originating external to the embryo may contribute to the initiation and progression of skeletal development when muscle development is abnormal.

Aarskog–Scott syndrome: Clinical update and report of nine novel mutations of the FGD1 gene

Mutations in the FGD1 gene have been shown to cause Aarskog-Scott syndrome (AAS), or facio-digito-genital dysplasia (OMIM#305400), an X-linked disorder characterized by distinctive genital and skeletal developmental abnormalities with a broad spectrum of clinical phenotypes. To date, 20 distinct mutations have been reported, but little phenotypic data are available on patients with molecularly confirmed AAS. In the present study, we report on our experience of screening for mutations in the FGD1 gene in a cohort of 60 European patients with a clinically suspected diagnosis of AAS. We identified nine novel mutations in 11 patients (detection rate of 18.33%), including three missense mutations (p.R402Q; p.S558W; p.K748E), four truncating mutations (p.Y530X; p.R656X; c.806delC; c.1620delC), one in-frame deletion (c.2020_2022delGAG) and the first reported splice site mutation (c.1935+3A>C). A recurrent mutation (p.R656X) was detected in three independent families. We did not find any evidence for phenotype-genotype correlations between type and position of mutations and clinical features. In addition to the well-established phenotypic features of AAS, other clinical features are also reported and discussed.

Conditional Disruption of Pkd1 in Osteoblasts Results in Osteopenia Due to Direct Impairment of Bone Formation

PKD1 (polycystin-1), the disease-causing gene for ADPKD, is widely expressed in various cell types, including osteoblasts, where its function is unknown. Although global inactivation of Pkd1 in mice results in abnormal skeletal development, the presence of polycystic kidneys and perinatal lethality confound ascertaining the direct osteoblastic functions of PKD1 in adult bone. To determine the role of PKD1 in osteoblasts, we conditionally inactivated Pkd1 in postnatal mature osteoblasts by crossing Oc (osteocalcin)-Cre mice with floxed Pkd1 (Pkd1(flox/m1Bei)) mice to generate conditional heterozygous (Oc-Cre;Pkd1(flox/+)) and homozygous (Oc-Cre;Pkd1(flox/m1Bei)) Pkd1-deficient mice. Cre-mediated recombination (Pkd1(Delta flox)) occurred exclusively in bone. Compared with control mice, the conditional deletion of Pkd1 from osteoblasts resulted in a gene dose-dependent reduction in bone mineral density, trabecular bone volume, and cortical thickness. In addition, mineral apposition rates and osteoblast-related gene expression, including Runx2-II (Runt-related transcription factor 2), osteocalcin, osteopontin, and bone sialoprotein, were reduced proportionate to the reduction of Pkd1 gene dose in bone of Oc-Cre;Pkd1(flox/+) and Oc-Cre;Pkd1(flox/m1Bei) mice. Primary osteoblasts derived from Oc-Cre;Pkd1(flox/m1Bei) displayed impaired differentiation and suppressed activity of the phosphatidylinositol 3-kinase-Akt-GSK3beta-beta-catenin signaling pathways. The conditional deletion of Pkd1 also resulted in increased adipogenesis in bone marrow and in osteoblast cultures. Thus, PKD1 directly functions in osteoblasts to regulate bone formation.

Osteo-Chondroprogenitor–Specific Deletion of the Selenocysteine tRNA Gene, Trsp, Leads to Chondronecrosis and Abnormal Skeletal Development: A Putative Model for Kashin-Beck Disease

Kashin-Beck disease, a syndrome characterized by short stature, skeletal deformities, and arthropathy of multiple joints, is highly prevalent in specific regions of Asia. The disease has been postulated to result from a combination of different environmental factors, including contamination of barley by mold mycotoxins, iodine deficiency, presence of humic substances in drinking water, and, importantly, deficiency of selenium. This multifunctional trace element, in the form of selenocysteine, is essential for normal selenoprotein function, including attenuation of excessive oxidative stress, and for the control of redox-sensitive molecules involved in cell growth and differentiation. To investigate the effects of skeletal selenoprotein deficiency, a Cre recombinase transgenic mouse line was used to trigger Trsp gene deletions in osteo-chondroprogenitors. Trsp encodes selenocysteine tRNA[Ser]Sec, required for the incorporation of selenocysteine residues into selenoproteins. The mutant mice exhibited growth retardation, epiphyseal growth plate abnormalities, and delayed skeletal ossification, as well as marked chondronecrosis of articular, auricular, and tracheal cartilages. Phenotypically, the mice thus replicated a number of the pathological features of Kashin-Beck disease, supporting the notion that selenium deficiency is important to the development of this syndrome.

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Reduction of complex ventricular ectopy and improvement in exercise capacity with flecainide therapy in Andersen-Tawil syndrome

Andersen-Tawil syndrome (ATS) is a rare inherited autosomal disorder characterized by the clinical triad of ventricular arrhythmias, hypokalaemic periodic paralyses, and skeletal developmental abnormalities, resulting in dysmorphic features. Although ATS patients have a high incidence of ventricular arrhythmias, the occurrence of sudden cardiac death is rare. In this report, we describe the successful use of flecainide in an ATS patient with a considerable ventricular arrhythmia burden who had not demonstrated any response to conventional beta-blocker therapy used in conjunction with potassium (K(+)) supplementation.

Coexistent manifestations of the Andersen-Tawil and Brugada syndromes

The Andersen-Tawil syndrome (ATS) is a rare inherited disorder characterized by periodic paralysis, ventricular arrhythmias, skeletal developmental abnormalities, and in most cases, prolongation of the QT interval (or more correctly, the QU interval). The Brugada syndrome (BS) similarly is an inherited disorder manifested by the unique electrocardiographic (ECG) abnormality of ST-segment elevation in leads V1 and V2, ventricular arrhythmias, and sudden cardiac death. This report details an unusual patient with manifestations of both syndromes, who was treated by the implantation of a defibrillator (implanted cardioverter/defibrillator).

Abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataracts in mice with a targeted disruption of the Nov (Ccn3) gene.

BackgroundSignals from the extracellular environment control many aspects of cell behaviour including proliferation, survival, differentiation, adhesion and migration. It is increasingly evident that these signals can be modulated by a group of matricellular proteins called the CCN family. CCN proteins have multiple domains through which they regulate the activities of a variety of signalling molecules including TGFβ, BMPs and integrins, thereby influencing a wide range of processes in development and disease. Whilst the developmental roles of CCN1 and CCN2 have been elucidated, very little is known about the function of CCN3 (NOV). To investigate this, we have generated mice carrying a targeted mutation in the Nov gene (Novdel3) which reveal for the first time its diverse functions in embryos and adults.ResultsBy replacing Nov exon 3 with a TKneomycin cassette, we have generated Novdel3-/- mice which produce no full length NOV protein and express at a barely detectable level a mutant NOV protein that lacks the VWC domain. In Novdel3-/- embryos, and to a lesser extent in Novdel3+/- embryos, development of the appendicular and axial skeleton was affected with enlarged vertebrae, elongated long bones and digits, delayed ossification, increased bone mineralization and severe joint malformations. Primary embryo fibroblasts from Novdel3-/- mutant embryos showed enhanced chondrogenesis and osteogenesis. Cardiac development was also influenced leading to enlargement and abnormal modelling of the endocardial cushions, associated with septal defects and delayed fusion. In adults, cardiomyopathy was apparent, with hypertrophy and calcification of the septum and left ventricle dilation. Muscle atrophy was seen by 5 months of age, associated with transdifferentiation to fat. Premature tissue degeneration was also seen in the lens, with cataracts present from 6 months.ConclusionWe have generated the first mice with a mutation in the Nov gene (Novdel3). Our data demonstrate that NOV is a regulator of skeletal and cardiac development, and implicates NOV in various disease processes including cardiomyopathy, muscle atrophy and cataract formation. Novdel3 mutants represent a valuable resource for studying NOV's role in the modulation and co-ordination of multiple signalling pathways that underpin organogenesis and tissue homeostasis.

CCN3: A novel function in vivo

Abnormal skeletal and cardiac development, cardiomyopathy, muscle atrophy and cataracts in mice with a targeted disruption of the Nov (Ccn3) gene Heath et al., BMC Dev Biol. 2008; 8: 18. Development, tissue regeneration and disease are regulated through complex interactions among molecules which signal to regulate cellular processes such as proliferation, survival, differentiation, adhesion and migration. The CCN family of matricellular proteins is a central player in regulating several of these signaling molecules (reviewed Leask and Abraham 2006). Thus CCN family members profoundly affect cellular behavior in development, wound healing, tissue homeostasis and in a range of pathologies, including fibrosis and cancer. There are six members of the CCN family: CCN1 (cysteine-rich 61, Cyr61), CCN2 (connective tissue growth factor, CTGF), CCN3 (nephroblastoma overexpressed gene, NOV), CCN4 (Wnt-induced secreted protein-1, WISP1), CCN5 (WISP2/rCOP-1) and CCN6 (WISP3). Their effects are mediated by four cysteine-rich conserved domains which are shared by all members of the family, with the exception of WISP2 which lacks the C-terminal domain (Bork 1993; Perbal 2004). Through these domains, CCN proteins interact with a variety of extra-cellular signaling molecules, thereby regulating their activities. Recent evidence has shown that, in spite of their structural similarity, CCN proteins have a diverse range of activities. In particular, results revealed using genetically-deficient mice and cells derived thereof have revealed essential non-redundant functions for CCN family members. Targeted disruption of CCN2/CTGF identified a role in coordinating chondrogenesis and angiogenesis (Ivkovic et al. 2003) while knock out mice lacking CCN1/CYR61 show that it is required for placental development and vascular integrity (Mo et al. 2002). Experiments using CCN2-deficient cells have shown that CCN2 is required for optimal adhesion, contraction, migration and gene transcription both basally and in response to fibronectin, transforming growth factor-beta 1 and micromass culture (Chen et al. 2004; Shi-wen et al. 2006; Kennedy et al. 2007; Nishida et al. 2007; Pala et al. 2008). CCN3/nov (nephroblastoma overexpressed) was initially identified in a chick nephroblastoma caused by the mouse adenovirus type 1 (MAV-1) retrovirus (Joliot et al. 1992). CCN3 is over-expressed in all MAV-induced avian nephroblastomas studied and deregulated in a variety of other tumor types, including Wilms’ tumors (Chevalier et al. 1998) and musculoskeletal tumors (Manara et al. 2002). Although these results suggest that CCN3 may contribute to tumorigenesis, whether CCN3 plays an essential role in vivo is unknown. To investigate the in vivo function of CCN3, Heath and colleagues (2008) generated mice carrying a targeted mutation of CCN3. These mice produce no full length NOV protein, but express at a barely detectable level a mutant NOV protein that lacks the van Willebrand domain. Mutating CCN3 resulted in abnormal skeletal and cardiac development, including joint abnormalities, cardiomyopathy, and premature tissue degeneration culminating in muscle atrophy and cataracts in adult mice. In development at E16.5, CCN3 expression was found in the mesenchyme surrounding cartilage condensations, and in the tendons and myotendinous junctions. Chondrocytes were enlarged and the region in which chondrocytes were present was enlarged. Ossification was severely impaired in mutant embryos. These results differed significantly from those seen in CCN2 knockout mice which die shortly after birth with severely malformed rib cages (Ivkovic et al. 2003). In these latter animals, delayed ossification was also observed, but these mice had enlarged pre-hypertrophic/hypertrophic zones and thinner bone collars. In contrast, in CCN3 mutant mice, the size of the prehypertrophic/hypertrophic zone was reduced, and the bone collars were increased in thickness. The basis of the phenotype of the CCN3-deficient mice was not explored mechanistically. It is unclear whether the phenotypes observed are due to the loss of full length CCN3, or to possible novel functions of the very low level of mutant CCN3 expression. Moreover, expression of key players in chondrogenesis and ossification (e.g., the sox family of transcription factors) were not examined. Moreover, it has been recently shown that CCN2, but not CCN3, is induced by Wnt3a, which plays a key role in signaling in chondrogenesis, osteoblastogenesis, and osteoclastogenesis (Si et al. 2006; Chen et al. 2008). An intriguing possibility exists that the differential results between the CCN2 and CCN3 animals may arise due to differential responses in expression to Wnt3a. Nonetheless, the results presented by Heath and colleagues (2008) are intriguing at they point to clear functional differences in vivo among the different CCN family members. These differences which may reflect divergences not only regarding the proteins with which they interact with, but also regarding the expression patterns in response to various stimuli in vivo.

A Lack of Thyroid Hormones Rather than Excess Thyrotropin Causes Abnormal Skeletal Development in Hypothyroidism

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).

Cell-Based Bone Tissue Engineering

To restore skeleton function in the field of orthopaedic and oral-maxillofacial surgery, bone tissue regeneration remains an important challenge. Spinal fusion, augmentation of fracture healing, and reconstruction of bone defects resulting from trauma, tumour, infections, biochemical disorders, or abnormal skeletal development are clinical situations in which surgical intervention is required. The types of graft materials available to treat such problems essentially include autologous bone (from the patient), allogeneic bone (from a donor), and demineralised bone matrices, as well as a wide range of synthetic biomaterials such as metals, ceramics, polymers, and composites. Until recently, the use of autologous bone grafts has been the number one choice for bone repair and regeneration [1–5]. A patient's own bone lacks immunogenicity and provides bone-forming cells, which are directly delivered at the implant site. Moreover, autologous bone grafts recruit mesenchymal cells and induce them to differentiate into osteogenic cells through exposure to osteoinductive growth factors [1,3,6,7]. Although there are many advantages to using autologous bone, there are major drawbacks to the harvesting procedure, and for centuries there has been a search for alternatives. The extra surgery involved in harvesting autologous bone causes morbidity at the donor site [1,3,6,8] and can cause post-operative continuous pain [3,9–11], hypersensitivity [3], pelvic instability [10–12], infection [6,9], and paresthesia [3,6]. These complications affect 10% to 30% of the patients [9]. Moreover, the amount of bone that can be collected is limited. As an alternative, the use of allografts (from human to human) eliminates the harvesting procedure and the quantity of available tissue is no longer an issue. Nevertheless, the quality of allografts is worse than that of autologous grafts. Allografts have a poor degree of cellularity, less revascularisation, and a higher resorption rate compared to autologous grafts [3,6], resulting in a slower rate of new bone tissue formation, as observed in several studies [11,13–15]. In addition, the immunogenic potential of these allografts and the risks of virus transmission to the recipient are serious disadvantages [2,14,16]. Although processing techniques such as demineralisation, freeze-drying, and irradiation have been shown to reduce the patient's immune response, processing also alters the structure of the graft and reduces its potential to induce bone healing (osteoinductivity), while the possibility of disease transmission still remains [3].