A novel duplication polymorphism in the FANCA promoter and its association with breast and ovarian cancer

The FANCA gene is one of the genes in which mutations lead to Fanconi anaemia, a rare autosomal recessive disorder characterised by congenital abnormalities, bone marrow failure, and predisposition to malignancy. FANCA is also a potential breast and ovarian cancer susceptibility gene. A novel allele was identified which has a tandem duplication of a 13 base pair sequence in the promoter region. Methods: We screened germline DNA from 352 breast cancer patients, 390 ovarian cancer patients and 256 normal controls to determine if the presence of either of these two alleles was associated with an increased risk of breast or ovarian cancer. Results: The duplication allele had a frequency of 0.34 in the normal controls. There was a nonsignificant decrease in the frequency of the duplication allele in breast cancer patients. The frequency of the duplication allele was significantly decreased in ovarian cancer patients. However, when malignant and benign tumours were considered separately, the decrease was only significant in benign tumours. Conclusion: The allele with the tandem duplication does not appear to modify breast cancer risk but may act as a low penetrance protective allele for ovarian cancer.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Fresh-frozen vs irradiated allograft bone in orthopaedic reconstructive surgery

The use of allograft bone is increasingly common in orthopaedic reconstruction procedures. The optimal method of preparation of allograft bone is subject of great debate. Proponents of fresh-frozen graft cite improved biological and biomechanical characteristics relative to irradiated material, whereas fear of bacterial or viral transmission warrants some to favour irradiated graft. Careful review of the literature is necessary to appreciate the influence of processing techniques on bone quality. Whereas limited clinical trials are available to govern the selection of appropriate bone graft, this review presents the argument favouring the use of fresh-frozen bone allograft as compared to irradiated bone.

Lateral bone density variations in the scoliotic spine

Adolescent Idiopathic Scoliosis (AIS) is the most common deformity of the spine, affecting 2-4% of the population. Previous studies have shown that the vertebrae in scoliotic spines undergo abnormal shape changes, however there has been little exploration of how AIS affects bone density distribution within the vertebrae. Existing pre-operative CT scans of 53 female idiopathic scoliosis patients with right-sided main thoracic curves were used to measure the lateral (right to left) bone density profile at mid-height through each vertebral body. This study demonstrated that AIS patients have a marked convex/concave asymmetry in bone density for vertebral levels at or near the apex of the scoliotic curve. To the best of our knowledge, the only previous studies of bone density distribution in AIS are those of Périé et al [1,2], who reported a coronal plane ‘mechanical migration’ of 0.54mm toward the concavity of the scoliotic curve in the lumbar apical vertebrae of 11 scoliosis patients. This is comparable to the value of 0.8mm (4%) in our study, especially since our patients had more severe scoliotic curves. From a bone adaptation perspective, these results suggest that the axial loading on the scoliotic spine is strongly asymmetric.

Effect of bone graft type on fusion rates following endoscopic anterior scoliosis correction

Bone graft is generally considered fundamental in achieving solid fusion in scoliosis correction and pseudarthrosis following instrumentation may predispose to implant failure. In endoscopic anterior-instrumented scoliosis surgery, autologous rib or iliac crest graft has been utilised traditionally but both techniques increase operative duration and cause donor site morbidity. Allograft bone and bone- morphogenetic-protein alternatives may improve fusion rates but this remains controversial. This study's objective was to compare two-year postoperative fusion rates in a series of patients who underwent endoscopic anterior instrumentation for thoracic scoliosis utilising various bone graft types. Significantly better rates of fusion occurred in endoscopic anterior instrumented scoliosis correction using femoral allograft compared to autologous rib-heads and iliac crest graft. This may be partly explained by the difficulty obtaining sufficient quantities of autologous graft. Lower fusion rates in the autologous graft group appeared to predispose to rod fracture although the clinical consequence of implant failure is uncertain.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Defining the flammability of cylindrical metal rods through characterization of the thermal effects of the ignition promoter

All relevant international standards for determining if a metallic rod is flammable in oxygen utilize some form of “promoted ignition” test. In this test, for a given pressure, an overwhelming ignition source is coupled to the end of the test sample and the designation flammable or nonflammable is based upon the amount burned, that is, a burn criteria. It is documented that (1) the initial temperature of the test sample affects the burning of the test sample both (a) in regards to the pressure at which the sample will support burning (threshold pressure) and (b) the rate at which the sample is melted (regression rate of the melting interface); and, (2) the igniter used affects the test sample by heating it adjacent to the igniter as ignition occurs. Together, these facts make it necessary to ensure, if a metallic material is to be considered flammable at the conditions tested, that the burn criteria will exclude any region of the test sample that may have undergone preheating during the ignition process. A two-dimensional theoretical model was developed to describe the transient heat transfer occurring and resultant temperatures produced within this system. Several metals (copper, aluminum, iron, and stainless steel) and ignition promoters (magnesium, aluminum, and Pyrofuze®) were evaluated for a range of oxygen pressures between 0.69 MPa (100 psia) and 34.5 MPa (5,000 psia). A MATLAB® program was utilized to solve the developed model that was validated against (1) a published solution for a similar system and (2) against experimental data obtained during actual tests at the National Aeronautics and Space Administration White Sands Test Facility. The validated model successfully predicts temperatures within the test samples with agreement between model and experiment increasing as test pressure increases and/or distance from the promoter increases. Oxygen pressure and test sample thermal diffusivity were shown to have the largest effect on the results. In all cases evaluated, there is no significant preheating (above about 38°C/100°F) occurring at distances greater than 30 mm (1.18 in.) during the time the ignition source is attached to the test sample. This validates a distance of 30 mm (1.18 in.) above the ignition promoter as a burn length upon which a definition of flammable can be based for inclusion in relevant international standards (that is, burning past this length will always be independent of the ignition event for the ignition promoters considered here. KEYWORDS: promoted ignition, metal combustion, heat conduction, thin fin, promoted combustion, burn length, burn criteria, flammability, igniter effects, heat affected zone.

Lateral bone density variations in the scoliotic spine

Adolescent Idiopathic Scoliosis (AIS) is the most common deformity of the spine, affecting 2-4% of the population. Previous studies have shown that the vertebrae in scoliotic spines undergo abnormal shape changes, however there has been little exploration of how scoliosis affects bone density distribution within the vertebrae. In this study, existing CT scans of 53 female idiopathic scoliosis patients with right-sided main thoracic curves were used to measure the lateral (right to left) bone density profile at mid-height through each vertebral body. Five key bone density profile measures were identified from each normalised bone density distribution, and multiple regression analysis was performed to explore the relationship between bone density distribution and patient demographics (age, height, weight, body mass index (BMI), skeletal maturity, time since Menarche, vertebral level, and scoliosis curve severity). Results showed a marked convex/concave asymmetry in bone density for vertebral levels at or near the apex of the scoliotic curve. At the apical vertebra, mean bone density at the left side (concave) cortical shell was 23.5% higher than for the right (convex) cortical shell, and cancellous bone density along the central 60% of the lateral path from convex to concave increased by 13.8%. The centre of mass of the bone density profile at the thoracic curve apex was located 53.8% of the distance along the lateral path, indicating a shift of nearly 4% toward the concavity of the deformity. These lateral bone density gradients tapered off when moving away from the apical vertebra. Multi-linear regressions showed that the right cortical shell peak bone density is significantly correlated with skeletal maturity, with each Risser increment corresponding to an increase in mineral equivalent bone density of 4-5%. There were also statistically significant relationships between patient height, weight and BMI, and the gradient of cancellous bone density along the central 60% of the lateral path. Bone density gradient is positively correlated with weight, and negatively correlated with height and BMI, such that at the apical vertebra, a unit decrease in BMI corresponds to an almost 100% increase in bone density gradient.

The effect of bone graft type on fusion rates following anterior thoracoscopic scoliosis correction

Bone graft is generally considered fundamental in achieving solid fusion in scoliosis correction and pseudarthrosis following instrumentation may predispose to implant failure. In thoracoscopic anterior-instrumented scoliosis surgery, autologous rib or iliac crest graft has been utilised traditionally but both techniques increase operative duration and cause donor site morbidity. Allograft bone and bone morphogenetic protein (BMP) alternatives may improve fusion rates but this remains controversial. This study's objective was to compare two-year postoperative fusion rates in a series of patients who underwent thoracoscopic anterior instrumentation for thoracic scoliosis utilising various bone graft types.

Multilineage differentiation potential of bone and cartilage cells derived from explant culture

To date, mesenchymal stem cells (MSCs) from various tissues have been reported, but the yield and differentiation potential of different tissue-derived MSCs is still not clear. This study was undertaken in an attempt to investigate the multilineage stem cell potential of bone and cartilage explant cultures in comparison with bone marrow derived mesenchymal stem cells (BMSCs). The results showed that the surface antigen expression of tissue-derived cells was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing markers related to adhesion (CD29, CD166) and stem cells (CD90, CD105). The tissue-derived cells were able to differentiate into osteoblast, chondrocyte and adipocyte lineage pathways when stimulated in the appropriate differentiating conditions. However, compared with BMSCs, tissue-derived cells showed less capacity for multilineage differentiation when the level of differentiation was assessed in monolayer culture by analysing the expression of tissue-specific genes by reverse transcription polymerase chain reaction (RT-PCR) and histology. In high density pellet cultures, tissue-derived cells were able to differentiate into chondrocytes, expressing chondrocyte markers such as proteoglycans, type II collagen and aggrecan. Taken together, these results indicate that cells derived from tissue explant cultures reserved certain degree of differentiation properties of MSCs in vitro.

Composite electrospun scaffolds for engineering tubular bone grafts

In this study, poly (e-caprolactone) [PCL] and its collagen composite blend (PCL=Col) were fabricated to scaffolds using electrospinning method. Incorporated collagen was present on the surface of the fibers, and it modulated the attachment and proliferation of pig bone marrow mesenchymal cells (pBMMCs). Osteogenic differentiation markers were more pronounced when these cells were cultured on PCL=Col fibrous meshes, as determined by immunohistochemistry for collagen type I, osteopontin, and osteocalcin. Matrix mineralization was observed only on osteogenically induced PCL=Col constructs. Long bone analogs were created by wrapping osteogenic cell sheets around the PCL=Col meshes to form hollow cylindrical cell-scaffold constructs. Culturing these constructs under dynamic conditions enhanced bone-like tissue formation and mechanical strength.We conclude that electrospun PCL=Col mesh is a promising material for bone engineering applications. Its combination with osteogenic cell sheets offers a novel and promising strategy for engineering of tubular bone analogs.