Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73342/1/j.1600-0722.2006.00288.x.pd

STIM1 R304W in mice causes subgingival hair growth and an increased fraction of trabecular bone

Calcium signaling plays a central role in bone development and homeostasis. Store operated calcium entry (SOCE) is an important calcium influx pathway mediated by calcium release activated calcium (CRAC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum calcium sensing protein important for SOCE. We generated a mouse model expressing the STIM1 R304W mutation, causing Stormorken syndrome in humans. Stim1R304W/R304W mice showed perinatal lethality, and the only three animals that survived into adulthood presented with reduced growth, low body weight, and thoracic kyphosis. Radiographs revealed a reduced number of ribs in the Stim1R304W/R304W mice. Microcomputed tomography data revealed decreased cortical bone thickness and increased trabecular bone volume fraction in Stim1R304W/R304W mice, which had thinner and more compact bone compared to wild type mice. The Stim1R304W/+ mice showed an intermediate phenotype. Histological analyses showed that the Stim1R304W/R304W mice had abnormal bone architecture, with markedly increased number of trabeculae and reduced bone marrow cavity. Homozygous mice showed STIM1 positive osteocytes and osteoblasts. These findings highlight the critical role of the gain-of-function (GoF) STIM1 R304W protein in skeletal development and homeostasis in mice. Furthermore, the novel feature of bilateral subgingival hair growth on the lower incisors in the Stim1R304W/R304W mice and 25 % of the heterozygous mice indicate that the GoF STIM1 R304W protein also induces an abnormal epithelial cell fate

One-Step Purification of Recombinant Human Amelogenin and Use of Amelogenin as a Fusion Partner

Amelogenin is an extracellular protein first identified as a matrix component important for formation of dental enamel during tooth development. Lately, amelogenin has also been found to have positive effects on clinical important areas, such as treatment of periodontal defects, wound healing, and bone regeneration. Here we present a simple method for purification of recombinant human amelogenin expressed in Escherichia coli, based on the solubility properties of amelogenin. The method combines cell lysis with recovery/purification of the protein and generates a >95% pure amelogenin in one step using intact harvested cells as starting material. By using amelogenin as a fusion partner we could further demonstrate that the same method also be can explored to purify other target proteins/peptides in an effective manner. For instance, a fusion between the clinically used protein PTH (parathyroid hormone) and amelogenin was successfully expressed and purified, and the amelogenin part could be removed from PTH by using a site-specific protease

Ultra-porous titanium oxide scaffold with high compressive strength

Highly porous and well interconnected titanium dioxide (TiO2) scaffolds with compressive strength above 2.5 MPa were fabricated without compromising the desired pore architectural characteristics, such as high porosity, appropriate pore size, surface-to-volume ratio, and interconnectivity. Processing parameters and pore architectural characteristics were investigated in order to identify the key processing steps and morphological properties that contributed to the enhanced strength of the scaffolds. Cleaning of the TiO2 raw powder removed phosphates but introduced sodium into the powder, which was suggested to decrease the slurry stability. Strong correlation was found between compressive strength and both replication times and solid content in the ceramic slurry. Increase in the solid content resulted in more favourable sponge loading, which was achieved due to the more suitable rheological properties of the ceramic slurry. Repeated replication process induced only negligible changes in the pore architectural parameters indicating a reduced flaw size in the scaffold struts. The fabricated TiO2 scaffolds show great promise as load-bearing bone scaffolds for applications where moderate mechanical support is required

Demographics of extra-articular calcaneal fractures: Including a review of the literature on treatment and outcome

Introduction: Extra-articular calcaneal fractures represent 25-40% of all calcaneal fractures and an even higher percentage of up to 60% is seen in children. A disproportionately small part of the literature on calcaneal fractures involves the extra-articular type. The aim of this study was to investigate the incidence of extra-articular calcaneal fractures in a Level 1 trauma centre, define the distribution of the various types of fractures and compare patient demographics between extra- and intra-articular calcaneal fractures. In addition the literature was reviewed for the most common types of extra-articular calcaneal fractures with regard to incidence, treatment and clinical outcome. Methods: The radiological records between 2003 and 2005 were reviewed for intra- and extra-articular calcaneal fractures. Patient gender-distribution and age were compared. A literature search was conducted for the treatment of extra-articular calcaneal fractures. Results: In this 3-year study period a total of 49 patients with 50 extra-articular calcaneal fractures and 91 patients with 101 intra-articular fractures were identified. The median age for the first group was 32.7 years, and for the second group 40.3 years; P = 0.04. Male predominance was significantly less pronounced for extra-articular (63%) compared with intra-articular fractures (79%; P = 0.04). Conclusion: One-third of all calcaneal fractures are extra-articular. Significant differences exist between the intra- and extra-articular groups, in terms of lower age and male-female ratio. The literature study shows inconsistencies in treatment options, but most extra-articular fractures are well manageable conservatively

Effects of enamel matrix derivative and transforming growth factor-β1 on human osteoblastic cells

<p>Abstract</p> <p>Background</p> <p>Extracellular matrix proteins are key factors that influence the regenerative capacity of tissues. The objective of the present study was to evaluate the effects of enamel matrix derivative (EMD), TGF-β1, and the combination of both factors (EMD+TGF-β1) on human osteoblastic cell cultures.</p> <p>Methods</p> <p>Cells were obtained from alveolar bone of three adult patients using enzymatic digestion. Effects of EMD, TGF-β1, or a combination of both were analyzed on cell proliferation, bone sialoprotein (BSP), osteopontin (OPN) and alkaline phosphatase (ALP) immunodetection, total protein synthesis, ALP activity and bone-like nodule formation.</p> <p>Results</p> <p>All treatments significantly increased cell proliferation compared to the control group at 24 h and 4 days. At day 7, EMD group showed higher cell proliferation compared to TGF-β1, EMD + TGF-β1 and the control group. OPN was detected in the majority of the cells for all groups, whereas fluorescence intensities for ALP labeling were greater in the control than in treated groups; BSP was not detected in all groups. All treatments decreased ALP levels at 7 and 14 days and bone-like nodule formation at 21 days compared to the control group.</p> <p>Conclusions</p> <p>The exposure of human osteoblastic cells to EMD, TGF-β1 and the combination of factors <it>in vitro </it>supports the development of a less differentiated phenotype, with enhanced proliferative activity and total cell number, and reduced ALP activity levels and matrix mineralization.</p

EMD in periodontal regenerative surgery modulates cytokine profiles: A randomised controlled clinical trial

The enamel matrix derivative (EMD) contains hundreds of peptides in different levels of proteolytic processing that may provide a range of biological effects of importance in wound healing. The aim of the present study was to compare the effect of EMD and its fractions on the cytokine profiles from human gingival fibroblasts in vitro and in gingival crevicular fluid (GCF) in a randomized controlled split-mouth clinical study (n = 12). Levels of cytokines in cell culture medium and in GCF were measured by Luminex over a 2-week period. In the clinical study, levels of pro-inflammatory cytokines and chemokines were increased, whereas the levels of transforming growth factor-α (TGF-α) and platelet-derived growth factor-BB (PDGF-BB) were reduced. The in vitro study showed that EMD and its high and low molecular weight fractions reduced the secretion of pro-inflammatory cytokines and chemokines compared to untreated cells. EMD had an effect on levels of cytokines related to fibroplasia, angiogenesis, inflammation and chemotaxis both in vitro and in vivo, however, the anti-inflammatory effect induced by EMD observed in the in vitro study could not be confirmed clinically

Tracking Endogenous Amelogenin and Ameloblastin In Vivo

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling