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Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects.

We describe here our experience in annotating the Drosophila melanogaster genome sequence, in the course of which we developed several new open-source software tools and a database schema to support large-scale genome annotation. We have developed these into an integrated and reusable software system for whole-genome annotation. The key contributions to overall annotation quality are the marshalling of high-quality sequences for alignments and the design of a system with an adaptable and expandable flexible architecture.

Several proteins in the bone matrix have been implicated in the regulation of mineral crystal formation and growth. To investigate the relationships between these proteins and the mineral phase at various stages of mineral maturation, fetal porcine calvariae and long bones were fragmented and the particles (20 microm) separated by density gradient sedimentation into fractions of increasing density (1.8 to >2.2 g/cm3). Samples from each fraction were analyzed by X-ray diffraction to obtain the average crystal size/strain and chemical composition. Other samples were sequentially extracted, first with 4.0 mol/L guanidium hydrochloride (GuHCl) (G1), then with 0.5 mol/L ethylene-diamine tetraacetic acid (EDTA) (E), and again with 4.0 mol/L Gu-HCI (G2), for analysis of proteins in different tissue compartments. Based on the mineral density distribution and crystal size, fetal porcine bone protein content was determined for tissue residue and each extract and the protein composition analyzed by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). Although the insoluble organic matrix decreased with mineral density the collagen and protein content remained fairly constant, representing approximately 10% of the tissue weight, except in the highest density fraction. Whereas the total extractable protein, representing predominantly noncollagenous proteins, did not show density-related differences, differences were observed for individual proteins on SDS-PAGE. Consistent with their presence in osteoid, the content of bone sialoprotein (BSP), tyrosine-rich acidic matrix protein (TRAMP), and a series of small proteins with cell attachment properties in the G1 extract decreased with mineral density, whereas TRAMP and BSP were increased in G2 extracts. Mineral-associated proteins, including alpha2HS-glycoprotein, BSP, osteopontin (OPN), and osteocalcin, increased with mineral density, whereas secreted protein acidic and rich in cysteine (SPARC)/osteonectin, and some minor proteins, appeared to decrease. Differences of individual proteins within and between the calvarial and long bones could be related to the role of these proteins in the formation and maturation of hydroxyapatite crystals. Collectively, these studies demonstrate mineral density-associated changes in protein composition that reflect a rapid maturation of mineral crystals in embryonic porcine bones.