Bond Strength and Adhesion Mechanisms of Novel Bone Adhesives.

Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs.

Reliable inkjet printing of chondrocytes and MSCs using reservoir agitation.

Drop-on-demand (DoD) inkjet printing has been explored for a range of applications, including those to selectively deposit cellular material, due to the high accuracy and scalability of such systems when compared with alternative bioprinting techniques. Despite this, there remain considerable limitations when handling cell suspensions due to the agglomeration and sedimentation of cells during printing, leading to a deterioration in jetting performance. The objective of this work was to design and assess the effectiveness of a custom agitation system to maintain cellular dispersion within the ink reservoir during printing. The cell printing performance of an inkjet printer was assessed with and without the use of a custom agitation system, with biological characterisation performed to characterise the impact of the agitator on cellular viability and function. Cell printing performance was retained over a 2 h printing period when incorporating an agitated reservoir, with a gradual reduction in performance observed under a non-agitated configuration. Cell assays indicated that the agitation process did not significantly affect the viability, metabolic activity or morphology of the mesenchymal stromal cell (MSC) or chondrocyte cell types. This study therefore provides a new methodology to increase process reliability within DoD printing platforms when jetting cellularised material.

Reactive jet impingement bioprinting of high cell density gels for bone microtissue fabrication.

Advances in three-dimensional cell cultures offer new opportunities in biomedical research and drug development. However, there are still challenges to overcome, including the lack of reliability, repeatability and complexity of tissues obtained by these techniques. In this study, we describe a new bioprinting system called reactive jet impingement (ReJI) for the bioprinting of cell-laden hydrogels. Droplets of gel precursor solutions are jetted at one another such that they meet and react in mid-air before the gel droplets fall to the substrate. This technique offers a combination of deposition rate, cell density and cell viability which is not currently matched by any other bioprinting technique. The importance of cell density is demonstrated in the development of bone microtissues derived from immortalised human bone marrow stem cells. The cells were printed with high viability within a collagen-alginate-fibrin gel, and tissue specific gene expression shows significantly higher tissue maturation rates using the ability of the ReJI system to deposit gels with a high cell density.

Osseointegration of porous apatite-wollastonite and poly(lactic acid) composite structures created using 3D printing techniques.

A novel apatite-wollastonite/poly(lactic acid) (AW/PLA) composite structure, which matches cortical and cancellous bone properties has been produced and evaluated in vitro and in vivo. The composites structure has been produced using an innovative combination of 3D printed polymer and ceramic macrostructures, thermally bonded to create a hybrid composite structure. In vitro cell assays demonstrated that the AW structure alone, PLA structure alone, and AW/PLA composite were all biocompatible, with the AW structure supporting the proliferation and osteogenic differentiation of rat bone marrow stromal cells. Within a rat calvarial defect model the AW material showed excellent osseointegration with the formation of new bone, and vascularisation of the porous AW structure, both when the AW was implanted alone and when it was part of the AW/PLA composite structure. However, the AW/PLA structure showed the largest amount of the newly formed bone in vivo, an effect which is considered to be a result of the presence of the osteoinductive AW structure stimulating bone growth in the larger pores of the adjacent PLA structure. The layered AW/PLA structure showed no signs of delamination in any of the in vitro or in vivo studies, a result which is attributed to good initial bonding between polymer and ceramic, slow resorption rates of the two materials, and excellent osseointegration. It is concluded that macro-scale composites offer an alternative route to the fabrication of bioactive bone implants which can provide a match to both cortical and cancellous bone properties over millimetre length scales.

Thermal stability and rheological properties of the 'non-stick' Caf1 biomaterial.

The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold. Caf1 is a protein polymer that is secreted from its host organism, Yersinia pestis, to enable escape from phagocytosis. In vitro, cells adhere poorly to the protein unless adhesion motifs are specifically introduced. Caf1 is a good candidate biomaterial due to its definable bioactivity, economical production and its ability to form hydrogels, through the use of cross-linkers. In this study, the thermostability of Caf1 was tested over a range of chemical conditions, and an initial characterisation of its rheological properties conducted in order to assess the suitability of Caf1 as a biomedical material. The results show that Caf1 retains its high thermostability even in harsh conditions such as extremes of pH, high salt concentrations and the presence of detergents. In solution, the concentrated polymer behaves as a complex viscous liquid. Due to these properties, Caf1 polymers are compatible with 3D bioprinting technologies and could be made to form a stimuli-responsive biomaterial that can alter its macrorheological properties in response to external factors. Caf1 biomaterials could therefore prove useful as 3D cell scaffolds for use in cell culture and wound repair.

Development of an arthroscopically compatible polymer additive layer manufacture technique.

This article describes a proof of concept study designed to evaluate the potential of an in vivo three-dimensional printing route to support minimally invasive repair of the musculoskeletal system. The study uses a photocurable material to additively manufacture in situ a model implant and demonstrates that this can be achieved effectively within a clinically relevant timescale. The approach has the potential to be applied with a wide range of light-curable materials and with development could be applied to create functionally gradient structures in vivo.

Temporary Single-Cell Coating for Bioprocessing with a Cationic Polymer.

Temporary single-cell coating is a useful tool for cell processing, allowing manipulation of cells to prevent cell attachment and agglomeration, before re-establishing normal cell function. In this work, a speckled coating method using a known polycation [poly(l-lysine), PLL] is described to induce cell surface electrostatic charges on three different cell types, namely, two bone cancer cell lines and fibroblasts. The morphology of the PLL speckled coating on the cell surface, internalization and metabolization of the polymer, and prevention of cellular aggregations are reported. Polymer concentration was found to be the key parameter controlling both capsule morphology and cell health. This approach allows a temporary cell coating over the course of 1-2 h, with cells exhibiting phenotypically normal behavior after ingesting and metabolizing the polymer. The process offers a fast and efficient alternative to aid single-cell manipulation for bioprocessing applications. Preliminary work on the application of PLL speckled cell coating in enabling reliable bioprinting is also presented.

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30.

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N(10) -formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed ß-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a ß-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.

The structure of classical swine fever virus N(pro): a novel cysteine Autoprotease and zinc-binding protein involved in subversion of type I interferon induction.

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro)) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro) is no longer active as a protease. The released N(pro) suppresses the induction of the host's type-I interferon-α/ß (IFN-α/ß) response. N(pro) binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/ß genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/ß response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro). N(pro) is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro) allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro) involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro)'s interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro), and its role in subversion of host immune response.

Gold nanoparticle-enhanced luminescence of silicon quantum dots co-encapsulated in polymer nanoparticles.

The preparation of two-component polymer composite nanoparticles encapsulating both Si quantum dots (SiQDs) and Au nanoparticles (AuNPs) by a single step miniemulsion polymerization of divinylbenzene is described. This simple and robust method affords well-defined polymer composite nanoparticles with mean diameters in a range of 100-200 nm and with narrow polydispersity indices as determined by dynamic light scattering and transmission electron microscopy. The successful encapsulation of AuNPs within poly(divinylbenzene) was confirmed by UV-visible spectroscopy and from TEM images. Plasmon-enhanced fluorescence of the luminescence of the SiQDs by AuNPs encapsulated within the polymer composite nanoparticles was evaluated by confocal microspectroscopy, and luminescence enhancements of up to 15 times were observed. These observations indicate that the luminescence of the SiQDs is enhanced by the proximity of the AuNPs. The polymer composite nanoparticles were successfully ink-jet printed onto a glass substrate, which demonstrates that these composites are processable in printing applications.

Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase.

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

The 2A resolution crystal structure of HetL, a pentapeptide repeat protein involved in regulation of heterocyst differentiation in the cyanobacterium Nostoc sp. strain PCC 7120.

The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N(2) during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10-15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0A resolution using sulfur anomalous scattering.

Structure of the central RNA recognition motif of human TIA-1 at 1.95A resolution.

T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95A resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1-RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein-protein interactions mediated by the RNP motifs of TIA-1-RRM2. Importantly, the TIA-1-RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding.

New analogs of 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 with conformationally restricted side chains: evaluation of biological activity and structural determination of VDR-bound conformations.

We have successfully prepared E- and Z- isomers of 17-20 dehydro analogs of 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17-20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17-20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17-20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17-C20 double bond. The Vit-III 17-20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity.

Operator-assisted harvesting of protein crystals using a universal micromanipulation robot.

High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 microm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 A structure demonstrating the feasibility of robotic protein crystal harvesting.

Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide.

We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.