Strategies for Improving Impaired Osseointegration in Compromised Animal Models.

Implant osseointegration is reduced in patients with systemic conditions that compromise bone quality, such as osteoporosis, disuse syndrome, and type 2 diabetes. Studies using rodent models designed to mimic these compromised conditions demonstrated reduced bone-to-implant contact (BIC) or a decline in bone mineral density. These adverse effects are a consequence of disrupted intercellular communication. A variety of approaches have been developed to compensate for the altered microenvironment inherent in compromised conditions, including the use of biologics and implant surface modification. Chemical and physical modification of surface properties at the microscale, mesoscale, and nanoscale levels to closely resemble the surface topography of osteoclast resorption pits found in bone has proven to be a highly effective strategy for improving implant osseointegration. The addition of hydrophilicity to the surface further enhances osteoblast response at the bone-implant interface. These surface modifications, applied either alone or in combination, improve osseointegration by increasing proliferation and osteoblastic differentiation of osteoprogenitor cells and enhancing angiogenesis while modulating osteoclast activity to achieve net new bone formation, although the specific effects vary with surface treatment. In addition to direct effects on surface-attached cells, the communication between bone marrow stromal cells and immunomodulatory cells is sensitive to these surface properties. This article reports on the advances in titanium surface modifications, alone and in combination with novel therapeutics in animal models of human disease affecting bone quality. It offers clinically translatable perspectives for clinicians to consider when using different surface modification strategies to improve long-term implant performance in compromised patients. This review supports the use of surface modifications, bioactive coatings, and localized therapeutics as pragmatic approaches to improve BIC and enhance osteogenic activity from both structural and molecular standpoints.

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

Human Bone Marrow Stromal Cell Exosomes Ameliorate Periodontitis.

Human bone marrow stromal cell (hBMSC)-derived exosomes are promising therapeutics for inflammatory diseases due to their unique microRNA (miRNA) and protein cargos. Periodontal diseases often present with chronicity and corresponding exuberant inflammation, which leads to loss of tooth support. In this study, we explored whether hBMSC exosomes can affect periodontitis progression. hBMSC exosomes were isolated from cell culture medium through sequential ultracentrifugation. miRNAs and proteins that were enriched in hBMSC exosomes were characterized by RNA sequencing and protein array, respectively. hBMSC exosomes significantly suppressed periodontal keystone pathogen Porphyromonas gingivalis-triggered inflammatory response in macrophages in vitro. Transcriptomic analysis suggested that exosomes exerted their effects through regulating cell metabolism, differentiation, and inflammation resolution. In vivo, weekly exosome injection into the gingival tissues reduced the tissue destruction and immune cell infiltration in rat ligature-induced periodontitis model. Collectively, these findings suggest that hBMSC-derived exosomes can potentially be used as a host modulation agent in the management of periodontitis.

Multi-omic based production strain improvement (MOBpsi) for bio-manufacturing of toxic chemicals.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

The Role of Lipid Chains as Determinants of Membrane Stability in the Presence of Styrene.

Biofermentative production of styrene from renewable carbon sources is crucially dependent on strain tolerance and viability at elevated styrene concentrations. Solvent-driven collapse of bacterial plasma membranes limits yields and is technologically restrictive. Styrene is a hydrophobic solvent that readily partitions into the membrane interior and alters membrane-chain order and packing. We investigate styrene incorporation into model membranes and the role lipid chains play as determinants of membrane stability in the presence of styrene. MD simulations reveal styrene phase separation followed by irreversible segregation into the membrane interior. Solid state NMR shows committed partitioning of styrene into the membrane interior with persistence of the bilayer phase up to 67 mol % styrene. Saturated-chain lipid membranes were able to retain integrity even at 80 mol % styrene, whereas in unsaturated lipid membranes, we observe the onset of a non-bilayer phase of small lipid aggregates in coexistence with styrene-saturated membranes. Shorter-chain saturated lipid membranes were seen to tolerate styrene better, which is consistent with observed chain length reduction in bacteria grown in the presence of small molecule solvents. Unsaturation at mid-chain position appears to reduce the membrane tolerance to styrene and conversion from cis- to trans-chain unsaturation does not alter membrane phase stability but the lipid order in trans-chains is less affected than cis.

Interactions of polymyxin B with lipopolysaccharide-containing membranes.

Bacterial resistance to antibiotics constantly remodels the battlefront between infections and antibiotic therapy. Polymyxin B, a cationic peptide with an anti-Gram-negative spectrum of activity is re-entering use as a last resort measure and as an adjuvant. We use fluorescence dequenching to investigate the role of the rough chemotype bacterial lipopolysaccharide from E. coli BL21 as a molecular facilitator of membrane disruption by LPS. The minimal polymyxin B/lipid ratio required for leakage onset increased from 5.9 × 10-4 to 1.9 × 10-7 in the presence of rLPS. We confirm polymyxin B activity against E. coli BL21 by the agar diffusion method and determined a MIC of 291 µg ml-1. Changes in lipid membrane stability and dynamics in response to polymyxin and the role of LPS are investigated by 31P NMR and high resolution 31P MAS NMR relaxation is used to monitor selective molecular interactions between polymyxin B and rLPS within bilayer lipid membranes. We observe a strong facilitating effect from rLPS on the membrane lytic properties of polymyxin B and a specific, pyrophosphate-mediated process of molecular recognition of LPS by polymyxin B.

Solid state NMR of membrane proteins: methods and applications.

Membranes of cells are active barriers, in which membrane proteins perform essential remodelling, transport and recognition functions that are vital to cells. Membrane proteins are key regulatory components of cells and represent essential targets for the modulation of cell function and pharmacological intervention. However, novel folds, low molarity and the need for lipid membrane support present serious challenges to the characterisation of their structure and interactions. We describe the use of solid state NMR as a versatile and informative approach for membrane and membrane protein studies, which uniquely provides information on structure, interactions and dynamics of membrane proteins. High resolution approaches are discussed in conjunction with applications of NMR methods to studies of membrane lipid and protein structure and interactions. Signal enhancement in high resolution NMR spectra through DNP is discussed as a tool for whole cell and interaction studies.

Regulation of inflammatory and catabolic responses to IL-1ß in rat articular chondrocytes by microRNAs miR-122 and miR-451.

OBJECTIVE: miR-122 stimulates proliferation of growth plate chondrocytes whereas miR-451 stimulates terminal differentiation and matrix turnover. Here, we examined the potential of these microRNA as regulators of articular chondrocytes using an in vitro model of osteoarthritis. METHODS: miR-122 and miR-451 presence in rat articular cartilage was assessed using the anterior cruciate ligament transection model of OA. In vitro testing used first passage rat articular chondrocytes (rArCs) that were transfected with lipofectamine (Lipo) and miR-122 or miR-451 for 24-h, then treated with 10 ng/mL IL-1ß in order to mimic an osteoarthritic environment. Conditioned media were collected and MMP13, PGE2 and OA-related cytokines were measured. Matrix vesicles were collected from cell layer lysates using ultra-centrifugation. Cells were treated with miR-122 or miR-451 inhibitors to verify miR-specific effects. RESULTS: Both miR-122 and miR-451 were increased in the OA articular cartilage compared to healthy tissue; rArCs expressed both microRNAs in MVs. miR-122 prevented IL-1ß-dependent increases in MMP-13 and PGE2, whereas miR-451 significantly increased the IL-1ß effect. Multiplex data indicated that miR-122 reduced the stimulatory effect of IL-1ß on IL-1α, IL-2, Il-4, IL-6, GM-CSF, MIP-1A, RANTES and VEGF. In contrast, IL-2, IL-4, IL-6, GM-CSF, and MIP-1A were increased by miR-451 while VEGF was decreased. Inhibiting miR-122 exacerbated the response to IL-1ß indicating endogenous levels of miR-122 were present. There were no differences in MMP-13 or PGE2 with miR-451 Locked Nucleic Acid (LNA) inhibitor treatment. CONCLUSIONS: Both miRs were elevated in OA in a rat bilateral anterior cruciate ligament transection (ACLT) model. miR-122 prevented, while miR-451 exacerbated the effects of IL-1ß on rArCs.

Membrane Protein Structure Determination and Characterisation by Solution and Solid-State NMR.

Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane proteins. Solution NMR inherits high resolution from small molecule analysis, providing insights from detergent solubilised proteins and small molecular assemblies. Solid-state NMR achieves high resolution in membrane samples through fast sample spinning or sample alignment. Recent developments in dynamic nuclear polarisation NMR allow signal enhancement by orders of magnitude opening new opportunities for expanding the applications of NMR to studies of native membranes and whole cells.

Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii.

The extracellular signal-regulated kinases (ERKs) serve as important determinants of cellular signal transduction pathways, and hence may play important roles during infections. Previous work suggested that putative ERK7 of Toxoplasma gondii is required for efficient intracellular replication of the parasite. However, the antigenic and immunostimulatory properties of TgERK7 protein remain unknown. The objective of this study was to produce a recombinant TgERK7 protein in vitro and to evaluate its effect on the induction of humoral and T cell-mediated immune responses against T. gondii infection in BALB/c mice. Immunization using TgERK7 mixed with Freund's adjuvants significantly increased the ratio of CD3e+CD4+ T/CD3e+CD8a+ T lymphocytes in spleen and elevated serum cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-12p70, IL-23, MCP-1, and TNF-α) in immunized mice compared to control mice. On the contrary, immunization did not induce high levels of serum IgG antibodies. Five predicted peptides of TgERK7 were synthesized and conjugated with KLH and used to analyze the antibody specificity in the sera of immunized mice. We detected a progressive increase in the antibody level only against TgERK7 peptide A (DEVDKHVLRKYD). Antibody raised against this peptide significantly decreased intracellular proliferation of T. gondii in vitro, suggesting that peptide A can potentially induce a protective antibody response. We also showed that immunization improved the survival rate of mice challenged with a virulent strain and significantly reduced the parasite cyst burden within the brains of chronically infected mice. Our data show that TgERK7-based immunization induced TgERK7 peptide A-specific immune responses that can impart protective immunity against T. gondii infection. The therapeutic potential of targeting ERK7 signaling pathway for future toxoplasmosis treatment is warranted.

Membrane Stability in the Presence of Methacrylate Esters.

Bioproduction of poly(methyl methacrylate) is a fast growing global industry that is limited by cellular toxicity of monomeric methacrylate intermediates to the producer strains. Maintaining high methacrylate concentrations during biofermentation, required by economically viable technologies, challenges bacterial membrane stability and cellular viability. Studying the stability of model lipid membranes in the presence of methacrylates offers unique molecular insights into the mechanisms of methacrylate toxicity, as well as into the fundamental structural bases of membrane assembly. We investigate the structure and stability of model membranes in the presence of high levels of methacrylate esters using solid-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS). Wide-line 31P NMR spectroscopy shows that butyl methacrylate (BMA) can be incorporated into the lipid bilayer at concentrations as high as 75 mol % without significantly disrupting membrane integrity and that lipid acyl chain composition can influence membrane tolerance and ability to accommodate BMA. Using high resolution 13C magic angle spinning (MAS) NMR, we show that the presence of 75 mol % BMA lowers the lipid main transition temperature by over 12 degrees, which suggests that BMA intercalates between the lipid chains, causing uncoupling of collective lipid motions that are typically dominated by chain trans-gauche isomerization. Potential uncoupling of the bilayer leaflets to accommodate a separate BMA subphase was not supported by the SAXS experiments, which showed that membrane thickness remained unchanged even at 80% BMA. Reduced X-ray scattering contrast at the polar/apolar interface suggests BMA localization in that region between the lipid molecules.

Timing Is Everything: Impact of Naturally Occurring Staphylococcus aureus AgrC Cytoplasmic Domain Adaptive Mutations on Autoinduction.

Mutations in the polymorphic Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated "colonization" mode.IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.

Molecular recognition of lipopolysaccharide by the lantibiotic nisin.

Nisin is a lanthionine antimicrobial effective against diverse Gram-positive bacteria and is used as a food preservative worldwide. Its action is mediated by pyrophosphate recognition of the bacterial cell wall receptors lipid II and undecaprenyl pyrophosphate. Nisin/receptor complexes disrupt cytoplasmic membranes, inhibit cell wall synthesis and dysregulate bacterial cell division. Gram-negative bacteria are much more tolerant to antimicrobials including nisin. In contrast to Gram-positives, Gram-negative bacteria possess an outer membrane, the major constituent of which is lipopolysaccharide (LPS). This contains surface exposed phosphate and pyrophosphate groups and hence can be targeted by nisin. Here we describe the impact of LPS on membrane stability in response to nisin and the molecular interactions occurring between nisin and membrane-embedded LPS from different Gram-negative bacteria. Dye release from liposomes shows enhanced susceptibility to nisin in the presence of LPS, particularly rough LPS chemotypes that lack an O-antigen whereas LPS from microorganisms sharing similar ecological niches with antimicrobial producers provides only modest enhancement. Increased susceptibility was observed with LPS from pathogenic Klebsiella pneumoniae compared to LPS from enteropathogenic Salmonella enterica and gut commensal Escherichia coli. LPS from Brucella melitensis, an intra-cellular pathogen which is adapted to invade professional and non-professional phagocytes, appears to be refractory to nisin. Molecular complex formation between nisin and LPS was studied by solid state MAS NMR and revealed complex formation between nisin and LPS from most organisms investigated except B. melitensis. LPS/nisin complex formation was confirmed in outer membrane extracts from E. coli.

To Boil an Egg: Substrate Binding Affects Critical Stability in Thermal Unfolding of Proteins.

Thermal unfolding of proteins is used extensively in screening of drug candidates because molecular interactions with ligands and substrates affect strongly protein stability, transition temperature, and cooperativity. We use synchrotron radiation circular dichroism to monitor the thermal evolution of secondary structure in proteins as they approach the melting point and the impact of substrate on their thermal behavior. Using Landau free energy expansion, we quantify transition strength and proximity to a critical point through the relative separation τ+ between the transition temperature Tm and the spinodal T+, obtained from the equation of state. The weakest transition was observed in lysozyme with τ+ = -0.0167 followed by holo albumin with τ+ = -0.0208 with the strongest transition in monomeric apo albumin τ+ = -0.0242. A structural transition at 45 °C in apo albumin leads to a noncooperative melt with τ+ = -0.00532 and amyloidogenic increase in beta content.

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser-sintered titanium alloy constructs.

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X-ray spectroscopy; and transmission electron microscopy (TEM) for high-resolution imaging. FIB was used to prepare thin sections of the cell-material interface for TEM, or for three-dimensional electron tomography. Cells were cultured on laser-sintered Ti-6Al-4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell-material cross-sectional interface at the single cell level across multiple high-resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell-material interface.

NMR of Membrane Proteins: Beyond Crystals.

Membrane proteins are essential for the flow of signals, nutrients and energy between cells and between compartments of the cell. Their mechanisms can only be fully understood once the precise structures, dynamics and interactions involved are defined at atomic resolution. Through advances in solution and solid state NMR spectroscopy, this information is now available, as demonstrated by recent studies of stable peripheral and transmembrane proteins. Here we highlight recent cases of G-protein coupled receptors, outer membrane proteins, such as VDAC, phosphoinositide sensors, such as the FAPP-1 pleckstrin homology domain, and enzymes including the metalloproteinase MMP-12. The studies highlighted have resulted in the determination of the 3D structures, dynamical properties and interaction surfaces for membrane-associated proteins using advanced isotope labelling strategies, solubilisation systems and NMR experiments designed for very high field magnets. Solid state NMR offers further insights into the structure and multimeric assembly of membrane proteins in lipid bilayers, as well as into interactions with ligands and targets. Remaining challenges for wider application of NMR to membrane structural biology include the need for overexpression and purification systems for the production of isotope-labelled proteins with fragile folds, and the availability of only a few expensive perdeuterated detergents.Step changes that may transform the field include polymers, such as styrene maleic acid, which obviate the need for detergent altogether, and allow direct high yield purification from cells or membranes. Broader demand for NMR may be facilitated by MODA software, which instantly predicts membrane interactive residues that can subsequently be validated by NMR. In addition, recent developments in dynamic nuclear polarization NMR instrumentation offer a remarkable sensitivity enhancement from low molarity samples and cell surfaces. These advances illustrate the current capabilities and future potential of NMR for membrane protein structural biology and ligand discovery.

Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A (13)C MAS NMR Study.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state (13)C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol (13)C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.

Craniosynostosis and Resynostosis: Models, Imaging, and Dental Implications.

Craniosynostosis occurs in approximately 1 in 2,000 children and results from the premature fusion of ≥1 cranial sutures. If left untreated, craniosynostosis can cause numerous complications as related to an increase in intracranial pressure or as a direct result from cranial deformities, or both. More than 100 known mutations may cause syndromic craniosynostosis, but the majority of cases are nonsyndromic, occurring as isolated defects. Most cases of craniosynostosis require complex cranial vault reconstruction that is associated with a high risk of morbidity. While the first operation typically has few complications, bone rapidly regrows in up to 40% of children who undergo it. This resynostosis typically requires additional surgical intervention, which can be associated with a high incidence of life-threatening complications. This article reviews work related to the dental and maxillofacial implications of craniosynostosis and discusses clinically relevant animal models related to craniosynostosis and resynostosis. In addition, information is provided on the imaging modalities used to study cranial defects in animals and humans.

Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation.

Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration.

Novel Osteogenic Ti-6Al-4V Device For Restoration Of Dental Function In Patients With Large Bone Deficiencies: Design, Development And Implementation.

Custom devices supporting bone regeneration and implant placement are needed for edentulous patients with large mandibular deficiencies where endosteal implantation is not possible. We developed a novel subperiosteal titanium-aluminum-vanadium bone onlay device produced by additive manufacturing (AM) and post-fabrication osteogenic micro-/nano-scale surface texture modification. Human osteoblasts produced osteogenic and angiogenic factors when grown on laser-sintered nano-/micro-textured surfaces compared to smooth surfaces. Surface-processed constructs caused higher bone-to-implant contact, vertical bone growth into disk pores (microCT and histomorphometry), and mechanical pull-out force at 5 and 10 w on rat calvaria compared to non surface-modified constructs, even when pre-treating the bone to stimulate osteogenesis. Surface-modified wrap-implants placed around rabbit tibias osseointegrated by 6 w. Finally, patient-specific constructs designed to support dental implants produced via AM and surface-processing were implanted on edentulous mandibular bone. 3 and 8 month post-operative images showed new bone formation and osseointegration of the device and indicated stability of the dental implants.