Disruption of Genes Encoding Putative Zwitterionic Capsular Polysaccharides of Diverse Intestinal Bacteroides Reduces the Induction of Host Anti-Inflammatory Factors.

Bacterial zwitterionic capsular polysaccharides (ZPS), such as polysaccharide A (PSA) of the intestinal commensal Bacteroides fragilis, have been shown to modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). We previously used a genomic screen to identify diverse host-associated bacteria with the predicted genetic capacity to produce ZPSs related to PSA of B. fragilis and hypothesized that genetic disruption (KO) of a key functional gene within these operons would reduce the anti-inflammatory activity of these bacteria. We found that ZPS-KO bacteria in two common gut commensals, Bacteroides uniformis and Bacteroides cellulosilyticus, had a reduced ability to induce Tregs and IL-10 in stimulations of human peripheral blood mononuclear cells (PBMCs). Additionally, we found that macrophage stimulated with either wildtype B. fragilis or B. uniformis produced significantly more IL-10 than KOs, indicating a potentially novel function of ZPS of shifting the cytokine response in macrophages to a more anti-inflammatory state. These findings support the hypothesis that these related ZPS may represent a shared strategy to modulate host immune responses.

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2.

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4+ and CD8+ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV1), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.

Elevated inflammatory fecal immune factors in men who have sex with men with HIV associate with microbiome composition and gut barrier function.

Introduction: People living with HIV infection (PLWH) exhibit elevated levels of gastrointestinal inflammation. Potential causes of this inflammation include HIV infection and associated immune dysfunction, sexual behaviors among men who have sex with men (MSM) and gut microbiome composition. Methods: To better understand the etiology of gastrointestinal inflammation we examined levels of 28 fecal soluble immune factors (sIFs) and the fecal microbiome in well-defined cohorts of HIV seronegative MSM (MSM-SN), MSM with untreated HIV infection (MSM-HIV) and MSM with HIV on anti-retroviral treatment (MSMART). Additionally, fecal solutes from these participants were used to stimulate T-84 colonic epithelial cells to assess barrier function. Results: Both MSM cohorts with HIV had elevated levels of fecal calprotectin, a clinically relevant marker of GI inflammation, and nine inflammatory fecal sIFs (GM-CSF, ICAM-1, IL-1ß, IL-12/23, IL-15, IL-16, TNF-ß, VCAM-1, and VEGF). Interestingly, four sIFs (GM-CSF, ICAM-1, IL-7 and IL-12/23) were significantly elevated in MSM-SN compared to seronegative male non-MSM. Conversely, IL-22 and IL-13, cytokines beneficial to gut health, were decreased in all MSM with HIV and MSM-SN respectively. Importantly, all of these sIFs significantly correlated with calprotectin, suggesting they play a role in GI inflammation. Principal coordinate analysis revealed clustering of fecal sIFs by MSM status and significant associations with microbiome composition. Additionally, fecal solutes from participants in the MSM-HIV cohort significantly decreased colonic transcellular fluid transport in vitro, compared to non-MSM-SN, and this decrease associated with overall sIF composition and increased concentrations of eight inflammatory sIFs in participants with HIV. Lastly, elevated levels of plasma, sCD14 and sCD163, directly correlated with decreased transcellular transport and microbiome composition respectively, indicating that sIFs and the gut microbiome are associated with, and potentially contribute to, bacterial translocation. Conclusion: Taken together, these data demonstrate that inflammatory sIFs are elevated in MSM, regardless of HIV infection status, and are associated with the gut microbiome and intestinal barrier function.

Intestinal microbial communities and Holdemanella isolated from HIV+/- men who have sex with men increase frequencies of lamina propria CCR5+ CD4+ T cells.

Men who have sex with men (MSM), regardless of HIV infection status, have an intestinal microbiome that is compositionally distinct from men who have sex with women (MSW) and women. We recently showed HIV-negative MSM have elevated levels of intestinal CD4+ T cells expressing CCR5, a critical co-receptor for HIV. Whether elevated expression of CCR5 is driven by the altered gut microbiome composition in MSM has not been explored. Here we used in vitro stimulation of gut Lamina Propria Mononuclear Cells (LPMCs) with whole intact microbial cells isolated from stool to demonstrate that fecal bacterial communities (FBCs) from HIV-positive/negative MSM induced higher frequencies of CCR5+ CD4+ T cells compared to FBCs from HIV-negative MSW and women. To identify potential microbial drivers, we related the frequency of CCR5+ CD4+ T cells to the abundance of individual microbial taxa in rectal biopsy of HIV-positive/negative MSM and controls, and Holdemanella biformis was strongly associated with increased frequency of CCR5+ CD4+ T cells. We used in vitro stimulation of gut LPMCs with the type strain of H. biformis, a second strain of H.biformis and an isolate of the closely related Holdemanella porci , cultured from either a HIV-positive or a HIV-negative MSM stool. H. porci elevated the frequency of both CCR5+ CD4+ T cells and the ratio of TNF-α/IL-10 Genomic comparisons of the 3 Holdemanella isolates revealed unique cell wall and capsular components, which may be responsible for their differences in immunogenicity. These findings describe a novel mechanism potentially linking intestinal dysbiosis in MSM to HIV transmission and mucosal pathogenesis.

Systems Analysis of Gut Microbiome Influence on Metabolic Disease in HIV-Positive and High-Risk Populations.

Poor metabolic health, characterized by insulin resistance and dyslipidemia, is higher in people living with HIV and has been linked with inflammation, antiretroviral therapy (ART) drugs, and ART-associated lipodystrophy (LD). Metabolic disease is associated with gut microbiome composition outside the context of HIV but has not been deeply explored in HIV infection or in high-risk men who have sex with men (HR-MSM), who have a highly altered gut microbiome composition. Furthermore, the contribution of increased bacterial translocation and associated systemic inflammation that has been described in HIV-positive and HR-MSM individuals has not been explored. We used a multiomic approach to explore relationships between impaired metabolic health, defined using fasting blood markers, gut microbes, immune phenotypes, and diet. Our cohort included ART-treated HIV-positive MSM with or without LD, untreated HIV-positive MSM, and HR-MSM. For HIV-positive MSM on ART, we further explored associations with the plasma metabolome. We found that elevated plasma lipopolysaccharide binding protein (LBP) was the most important predictor of impaired metabolic health and network analysis showed that LBP formed a hub joining correlated microbial and immune predictors of metabolic disease. Taken together, our results suggest the role of inflammatory processes linked with bacterial translocation and interaction with the gut microbiome in metabolic disease among HIV-positive and -negative MSM.IMPORTANCE The gut microbiome in people living with HIV (PLWH) is of interest since chronic infection often results in long-term comorbidities. Metabolic disease is prevalent in PLWH even in well-controlled infection and has been linked with the gut microbiome in previous studies, but little attention has been given to PLWH. Furthermore, integrated analyses that consider gut microbiome, together with diet, systemic immune activation, metabolites, and demographics, have been lacking. In a systems-level analysis of predictors of metabolic disease in PLWH and men who are at high risk of acquiring HIV, we found that increased lipopolysaccharide-binding protein, an inflammatory marker indicative of compromised intestinal barrier function, was associated with worse metabolic health. We also found impaired metabolic health associated with specific dietary components, gut microbes, and host and microbial metabolites. This study lays the framework for mechanistic studies aimed at targeting the microbiome to prevent or treat metabolic endotoxemia in HIV-infected individuals.

Correction to: An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

Following publication of the original article [1], the authors reported an error in Fig. 2. The original Fig. 2 has been incorrectly replaced with the Supplementary Fig. 2. The correct Fig. 2 is presented here.

Can gut microbiota of men who have sex with men influence HIV transmission?

Gaining a complete understanding of transmission risk factors will assist in efforts to reduce new HIV infections, especially within the disproportionally affected population of men who have sex with men (MSM). We recently reported that the fecal microbiota of MSM elevates immune activation in gnotobiotic mice and enhances HIV infection in vitro over that of fecal microbiota from men who have sex with women. We also demonstrated elevation of the gut homing marker CD103 (integrin αE) on CD4+ T cells by MSM-microbiota. Here we provide additional evidence that the gut microbiota is a risk factor for HIV transmission in MSM by showing elevated frequencies of the HIV co-receptor CCR5 on CD4+ T cells in human rectosigmoid colon biopsies. We discuss our interest in specific MSM-associated bacteria and propose the influx of CD103+ and CCR5+ CD4+ T cells into the colon as a potential link between the MSM microbiota and HIV transmission.

Human mesenchymal stem cell morphology, migration, and differentiation on micro and nano-textured titanium.

Orthopedic implants rely on facilitating a robust interaction between the implant material surface and the surrounding bone tissue. Ideally, the interface will encourage osseointegration with the host bone, resulting in strong fixation and implant stability. However, implant failure can occur due to the lack of integration with bone tissue or bacterial infection. The chosen material and surface topography of orthopedic implants are key factors that influence the early events following implantation and may ultimately define the success of a device. Early attachment, rapid migration and improved differentiation of stem cells to osteoblasts are necessary to populate the surface of biomedical implants, potentially preventing biofilm formation and implant-associated infection. This article explores these early stem cell specific events by seeding human mesenchymal stem cells (MSCs) on four clinically relevant materials: polyether ether ketone (PEEK), Ti6Al4V (smooth Ti), macro-micro rough Ti6Al4V (Endoskeleton®), and macro-micro-nano rough Ti6Al4V (nanoLOCK®). The results demonstrate the incorporation of a hierarchical macro-micro-nano roughness on titanium produces a stellate morphology typical of mature osteoblasts/osteocytes, rapid and random migration, and improved osteogenic differentiation in seeded MSCs. Literature suggests rapid coverage of a surface by stem cells coupled with stimulation of bone differentiation minimizes the opportunity for biofilm formation while increasing the rate of device integration with the surrounding bone tissue.

VOLARE: visual analysis of disease-associated microbiome-immune system interplay.

BACKGROUND: Relationships between specific microbes and proper immune system development, composition, and function have been reported in a number of studies. However, researchers have discovered only a fraction of the likely relationships. "Omic" methodologies such as 16S ribosomal RNA (rRNA) sequencing and time-of-flight mass cytometry (CyTOF) immunophenotyping generate data that support generation of hypotheses, with the potential to identify additional relationships at a level of granularity ripe for further experimentation. Pairwise linear regressions between microbial and host immune features provide one approach for quantifying relationships between "omes", and the differences in these relationships across study cohorts or arms. This approach yields a top table of candidate results. However, the top table alone lacks the detail that domain experts such as microbiologists and immunologists need to vet candidate results for follow-up experiments. RESULTS: To support this vetting, we developed VOLARE (Visualization Of LineAr Regression Elements), a web application that integrates a searchable top table, small in-line graphs illustrating the fitted models, a network summarizing the top table, and on-demand detailed regression plots showing full sample-level detail. We applied VOLARE to three case studies-microbiome:cytokine data from fecal samples in human immunodeficiency virus (HIV), microbiome:cytokine data in inflammatory bowel disease and spondyloarthritis, and microbiome:immune cell data from gut biopsies in HIV. We present both patient-specific phenomena and relationships that differ by disease state. We also analyzed interaction data from system logs to characterize usage scenarios. This log analysis revealed that users frequently generated detailed regression plots, suggesting that this detail aids the vetting of results. CONCLUSIONS: Systematically integrating microbe:immune cell readouts through pairwise linear regressions and presenting the top table in an interactive environment supports the vetting of results for scientific relevance. VOLARE allows domain experts to control the analysis of their results, screening dozens of candidate relationships with ease. This interactive environment transcends the limitations of a static top table.

Gut microbiota from high-risk men who have sex with men drive immune activation in gnotobiotic mice and in vitro HIV infection.

Men who have sex with men (MSM) have differences in immune activation and gut microbiome composition compared with men who have sex with women (MSW), even in the absence of HIV infection. Gut microbiome differences associated with HIV itself when controlling for MSM, as assessed by 16S rRNA sequencing, are relatively subtle. Understanding whether gut microbiome composition impacts immune activation in HIV-negative and HIV-positive MSM has important implications since immune activation has been associated with HIV acquisition risk and disease progression. To investigate the effects of MSM and HIV-associated gut microbiota on immune activation, we transplanted feces from HIV-negative MSW, HIV-negative MSM, and HIV-positive untreated MSM to gnotobiotic mice. Following transplant, 16S rRNA gene sequencing determined that the microbiomes of MSM and MSW maintained distinct compositions in mice and that specific microbial differences between MSM and MSW were replicated. Immunologically, HIV-negative MSM donors had higher frequencies of blood CD38+ HLADR+ and CD103+ T cells and their fecal recipients had higher frequencies of gut CD69+ and CD103+ T cells, compared with HIV-negative MSW donors and recipients, respectively. Significant microbiome differences were not detected between HIV-negative and HIV-positive MSM in this small donor cohort, and immune differences between their recipients were trending but not statistically significant. A larger donor cohort may therefore be needed to detect immune-modulating microbes associated with HIV. To investigate whether our findings in mice could have implications for HIV replication, we infected primary human lamina propria cells stimulated with isolated fecal microbiota, and found that microbiota from MSM stimulated higher frequencies of HIV-infected cells than microbiota from MSW. Finally, we identified several microbes that correlated with immune readouts in both fecal recipients and donors, and with in vitro HIV infection, which suggests a role for gut microbiota in immune activation and potentially HIV acquisition in MSM.

Endplate Deformation Due to Open and Strutted Intervertebral Devices.

BACKGROUND: There is an absence of work on vertebral endplate response to peripheral loading following disc removal and interbody placement. Endplate deflection into the interbody space may impart beneficial strain on the developing fusion mass, influencing bone formation and remodeling. The aim of this study was to verify endplate deformation due to peripheral loading using a custom transducer and to investigate whether endplate motion is inhibited by implant design. METHODS: A total of 14 porcine (L4, L5) vertebrae were assigned to open or strutted implant designs. A custom transducer was placed on the endplate while 500 N was applied to the implant at 1 Hz for 500 cycles. Endplate motion was acquired for each time point and averaged among specimens of the same design. The rates and magnitudes of endplate deformation were compared between implant designs using unpaired t tests. RESULTS: Peripheral loading of both implant designs resulted in endplate deflection into the interbody space. The open implant design demonstrated an increased rate and magnitude of endplate deformation when compared with strutted implants. CONCLUSION: Interbody cage design directly influences the dynamic motion of the vertebral endplate during cyclic loading. A larger, faster deflection of the endplate could increase the strain rate, duration, and magnitude on the developing interbody fusion mass. These parameters of dynamic strain have been correlated with increased bone formation and remodeling. CLINICAL RELEVANCE: Unimpeded endplate deformation in an open cage design could impart a strain pattern on the developing fusion mass that increases bone formation and remodeling, ultimately leading to a faster and stronger fusion.

An exploration of Prevotella-rich microbiomes in HIV and men who have sex with men.

BACKGROUND: Gut microbiome characteristics associated with HIV infection are of intense research interest but a deep understanding has been challenged by confounding factors across studied populations. Notably, a Prevotella-rich microbiome described in HIV-infected populations is now understood to be common in men who have sex with men (MSM) regardless of HIV status, but driving factors and potential health implications are unknown. RESULTS: Here, we further define the MSM-associated gut microbiome and describe compositional differences between the fecal microbiomes of Prevotella-rich MSM and non-MSM that may underlie observed pro-inflammatory properties. Furthermore, we show relatively subtle gut microbiome changes in HIV infection in MSM and women that include an increase in potential pathogens that is ameliorated with antiretroviral therapy (ART). Lastly, using a longitudinal cohort, we describe microbiome changes that happen after ART initiation. CONCLUSIONS: This study provides an in-depth characterization of microbiome differences that occur in a US population infected with HIV and demonstrates the degree to which these differences may be driven by lifestyle factors, ART, and HIV infection itself. Understanding microbiome compositions that occur with sexual behaviors that are high risk for acquiring HIV and untreated and ART-treated HIV infection will guide the investigation of immune and metabolic functional implications to ultimately target the microbiome therapeutically.

Afferent neural feedback overrides the modulating effects of arousal, hypercapnia and hypoxaemia on neonatal cardiorespiratory control.

KEY POINTS: Evidence obtained at whole animal, organ-system, and cellular and molecular levels suggests that afferent volume feedback is critical for the establishment of adequate ventilation at birth. As a result of the irreversible nature of the vagal ablation studies performed to date, it was difficult to quantify the roles of afferent volume input, arousal and changes in blood gas tensions on neonatal respiratory control. During reversible perineural vagal block, profound apnoeas and hypoxaemia and hypercarbia were observed, necessitating the termination of perineural blockade. Respiratory depression and apnoeas were independent of sleep state. We demonstrate that profound apnoeas and life-threatening respiratory failure in vagally denervated animals do not result from a lack of arousal or hypoxaemia. A change in sleep state and concomitant respiratory depression result from a lack of afferent volume feedback, which appears to be critical for the maintenance of normal breathing patterns and adequate gas exchange during the early postnatal period. ABSTRACT: Afferent volume feedback plays a vital role in neonatal respiratory control. Mechanisms for the profound respiratory depression and life-threatening apnoeas observed in vagally denervated neonatal animals remain unclear. We investigated the roles of sleep states, hypoxic-hypercapnia and afferent volume feedback on respiratory depression using reversible perineural vagal block during the early postnatal period. Seven lambs were instrumented during the first 48 h of life to record/analyse sleep states, diaphragmatic electromyograph, arterial blood gas tensions, systemic arterial blood pressure and rectal temperature. Perineural cuffs were placed around the vagi to attain reversible blockade. Postoperatively, during the awake state, both vagi were blocked using 2% xylocaine for up to 30 min. Compared to baseline values, pHa , Pao2 and Sao2 decreased and Paco2 increased during perineural blockade (P < 0.05). Four of seven animals exhibited apnoeas of ≥20 s requiring the immediate termination of perineural blockade. Breathing rates decreased from the baseline value of 53 ± 12 to 24 ± 20 breaths min-1 during blockade despite an increased Paco2 (P < 0.001). Following blockade, breathing patterns returned to baseline values despite marked hypocapnia ( Paco2 33 ± 3 torr; P = 0.03). Respiratory depression and apnoeas were independent of sleep states. The present study provides the much needed physiological evidence indicating that profound apnoeas and life-threatening respiratory failure in vagally denervated animals do not result from a lack of arousal or hypoxaemia. Rather, a change in sleep state and concomitant respiratory depression result from a lack of afferent volume feedback, which appears to be critical for the maintenance of normal breathing patterns and adequate gas exchange during the early postnatal period.

Fecal Microbiota Composition Drives Immune Activation in HIV-infected Individuals.

The inflammatory properties of the enteric microbiota of Human Immunodeficiency Virus (HIV)-infected individuals are of considerable interest because of strong evidence that bacterial translocation contributes to chronic immune activation and disease progression. Altered enteric microbiota composition occurs with HIV infection but whether altered microbiota composition or increased intestinal permeability alone drives peripheral immune activation is controversial. To comprehensively assess the inflammatory properties of HIV-associated enteric microbiota and relate these to systemic immune activation, we developed methods to purify whole fecal bacterial communities (FBCs) from stool for use in in vitro immune stimulation assays with human cells. We show that the enteric microbiota of untreated HIV-infected subjects induce significantly higher levels of activated monocytes and T cells compared to seronegative subjects. FBCs from anti-retroviral therapy (ART)-treated HIV-infected individuals induced intermediate T cell activation, indicating an only partial correction of adaptive immune cell activation capacity of the microbiome with ART. In vitro activation levels correlated with activation levels and viral load in blood and were particularly high in individuals harboring specific gram-positive opportunistic pathogens. Blockade experiments implicated Tumor Necrosis Factor (TNF)-α and Toll-Like Receptor-2 (TLR2), which recognizes peptidoglycan, as strong mediators of T cell activation; This may contradict a previous focus on lipopolysaccharide as a primary mediator of chronic immune activation. These data support that increased inflammatory properties of the enteric microbiota and not increased permeability alone drives chronic inflammation in HIV.

Diverse Intestinal Bacteria Contain Putative Zwitterionic Capsular Polysaccharides with Anti-inflammatory Properties.

Zwitterionic capsular polysaccharides (ZPSs) are bacterial products that modulate T cells, including inducing anti-inflammatory IL-10-secreting T regulatory cells (Tregs). However, only a few diverse bacteria are known to modulate the host immune system via ZPS. We present a genomic screen for bacteria encoding ZPS molecules. We identify diverse host-associated bacteria, including commensals and pathogens with known anti-inflammatory properties, with the capacity to produce ZPSs. Human mononuclear cells stimulated with lysates from putative ZPS-producing bacteria induce significantly greater IL-10 production and higher proportions of Tregs than lysates from non-ZPS-encoding relatives or a commensal strain of Bacteroides cellulosilyticus in which a putative ZPS biosynthetic operon was genetically disrupted. Similarly, wild-type B. cellulosilyticus DSM 14838, but not a close relative lacking a putative ZPS, attenuated experimental colitis in mice. Collectively, this screen identifies bacterial strains that may use ZPSs to interact with the host as well as those with potential probiotic properties.

Long-distance communication facilitates cooperation among wild spotted hyaenas, Crocuta crocuta.

Calls that catalyse group defence, as in the mobbing of predators, appear to facilitate cooperation by recruiting receivers to act collectively. However, even when such signals reliably precede cooperative behaviour, the extent to which the calls function as recruitment signals is unclear. Calls might simply arouse listeners' attention, setting off a cascade of independent responses to the threat. By contrast, they might convey information, for example, about signaller identity and the nature of a threat that affects receivers' decisions to participate. We explored this distinction by investigating a possible long-distance recruitment call used by spotted hyaenas. These social carnivores live in fission-fusion clans and individuals disperse widely within their territories. Putative recruitment calls must therefore attract receivers that are distant from the inciting threat and free to opt out of risky collective aggression. Hyaenas compete with lions over food, and neighbouring clans sometimes engage in violent border clashes. These high-stakes contests are decided based on numerical asymmetries, so hyaenas can only protect critical resources if the dispersed clan can converge quickly at conflict sites. We recorded and analysed whoop bouts produced in multiple contexts and found that bouts produced in response to signs of lion-hyaena conflict had shorter inter-whoop intervals than spontaneous 'display' bouts. In subsequent field playback experiments, resting hyaenas were significantly more likely to move in response to 'recruitment' bouts with shortened intervals than to otherwise identical 'display' bouts. Whereas only stimulus type predicted movement, lower-ranked subjects responded most quickly, perhaps because their feeding opportunities depend on arriving early at any kill site. Results demonstrate that hyaenas possess a signal that can reliably recruit allies across long distances, despite moderating effects of individual circumstances on the strength of receivers' responses.

Implant materials generate different peri-implant inflammatory factors: poly-ether-ether-ketone promotes fibrosis and microtextured titanium promotes osteogenic factors.

STUDY DESIGN: An in vitro study examining factors produced by human mesenchymal stem cells on spine implant materials. OBJECTIVE: The aim of this study was to examine whether the inflammatory microenvironment generated by cells on titanium-aluminum-vanadium (Ti-alloy, TiAlV) surfaces is affected by surface microtexture and whether it differs from that generated on poly-ether-ether-ketone (PEEK). SUMMARY OF BACKGROUND DATA: Histologically, implants fabricated from PEEK have a fibrous connective tissue surface interface whereas Ti-alloy implants demonstrate close approximation with surrounding bone. Ti-alloy surfaces with complex micron/submicron scale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation whereas PEEK favors fibrous tissue formation. METHODS: Human mesenchymal stem cells were cultured on tissue culture polystyrene, PEEK, smooth TiAlV, or macro-/micro-/nano-textured rough TiAlV (mmnTiAlV) disks. Osteoblastic differentiation and secreted inflammatory interleukins were assessed after 7 days. Fold changes in mRNAs for inflammation, necrosis, DNA damage, or apoptosis with respect to tissue culture polystyrene were measured by low-density polymerase chain reaction array. Data were analyzed by analysis of variance, followed by Bonferroni's correction of Student's t-test. RESULTS: Cells on PEEK upregulated mRNAs for chemokine ligand-2, interleukin (IL) 1ß, IL6, IL8, and tumor necrosis factor. Cells grown on the mmnTiAlV had an 8-fold reduction in mRNAs for toll-like receptor-4. Cells grown on mmnTiAlV had reduced levels of proinflammatory interleukins. Cells on PEEK had higher mRNAs for factors strongly associated with cell death/apoptosis, whereas cells on mmnTiAlV exhibited reduced cytokine factor levels. All results were significant (P < 0.05). CONCLUSION: These results suggest that fibrous tissue around PEEK implants may be due to several factors: reduced osteoblastic differentiation of progenitor cells and production of an inflammatory environment that favors cell death via apoptosis and necrosis. Ti alloy surfaces with complex macro/micro/nanoscale roughness promote osteoblastic differentiation and foster a specific cellular environment that favors bone formation. LEVEL OF EVIDENCE: N/A.

Osteoblast lineage cells can discriminate microscale topographic features on titanium-aluminum-vanadium surfaces.

Titanium (Ti) and Ti alloys are used in orthopaedic/spine applications where biological implant fixation, or osseointegration, is required for long-term stability. These implants employ macro-scale features to provide mechanical stability until arthrodesis, features that are too large to influence healing at the cellular level. Micron-scale rough Ti alloy (Ti-6Al-4V) increases osteoblastic differentiation and osteogenic factor production in vitro and increases in vivo bone formation; however, effects of overall topography, including sub-micron scale and nanoscale features, on osteoblast lineage cells are less well appreciated. To address this, Ti6Al4V surfaces with macro/micro/nano-textures were generated using sand blasting and acid etching that had comparable average roughness values but differed in other roughness parameters (total roughness, profile roughness, maximum peak height, maximum valley depth, root-mean-squared roughness, kurtosis, skewness) (#5, #9, and #12). Human mesenchymal stem cells (HMSCs) and normal human osteoblasts (NHOst) were cultured for 7 days on the substrates and then analyzed for alkaline phosphatase activity and osteocalcin content, production of osteogenic local factors, and integrin subunit expression. All three surfaces supported osteoblastic differentiation of HMSCs and further maturation of NHOst cells, but the greatest response was seen on the #9 substrate, which had the lowest skewness and kurtosis. The #9 surface also induced highest expression of α2 and ß1 integrin mRNA. HMSCs produced highest levels of ITGAV on #9, suggesting this integrin may play a role for early lineage cells. These results indicate that osteoblast lineage cells are sensitive to specific micro/nanostructures, even when overall macro roughness is comparable and suggest that skewness and kurtosis are important variables.

A bidirectional model of postural sway using force plate data.

This work was designed to expand on our previous anterior-posterior postural control model to include medial-lateral sway of unperturbed posture during quiet standing. The bidirectional model simulates two decoupled inverted pendulums, each restricted to sway in either the anterior-posterior (AP) direction (ankle strategy) or medial-lateral (ML) direction (hip strategy), and each controlled by a Proportional-Integral-Derivative (PID) controller. Postural data was collected from 31 healthy participants under different sensory test conditions: eyes closed, eyes open, and eyes open with real-time visual feedback. Simulation iterations of the bidirectional model were run for each sensory test condition to adjust the PID controller parameters until modeled sway metrics did not differ significantly from experimental metrics at p ≤ 0.01. Simulations did not show significant changes in the AP sway controller parameters among the 3 sensory test conditions. The model did show significant changes in ML sway controller parameters, namely stiffness and time delay. Significant differences were also seen in the experimental sway metrics under the three different sensory test conditions. The multi-sensory evaluation and bidirectional sway model offer unique insight for further exploration of postural pathology, control mechanisms and planar coupling that includes both ankle and hip strategies.

Rough titanium alloys regulate osteoblast production of angiogenic factors.

BACKGROUND CONTEXT: Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin α2ß1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK. PURPOSE: The purpose of this study was to determine if osteoblasts generate an environment that supports angiogenesis and reduces osteoclastic activity when grown on smooth titanium alloy, rough titanium alloy, or PEEK. STUDY DESIGN: This in vitro study compared angiogenic factor production and integrin gene expression of human osteoblast-like MG63 cells cultured on PEEK or titanium-aluminum-vanadium (titanium alloy). METHODS: MG63 cells were grown on PEEK, smooth titanium alloy, or rough titanium alloy. Osteogenic microenvironment was characterized by secretion of osteoprotegerin and transforming growth factor beta-1 (TGF-ß1), which inhibit osteoclast activity and angiogenic factors including vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and angiopoietin-1 (ANG-1). Expression of integrins, transmembrane extracellular matrix recognition proteins, was measured by real-time polymerase chain reaction. RESULTS: Culture on titanium alloy stimulated osteoprotegerin, TGF-ß1, VEGF-A, FGF-2, and angiopoietin-1 production, and levels were greater on rough titanium alloy than on smooth titanium alloy. All factors measured were significantly lower on PEEK than on smooth or rough titanium alloy. Culture on titanium alloy stimulated expression of messenger RNA for integrins that recognize Type I collagen in comparison with PEEK. CONCLUSIONS: Rough titanium alloy stimulated cells to create an osteogenic-angiogenic microenvironment. The osteogenic-angiogenic responses to titanium alloy were greater than PEEK and greater on rough titanium alloy than on smooth titanium alloy. Surface features regulated expression of integrins important in collagen recognition. These factors may increase bone formation, enhance integration, and improve implant stability in interbody spinal fusions.