Ursodeoxycholic acid alleviates sepsis-induced lung injury by blocking PANoptosis via STING pathway.

Acute lung injury (ALI), a progressive lung disease mostly caused by sepsis, is characterized by uncontrolled inflammatory responses, increased oxidative stress, pulmonary barrier dysfunction, and pulmonary edema. Ursodeoxycholic acid (UDCA) is a natural bile acid with various pharmacological properties and is extensively utilized in clinical settings for the management of hepatobiliary ailments. Nonetheless, the potential protective effects and mechanism of UDCA on sepsis-induced lung injuries remain unknown. In this study, we reported that UDCA effectively inhibited pulmonary edema, inflammatory cell infiltration, pro-inflammatory cytokines production, and oxidative stress. Furthermore, UDCA treatment significantly alleviated the damage of pulmonary barrier and enhanced alveolar fluid clearance. Importantly, UDCA treatment potently suppressed PANoptosis-like cell death which is demonstrated by the block of apoptosis, pyroptosis, and necroptosis. Mechanistically, UDCA treatment prominently inhibited STING pathway. And the consequential loss of STING substantially impaired the beneficial effects of UDCA treatment on the inflammatory response, pulmonary barrier, and PANoptosis. These results indicate that STING plays a pivotal role in the UDCA treatment against sepsis-induced lung injury. Collectively, our findings show that UDCA treatment can ameliorate sepsis-induced lung injury and verified a previously unrecognized mechanism by which UDCA alleviated sepsis-induced lung injury through blocking PANoptosis-like cell death via STING pathway.

Deciphering Cell-Cell Interactions with Integrative Single-Cell Secretion Profiling.

Cell-cell interactions are the fundamental behaviors to regulate cellular activities. A comprehensive evaluation of intercellular interactions requires direct profiling of various signaling behaviors simultaneously at the single-cell level, which remains lacking. Herein, an integrative single-cell secretion analysis platform is presented to profile different secreted factors (four proteins, three extracellular vesicles (EV) phenotypes), spatial distances, and migration information (distances and direction) simultaneously from high-throughput paired single cells using an antibody-barcode microchip. Applying the platform to analyze the tumor-stromal and tumor-immune interactions with the human oral squamous cell carcinoma (OSCC) cell lines and primary OSCC cells reveals that the initial distances between cells would determine their migratory distances and direction to approach stable organization. The cell-cell in close proximity enhances protein secretions while attenuating EV secretions. Migration has a more profound correlation with protein secretions than EV secretions, in which absolute migration distance affects protein secretions significantly but not the direction. These findings highlight the significance of spatial organization in regulating cell signaling behaviors and demonstrate that the integrative single-cell secretion profiling platform is well-suited for a comprehensive dissection of intercellular communication and interactions, providing new avenues for understanding cell-cell interaction biology and how different signaling behaviors coordinate within the tumor microenvironment.

Osteogenic and long-term antibacterial properties of Sr/Ag-containing TiO2 microporous coating in vitro and in vivo.

Bacterial infection and poor osseointegration are two critical issues that need to be solved for long-term use of titanium implants. As such, Sr/Ag-containing TiO2 microporous coatings were prepared on a Ti alloy surface in the current study via a single-step microarc oxidation technique. The coatings showed both good cytocompatibility in vitro and biosafety in vivo. Sr/Ag incorporation brought no significant change in the surface micromorphology and physicochemical properties, but endowed the coating with strong osteogenic activity and long-term antibacterial capability in vitro. Furthermore, the osteogenic and antibacterial capability of the coating was also confirmed in vivo. In a rat osseointegration model, new bone formation, implant-bone contact, removal torque and bone mineralization were all significantly increased in the M-Sr/Ag group when compared with those in group M, although they were slightly lower than those in group M-Sr. In a periimplantitis model, no rats suffered infection in the M-Sr/Ag group after 3 months of osseointegration and 5 weeks of bacterial inoculation period, when compared to 100% and 75% infection rates in M and M-Sr groups, respectively. In addition, active bone remodeling and many mesenchymal cells were observed in the M-Sr group, suggesting good bone regeneration potential in Sr-containing coatings in the case of controlled periimplantitis. Overall, the Sr/Ag-containing TiO2 microporous coating is valuable for preventing periimplantitis and improving implant reosseointegration, and is therefore promising for long-term and high quality use of titanium implants.

The effect of strontium content on physicochemical and osteogenic property of Sr/Ag-containing TiO2 microporous coatings.

Strontium (Sr) is the most common element introduced into TiO2 coatings to strengthen the osteogenic property of titanium implants. However, the optimal Sr content and its effect on osteogenic and physicochemical properties of the coatings need to be clarified. In the current study, TiO2 microporous coatings with different contents of Sr (9.64-21.25 wt %) and silver (Ag) (0.38-0.75 wt %) were prepared via micro-arc oxidation technique. Sr contents did not change physicochemical properties of the coatings, including surface microstructure, micropore size and distribution, phase composition, roughness and hydrophilicity. Meanwhile, higher Sr contents (18.23-21.25 wt %) improved cytocompatibility, proliferation and alkaline phosphatase (ALP) activity of preosteoblasts, even the coatings underwent 30 days' PBS immersion. Furthermore, higher Sr contents facilitated preosteoblast growth and spreading, which are essential for their proliferation and osteogenic differentiation. Therefore, it is promising to incorporate higher Sr content (18.23-21.25 wt %) within TiO2 microporous coatings to improve their osteogenic capability.

Mapping secretome-mediated interaction between paired neuron-macrophage single cells.

Neuron-immune interaction through secreted factors contributes significantly to the complex microenvironment in the central nervous system that could alter cell functionalities and fates in both physiological and pathological conditions, which remains poorly characterized at the single-cell level. Herein, using a spatially patterned antibody barcode microchip, we realized the mapping of 12 different secretomes, covering cytokines, neurotrophic factors (NFs), and neuron-derived exosomes (NDEs) from high-throughput, paired single cells (≥ 600) simultaneously under normal conditions and an Alzheimer's disease (AD) model induced with amyloid beta protein 1-42 (Aß1-42). We applied the platform to analyze the secretion profiles from paired neuron-macrophage and neuron-microglia single cells with human cell lines. We found that pairwise neuron-macrophage interaction would trigger immune responses and attenuate neuron cells' secretion, while neuron-microglia interaction generally results in opposite outcomes in secretion. When neuron cells are induced with Aß1-42 protein into the AD model, both neuron-macrophage and neuron-microglia interactions lead to increased cytokines and NDEs and decreased NFs. Further analysis of AD patients' serum showed that NDEs were significantly higher in patients' samples than in the control group, validating our observation from the interaction assay. Furthermore, we resolved previously undifferentiated heterogeneity underlying the secretions from single-neuron cells. We found that the NDE and NF secretion was less dependent on the paracrine signaling between one another and that secretions from neuron cells would attenuate after differentiation with Aß1-42. This study demonstrates the mapping of the different secretomes from paired neuron-immune single cells, providing avenues for understanding how neurons and immune cells interact through the complex secretome network.

Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics.

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 µL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.

Core fucosylation involvement in the paracrine regulation of proteinuria-induced renal interstitial fibrosis evaluated with the use of a microfluidic chip.

Proteinuria is a clinical manifestation of chronic kidney disease that aggravates renal interstitial fibrosis (RIF), in which injury of peritubular microvessels is an important event. However, the changes in peritubular microvessels induced by proteinuria and their molecular mechanisms remain unclear. Thus, we aimed to develop a co-culture microfluidic device that contains renal tubules and peritubular microvessels to create a proteinuria model. We found that protein overload in the renal tubule induced trans-differentiation and apoptosis of endothelial cells (ECs) and pericytes. Moreover, profiling of secreted proteins in this model revealed that a paracrine network between tubules and microvessels was activated in proteinuria-induced microvascular injury. Multiple cytokine receptors in this paracrine network were core-fucosylated. Inhibition of core fucosylation significantly reduced ligand-receptor binding ability and blocked downstream pathways, alleviating trans-differentiation and apoptosis of ECs and pericytes. Furthermore, the protective effect of genetic FUT8 deficiency on proteinuria overload-induced RIF and pericyte-myofibroblast trans-differentiation was validated in FUT8 knockout heterozygous mice. In conclusion, we constructed and used a multiple-unit integrated microfluidic device to uncover the mechanism of proteinuria-induced RIF. Furthermore, FUT8 may serve as a hub-like therapeutic target to alleviate peritubular microvascular injury in RIF. STATEMENT OF SIGNIFICANCE: In this study, we constructed a multiple-unit integrated renal tubule-vascular chip. We reproduced human proteinuria on the chip and found that multiple receptors were modified by FUT8-catalyzed core fucosylation (CF) involved in the cross-talk between renal tubules and peritubular microvessels in proteinuria-induced RIF, and inhibiting the FUT8 of receptors could block the tubule-microvessel paracrine network and reverse the damage of peritubular microvessels and renal interstitial fibrosis. This tubule-vascular chip may provide a prospective platform to facilitate future investigations into the mechanisms of kidney diseases, and target-FUT8 inhibition may be an innovative and potential therapeutic strategy for RIF induced by proteinuria.

Tanreqing Inhibits LPS-Induced Acute Lung Injury In Vivo and In Vitro Through Downregulating STING Signaling Pathway.

Acute lung injury (ALI) is a common life-threatening lung disease, which is mostly associated with severe inflammatory responses and oxidative stress. Tanreqing injection (TRQ), a Chinese patent medicine, is clinically used for respiratory-related diseases. However, the effects and action mechanism of TRQ on ALI are still unclear. Recently, STING as a cytoplasmic DNA sensor has been found to be related to the progress of ALI. Here, we showed that TRQ significantly inhibited LPS-induced lung histological change, lung edema, and inflammatory cell infiltration. Moreover, TRQ markedly reduced inflammatory mediators release (TNF-α, IL-6, IL-1ß, and IFN-ß). Furthermore, TRQ also alleviated oxidative stress, manifested by increased SOD and GSH activities and decreased 4-HNE, MDA, LDH, and ROS activities. In addition, we further found that TRQ significantly prevented cGAS, STING, P-TBK, P-P65, P-IRF3, and P-IκBα expression in ALI mice. And we also confirmed that TRQ could inhibit mtDNA release and suppress signaling pathway mediated by STING in vitro. Importantly, the addition of STING agonist DMXAA dramatically abolished the protective effects of TRQ. Taken together, this study indicated that TRQ alleviated LPS-induced ALI and inhibited inflammatory responses and oxidative stress through STING signaling pathway.

[Research progress on active components and mechanism of Isatidis Radix for influenza virus].

Isatidis Radix is the dried root of the Isatis indigotica, with pharmacological effects such as heat-clearing and detoxification, cooling blood and pharyngeal relief, antibacterial and anti-inflammatory effects. It is often used clinically to prevent and treat influenza and other diseases. In this paper, relevant domestic and foreign literatures in recent years were summarized, and it was found that Isatidis Radix lignans, indole alkaloids, polysaccharides, etc. were the main active components against influenza virus. Then its pharmacological effects and the mechanism of action were reviewed, providing a basis for in-depth research on the antiviral effect of Isatidis Radix.

Natural product derived phytochemicals in managing acute lung injury by multiple mechanisms.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) as common life-threatening lung diseases with high mortality rates are mostly associated with acute and severe inflammation in lungs. With increasing in-depth studies of ALI/ARDS, significant breakthroughs have been made, however, there are still no effective pharmacological therapies for treatment of ALI/ARDS. Especially, the novel coronavirus pneumonia (COVID-19) is ravaging the globe, and causes severe respiratory distress syndrome. Therefore, developing new drugs for therapy of ALI/ARDS is in great demand, which might also be helpful for treatment of COVID-19. Natural compounds have always inspired drug development, and numerous natural products have shown potential therapeutic effects on ALI/ARDS. Therefore, this review focuses on the potential therapeutic effects of natural compounds on ALI and the underlying mechanisms. Overall, the review discusses 159 compounds and summarizes more than 400 references to present the protective effects of natural compounds against ALI and the underlying mechanism.

A liver-on-a-chip for hepatoprotective activity assessment.

Hepatoprotectant is critical for the treatment of liver disease. This study first reported the application of a liver chip in the hepatoprotective effect assessment. We first established a biomimetic sinusoid-on-a-chip by laminating four types of hepatic cell lines (HepG2, HUVEC, LX-2, and U937 cells) in a single microchannel with the help of laminar flow in the microchannel and some micro-fences. This chip was straightforward to fabricate and operate and was able to be long-term cultured. It also demonstrated better hepatic activity (cell viability, albumin synthesis, urea secretion, and cytochrome P450 enzyme activities) over the traditional planar cell culture model. Then, we loaded three hepatoprotectants (tiopronin, bifendatatum, and glycyrrhizinate) into the chip followed by the addition of acetaminophen as a toxin. We successfully observed the hepatoprotective effect of these hepatoprotectants in the chip, and we also found that bifendatatum predominantly reduced alanine transaminase secretion, tiopronin predominantly reduced lactate dehydrogenase secretion, and glycyrrhizinate predominantly reduced aspartate transaminase secretion, which revealed the different mechanisms of these hepatoprotectants and provided a clue for following molecular biological study of the protecting mechanism.

The rs6427384 and rs6692977 Single Nucleotide Polymorphisms of the Fc Receptor-Like 5 (FCRL5) Gene and the Risk of Ankylosing Spondylitis: A Case Control Study in a Single Center in China.

BACKGROUND The study aimed to explore the genetic association of Fc receptor-like 5 (FCRL5) gene variants (rs6427384 and rs6692977) with ankylosing spondylitis risk in Chinese Han population. MATERIAL AND METHODS Genotyping for FCRL5 gene variations rs6427384 and rs6692977 was implemented among 130 ankylosing spondylitis cases and 135 healthy persons, through polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Frequency dissimilarity for 2 polymorphisms was compared between 2 groups using chi-square test. The association strength of FCRL5 gene polymorphism with ankylosing spondylitis risk was estimated by odds ratios with 95% confidence intervals. RESULTS The frequencies of rs6427384 CC genotype and C allele were significantly lower in the case group than that in the control group (P<0.05), which suggested that C allele of rs6427384 polymorphism might offer protection against ankylosing spondylitis onset. Whereas only 2 genotypes of rs6692977 were detected in the control group, and no significant association was found with ankylosing spondylitis susceptibility. CONCLUSIONS FCRL5 gene polymorphism rs6427384 was correlated to ankylosing spondylitis occurrence among Chinese Han population, while rs6692977 was not.

Assessment of thoracic volume changes after the collapse of lateral rib fractures based on chest computed tomography data: computer simulation and a multiple variable linear regression analysis.

BACKGROUND: Chest blunt trauma (CBT) and the resultant rib fractures often lead to thoracic collapse. The purpose of this study was to explore the effect of displacement of the rib fracture and thoracic collapse on the thoracic volume by using normal chest CT data. METHODS: In this retrospective study, seven consecutive normal participants were selected from our hospital between June and July 2018. Normal thoracic models were reconstructed, followed by simulation of lateral fractures through the 4th to 9th ribs under three collapse modes with 1-5 cm of collapse. The thoracic collapse models (n = 630) were reconstructed using 3Dmax 2014. We calculated the thoracic volume and reduction percentage for each thoracic collapse model. Linear regression-based comparisons of thoracic volume reductions were performed. RESULTS: In all three collapse modes, the degree of the collapse was linearly correlated with the mean thoracic volume reduction. The reduction percentage in the posterior collapse mode was higher than that in the anterior collapse mode (P < 0.001). The largest volume reductions in the anterior, posterior, and simultaneous collapse models were in the 6th rib fracture model (P < 0.001), 8th rib fracture model (P < 0.001), and 7th rib fracture model (P < 0.001), respectively. CONCLUSIONS: The influences of rib fracture displacement and collapse on the thoracic volume in the 6th through 8th ribs are critical in lateral rib fractures. For patients with 6th to 8th rib fractures and posterior rib collapse, surgical intervention to restore thoracic volume may be more essential.

A novel micro-injection droplet microfluidic system for studying locomotive behavior responses to Cu2+ induced neurotoxin in individual C.elegans.

Analysis of C.elegans by droplet microfluidics has been widely used in study of locomotive behavior responses to neurotoxicity due to the capacity of high-throughput manipulating single cells. However, it has been difficult to manipulate droplets flexibly and actively on account of the limitation of the dimension of individual C. elegans droplets. In this study, a novel MiDMS (Micro-injection Droplet Microfluidic System) was proposed, which consisted of three parts: single C. elegans droplet generator, droplets drug micro-injection channels and drug-incubation observation array. Individual C.elegans droplets were produced initially by regulating the flow rates between oil and water phase as well as the concentration of C.elegans in suspension. Then, the drug solution was precisely injected into each C.elegans droplet, which by electricity induced surface tension of droplet changing. In addition, the effect of neurotoxic Cu2+ on locomotive behavior of C. elegans was evaluated at single cell resolution. The results showed that the neurotoxicity induced behavioral disorder of the C. elegans was more obvious with the increase of Cu2+ concentration or treatment time, and these dose-effect and time-effect relationship in MiDMS were similar as in petri dish. This study will provide a powerful platform for the study of the response of C. elegans to quantitative drug at single cell resolution.

Engineered Liver-on-a-Chip Platform to Mimic Liver Functions and Its Biomedical Applications: A Review.

Hepatology and drug development for liver diseases require in vitro liver models. Typical models include 2D planar primary hepatocytes, hepatocyte spheroids, hepatocyte organoids, and liver-on-a-chip. Liver-on-a-chip has emerged as the mainstream model for drug development because it recapitulates the liver microenvironment and has good assay robustness such as reproducibility. Liver-on-a-chip with human primary cells can potentially correlate clinical testing. Liver-on-a-chip can not only predict drug hepatotoxicity and drug metabolism, but also connect other artificial organs on the chip for a human-on-a-chip, which can reflect the overall effect of a drug. Engineering an effective liver-on-a-chip device requires knowledge of multiple disciplines including chemistry, fluidic mechanics, cell biology, electrics, and optics. This review first introduces the physiological microenvironments in the liver, especially the cell composition and its specialized roles, and then summarizes the strategies to build a liver-on-a-chip via microfluidic technologies and its biomedical applications. In addition, the latest advancements of liver-on-a-chip technologies are discussed, which serve as a basis for further liver-on-a-chip research.

A liver-chip-based alcoholic liver disease model featuring multi-non-parenchymal cells.

Non-parenchymal cells play a key role in the occurrence and development of alcoholic liver disease. However, this cellular behaviour has not been fully characterized, and it is inconvenient to observe in traditional in vitro alcoholic liver disease (ALD) models and animal models. Herein we developed a demountable liver-on-chip device for investigation of pathophysiological process of individual non-parenchymal cells in alcohol induced ALD. This liver-device comprised of HepG2, LX-2, EAhy926 and U937 cells, which were ordered in a physiological distribution under perfuse. This device allows improved HepG2 cells activities and maintained high liver functions which including albumin synthesis and urea secretion. This novel liver-device is able to recreate the damage process of hepatic non-parenchymal cell lines induced by alcohol, and to understand the intercellular communication between different types of hepatic cells during ALD by measuring multiple biomarkers of each types of hepatic non-parenchymal cell lines, including Ve-cadherin, eNOS, VEGF and α-SMA. The proposed liver-device is able to further studies of pathological analysis and drug- and toxicity-screening.

Epimedin C Protects H2O2-Induced Peroxidation Injury by Enhancing the Function of Endothelial Progenitor HUVEC Populations.

Endothelial cell injury and apoptosis induced by oxidative stress serve important roles in many vascular diseases. The repair of endothelial cell vascular injury relies on the function of local endothelial progenitor cells (EPCs). Our previous study indicated that epimedin C, a major flavonoid derived from Herba epimedii (yin yang huo), could promote vascularization by inducing endothelial-like differentiation of mesenchymal stem cells C3H/10T1/2 both in vivo and in vitro. In view of the significant cardiovascular protective effects of Herba epimedii, we detected a protective effect of epimedin C on hydrogen peroxide (H2O2)-induced peroxidation injury in human umbilical vein endothelial cells (HUVECs) and the role of EPC in this process. The results show that epimedin C increased the expression of the stem cell marker, CD34 and PROM1, and subsequently enhanced the expression and function of vascular endothelial growth factor and matrix metalloproteinase (MMP)-2 in local vascular endothelial cells. In conclusion, epimedin C protects H2O2-induced peroxidation injury by enhancing the function of endothelial progenitor HUVEC populations.

A cell lines derived microfluidic liver model for investigation of hepatotoxicity induced by drug-drug interaction.

The poor metabolic ability of cell lines fails to meet the requirements of an in vitro model for drug interaction testing which is crucial for the development or clinical application of drugs. Herein, we describe a liver sinusoid-on-a-chip device composed of four kinds of transformed cell lines (HepG2 cells, LX-2 cells, EAhy926 cells, and U937 cells) that were ordered in a physiological distribution with artificial liver blood flow and biliary efflux flowing in the opposite direction. This microfluidic device applied three-dimensional culturing of HepG2 cells with high density (107 ml-1), forming a tightly connected monolayer of EAhy926 cells and achieving the active transport of drugs in HepG2 cells. Results showed that the device maintained synthetic and secretory functions, preserved cytochrome P450 1A1/2 and uridine diphosphate glucuronyltransferase enzymatic activities, as well as sensitivity of drug metabolism. The cell lines derived device enables the investigation of a drug-drug interaction study. We used it to test the hepatotoxicity of acetaminophen and the following combinations: "acetaminophen + rifampicin," "acetaminophen + omeprazole," and "acetaminophen + ciprofloxacin." The variations in hepatotoxicity of the combinations compared to acetaminophen alone, which is not found in a 96-well plate model, in the device were -17.15%, 14.88%, and -19.74%. In addition, this result was similar to the one tested by the classical primary hepatocyte plate model (-13.22%, 13.51%, and -15.81%). Thus, this cell lines derived liver model provides an alternative to investigate drug hepatotoxicity, drug-drug interaction.

Measurement of Carcinoembryonic Antigen in Clinical Serum Samples Using a Centrifugal Microfluidic Device.

Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker used in clinical applications. The primarily clinical method for measuring CEA is based on chemiluminescence in serum during enzyme-linked immunosorbent assays (ELISA) in 96-well plates. However, this multi-step process requires large and expensive instruments, and takes a long time. In this study, a high-throughput centrifugal microfluidic device was developed for detecting CEA in serum without the need for cumbersome washing steps normally used in immunoreactions. This centrifugal microdevice contains 14 identical pencil-like units, and the CEA molecules are separated from the bulk serum for subsequent immunofluorescence detection using density gradient centrifugation in each unit simultaneously. To determine the optimal conditions for CEA detection in serum, the effects of the density of the medium, rotation speed, and spin duration were investigated. The measured values from 34 clinical serum samples using this high-throughput centrifugal microfluidic device showed good agreement with the known values (average relative error = 9.22%). These results indicate that the high-throughput centrifugal microfluidic device could provide an alternative approach for replacing the classical method for CEA detection in clinical serum samples.

Combined antibacterial and osteogenic in situ effects of a bifunctional titanium alloy with nanoscale hydroxyapatite coating.

To resolve the problems of bacterial infections and the low efficiency of osteogenesis of implanted titanium alloys in clinical dental and bone therapy, we developed a bifunctional titanium alloy (Ti) with a nano-hydroxyapatite (HA) coating (HBD + BMP/HA-Ti), which enables the sustained release of the natural antimicrobial peptide human ß-defensin 3 (HBD-3) and bone morphogenetic protein-2 (BMP-2). Due to the poriferous nano-sized structure of the HA coating with a 20-30 µm thickness, the HBD + BMP/HA-Ti material had a high encapsulation efficiency (>74%) and exhibited synchronized slow release of HBD-3 and BMP-2. In an antibacterial test, HBD + BMP/HA-Ti prevented the growth of bacteria in an inoculated medium, and its surface remained free from viable bacteria after a continuous incubation with Gram-negative and Gram-positive strains for 7 days. Furthermore, good adhesion, proliferation and osteogenic differentiation of hBMSCs in contact with HBD + BMP/HA-Ti were achieved in 7 days. Therefore, the bifunctional titanium alloy HBD + BMP/HA-Ti has a great potential for eventual applications in the protection of implants against bacteria in the orthopaedic and dental clinic.