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Antibiotics are among the most used weapons in fighting microbial infections and have greatly improved the quality of human life. However, bacteria can eventually evolve to exhibit antibiotic resistance to almost all prescribed antibiotic drugs. Photodynamic therapy (PDT) develops little antibiotic resistance and has become a promising strategy in fighting bacterial infection. To augment the killing effect of PDT, the conventional strategy is introducing excess ROS in various ways, such as applying high light doses, high photosensitizer concentrations, and exogenous oxygen. In this study, we report a metallacage-based PDT strategy that minimizes the use of ROS by jointly using gallium-metal organic framework rods to inhibit the production of bacterial endogenous NO, amplify ROS stress, and enhance the killing effect. The augmented bactericidal effect was demonstrated both in vitro and in vivo. This proposed enhanced PDT strategy will provide a new option for bacterial ablation.
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Fotoquimioterapia , Humanos , Especies Reactivas de Oxígeno/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , BacteriasRESUMEN
Excessive production of reactive oxygen species (ROS) and inflammation are the key problems that impede diabetic wound healing. In particular, dressings with ROS scavenging capacity play a crucial role in the process of chronic wound healing. Herein, Zr-based large-pore mesoporous metal-organic frameworks (mesoMOFs) were successfully developed for the construction of spatially organized cascade bioreactors. Natural superoxide dismutase (SOD) and an artificial enzyme were spatially organized in these hierarchical mesoMOFs, forming a cascade antioxidant defense system, and presenting efficient intracellular and extracellular ROS scavenging performance. In vivo experiments demonstrated that the SOD@HMUiO-MnTCPP nanoparticles (S@M@H NPs) significantly accelerated diabetic wound healing. Transcriptomic and western blot results further indicated that the nanocomposite could inhibit fibroblast senescence and ferroptosis as well as the stimulator of interferon genes (STING) signaling pathway activation in macrophages mediated by mitochondrial oxidative stress through ROS elimination. Thus, the biomimetic multi-enzyme cascade catalytic system with spatial ordering demonstrated a high potential for diabetic wound healing, where senescence, ferroptosis, and STING signaling pathways may be potential targets.
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Inflamación , Estructuras Metalorgánicas , Especies Reactivas de Oxígeno , Cicatrización de Heridas , Cicatrización de Heridas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Ratones , Superóxido Dismutasa/metabolismo , Porosidad , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células RAW 264.7 , Masculino , Ferroptosis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Diabetes Mellitus Experimental , Nanopartículas/química , Humanos , Antioxidantes/farmacología , Nanocompuestos/química , Proteínas de la MembranaRESUMEN
Qu-aroma is of great significance for evaluation the quality of Daqu starter. This study aimed to decode the Qu-aroma of medium-temperature Daqu (MT-Daqu) via "top-down" and "bottom-up" approaches. Firstly, 52 aroma descriptors were defined to describe the MT-Daqu aroma by quantitative descriptive analysis. Secondly, 193 volatile organic compounds (VOCs) were identified from 42 MT-Daqu samples by HS-SPME-GC-MS, and 43 dominant VOCs were screened out by frequence of occurrence or abundance. By Thin Film (TF)-SPME-GC-O-MS, 27 odors and 90 VOCs were detected in MT-Daqu mixture, and 14 odor-active VOCs were screened out by odor intensity. Thirdly, a five-level MT-Daqu aroma wheel was constructed by matching 52 aroma descriptors and 37 aroma-active VOCs. Finally, Qu-aroma of MT-Daqu was reconstructed with 37 aroma-active VOCs and evaluated by omission experiments. Hereinto, 26 key aroma-active VOCs were determined by OAV value ≥1, including isovaleric acid, 1-hexanol, isovaleraldehyde, 2-octanone, trimethylpyrazine, γ-nonalactone, 4-vinylguaiacol, etc.
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Cromatografía de Gases y Espectrometría de Masas , Odorantes , Compuestos Orgánicos Volátiles , Compuestos Orgánicos Volátiles/química , Odorantes/análisis , Humanos , Adulto , Masculino , Femenino , Microextracción en Fase Sólida , Temperatura , Gusto , Aromatizantes/química , Adulto Joven , OlfatoRESUMEN
The specific mechanisms underlying bacteria-triggered cell death and osteogenic dysfunction in host bone marrow mesenchymal stem cells (BMSCs) remain unclear, posing a significant challenge to the repair of infected bone defects. This study identifies ferroptosis as the predominant cause of BMSCs death in the infected bone microenvironment. Mechanistically, the bacteria-induced activation of the innate immune response in BMSCs leads to upregulation and phosphorylation of interferon regulatory factor 7 (IRF7), thus facilitating IRF7-dependent ferroptosis of BMSCs through the transcriptional upregulation of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4). Moreover, it is found that intervening in ferroptosis can partially rescue cell injuries and osteogenic dysfunction. Based on these findings, a hydrogel composite 3D-printed scaffold is designed with reactive oxygen species (ROS)-responsive release of antibacterial quaternized chitosan and sustained delivery of the ferroptosis inhibitor Ferrostatin-1 (Fer-1), capable of eradicating pathogens and promoting bone regeneration in a rat model of infected bone defects. Together, this study suggests that ferroptosis of BMSCs is a promising therapeutic target for infected bone defect repair.
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Background: Pure magnesium-based ortho-implants have a number of advantages. However, vital parameters like degradation rate and biocompatibility still call for significant improvement. Methods: In this study, poly (1,3-trimethylene carbonate) (PTMC) and polydopamine (PDA) bilayer and micro arc oxidation composite coatings were prepared successively on magnesium surface by immersion method and microarc oxidation. Its corrosion resistance and biocompatibility were evaluated by in vitro corrosion tests, cellular compatibility experiments, and in vivo animal experiments. Results: In vitro experiments demonstrated that the composite coating provides excellent corrosion protection and biocompatibility. Animal studies demonstrated that the composite coating slowed the degradation of the implant and was not toxic to animal viscera. Conclusion: In conclusion, the inorganic-organic composite coating proposed in this study provided good corrosion resistance and enhanced biocompatibility for pure magnesium implants. The translational potential of this article: The translational potential of this article is to develop an anti-corrosion composite coating on a pure magnesium surface and to verify the viability of its use in animal models. It is hoped to open up a new approach to the design of new degradable orthopedic magnesium-based implants.
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Recent studies on osteosarcoma and matrix stiffness are still mostly performed in a 2D setting, which is distinct from in vivo conditions. Therefore, the results from the 2D models may not reflect the real effect of matrix stiffness on cell phenotype. Here, we employed a 3D bioprinted osteosarcoma model, to study the effect of matrix stiffness on osteosarcoma cells. Through density adjustment of GelMA, we constructed three osteosarcoma models with distinct matrix stiffnesses of 50, 80, and 130 kPa. In this study, we found that osteosarcoma cells proliferated faster, migrated more actively, had a more stretched morphology, and a lower drug sensitivity in a softer 3D matrix. When placed in a stiffer matrix, osteosarcoma cells secrete more MMP and VEGF, potentially to fight for survival and attract vascular invasion. Transcriptomic analysis showed that matrix stiffness could impact the signaling pathway of integrin α5-MAPK. The transplantation of 3D printed models in nude mice showed that cells encapsulated in the softer hydrogel were more likely to form subcutaneous tumors. These results suggest that matrix stiffness plays an important role in the development of osteosarcoma in a 3D environment and that inhibition of integrin α5 could block the signal transduction of matrix stiffness.
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Neoplasias Óseas , Osteosarcoma , Ratones , Animales , Hidrogeles/farmacología , Gelatina , Biomimética/métodos , Ratones Desnudos , Integrina alfa5 , Impresión TridimensionalRESUMEN
Senile osteoporosis is characterized by age-related bone loss and bone microarchitecture deterioration. However, little is known to date about the mechanism that maintains bone homeostasis during aging. In this study, we identify adenosine monophosphate-activated protein kinase alpha 1 (AMPKα1) as a critical factor regulating the senescence and lineage commitment of mesenchymal stem cells (MSCs). A phospho-mutant mouse model shows that constitutive AMPKα1 activation prevents age-related bone loss and promoted MSC osteogenic commitment with increased bone-derived insulin-like growth factor 1 (IGF-1) secretion. Mechanistically, upregulation of IGF-1 signalling by AMPKα1 depends on cAMP-response element binding protein (CREB)-mediated transcriptional regulation. Furthermore, the essential role of the AMPKα1/IGF-1/CREB axis in promoting aged MSC osteogenic potential is confirmed using three-dimensional (3D) culture systems. Taken together, these results can provide mechanistic insight into the protective effect of AMPKα1 against skeletal aging by promoting bone-derived IGF-1 secretion.
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Factor I del Crecimiento Similar a la Insulina , Osteoporosis , Ratones , Animales , Factor I del Crecimiento Similar a la Insulina/metabolismo , Huesos/metabolismo , Envejecimiento/metabolismo , Osteogénesis , Osteoporosis/prevención & controlRESUMEN
BACKGROUND: Although biomedical implants have been widely used in orthopedic treatments, two major clinical challenges remain to be solved, one is the bacterial infection resulting in biofilm formation, and the other is aseptic loosening during implantation due to over-activated osteoclastogenesis. These factors can cause many clinical issues and even lead to implant failure. Thus, it is necessary to endow implants with antibiofilm and aseptic loosening-prevention properties, to facilitate the integration between implants and bone tissues for successful implantation. To achieve this goal, this study aimed to develop a biocompatible titanium alloy with antibiofilm and anti-aseptic loosening dual function by utilizing gallium (Ga) as a component. METHODS: A series of Ti-Ga alloys were prepared. We examined the Ga content, Ga distribution, hardness, tensile strength, biocompatibility, and anti-biofilm performance in vitro and in vivo. We also explored how Ga3+ ions inhibited the biofilm formation of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and osteoclast differentiation. RESULTS: The alloy exhibited outstanding antibiofilm properties against both S. aureus and E. coli in vitro and decent antibiofilm performance against S. aureus in vivo. The proteomics results demonstrated that Ga3+ ions could disturb the bacterial Fe metabolism of both S. aureus and E. coli, inhibiting bacterial biofilm formation. In addition, Ti-Ga alloys could inhibit receptor activator of nuclear factor-κB ligand (RANKL)-dependent osteoclast differentiation and function by targeting iron metabolism, then suppressing the activation of the NF-κB signaling pathway, thus, showing their potential to prevent aseptic loosening. CONCLUSION: This study provides an advanced Ti-Ga alloy that can be used as a promising orthopedic implant raw material for various clinical scenarios. This work also revealed that iron metabolism is the common target of Ga3+ ions to inhibit biofilm formation and osteoclast differentiation.
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With the abuse and misuse of antibiotics, antimicrobial resistance has become a challenging issue in the medical system. Iatrogenic and non-iatrogenic infections caused by multidrug-resistant (MDR) pathogens pose serious threats to global human life and health because the efficacy of traditional antibiotics has been greatly reduced and the resulting socio-economic burden has increased. It is important to find and develop non-antibiotic-dependent antibacterial strategies because the development of new antibiotics can hardly keep pace with the emergence of resistant bacteria. Gallium (III) is a multi-target antibacterial agent that has an excellent antibacterial activity, especially against MDR pathogens; thus, a gallium (III)-based treatment is expected to become a new antibacterial strategy. However, some limitations of gallium ions as antimicrobials still exist, including low bioavailability and explosive release. In recent years, with the development of nanomaterials and clathrates, the progress of manufacturing technology, and the emergence of synergistic antibacterial strategies, the antibacterial activities of gallium have greatly improved, and the scope of application in medical systems has expanded. This review summarizes the advancement of current optimization for these key factors. This review will enrich the knowledge about the efficiency and mechanism of various gallium-based antibacterial agents and provide strategies for the improvement of the antibacterial activity of gallium-based compounds.
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Current in vitro models for osteosarcoma investigation and drug screening, including two-dimensional (2D) cell culture and tumour spheroids (i.e. cancer stem-like cells), lack extracellular matrix (ECM). Therefore, results from traditional models may not reflect real pathological processes in genuine osteosarcoma histological structures. Here, we report a three-dimensional (3D) bioprinted osteosarcoma model (3DBPO) that contains osteosarcoma cells and shrouding ECM analogue in a 3D frame. Photo-crosslinkable bioinks composed of gelatine methacrylamide and hyaluronic acid methacrylate mimicked tumour ECM. We performed multi-omics analysis, including transcriptomics and DNA methylomics, to determine differences between the 3DBPO model and traditional models. Compared with 2D models and tumour spheroids, our 3DBPO model showed significant changes in cell cycle, metabolism, adherens junctions, and other pathways associated with epigenetic regulation. The 3DBPO model was more sensitive to therapies targeted to the autophagy pathway. We showed that simulating ECM yielded different osteosarcoma cell metabolic characteristics and drug sensitivity in the 3DBPO model compared with classical models. We suggest 3D printed osteosarcoma models can be used in osteosarcoma fundamental and translational research, which may contribute to novel therapeutic strategy discovery.
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Infections caused by drug-resistant bacteria pose a great threat to human health. Non-antibiotic-dependent antibacterial strategies have become the focus of research. Among them, chemical dynamic treatment-based (CDT) therapeutic systems, which catalyze the production of hydroxyl radicals by enzymes, have achieved tremendous success for antibacterial purposes. However, limited kinetics of the Fenton reaction, poor permeability, and short half-life of hydroxyl radicals compromise the antibacterial effects of CDT. In addition, difficulties in the early diagnosis of infection lead to drug abuse and delayed treatment. Herein, a polydopamine coated ferrous sulfide theranostic platform adsorbing a hypochlorite responsive probe with photothermal treatment (PTT) enhanced CDT was synthesized. The probe component was used for the early diagnosis of infection. PTT not only inactivated bacteria by hyperthermia but also accelerated the Fenton reaction to produce more ·OH. In vitro antibacterial experiments demonstrated that the multifunctional theranostic platform has a broad antibacterial spectrum, including methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Escherichia coli (DR E. coli), and Pseudomonas aeruginosa (P. aeruginosa). In addition, in vivo antibacterial experiments demonstrated that nanoparticles could effectively rescue S. aureus-infected full-thickness skin defects with negligible cytotoxicity. This study proposes an efficient and multifunctional theranostic platform for bacterial infection, providing an effective synergistic antibacterial strategy for the treatment of antibiotic resistance. STATEMENT OF SIGNIFICANCE: An infection responsive theranostic platform (ClO- probe@FeS@PDA) is prepared. ·CDT is enhanced prominently by PTT at a relative low temperature. · FeS@PDA exhibits good antibacterial performance against drug resistant bacteria in vitro and in vivo.
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Staphylococcus aureus Resistente a Meticilina , Staphylococcus aureus , Antibacterianos/química , Antibacterianos/farmacología , Bacterias , Escherichia coli , Humanos , Indoles , Fototerapia , Polímeros , Medicina de Precisión , Nanomedicina TeranósticaRESUMEN
Diabetic osteoporosis (DOP) is the leading complication continuously threatening the bone health of patients with diabetes. A key pathogenic factor in DOP is loss of osteocyte viability. However, the mechanism of osteocyte death remains unclear. Here, we identified ferroptosis, which is iron-dependent programmed cell death, as a critical mechanism of osteocyte death in murine models of DOP. The diabetic microenvironment significantly enhanced osteocyte ferroptosis in vitro, as shown by the substantial lipid peroxidation, iron overload, and aberrant activation of the ferroptosis pathway. RNA sequencing showed that heme oxygenase-1 (HO-1) expression was notably upregulated in ferroptotic osteocytes. Further findings revealed that HO-1 was essential for osteocyte ferroptosis in DOP and that its promoter activity was controlled by the interaction between the upstream NRF2 and c-JUN transcription factors. Targeting ferroptosis or HO-1 efficiently rescued osteocyte death in DOP by disrupting the vicious cycle between lipid peroxidation and HO-1 activation, eventually ameliorating trabecular deterioration. Our study provides insight into DOP pathogenesis, and our results provide a mechanism-based strategy for clinical DOP treatment.
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BACKGROUND: Clinic treatment of diabetic foot ulcers (DFUs) is considerably challenging. Impaired wound healing may be caused by poor vascularization and dysfunction of the extracellular matrix, which leads to poor re-epithelialization and increased risk of infection. In this study, we evaluated the treatment potential of a functional dressing comprising quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan) and magnesium (Mg) on DFUs. METHODS: Dressings were prepared by vacuum freeze-drying. The cellular proliferation, migration, and angiogenesis potential of the functional dressings were determined in vitro. Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) and methicillin-resistant Staphylococcus epidermidis 287 (MRSE287) were used to evaluate the antibacterial efficiency of the dressings. Finally, a diabetic rat model with infected wounds was used to further evaluate the effects of functional dressings on the healing of DFUs. RESULTS: Functional dressings facilitated the migration of human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs), while also stimulating angiogenesis in HUVECs. Additionally, the functional dressing could effectively eradicate MRSA and MRSE, exhibiting excellent antibacterial ability against drug-resistant bacteria. The results of in vivo microbiological and histological tests demonstrated effective anti-infection ability and wound-healing potential of this functional dressing. CONCLUSIONS: The dual-functional dressing exhibited wound-healing ability and anti-infection efficiency, demonstrating potential application prospects in DFU treatment. TRANSLATIONAL POTENTIAL OF THIS ARTICLE: As one of the common and serious complications of diabetes, DFUs do not heal easily, causing great suffering to patients. Therefore, improvement in the prognosis of DFUs is a crucial clinical need. The dual-functional dressing prepared in this study was proven to improve the treatment of DFUs, both in vitro and in vivo. Considering its urgent clinical necessity and good biocompatibility of its raw materials, such as alginate, Mg, and chitosan derivatives, this dual-functional dressing presents good prospects for clinical translation.
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Osteosarcoma has a poor prognosis, and the poor understanding of the genetic drivers of osteosarcoma hinders further improvement in therapeutic approaches. Transcription factor forkhead box P1 (FOXP1) is a crucial modulator in skeletal development and aging. Here, we determined the role and regulatory mechanisms of FOXP1 in osteosarcoma. Higher FOXP1 expression correlated with malignancy in both osteosarcoma cell lines and clinical biopsies. FOXP1 overexpression and knockdown in osteosarcoma cell lines revealed that FOXP1 promoted proliferation, tumor sphere formation, migration and invasion, and inhibited anoikis. Mechanistically, FOXP1 acted as a repressor of P21 and RB (retinoblastoma protein) transcription, and directly interacted with the tumor suppressor p53 to inhibit its activity. Extracellular signal-regulated kinase/c-Jun N-terminal kinase (ERK/JNK) signaling and c-JUN/c-FOS transcription factors were found to be upstream activators of FOXP1. Moreover, FOXP1 silencing via lentivirus or adeno-associated virus (AAV)-mediated delivery of shRNA suppressed osteosarcoma development and progression in cell-derived and patient-derived xenograft animal models. Taken together, we demonstrate that FOXP1, which is transactivated by ERK/JNK-c-JUN/c-FOS, drives osteosarcoma development by regulating the p53-P21/RB signaling cascade, suggesting that FOXP1 is a potential target for osteosarcoma therapy.
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Neoplasias Óseas/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Factores de Transcripción Forkhead/genética , Osteosarcoma/genética , Proteínas Represoras/genética , Proteína de Retinoblastoma/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Factores de Transcripción Forkhead/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Current treatments for diabetic ulcers (DUs) remain unsatisfactory due to the risk of bacterial infection and impaired angiogenesis during the healing process. The increased degradation of polyubiquitinated hypoxia-inducible factor-1α (HIF-1α) compromises wound healing efficacy. Therefore, the maintenance of HIF-1α protein stability might help treat DU. Nitric oxide (NO) is an intrinsic biological messenger that functions as a ubiquitination flow repressor and antibacterial agent; however, its clinical application in DU treatment is hindered by the difficulty in controlling NO release. Here, an intelligent near-infrared (NIR)-triggered NO nanogenerator (SNP@MOF-UCNP@ssPDA-Cy7/IR786s, abbreviated as SNP@UCM) is presented. SNP@UCM represses ubiquitination-mediated proteasomal degradation of HIF-1α by inhibiting its interaction with E3 ubiquitin ligases under NIR irradiation. Increased HIF-1α expression in endothelial cells by SNP@UCM enhances angiogenesis in wound sites, promoting vascular endothelial growth factor (VEGF) secretion and cell proliferation and migration. SNP@UCM also enables early detection of wound infections and ROS-mediated killing of bacteria. The potential clinical utility of SNP@UCM is further demonstrated in infected full-thickness DU model under NIR irradiation. SNP@UCM is the first reported HIF-1α-stabilizing advanced nanomaterial, and further materials engineering might offer a facile, mechanism-based method for clinical DU management.
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Materiales Biocompatibles/química , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Óxido Nítrico/metabolismo , Cicatrización de Heridas , Materiales Biocompatibles/farmacología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Pie Diabético/microbiología , Pie Diabético/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Rayos Infrarrojos , Nanopartículas del Metal/química , Estructuras Metalorgánicas/química , Neovascularización Fisiológica/efectos de los fármacos , Nitroprusiato/química , Medicina de Precisión , Estabilidad Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Staphylococcus aureus/efectos de los fármacos , Ubiquitinación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Large bone defects face a high risk of infection, which can also lead to bone homeostasis disorders. This seriously hinders the bone healing process; therefore, the help of a dual-functional scaffold that has both anti-infection and bone-homeostasis-regulating capacities is needed in the treatment of infected bone defects. In this study, a 3D printed dual-functional scaffold composed of poly-ε-caprolactone (PCL), mesoporous bioactive glasses (MBG), and gallium (Ga) was produced. In vitro experiments demonstrated the excellent antibacterial ability of the PCL/MBG/Ga scaffold against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). The scaffold also significantly inhibited osteoclastic activity and promoted osteogenic differentiation. Furthermore, a rabbit model with an infected bone defect in the radius was used to evaluate the in vivo bone healing capability of PCL/MBG/Ga. The results demonstrate that the PCL/MBG/Ga scaffold can significantly accelerate bone healing and prevent bone resorption, suggesting its potential for application in repairing infected bone defects.
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Antiinfecciosos/uso terapéutico , Huesos/patología , Infecciones por Escherichia coli/tratamiento farmacológico , Galio/química , Homeostasis , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Impresión Tridimensional , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Regeneración Ósea , Huesos/microbiología , Conejos , Infecciones Estafilocócicas/microbiología , Andamios del Tejido , Cicatrización de HeridasRESUMEN
Large bone defects face a high risk of pathogen exposure due to open wounds, which leads to high infection rates and delayed bone union. To promote successful repair of infectious bone defects, fabrication of a scaffold with dual functions of osteo-induction and bacterial inhibition is required. This study describes creation of an engineered progenitor cell line (C3H10T1/2) capable of doxycycline (DOX)-mediated release of bone morphogenetic protein-2 (BMP2). Three-dimensional bioprinting technology enabled creation of scaffolds, comprising polycaprolactone/mesoporous bioactive glass/DOX and bioink, containing these engineered cells. In vivo and in vitro experiments confirmed that the scaffold could actively secrete BMP2 to significantly promote osteoblast differentiation and induce ectopic bone formation. Additionally, the scaffold exhibited broad-spectrum antibacterial capacity, thereby ensuring the survival of embedded engineered cells when facing high risk of infection. These findings demonstrated the efficacy of this bioprinted scaffold to release BMP2 in a controlled manner and prevent the occurrence of infection; thus, showing its potential for repairing infectious bone defects.
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Osteocytes, essential regulators of bone homeostasis, are embedded in the mineralized bone matrix. Given the spatial arrangement of osteocytes, bioprinting represents an ideal method to biofabricate a 3D osteocyte network with a suitable surrounding matrix similar to native bone tissue. Here, we reported a 3D bioprinted osteocyte-laden hydrogel for biomimetic mineralization in vitro with exceptional shape fidelity, a high cell density (107 cells per ml) and high cell viability (85%-90%). The bioinks were composed of biomimetic modified biopolymers, namely, gelatine methacrylamide (GelMA) and hyaluronic acid methacrylate (HAMA), with or without type I collagen. The osteocyte-laden constructs were printed and cultured in mineralization induction media. After 28 d, increased dendritic cell connections and enhanced mineralized matrix production were observed after the addition of type I collagen. These results were further confirmed by the expression of osteocyte-related genes, markers of osteocyte morphology (Connexin43 and E11/Podoplanin), markers of mineralization (dentin matrix acidic phosphoprotein 1 (Dmp1)) and the cellular response to parathyroid hormone (PTH). Moreover, the 3D bioprinting constructs outperformed the 2D monolayer culture and they were at least comparable to 3D casted hydrogels in mimicking the natural osteocyte phenotype. All results indicated that the 3D bioprinting osteocyte network shows promise for mechanistic studies and pharmaceutical screening in vitro.
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Biomimética , Bioimpresión , Calcificación Fisiológica/fisiología , Osteocitos/citología , Animales , Calcificación Fisiológica/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hidrogeles/química , Tinta , Osteocitos/efectos de los fármacos , Hormona Paratiroidea/farmacología , Impresión Tridimensional , Ratas , Andamios del Tejido/químicaRESUMEN
Sufficient vascularization is quite important for preventing cell death and promoting host integration during the repair of the critical sized bone defects. Porous structure providing enough space for the ingrowth of vessels is an essential consideration during the scaffold's development. In this study, we designed and fabricated three kinds of porous structured scaffolds based on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), such as mono-structured PHBHHx scaffolds with macro pores (PH-1), di-structured PHBHHx scaffolds with macro-meso pores (PHS-2), and tri-structured PHBHHx scaffolds with macro-micro-meso pores (PHS-3), respectively. In vitro effects of the hierarchical porous scaffolds on human umbilical vein endothelial cells (HUVECs), such as cell attachment, glucose and lactate detection, relative gene expressions of endothelial markers were investigated. The PHS-3 scaffolds exhibited preferential potency of inducing better angiogenesis in vitro. Consequently, the hierarchical porous scaffolds were applied to load rhBMP-2 and repair the critical sized bone defect (15 mm) in rabbits. Microangiography analysis by three dimensional micro-computed tomographic (micro-CT) demonstrated that the volume of blood vessels within the defect area was higher in the rhBMP-2 loaded PHS-3 (PHS-3/rhBMP-2) than that in other rhBMP-2 loaded porous scaffolds with simplex or double scaled pores (PH-1/rhBMP-2 or PHS-2/rhBMP-2) at 4 weeks and 8 weeks, which implied that multi-level porous structure was conducive to nutrition transmission and revascularization. Further investigations of orthotopic bone formation by micro-CT, histological and immunohistochemistry analysis confirmed the most accelerated new bone formation rate in the PHS-3/rhBMP-2 group. The maximum load value of the regenerated bone induced by PHS-3/rhBMP-2 at 12 weeks was 258.47 ± 14.77 N which did not show significant difference from the normal bone of 268.81 ± 12.05 N. These results highlighted that introducing multi-level pores into the biocompatible scaffolds may be an effective approach to promote angiogenesis and bone regeneration.
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Ácido 3-Hidroxibutírico/química , Regeneración Ósea , Caproatos/química , Regulación de la Expresión Génica , Neovascularización Fisiológica , Osteogénesis , Andamios del Tejido/química , Animales , Antígenos de Diferenciación/biosíntesis , Proteína Morfogenética Ósea 2/química , Adhesión Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Porosidad , ConejosRESUMEN
Accumulating evidence suggests that activation of proinflammatory M1-type macrophages in the synovium plays a vital role in the progression of osteoarthritis (OA). Redundant nitric oxide (NO) and hydrogen peroxide (H2O2) are key factors that drive macrophages to polarize to the M1 type. Herein, modified zeolitic imidazolate framework-8 (ZIF-8) nanoparticles (NPs) have been synthesized. By regulating intracellular gases and reprogramming the metabolism phenotype, modified NPs transformed macrophage polarization from proinflammatory M1 to anti-inflammatory M2 phenotype. Specifically, S-methylisothiourea hemisulfate salt was loaded into ZIF-8 NPs to inhibit inducible nitric oxide synthase, hence reducing NO production. Catalase was encapsulated to catalyze the production of oxygen (O2) from H2O2. Results demonstrated that modified NPs were capable of catalyzing H2O2 to produce O2 and eliminate NO, hence inhibiting hypoxia-inducible factor 1α, further rescuing mitochondrial function. Moreover, anti-CD16/32 antibody modification could prolong the retention time of NPs in knee joints of OA mice with anterior cruciate ligament transection. More significantly, modified NPs suppressed M1 macrophages and up-regulated M2 macrophage infiltration in the synovium, further inhibiting cartilage degeneration. This ZIF-8 NP-based gas regulation and metabolic reprogramming strategy may pave a new avenue for OA treatment.