OBJECTIVE: To investigate the antioxidant and osteogenic induction capabilities of calcium phosphate nanoflowers (hereinafter referred to as nanoflowers) in vitro at different concentrations. METHODS: Nanoflowers were prepared using gelatin, tripolyphosphate, and calcium chloride. Their morphology, microstructure, elemental composition and distribution, diameter, and molecular constitution were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and energy-dispersive spectroscopy. Femurs and tibias were harvested from twelve 4-week-old Sprague Dawley rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using the whole bone marrow adherent method, followed by passaging. The third passage cells were identified as stem cells by flow cytometry and then co-cultured with nanoflowers at concentrations of 0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, and 3.6 mg/mL. Cell counting kit 8 (CCK-8) assay was performed to screen for the optimal concentration that demonstrated the best cell viability, which was subsequently used as the experimental concentration for further studies. After co-culturing BMSCs with the screened concentration of nanoflowers, the biocompatibility of the nanoflowers was verified through live/dead cell staining, scratch assay, and cytoskeleton staining. The antioxidant capacity was assessed by using reactive oxygen species (ROS) fluorescence staining. The in vitro osteoinductive ability was evaluated via alkaline phosphatase (ALP) staining, alizarin red staining, and immunofluorescence staining of osteocalcin (OCN) and Runt-related transcription factor 2 (RUNX2). All the above indicators were compared with the control group of normally cultured BMSCs without the addition of nanoflowers. RESULTS: Scanning electron microscopy revealed that the prepared nanoflowers exhibited a flower-like structure; transmission electron microscopy scans discovered that the nanoflowers possessed a multi-layered structure, and high-magnification images displayed continuous atomic arrangements, with the nanoflower diameter measuring (2.00±0.25) μm; energy-dispersive spectroscopy indicated that the nanoflowers contained elements such as C, N, O, P, and Ca, which were uniformly distributed across the flower region; Fourier transform infrared spectroscopy analyzed the absorption peaks of each component, demonstrating the successful preparation of the nanoflowers. Through CCK-8 screening, the concentrations of 0.8, 1.2, and 1.6 mg/mL were selected for subsequent experiments. The live/dead cell staining showed that nanoflowers at different concentrations exhibited good cell compatibility, with the 1.2 mg/mL concentration being the best (P<0.05). The scratch assay results indicated that the cell migration ability in the 1.2 mg/mL group was superior to the other groups (P<0.05). The cytoskeleton staining revealed that the cell morphology was well-extended in all concentration groups, with no significant difference compared to the control group. The ROS fluorescence staining demonstrated that the ROS fluorescence in all concentration groups decreased compared to the control group after lipopolysaccharide induction (P<0.05), with the 1.2 mg/mL group showing the weakest fluorescence. The ALP staining showed blue-purple nodular deposits around the cells in all groups, with the 1.2 mg/mL group being significantly more prominent. The alizarin red staining displayed orange-red mineralized nodules around the cells in all groups, with the 1.2 mg/mL group having more and denser nodules. The immunofluorescence staining revealed that the expressions of RUNX2 and OCN proteins in all concentration groups increased compared to the control group, with the 1.2 mg/mL group showing the strongest protein expression (P<0.05). CONCLUSION The study successfully prepares nanoflowers, among which the 1.2 mg/mL nanoflowers exhibits excellent cell compatibility, antioxidant properties, and osteogenic induction capability, demonstrating their potential as an artificial bone substitute material.
Animals ; Osteogenesis/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Calcium Phosphates/pharmacology* ; Rats, Sprague-Dawley ; Rats ; Antioxidants/chemistry* ; Cells, Cultured ; Cell Differentiation/drug effects* ; Nanostructures/chemistry* ; Tissue Engineering/methods* ; Bone Marrow Cells/cytology* ; Coculture Techniques ; Tissue Scaffolds/chemistry* ; Male ; Biocompatible Materials/chemistry* ; Cell Survival ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Proliferation

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.
Coculture Techniques/methods* ; Humans ; Fusobacterium nucleatum/physiology* ; Gingiva/microbiology* ; Keratinocytes/microbiology* ; Host Microbial Interactions ; Mouth/microbiology* ; Host-Pathogen Interactions ; Epithelial Cells/microbiology* ; Cells, Cultured ; Porphyromonas gingivalis

The oral and maxillofacial region comprises a variety of organs made up of multiple soft and hard tissue, which are anatomically vulnerable to the pathogenic factors of trauma, inflammation, and cancer. The studies of this intricate entity have been long-termly challenged by a lack of versatile preclinical models. Recently, the advancements in the organoid industry have provided novel strategies to break through this dilemma. Here, we summarize the existing biological and engineering approaches that were employed to generate oral and maxillofacial organoids. Then, we detail the use of modified co-culture methods, such as cell cluster co-inoculation and air-liquid interface culture technology to reconstitute the vascular network and immune microenvironment in assembled organoids. We further retrospect the existing oral and maxillofacial assembled organoids and their potential to recapitulate the homeostasis in parental tissues such as tooth, salivary gland, and mucosa. Finally, we discuss how the next-generation organoids may benefit to regenerative and precision medicine for treatment of oral-maxillofacial illness.
Organoids ; Humans ; Tissue Engineering/methods* ; Coculture Techniques ; Regenerative Medicine ; Mouth

PURPOSE: Several studies have shown that the oral cavity is a secondary location for Helicobacter pylori colonization and that H. pylori is associated with the severity of periodontitis. This study investigated whether H. pylori had an effect on the periodontium. We established an invasion model of a standard strain of H. pylori in human periodontal ligament fibroblasts (hPDLFs), and evaluated the effects of H. pylori on cell proliferation and cell cycle progression. METHODS: Different concentrations of H. pylori were used to infect hPDLFs, with 6 hours of co-culture. The multiplicity of infection in the low- and high-concentration groups was 10:1 and 100:1, respectively. The Cell Counting Kit-8 method and Ki-67 immunofluorescence were used to detect cell proliferation. Flow cytometry, quantitative real-time polymerase chain reaction, and western blots were used to detect cell cycle progression. In the high-concentration group, the invasion of H. pylori was observed by transmission electron microscopy. RESULTS: It was found that H. pylori invaded the fibroblasts, with cytoplasmic localization. Analyses of cell proliferation and flow cytometry showed that H. pylori inhibited the proliferation of periodontal fibroblasts by causing G2 phase arrest. The inhibition of proliferation and G2 phase arrest were more obvious in the high-concentration group. In the low-concentration group, the G2 phase regulatory factors cyclin dependent kinase 1 (CDK1) and cell division cycle 25C (Cdc25C) were upregulated, while cyclin B1 was inhibited. However, in the high-concentration group, cyclin B1 was upregulated and CDK1 was inhibited. Furthermore, the deactivated states of tyrosine phosphorylation of CDK1 (CDK1-Y15) and serine phosphorylation of Cdc25C (Cdc25C-S216) were upregulated after H. pylori infection. CONCLUSIONS: In our model, H. pylori inhibited the proliferation of hPDLFs and exerted an invasive effect, causing G2 phase arrest via the Cdc25C/CDK1/cyclin B1 signaling cascade. Its inhibitory effect on proliferation was stronger in the high-concentration group.
Blotting, Western ; CDC2 Protein Kinase ; Cell Count ; Cell Cycle ; Cell Proliferation ; Coculture Techniques ; Colon ; Cyclin B1 ; Cytoplasm ; Fibroblasts ; Flow Cytometry ; Fluorescent Antibody Technique ; G2 Phase ; Helicobacter pylori ; Helicobacter ; Humans ; Methods ; Microscopy, Electron, Transmission ; Mouth ; Periodontal Ligament ; Periodontitis ; Periodontium ; Phosphorylation ; Real-Time Polymerase Chain Reaction ; Serine ; Tyrosine

BACKGROUND: With the popularity of laparoscopic cholecystectomy, common bile duct injury has been reported more frequently. There is no perfect method for repairing porcine biliary segmental defects.METHODS: After the decellularization of human arterial blood vessels, the cells were cultured with GFP⁺ (carry green fluorescent protein) porcine bile duct epithelial cells. The growth and proliferation of porcine bile duct epithelial cells on the human acellular arterial matrix (HAAM) were observed by hematoxylin-eosin (HE) staining, electron microscopy, and immunofluorescence. Then, the recellularized human acellular arterial matrix (RHAAM) was used to repair biliary segmental defects in the pig. The feasibility of it was detected by magnetic resonance cholangiopancreatography, liver function and blood routine changes, HE staining, immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blot.RESULTS: After 4 weeks (w) of co-culture of HAAM and GFP? porcine bile duct epithelial cells, GFP⁺ porcine bile duct epithelial cells grew stably, proliferated, and fused on HAAM. Bile was successfully drained into the duodenum without bile leakage or biliary obstruction. Immunofluorescence detection showed that GFP-positive bile duct cells could still be detected after GFP-containing bile duct cells were implanted into the acellular arterial matrix for 8 w. The implanted bile duct cells can successfully resist bile invasion and protect the acellular arterial matrix until the newborn bile duct is formed.CONCLUSION: The RHAAM can be used to repair biliary segmental defects in pigs, which provides a new idea for the clinical treatment of common bile duct injury.
Bile ; Bile Ducts ; Blood Vessels ; Blotting, Western ; Cholangiopancreatography, Magnetic Resonance ; Cholecystectomy, Laparoscopic ; Coculture Techniques ; Common Bile Duct ; Duodenum ; Epithelial Cells ; Fluorescent Antibody Technique ; Humans ; Infant, Newborn ; Liver ; Methods ; Microscopy, Electron ; Polymerase Chain Reaction ; Swine ; Tissue Engineering

Two new polyketides, chinoketides A and B (1 – 2) with a known compound xylarphthalide A (3), were isolated from the solid medium of the endophytes from the leaves of the relic plant Distylium chinense with the “black-box” co-culture method, and the structures of two new compounds were elucidated by NMR, MS and CD spectra. And the absolute configurations of chinoketides A (1) and B (2) were determined as 2R,3R,8S and 5R,6S by calculating their ECD spectra to compare with the experimental CD spectra. Finally, the antimicrobial activities were evaluated to Erwinia carotovora sub sp. Carotovora (Jones) Bersey et al, and the results showed that compounds 1 – 3 displayed the antimicrobial activities with MIC value at 20.5, 30.4 and 10.2 µg/mL.
Coculture Techniques ; Endophytes ; Methods ; Pectobacterium carotovorum ; Plants ; Polyketides

Transplantation of bone marrow derived stem cells (BMSCs) has been reported inhibits liver fibrosis. Several in vitro studies by co-culturing BMSCs and hepatic stellate cells (HSCs) indirectly or directly in 2D models showed inhibition of HSC as the key player in liver fibrosis. In this study, we investigated direct effect of BMSCs on HSCs by co-culturing BMSCs and HSCs in 3D model as it represents the liver microenvironment with intricate cell-cell and cell-matrix interactions. Primary isolated rat HSCs and BMSCs were directly co-cultured at 1:1 ratio with hanging drop method. The monoculture of rat HSCs served as positive control. Mono-culture and co-culture samples were harvested on day 3, 5 and 7 for histological analysis. The samples were analyzed for extracellular matrix deposition by Masson's Trichrome staining, tenascin-C immunocytochemistry, resting HSC's state as shown by positive Oil Red O stained cells. Our results indicated CD90+CD34− BMSCs anti-liver fibrosis potency as evidenced by higher proportion of Oil Red O-positive cells in the co-culture group compared to the monoculture group and the significant decrease in extracellular matrix deposition as well as the decrease in tenascin-C expression in the co-culture group (p<0.05) compared to the monoculture group. These findings demonstrate that BMSCs have a potential therapeutic effect against liver fibrotic process through their capacity to inhibit HSCs activation and their effect in minimizing extracellular matrix deposition.
Animals ; Bone Marrow* ; Coculture Techniques ; Extracellular Matrix* ; Fibrosis* ; Hepatic Stellate Cells* ; Immunohistochemistry ; In Vitro Techniques ; Liver ; Liver Cirrhosis ; Methods ; Rats ; Stem Cells* ; Tenascin

BACKGROUND/OBJECTIVES: Helicobacter pylori (H. pylori) colonization of the stomach mucosa and duodenum is the major cause of acute and chronic gastroduodenal pathology in humans. Efforts to find effective anti-bacterial strategies against H. pylori for the non-antibiotic control of H. pylori infection are urgently required. In this study, we used whey to prepare glycomacropeptide (GMP), from which sialic acid (G-SA) was enzymatically isolated. We investigated the anti-bacterial effects of G-SA against H. pylori in vitro and in an H. pylori-infected murine model. MATERIALS/METHODS: The anti-bacterial activity of G-SA was measured in vitro using the macrodilution method, and interleukin-8 (IL-8) production was measured in H. pylori and AGS cell co-cultures by ELISA. For in vivo study, G-SA 5 g/kg body weight (bw)/day and H. pylori were administered to mice three times over one week. After one week, G-SA 5 g/kg bw/day alone was administered every day for one week. Tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-10 levels were measured by ELISA to determine the anti-inflammatory effects of G-SA. In addition, real-time PCR was performed to measure the genetic expression of cytotoxin-associated gene A (cagA). RESULTS: G-SA inhibited the growth of H. pylori and suppressed IL-8 production in H. pylori and in AGS cell co-cultures in vitro. In the in vivo assay, administration of G-SA reduced levels of IL-1β and IL-6 pro-inflammatory cytokines whereas IL-10 level increased. Also, G-SA suppressed the expression of cagA in the stomach of H. pylori-infected mice. CONCLUSION: G-SA possesses anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect in an experimental H. pylori-infected murine model. G-SA has potential as an alternative to antibiotics for the prevention of H. pylori infection and H. pylori-induced gastric disease prevention.
Animals ; Anti-Bacterial Agents ; Body Weight ; Coculture Techniques ; Colon ; Cytokines ; Duodenum ; Enzyme-Linked Immunosorbent Assay ; Helicobacter pylori ; Helicobacter* ; Humans ; In Vitro Techniques ; Interleukin-10 ; Interleukin-6 ; Interleukin-8 ; Methods ; Mice ; Mucous Membrane ; N-Acetylneuraminic Acid* ; Necrosis ; Pathology ; Real-Time Polymerase Chain Reaction ; Stomach ; Stomach Diseases ; Whey

OBJECTIVETo investigate the effects of polyunsaturated fatty acids (PUFA) ω-3 and ω-6, and their middle metabolites PGE2 and PGE3 on angiogenesis formation of gastric cancer, and to explore associated mechanism.

METHODSThe effects of ω-3, ω-6, PGE2, PGE3 on the proliferation and migration of human umbilical vein endothelial cell (HUVEC) were measured by proliferation and migration assay respectively. The angiogenesis assay in vivo was used to measure the effects of ω-3, ω-6, PGE2 and PGE3 on neovascularization. In all the assays, groups without ω-3, ω-6, PGE2 and PGE3 were designed as the control.

RESULTSWith the increased concentration of ω-6 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs enhanced, and the number of migration cells also increased from 28.2±3.0 to 32.8±2.1, which was higher than control group (21.2±3.2) respectively (both P<0.05). With the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the proliferation ability of HUVECs was inhibited, and the number of migration cells decreased from 15.8±2.0 to 11.0±2.1, which was lower than control group (22.1±3.0) respectively (both P<0.05). In the angiogenesis assay, compared with control group (standard number: 43 721±4 654), the angiogenesis ability of HUVECs was significantly enhanced by ω-6 in concentration-dependent manner (1 μmol/L group: 63 238±4 795, 10 μmol/L group: 78 166±6 123, all P<0.01). Meanwhile, with the increased concentration of ω-3 from 1 μmol/L to 10 μmol/L, the angiogenesis ability was significantly decreased from 30 129±3 102 to 20 012±1 541(all P<0.01). The proliferation and migration ability of HUVECs were significantly promoted by ω-6 metabolites PGE2 (P<0.05) in a concentration-dependent manner. In contrast, ω-3 metabolites PGE3 significantly inhibited the proliferation and migration ability of HUVECs in a concentration-dependent manner (all P<0.05). After rofecoxib (a COX-2 specific inhibitor) inhibited the expression of COX-2, the expression level of PGE2 was significantly decreased in a dose-dependent manner. In co-culture system, whose gastric cancer cells expressed positive COX-2, ω-6 could increase angiogenesis of gastric cancer cells(P<0.01), but ω-3 could inhibit such angiogenesis(P<0.01). In co-culture system, whose gastric cancer cells did not express COX-2, ω-3 could inhibit the angiogenesis of gastric cancer cells (P<0.05), but ω-6 had no effect on angiogenesis.

CONCLUSIONSThe PUFA ω-6 can enhance the angiogenesis via the promotion of proliferation and migration of HUVECs, and COX-2 and PGE2 may play an important role in this process, whereas, the ω-3 can inhibit the angiogenesis through its middle metabolites PGE3 to inhibit the proliferation and migration of HUVECs. Results of this experiment may provide a new approach to inhibit and prevent the spread of gastric cancer.

Alprostadil ; analogs & derivatives ; pharmacology ; Angiogenesis Inducing Agents ; metabolism ; pharmacology ; Angiogenesis Inhibitors ; pharmacology ; Cell Count ; methods ; Cell Line, Tumor ; drug effects ; physiology ; Cell Migration Assays ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Coculture Techniques ; Cyclooxygenase 2 ; pharmacology ; Dinoprostone ; metabolism ; pharmacology ; Fatty Acids, Omega-3 ; pharmacology ; Fatty Acids, Omega-6 ; metabolism ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; physiology ; Humans ; Lactones ; pharmacology ; Neovascularization, Pathologic ; physiopathology ; Stomach Neoplasms ; physiopathology ; Sulfones ; pharmacology

The direct lineage conversion of fibroblasts into neuronal or neural precursor cells (NPCs) has become a hot issue in recent years as an attractive approach in the field of stem cell regenerative medicine. In this study, we adopted the stromal feeder co-culture method during the early conversion period to enhance conversion efficiency. Stromal cells are often used in directed differentiation of dopaminergic (DA) neurons from pluripotent stem cells. We co-cultured rat embryonic fibroblasts (REFs) on γ-irradiated sonic hedgehog-overexpressing MS5 stromal (MS5-SHH) cells after transduction with Brn2, Ascl1, Myt1L, and BclxL-GFP (BAMXGFP) transcription factors to REFs. One week after co-culture, transduced cells (GFP+ cells) that proliferated on MS5-SHH cells were separated from MS5-SHH cells through a 40 µm cell strainer. Subsequently, the converted cells (GFP+ cells) were expanded on fibronectin-coated culture plates in NPC expansion medium. The induced NPCs (iNPCs) expressed NPC potential (NESTIN+/SOX2+) earlier than seen with non-co-culture methods and were efficiently differentiated into DA neurons by overexpression of Nurr1 and Foxa2 genes, which are specific transcription factors for midbrain DA neuron development. These observations indicated that direct conversion to NPCs using an MS5 stromal cells co-culture method is a suitable technique for efficient generation of iNPC/DA neurons from fibroblasts.
Animals ; Coculture Techniques ; Dopaminergic Neurons ; Fibroblasts* ; Mesencephalon ; Methods ; Neurons ; Pluripotent Stem Cells ; Rats ; Regenerative Medicine ; Stem Cells ; Stromal Cells ; Transcription Factors