Modulation of macrophage polarization by secondary cross-linked hyaluronan-dopamine hydrogels.

The inflammatory response plays a critical role in standard tissue repair processes, wherein active modulation of macrophage polarization is necessary for wound healing. Dopamine, a mussel-inspired bioactive material, is widely involved in wound healing, neural/bone/myocardial regeneration, and more. Recent studies indicated that dopamine-modified biomaterials can potentially alter macrophages polarization towards a pro-healing phenotype, thereby enhancing tissue regeneration. Nevertheless the immunoregulatory activity of dopamine on macrophage polarization remains unclear. This study introduces a novel interpenetrating hydrogel to bridge this research gap. The hydrogel, combining varying concentrations of oxidized dopamine with hyaluronic acid hydrogel, allows precise regulation of mechanical properties, antioxidant bioactivity, and biocompatibility. Surprisingly, both in vivo and in vitro outcomes demonstrated that dopamine concentration modulates macrophage polarization, but not linearly. Lower concentration (2 mg/mL) potentially decrease inflammation and facilitate M2 type macrophage polarization. In contrast, higher concentration (10 mg/mL) exhibited a pro-inflammatory tendency in the late stages of implantation. RNA-seq analysis revealed that lower dopamine concentrations induced the M1/M2 transition of macrophages by modulating the NF-κB signaling pathway. Collectively, this research offers valuable insights into the immunoregulation effects of dopamine-integrated biomaterials in tissue repair and regeneration.

A unidirectional water-transport antibacterial bilayer nanofibrous dressing based on chitosan for accelerating wound healing.

Excessive accumulation of exudate from wounds often causes infection and hinders skin regeneration. To handle wound exudate quickly and prevent infection, we developed an antibacterial Janus nanofibrous dressing with a unidirectional water-transport function. The dressing consists of a hydrophilic chitosan aerogel (CS-A) as the outer layer and a hydrophobic laurylated chitosan (La-CS) nanofibrous membrane as the inner layer. These dressings achieved excellent liquid absorption performance (2987.8 ±â€¯123.5 %), air and moisture permeability (997.8 ±â€¯23.1 g/m2/day) and mechanical strength (5.1 ±â€¯2.6 MPa). This performance was obtained by adjusting the density of CS-A and the thickness of the La-CS membrane. Moreover, the dressing did not induce significant toxicity to cells and can prevent bacterial aggregation and infection at the wound site. Animal experiments showed that the dressing can shorten the inflammatory phase, enhance blood vessel generation, and accelerate collagen deposition, thus promoting wound healing. Overall, these results suggest that this Janus dressing is a promising material for clinical wound care.

Emergent Three-Dimensional Electric Dipole Sinewave in Bulk Perovskite Oxides.

The magnetic and electric dipoles of ferroics play a central role in their fascinating properties. In particular, topological configurations have shown promising potential for use in novel electromechanical and electronic devices. Magnetic configurations from simple collinear to complex topological are well-documented. In contrast, many complex topological features in the electric counterpart remain unexplored. Here, we report the first example of three-dimensional electric dipole sinewave topological structure in a PbZrO3-based bulk perovskite, which presents an interesting triple-hysteresis loop macroscopically. This polar configuration consists of two orthogonal sinewave electric dipole modulations decoded from a polar incommensurate phase by advanced diffraction and atomic-resolution imaging techniques. The resulting topology is unraveled to be the competition between the antiferroelectric and ferroelectric states, stabilized by the modulation of the Pb 6s2 lone pair and the antiferrodistortive effect. These findings further reinforce the similarity of the magnetic and electric topologies.

A DNA-inspired injectable adhesive hydrogel with dual nitric oxide donors to promote angiogenesis for enhanced wound healing.

Chronic diabetic wounds are a severe complication of diabetes, often leading to high treatment costs and high amputation rates. Numerous studies have revealed that nitric oxide (NO) therapy is a promising option because it favours wound revascularization. Here, base-paired injectable adhesive hydrogels (CAT) were prepared using adenine- and thymine-modified chitosan (CSA and CST). By further introducing S-nitrosoglutathione (GSNO) and binary l-arginine (bArg), we obtained a NO sustained-release hydrogel (CAT/bArg/GSON) that was more suitable for the treatment of chronic wounds. The results showed that the expression of HIF-1α and VEGF was upregulated in the CAT/bArg/GSON group, and improved blood vessel regeneration was observed, indicating an important role of NO. In addition, the research findings revealed that following treatment with the CAT/bArg/GSON hydrogel, the viability of Staphylococcus aureus and Escherichia coli decreased to 14 ± 2 % and 6 ± 1 %, respectively. Moreover, the wound microenvironment was improved, as evidenced by a 60 ± 1 % clearance of DPPH. In particular, histological examination and immunohistochemical staining results showed that wounds treated with CAT/bArg/GSNO exhibited denser neovascularization, faster epithelial tissue regeneration, and thicker collagen deposition. Overall, this study proposes an effective strategy to prepare injectable hydrogel dressings with dual NO donors. The functionality of CAT/bArg/GSON has been thoroughly demonstrated in research on chronic wound vascular regeneration, indicating that CAT/bArg/GSON could be a potential option for promoting chronic wound healing. STATEMENT OF SIGNIFICANCE: This article prepares a chitosan hydrogel utilizing the principle of complementary base pairing, which offers several advantages, including good adhesion, biocompatibility, and flow properties, making it a good material for wound dressings. Loaded GSNO and bArg can steadily release NO and l-arginine through the degradation of the gel. Then, the released l-arginine not only possesses antioxidant properties but can also continue to generate a small amount of NO under the action of NOS. This design achieves a sustained and stable supply of NO at the wound site, maximizing the angiogenesis-promoting and antibacterial effects of NO. More neovascularization and abundant collagen were observed in the regenerated tissues. This study provides an effective repair hydrogel material for diabetic wound.

The Optimal Layer for Breast Augmentation in an Autologous Fat Grafting Murine Model.

BACKGROUND: Fat grafting is an effective procedure for breast augmentation, but the variations in this technique result in unpredictable fat retention. Therefore, animal models are needed to simulate the operation and the optimal layer for fat retention. OBJECTIVES: An autologous fat grafting murine model for breast augmentation was built to detect a new layer for fat grafting in the chest. METHODS: The left side of the female rat inguinal fat flap was harvested, dissected into small pieces, and autotransplanted into 3 different layers of the breast. Retention rate and hematoxylin and eosin (H&E) staining were measured at 1, 4, 8 12, and 16 weeks. Immunofluorescence staining was utilized to detect adipocytes and endothelial cells, and immunohistochemistry was conducted to evaluate the expression of integrins ß1 and α6. RESULTS: The volume of fat grafts slightly grew in the intramuscular and submuscular layers at Week 4. Retention rates in the subcutaneous layer and submuscular layer were significantly higher than the intramuscular layer at Week 16. H&E staining showed that oil cysts existed in the subcutaneous layer throughout the 16 weeks. At the terminal time point, well-vascularized mature adipose structures were observed in intramuscular and submuscular layers, with smaller adipocytes in intramuscular layers. Immunohistochemistry analysis showed that integrin ß1 was identically expressed in every adipocyte in all the layers, whereas integrin α6 selectively expressed in bigger adipocytes in the intramuscular layer. The expression intensities of integrin ß1 and α6 were significantly higher in the intramuscular layer than in the subcutaneous and submuscular layers. CONCLUSIONS: The angiogenic and moderate mechanical environment makes the submuscular layer the optimal layer for fat retention.

Guanidinylated bioactive chitosan-based injectable hydrogels with pro-angiogenic and mechanical properties for accelerated wound closure.

Wound healing is a complex process involving the concerted action of many genes and signaling pathways, with angiogenesis being crucial for expediting wound closure. Dressings that possess pro-angiogenic properties are increasingly recognized as attractive candidates for wound care. Drawing inspiration from the active closure of wounds in embryos, we have developed a thermo-responsive hydrogel with mechanoactive properties, combining vascular regeneration and skin wound contraction to accelerate healing. The significant improvement in vascular reconstruction is attributed to the synergistic effect of arginine and deferoxamine (DFO) released from the hydrogels. Additionally, the contraction force of the hydrogel actively promotes skin closure in wounds. Remarkably, groups treated with hydroxybutyl chitosan methacrylate combined with arginine (HBC_m_Arg/DFO) exhibited increased vascularization, and greater wound maturity, leading to enhanced healing. These results highlight the synergistic impact of pro-angiogenic and mechanical properties of the HBC_m_Arg/DFO hydrogel in accelerating wound healing in rats.

Pulmonary Artery Stenosis in Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital cardiac defect. The survival rate after primary complete repair is high (98-100%); however, pulmonary artery stenosis (PAS) is not uncommon after TOF repair, and severe PAS aggravates pulmonary regurgitation, resulting in right ventricle dilation, ventricular arrhythmia, and possibly death. PAS in TOF can be congenital due to hypoplasia or coarctation or can be acquired secondary to a surgical procedure. The latter may be caused by an exogenous conduit implant, compression from the adjacent enlarged ascending aorta, or outflow tract dilation after transannular patch repair. PAS can also be caused by the pulmonary artery plasty strategy itself. Here, the intrinsic mechanisms underlying PAS and pulmonary artery plasty techniques and strategies are reviewed to provide guidance for surgeons.

Phase Change Material-Embedded Multifunctional Janus Nanofiber Dressing with Directional Moisture Transport, Controlled Release of Anti-Inflammatory Drugs, and Synergistic Antibacterial Properties.

Wound healing is a systematic and complex process that involves various intrinsic and extrinsic factors affecting different stages of wound repair. Therefore, multifunctional wound dressings that can modulate these factors to promote wound healing are in high demand. In this work, a multifunctional Janus electrospinning nanofiber dressing with antibacterial and anti-inflammatory properties, controlled release of drugs, and unidirectional water transport was prepared by depositing coaxial nanofibers on a hydrophilic poly(ε-caprolactone)@polydopamine-ε-polyl-lysine (PCL@PDA-ε-PL) nanofiber membrane. The coaxial nanofiber was loaded with the phase change material lauric acid (LA) in the shell layer and anti-inflammatory ibuprofen (IBU) in the core layer. Among them, LA with a melting point of 43 °C served as a phase change material to control the release of IBU. The phase transition of LA was induced by near-infrared (NIR) irradiation that triggered the photothermal properties of PDA. Moreover, the Janus nanofiber dressing exhibited synergistic antimicrobial properties for Escherichia coli and Staphylococcus aureus due to the photothermal properties of PDA and antibacterial ε-PL. The prepared Janus nanofiber dressing also exhibited anti-inflammatory activity and biocompatibility. In addition, the Janus nanofiber dressing had asymmetric wettability that enabled directional water transport, thereby draining excessive wound exudate. The water vapor transmission test indicated that the Janus nanofiber dressing had good air permeability. Finally, skin wound healing evaluation in rats confirmed its efficacy in promoting wound healing. Therefore, this strategy of designing and manufacturing a multifunctional Janus nanofiber dressing had great potential in wound healing applications.

Cell sheet-basedin vitrobone defect model for long term evaluation of bone repair materials.

Development of tissue-engineeredin vitrohuman bone defect models for evaluation of bone repair materials (BRMs) is a promising approach for addressing both translational and ethical concerns regarding animal models. In this study, human bone marrow mesenchymal stem cell sheets were stacked to form a periosteum like tissue. HE staining showed a cell-dense, multilayered structure. BRMs were implanted in the defect area of the three-dimensional (3D) model. The CCK-8 test demonstrated that the 3D model was stronger in resisting the cytotoxicity of three kinds of commercial BRMs than the 2D culture model, which was consistent within vivoresults. After 28 d implantation in the 3D model, western blot and RT-qPCR showed that three materials induced increased expressions of RUNX2, OSX, OCN, OPN, while Materials B and C seemed to have stronger osteoinductivity than A.In vivoexperiments also confirmed the osteoinductivity of the BRMs after 28 and 182 d implantation. Alizarin red staining proved that the mineralized nodules of Materials B and C were more than that of A. The differences of osteogenic properties among three BMRs might be attributed to calcium ion release. This cell sheet-based bone tissue model can resist cytotoxicity of BRMs, demonstrating the priority of long-term evaluation of osteoinductivity of BRMs. Further, the osteoinduction results of the 3D model corresponded to that ofin vivoexperiments, suggesting this model may have a potential to be used as a novel tool for rapid, accurate evaluation of BRMs, and thus shorten their research and development process.

Nrf2-mediated therapeutic effects of dietary flavones in different diseases.

Oxidative stress (OS) is a pathological status that occurs when the body's balance between oxidants and antioxidant defense systems is broken, which can promote the development of many diseases. Nrf2, a redox-sensitive transcription encoded by NFE2L2, is the master regulator of phase II antioxidant enzymes and cytoprotective genes. In this context, Nrf2/ARE signaling can be a compelling target against OS-induced diseases. Recently, natural Nrf2/ARE regulators like dietary flavones have shown therapeutic potential in various acute and chronic diseases such as diabetes, neurodegenerative diseases, ischemia-reperfusion injury, and cancer. In this review, we aim to summarize nrf2-mediated protective effects of flavones in different conditions. Firstly, we retrospected the mechanisms of how flavones regulate the Nrf2/ARE pathway and introduced the mediator role Nrf2 plays in inflammation and apoptosis. Then we review the evidence that flavones modulated Nrf2/ARE pathway to prevent diseases in experimental models. Based on these literature, we found that flavones could regulate Nrf2 expression by mechanisms below: 1) dissociating the binding between Nrf2 and Keap1 via PKC-mediated Nrf2 phosphorylation and P62-mediated Keap1 autophagic degradation; 2) regulating Nrf2 nuclear translocation by various kinases like AMPK, MAPKs, Fyn; 3) decreasing Nrf2 ubiquitination and degradation via activating sirt1 and PI3K/AKT-mediated GSK3 inhibition; and 4) epigenetic alternation of Nrf2 such as demethylation at the promoter region and histone acetylation. In conclusion, flavones targeting Nrf2 can be promising therapeutic agents for various OS-related disorders. However, there is a lack of investigations on human subjects, and new drug delivery systems to improve flavones' treatment efficiency still need to be developed.

Deciphering the contributions of cuproptosis in the development of hypertrophic scar using single-cell analysis and machine learning techniques.

Hypertrophic scar (HS) is a chronic inflammatory skin disease characterized by excessive deposition of extracellular matrix, but the exact mechanisms related to its formation remain unclear, making it difficult to treat. This study aimed to investigate the potential role of cuproptosis in the information of HS. To this end, we used single-cell sequencing and bulk transcriptome data, and screened for cuproptosis-related genes (CRGs) using differential gene analysis and machine learning algorithms (random forest and support vector machine). Through this process, we identified a group of genes, including ATP7A, ULK1, and MTF1, as novel therapeutic targets for HS. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to confirm the mRNA expression of ATP7A, ULK1, and MTF1 in both HS and normal skin (NS) tissues. We also constructed a diagnostic model for HS and analyzed the immune infiltration characteristics. Additionally, we used the expression profiles of CRGs to perform subgroup analysis of HS. We focused mainly on fibroblasts in the transcriptional profile at single-cell resolution. By calculating the cuproptosis activity of each fibroblast, we found that cuproptosis activity of normal skin fibroblasts increased, providing further insights into the pathogenesis of HS. We also analyzed the cell communication network and transcription factor regulatory network activity, and found the existence of a fibroblast-centered communication regulation network in HS, where cuproptosis activity in fibroblasts affects intercellular communication. Using transcription factor regulatory activity network analysis, we obtained highly active transcription factors, and correlation analysis with CRGs suggested that CRGs may serve as potential target genes for transcription factors. Overall, our study provides new insights into the pathophysiological mechanisms of HS, which may inspire new ideas for the diagnosis and treatment.

[Research progress of allogeneic abdominal wall transplantation].

Objective: To summarize the research progress of surgical technique and immunosuppressive regimen of abdominal wall vascularized composite allograft transplantation in animals and clinical practice. Methods: The literature on abdominal wall transplantation at home and abroad in recent years was extensively reviewed and analyzed. Results: This review includes animal and clinical studies. In animal studies, partial or total full-thickness abdominal wall transplantation models have been successfully established by researchers. Also, the use of thoracolumbar nerves has been described as an important method for functional reconstruction and prevention of long-term muscle atrophy in allogeneic abdominal wall transplantation. In clinical studies, researchers have utilized four revascularization techniques to perform abdominal wall transplantation, which has a high survival rate and a low incidence of complications. Conclusion: Abdominal wall allotransplantation is a critical reconstructive option for the difficulty closure of complex abdominal wall defects. Realizing the recanalization of the nerve in transplanted abdominal wall to the recipient is very important for the functional recovery of the allograft. The developments of similar research are beneficial for the progress of abdominal wall allotransplantation.

Revealing the roles of glycosphingolipid metabolism pathway in the development of keloid: a conjoint analysis of single-cell and machine learning.

Keloid is a pathological scar formed by abnormal wound healing, characterized by the persistence of local inflammation and excessive collagen deposition, where the intensity of inflammation is positively correlated with the size of the scar formation. The pathophysiological mechanisms underlying keloid formation are unclear, and keloid remains a therapeutic challenge in clinical practice. This study is the first to investigate the role of glycosphingolipid (GSL) metabolism pathway in the development of keloid. Single cell sequencing and microarray data were applied to systematically analyze and screen the glycosphingolipid metabolism related genes using differential gene analysis and machine learning algorithms (random forest and support vector machine), and a set of genes, including ARSA,GBA2,SUMF2,GLTP,GALC and HEXB, were finally identified, for which keloid diagnostic model was constructed and immune infiltration profiles were analyzed, demonstrating that this set of genes could serve as a new therapeutic target for keloid. Further unsupervised clustering was performed by using expression profiles of glycosphingolipid metabolism genes to discover keloid subgroups, immune cells, inflammatory factor differences and the main pathways of enrichment between different subgroups were calculated. The single-cell resolution transcriptome landscape concentrated on fibroblasts. By calculating the activity of the GSL metabolism pathway for each fibroblast, we investigated the activity changes of GSL metabolism pathway in fibroblasts using pseudotime trajectory analysis and found that the increased activity of the GSL metabolism pathway was associated with fibroblast differentiation. Subsequent analysis of the cellular communication network revealed the existence of a fibroblast-centered communication regulatory network in keloids and that the activity of the GSL metabolism pathway in fibroblasts has an impact on cellular communication. This contributes to the further understanding of the pathogenesis of keloids. Overall, we provide new insights into the pathophysiological mechanisms of keloids, and our results may provide new ideas for the diagnosis and treatment of keloids.

Preparation of a 3D printable high-performance GelMA hydrogel loading with magnetic cobalt ferrite nanoparticles.

Osteosarcoma remains a worldwide concern due to the poor effectiveness of available therapies in the clinic. Therefore, it is necessary to find a safe and effective therapy to realize the complete resection of osteosarcoma and reconstruction of the bone defect. Magnetic hyperthermia based on magnetic nanoparticles can kill tumor cells by raising the temperature without causing the side effects of conventional cancer treatments. This research aims to design a high-performance magnetic hydrogel composed of gelatin methacrylate and highly magnetic cobalt ferrite (CFO) nanoparticles for osteosarcoma treatment. Specifically, CFO is surface functionalized with methacrylate groups (MeCFO). The surface modified CFO has good biocompatibility and stable solution dispersion ability. Afterward, MeCFO nanoparticles are incorporated into GelMA to fabricate a three-dimensional (3D) printable MeCFO/GelMA magnetic hydrogel and then photocross-linked by UV radiation. MeCFO/GelMA hydrogel has high porosity and swelling ability, indicating that the hydrogel possesses more space and good hydrophily for cell survival. The rheological results showed that the hydrogel has shear thinning property, which is suitable as a bioprinting ink to produce desired structures by a 3D printer. Furthermore, 50 µg/mL MeCFO not only decreases the cell activity of osteosarcoma cells but also promotes the osteogenic differentiation of mBMSCs. The results of the CCK-8 assay and live/dead staining showed that MeCFO/GelMA hydrogel had good cytocompatibility. These results indicated that MeCFO/GelMA hydrogel with potential antitumor and bone reconstruction functions is a promising therapeutic strategy after osteosarcoma resection.

A wet-adhesive carboxymethylated yeast ß-glucan sponge with radical scavenging, bacteriostasis and anti-inflammatory functions for rapid hemostasis.

Local hemostats still face obstacles to efficiently achieving hemostasis and promoting wound healing. Herein, a series of multifunctional well-degradable hemostatic sponges based-on carboxymethylated yeast ß-glucan (CMYG) were fabricated by lyophilization. The porous CMYG sponge not only could absorb blood quickly (44.12 g/g), but also possessed unexpected tissue adhesion (∼30 kPa), and it represented good biocompatibility in vitro on fibroblasts and red blood cells. Notably, compared with the commercial Celox™, the CMYG sponge achieved more rapid hemostasis and significantly reduced blood loss in liver injury rat models by rapid wound block. Interestingly, the developed sponge showed an outstanding effect on antioxidant, anti-infection, anti-inflammatory, and cell proliferation, which are beneficial for further wound repair. Overall, these results suggest that the CMYG sponge is a promising candidate for the clinical management of uncontrollable hemorrhage and the further development of wound dressing materials throughout skin defect repair.

What Determines the Critical Electric Field of AFE-to-FE in Pb(Zr,Sn,Ti)O3-Based Perovskites?

Electric-field-induced antiferroelectric-ferroelectric (AFE-FE) phase transition is a prominent feature of antiferroelectric (AFE) materials. The critical electric field of this phase transition is crucial for the device performance of AEFs in many applications, but the determining factor of the critical electric field is still unclear. Here, we have established the correlation between the underlying structure and the critical electric field by using in situ synchrotron X-ray diffraction and high-resolution neutron diffraction in Pb(Zr,Sn,Ti)O3-based antiferroelectrics. It is found that the critical electric field is determined by the angle between the average polarization vector in the incommensurate AFE state and the [111]P polarization direction in the rhombohedral FE state. A large polarization rotation angle gives rise to a large critical electric field. Further, density functional theory (DFT) calculations corroborate that the lower energy is required for driving a smaller angle polarization rotation. Our discovery will offer guidance to optimize the performance of AFE materials.

Macromolecular crowding facilitates rapid fabrication of intact, robust cell sheets.

OBJECTIVES: To develop a rapid and simple method to fabricate intact, robust cell sheets from common cell culture dishes by combination of a macromolecular crowding (MMC) reagent and vitamin C. RESULTS: It was found that 3T3 fibroblasts or human bone marrow mesenchymal stem cells (hBMSCs) and their secreted cell derived extracellular matrices could be easily detached as intact cell sheets under gently pipetting after treated by MMC and vitamin C for 4 days. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing 10 × 104 cells/cm2 HepG2 cells on top of confluent 3T3 fibroblast layers. What's more, MMC induced hBMSC cell sheets demonstrated 1.9 times larger area and 1.6 times greater cell number than that of cell sheets harvested from temperature-responsive cell culture dishes. CONCLUSION: MMC based method make it possible to fabricate various types of cell sheets more conveniently, economically, and thus may facilitate wide application of cell sheet technology.

Ultrathin and handleable nanofibrous net as a novel biomimetic basement membrane material for endothelial barrier formation.

Many strategies have been adopted to develop porous membranes to reconstitute basement membrane in vitro, which play a key role in the development of in vitro biomimetic models. However, the development of an artificial basement membrane combines cytocompatibility and nano-thickness is still challenging. Herein, a monolayer nanofibrous net patch was fabricated by combining microfabrication and electrospinning as a biomimetic basement membrane material, which was demonstrated for endothelial barrier formation. The nanofibrous net patches with different fiber densities were obtained by controlling electrospinning time. The net was with high porosity and ultrathin thickness approximate to the diameter of nanofibers, which is comparable to that of the native basement membrane. The morphology, proliferation and cell-cell/cell-substrate interactions of endothelial cells on the nanofibrous nets were studied and compared with track-etched polycarbonate membrane and traditional multilayer nanofibers membrane. In addition, the results of TEER measurement and permeability test demonstrated that the endothelial barrier formed on the nanofibrous net patch displayed stronger barrier integrity and function. Therefore, the proposed nanofibrous net patch shows great potential as a novel biomimetic basement membrane, which is promising to be applied for in vitro tissue mimetic applications.

[Long term maintenance of cytochrome P450 activity in a cell sheet-based three-dimensional human hepatic model].

Primary human hepatocytes (PHH) are the gold standard of in vitro human liver model for drug screening. However, a problem of culturing PHH in vitro is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) "sandwich" co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×106 /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D in vitro liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.

Development of an in vitro insulin resistance dissociated model of hepatic steatosis by co-culture system.

The evidence shows that there is an associated relationship between hepatosteatosis and insulin resistance. While some existing genetic induction animal and patient models challenge this relationship, indicating that hepatosteatosis is dissociated from insulin resistance. However, the molecular mechanisms of this dissociation remain poorly understood due to a lack of available, reliable, and simplistic setup models. Currently, we used primary rat hepatocytes (rHPCs), co-cultured with rat hepatic stellate cells (HSC-T6) or human foreskin fibroblast cells (HFF-1) in stimulation with high insulin and glucose, to develop a model of steatosis charactered as dissociated lipid accumulation from insulin resistance. Oil-Red staining significantly showed intracellular lipid accumulated in the developed model. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c) and elongase of very-long-chain fatty acids 6 (ELOVL6), key genes responsible for lipogenesis, were detected and obviously increased in this model. Inversely, the insulin resistance related genes expression included phosphoenolpyruvate carboxykinase 1 (PCK1), pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), and glucose-6-phosphatase (G6pase) were decreased, suggesting a dissociation relationship between steatosis and insulin resistance in the developed model. As well, the drug metabolism of this developed model was investigated and showed up-regulation of cytochrome P450 3A (CYP3A) and down-regulation of cytochrome P450 2E1 (CYP2E1) and cytochrome P450 1A2 (CYP1A2). Taken together, those results demonstrate that the in vitro model of dissociated steatosis from insulin resistance was successfully created by our co-cultured cells in high insulin and glucose medium, which will be a potential model for investigating the mechanism of insulin resistance dissociated steatosis, and discovering a novel drug for its treatment.