Tissue engineering. Part C, Methods最新文献

This study describes the development of a three-dimensional (3D) oral mucosal model (OMM) to investigate how oral tissues respond to masticatory forces. The OMMs replicated key features of human oral mucosa, such as stratified keratinocyte telomerase-immortalized gingival keratinocytes (TIGK) layers and fibroblast-populated collagen matrices. Cyclical mechanical forces (0-10 N) for 2 h applied to the model caused force-dependent changes in the histological structure, including thinning of the epithelium and collagen matrix and cell displacement at higher forces. Lactate dehydrogenase (LDH) cytotoxicity assays revealed that 10 N forces led to significant cell damage (about 50% cell death) in TIGK monolayers, whereas lower forces (1-5 N) caused minimal damage. OMMs showed reduced cell death (∼15% at 10 N), indicating better resilience presumably due to their 3D architecture. Additionally, force-dependent increases in the release of the proinflammatory cytokines IL-6 and IL-8 were observed, with lower responses in OMMs compared with monolayer cultures. This study demonstrates that OMMs can be used to model the effects of masticatory forces on the response of the oral mucosa in denture wearers and has been utilized to investigate the effects of a denture adhesive on the inflammatory response of the OMM to pressure.

Cytometry by time-of-flight (CyTOF) enables comprehensive immune profiling for translational research. However, challenges such as signal variability, nonspecific binding, and antibody incompatibility can compromise data quality. This study presents an optimized CyTOF staining protocol for human peripheral blood mononuclear cells and bone marrow aspiration concentrate samples, addressing these challenges by refining antibody conjugation with polymer X8, saponin use, and fixation protocols. Preliminary data indicate improved staining for key markers (CD14, CD16, and CD19), enhancing signal consistency and clarity. These findings advance the utility of CyTOF in orthopaedic research and immune profiling for diseases such as osteonecrosis of the femoral head.

Bioengineering aims to develop biomaterials that closely mimic the native extracellular matrix (ECM) to support tissue regeneration. This study presents a detailed protocol for producing hydrogels derived from decellularized bovine placental cotyledons. Bovine placentas at 4-5 months of gestation (n = 10) were subjected to vascular perfusion with increasing concentrations of sodium dodecyl sulfate (0.01-1%) and Triton X-100 (1%), which effectively removed cellular components. Decellularization efficacy was confirmed by histological (hematoxylin and eosin and 4',6-diamidino-2-phenylindole [DAPI] staining), molecular, and structural analyses, including residual genomic DNA quantification averaging 9.1 ng/mg of dry tissue. The ECM scaffolds were enzymatically digested using 0.1% (w/v) pepsin in 0.01 M HCl and reconstituted with sodium alginate at concentrations of 5%, 8%, 10%, and 12% (w/v). Crosslinking was achieved with 1% calcium chloride. Among the tested formulations, hydrogels containing 12% alginate demonstrated greater mechanical stability and preserved three-dimensional architecture, including interconnected porosity, as evidenced by scanning electron microscopy. Cytocompatibility was evaluated by culturing canine adipose-derived mesenchymal stem cells on both decellularized biomaterials and hydrogels. DAPI staining revealed nuclei after 7 and 25 days of culture, indicating cell presence and distribution throughout the constructs. These results indicate that bovine cotyledon-derived ECM hydrogels maintain structural and biochemical features favorable for cell interaction and may serve as adaptable platforms for tissue engineering, dermal repair, and three-dimensional cell culture.

Fibrosis causes altered tissue structure and function in multiple organs due to a complex interplay between inflammatory cells, myofibroblasts, and extracellular matrix (ECM) components. While it is known that T cells play a role in tissue fibrosis, it remains unclear how they modulate cellular interactions to activate fibrogenesis. Since conventional monolayer cell cultures do not mimic the tissue complexity and cellular heterogeneity in the fibrotic tissue environment, there is a need to bridge the gap between monolayer cultures and in vivo animal studies of fibrosis by providing a more predictive 3D model for preclinical drug screening and mechanistic studies of fibrotic diseases. We have developed 3D skin-like tissues harboring blood-derived human T cells that offer a model to better understand the role these cells play in the pathogenesis of tissue fibrosis. In the current study, we constructed skin-like tissues harboring T cells, fibroblasts, macrophages, and keratinocytes and analyzed them using tissue analysis and single-cell RNA sequencing (scRNA-seq). Skin-like tissues constructed with fully autologous cells (donor-matched fibroblasts and T cells) or nonautologous cells (mismatched fibroblasts and T cells) derived from patients with scleroderma (SSc) demonstrated normal distribution of tissue markers of epithelial differentiation and proliferation. T cells in these tissues were viable and functional as seen by elevated IL-6 production by enzyme-linked immunosorbent assay, expression of alpha smooth muscle actin in fibroblasts, and scRNA-seq. We used scRNA-seq to identify five distinct T cell subpopulations: CD8 T cells (identified by KLRK1 and CD8A), proliferating CD4 T cells (identified by PCNA, MKI67, and CD4), activated CD4 T cells (identified by IL2RA, RORA, and CD4), naïve CD4 T cells (identified by CCR7 and CD4), and Th17 CD4 T cells (identified by KLRB1, RORA, IL2RA, and CD4). Fabrication of complex 3D tissues are an important step toward establishing tissue engineering approaches to study fibrosis in multiple diseases, including SSc, idiopathic pulmonary fibrosis, as well as liver and kidney fibrosis. Understanding the roles of T cells in the ECM environment and their interactions with fibroblasts will support the development of novel treatments to reverse fibrosis and restore normal tissue and organ function.

Tissue-engineered vascular grafts (TEVGs) are emerging as promising alternatives to synthetic grafts, particularly in pediatric cardiovascular surgery. While TEVGs have demonstrated growth potential, compliance, and resistance to calcification, their functional integration into the circulation, especially their ability to respond to physiological stimuli, remains underexplored. Vasoreactivity, the dynamic contraction or dilation of blood vessels in response to vasoactive agents, is a key property of native vessels that affects systemic hemodynamics and long-term vascular function. This study aimed to develop and validate an in vivo protocol to assess the vasoreactive capacity of TEVGs implanted as inferior vena cava (IVC) interposition grafts in a large animal model. Bone marrow-seeded TEVGs were implanted in the thoracic IVC of Dorset sheep. A combination of intravascular ultrasound (IVUS) imaging and invasive hemodynamic monitoring was used to evaluate vessel response to norepinephrine (NE) and sodium nitroprusside (SNP). Cross-sectional luminal area changes were measured using a custom Python-based software package (VIVUS) that leverages deep learning for IVUS image segmentation. Physiological parameters including blood pressure, heart rate, and cardiac output were continuously recorded. NE injections induced significant, dose-dependent vasoconstriction of TEVGs, with peak reductions in luminal area averaging ∼15% and corresponding increases in heart rate and mean arterial pressure. Conversely, SNP did not elicit measurable vasodilation in TEVGs, likely due to structural differences in venous tissue, the low-pressure environment of the thoracic IVC, and systemic confounders. Overall, the TEVGs demonstrated active, rapid, and reversible vasoconstrictive behavior in response to pharmacologic stimuli. This study presents a novel in vivo method for assessing TEVG vasoreactivity using real-time imaging and hemodynamic data. TEVGs possess functional vasoactivity, suggesting they may play an active role in modulating venous return and systemic hemodynamics. These findings are particularly relevant for Fontan patients and other scenarios where dynamic venous regulation is critical. Future work will compare TEVG vasoreactivity with native veins and synthetic grafts to further characterize their physiological integration and potential clinical benefits.

The nasopharynx constitutes a critical component of the respiratory tract. Nasopharyngeal diseases are closely related to nasopharyngeal epithelial cells (NECs). Considering the current paucity of appropriate cell models for studying nasopharynx-related diseases, there is an urgent need to develop a simple and efficient method for the long-term culture and robust expansion of primary NECs. In this study, we employed the NEC medium supplemented with Wnt3a, R-spondin, Noggin, and other growth factors to stimulate the proliferation of nasopharyngeal epithelial stem cells and maintain their self-renewal state, enabling long-term culture. Leveraging this strategy, we successfully developed a simplified and efficient method for long-term culture of primary murine NECs. The NEC medium provided a selective advantage for stably expanding cytokeratin 5- and epithelial membrane antigen-positive epithelial cells rather than alpha-smooth muscle actin-marked fibroblasts and prevented epithelial-mesenchymal transition as evidenced by continuously strong E-cadherin expression and being negative for vimentin. The established NEC line exhibited stable long-term proliferation with no evident signs of senescence. We also confirmed the nontumorigenic nature of the established nasopharyngeal cell line in mice. Our findings from this study provided a valuable cellular tool for investigating nasopharyngeal epithelial-related diseases and developing therapeutic strategies.

Platelet-rich plasma (PRP) was prepared from goat blood using a modified Landesberg method. A PRP/calcium phosphate bone cement (CPC) composite paste was then prepared by combining PRP with injectable CPC, whereby the platelet counts in PRP increased by about 5.9-fold compared to that in the whole blood. Additionally, the levels of PDGF-AB, TGF-β, and VEGF in PRP were significantly higher than those in the whole blood. The new PRP/CPC composite exhibited significantly better injectability, initial setting time, final setting time, and washout resistance compared with CPC alone. A lumbar vertebral defect model was established in 18 Hainan indigenous male black goats via a retroperitoneal approach. Six lumbar vertebrae from each goat were randomized to three groups: a control group receiving normal saline, a CPC group using CPC paste alone, and a PRP/CPC group treated with the autologous PRP/CPC composite paste. The goats were maintained under standard feeding conditions postoperatively. Six goats were euthanized at 1, 3, and 6 months after operation to obtain vertebral specimens for assessment of vertebral strength and stiffness. Digital radiographical imaging at 6 months after operation showed that the vertebrae had normal growth and morphology in all groups. At 1, 3, and 6 months after operation, the vertebral strength and stiffness in PRP/CPC group were significantly greater than those in CPC-alone group. In addition, both vertebral strength and stiffness showed further improvement with the extension of postoperative recovery time. The PRP/CPC composite exhibited commendable rheological properties, and its application in repair of vertebral bone defects yielded favorable biomechanical properties. Furthermore, the new autologous PRP/CPC composite showed excellent biocompatibility and tissue repair capability and may prove to be a suitable candidate for repair of load-bearing bone defects, particularly those present in vertebrae.

Advanced tissue-engineered respiratory models are essential for studying drug or cosmetic toxicity, infection biology and xenobiotic metabolism. Here, we investigated a polyamide 6 (PA6)-based electrospun stromal scaffold as a substitute for porcine-derived small intestinal submucosa (SIS) to build human airway mucosa tissue models at the air-liquid interface. We demonstrate that the porous PA6 scaffold supports extracellular matrix production by human nasal fibroblasts and facilitates the complete differentiation of respiratory epithelial cells to the mucociliary phenotype. These models reduce reliance on animal-derived materials, improve reproducibility, and minimize potential interference from animal-derived antigens and pathogens. Both PA6- and SIS-based models promote fibroblast migration, epithelial differentiation, and the expression of key xenobiotic metabolizing enzymes. They exhibit comparable epithelial barrier integrity and susceptibility to influenza A virus infections. These findings establish PA6 scaffolds as a suitable, animal-free alternative to the SIS to build human airway mucosa tissue models.

Currently, the evaluation of in vivo antimicrobial efficacy predominantly relies on endpoint detection methods, such as Colony Forming Units (CFU) counting and histopathological staining following animal sacrifice, to assess the antimicrobial properties of materials. These traditional detection methods struggle to capture real-time changes in infection status during treatment. This study proposes a novel strategy utilizing lipophilic near-infrared dye (e.g., DIR, [1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide]) for bacterial fluorescent labeling, combined with In Vivo Imaging System (IVIS) technology, to achieve real-time monitoring of dynamic changes in bacterial infection in localized infection models. Following local injection of stained bacteria, IVIS imaging revealed temporal changes in fluorescence signals within infected areas, which were further utilized to evaluate the in vivo efficacy of antimicrobial biomaterials. We have effectively validated this approach in a rat bone defect infection model. Additionally, this method can be used in conjunction with micro-CT to enable three-dimensional observation. Experimental results demonstrate that this approach intuitively reflects the immediate effects of antimicrobial treatment and facilitates precise quantitative analysis, providing technical support for in vivo detection of antimicrobial efficacy.

Vascular tissue engineering technology uses tubular viscoelastic materials as intermediaries to transmit the mechanical stimuli required for the construction of vascular grafts. However, most existing studies rely on elastic models, which fail to capture the time-dependent nature of viscoelastic materials. Moreover, the long fabrication cycles, high costs, and complex parameter measurements in tissue engineering pose significant challenges to experimental approaches. There is thus an urgent need to develop a viscoelastic mechanical model that combines physical interpretability, computational efficiency, and predictive accuracy, enabling precise characterization of material responses and unified quantification across experimental platforms. Here, we propose an error-corrected linear solid (ECLS) model with an embedded correction term to address the predictive deviations of conventional models in nonlinear viscoelastic scenarios. Instead of expanding the traditional model structure, the ECLS incorporates an error correction method that improves predictive performance while maintaining structural simplicity. Experiments were conducted on three representative viscoelastic materials-silicone rubber, polyurethane, and polytetrafluoroethylene-to acquire time-resolved response data through stress relaxation and creep tests. The fitting performance was quantitatively evaluated using the Euclidean norm and the Akaike information criterion, enabling a systematic comparison between the ECLS model and three classical models (Kelvin-Voigt, Maxwell, and standard linear solid [SLS]). The results show that the ECLS model exhibits higher predictive accuracy over a wide time range, with an average goodness of fit (R²) of 0.99, representing an improvement of ∼6% compared to the SLS model. Furthermore, the Root Mean Square Error (RMSE) and Mean Absolute Error (MAE) of the ECLS model are at least one order of magnitude lower than those of the traditional models, significantly improving the description of nonlinear viscoelastic behavior and providing more accurate predictions of material viscoelastic mechanical behavior. Therefore, the ECLS model not only improves the modeling accuracy of viscoelastic behavior but also establishes a unified and scalable framework for predicting and optimizing the mechanical performance of tissue-engineered vessels, expanding the application potential of mechanical modeling in bioreactor design and biomaterials development.