Tissue engineering. Part C, Methods最新文献

Tendon stem/progenitor cell (TSPC) senescence is often associated with age-dependent tendon diseases and greatly reduces the capacities for tendon repair and replacement. Exosomes contain bioactive molecules and have been increasingly used in regenerative medicine. In the present study, we demonstrated the antiaging effects of young exosomes from circPVT1-overexpressing TSPCs at early passages (circPVT1-exo). These exosomes attenuated the phenotypes of aged TSPCs at late passages (L-TSPCs) by enhancing self-renewal and proliferation abilities, suppressing cell senescence, maintaining their tenogenic capacity, and weakening their osteogenic differentiation. Mechanistically, circPVT1-exo inhibited the NF-κB pathway and increased SIRT1 expression in L-TSPCs. Knockdown of SIRT1 reversed these effects as evidenced by increased senescence, decreased proliferation, and tenogenic differentiation. These results suggest that circPVT1-exo may ameliorate aging-impaired TSPC function by modulating the SIRT1/NF-κB pathway, suggesting that circPVT1-exo has therapeutic potential for age-related diseases.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

This work employs nitrogen plasma immersion ion implantation (PIII) to modify electrospinning polylactic acid membranes and immobilizes basic fibroblast growth factors (bFGF) by forming crosslinking bonds. The study investigates the modified membranes' surface characteristics and the stimulatory effects of crosslinked bFGF polylactic acid membranes on osteoblast and fibroblast proliferation. The PIII process occurs under low vacuum conditions and is controlled by processing time and power pulse width. The experimental results indicate that, within a 400-second N₂-PIII treatment, the spun fibers remain undamaged, demonstrating an increase in hydrophilicity (from 117° to 38°/36°) and nitrogen content (from 0% to 7.54%/8.05%). X-ray photoelectron spectroscopy analysis suggests the formation of a C-N-C=O crosslinked bond. Cell culture and activity assessments indicate that the PIII-treated and crosslinked bFGF film exhibits significantly higher cell growth activity (p < 0.05) than the untreated group. These intergroup differences are attributed to the surface crosslinking bond content. In osteogenic induction, the results for each day show that the treated group performs better. However, the intergroup disparities within the crosslinked bFGF group disappear with prolonged culture time due to the rapid osteogenesis prompted by bFGF. The findings suggest that PIII treatment of electrospinning polylactic acid membranes holds promise in promoting osteogenesis in bone tissue scaffolds.

Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel^® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

Traumatic injuries may result in the formation of soft tissue adhesions between peripheral nerves and surrounding soft tissue. These soft tissue adhesions lead to compression and ischemic stress within fascicles due to nonpliability of adhered scar tissue, and nerve tension due to loss of nerve gliding from scar tethering. These changes in the soft tissue bed surrounding the nerve may result in axon degeneration and neuroma-in-continuity. Preclinical models that simulate clinically relevant levels of scar in the nerve environment may be impactful to the development of surgical techniques and treatments to prevent adhesions. This study presents the results of a rodent model with an induced indirect nerve injury by (1) thermal insult to the soft tissue bed surrounding the nerve and (2) air-drying the surrounding soft tissue bed of the nerve. Our findings suggest that inducing an injury of the soft tissue bed results in increased intraneural scar and extraneural adhesions to the nerve compared to a sham procedure. Thermal induced injuries showed more macrophages and changes in nerve health compared to air-dried induced injuries. The changes in the nerves of the induced injury groups, specifically the thermal injury group, may be meaningful for evaluating treatments for nontransected nerve injuries.

Cardiac fibrosis is characterized by pathological proliferation and activation of cardiac fibroblasts to myofibroblasts. Inhibition and reverse of transdifferentiation of cardiac fibroblasts to myofibroblasts is a potential strategy for cardiac fibrosis. Despite substantial progress, more effort is needed to discover effective drugs to improve and reverse cardiac fibrosis. The main reason for the slow development of antifibrotic drugs is that the traditional polystyrene culture platform does not recapitulate the microenvironment where cells reside in tissues. In this study, we propose an in vitro cardiac fibrotic model by seeding electrospun yarn scaffolds with cardiac fibroblasts. Our results show that yarn scaffolds allow three-dimensional growth of cardiac fibroblasts, promote extracellular matrix (ECM) deposition, and induce the transdifferentiation of cardiac fibroblasts to myofibroblasts. Exogenous transforming growth factor-β1 further promotes cardiac fibroblast activation and ECM deposition, which makes it a suitable fibrotic model to predict the antifibrotic potential of drugs. By using this platform, we demonstrate that both Honokiol (HKL) and Pirfenidone (PFD) show potential in antifibrosis to some extent. HKL is more efficient in antifibrosis than PFD as revealed by biochemical composition, gene, and molecular analyses as well as histological and biomechanical analysis. The electrospun yarn scaffold provides a novel platform for constructing in vitro fibrotic models to study cardiac fibrosis and to predict the antifibrotic efficacy of novel drugs.

Due to a growing need in visualizing human pluripotent stem cell-derived organoids from recent advancements in the field, an efficient bulk-processing application is necessary to provide preprocessing and image analysis services. In this study, we developed Organalysis, a high-accuracy, multifunctional, and accessible application that meets these needs by providing the functionality of image manipulation and enhancement, organoid area and intensity calculation, fractal analysis, noise removal, and feature importance computation. The image manipulation feature includes brightness and contrast adjustment. The area and intensity calculation computes six values for each image: organoid area, total image area, percentage of the image covered by organoid, the total intensity of organoid, the total intensity of organoid-by-organoid area, and total intensity of organoid by total image area. The fractal analysis function computes the fractal dimension value for each image. The noise removal function removes superfluous marks from the input images, such as bubbles and other unwanted noise. The feature importance function trains a lasso-regularized linear regression machine learning algorithm to identify cardiac growth factors that are the strongest determinants for cell differentiation. The batch processing of this application further builds on existing services like ImageJ to provide a more convenient way to process multiple images. Collectively, the versatility and preciseness of Organalysis demonstrate novelty, since no other current imaging software combines the capability of batch processing and the breadth of feature analysis. Therefore, Organalysis provides unique functions in cardiac organoid research and proves to be invaluable in regenerative medicine.

Adipose Stem Cell Tissue Engineering (ASCTE) has emerged as a promising field of research in recent years. To gain comprehensive insights into this field, we conducted a comprehensive bibliometric analysis using Web of Science Core Collection and various bibliometric tools, including CiteSpace, VOS viewer, and R-Bibliometrix. Our analysis focuses on the historical development and evolution of active topics in ASCTE from a time-dynamics perspective, covering 4522 publications, 3924 academic institutions, and 873 journals, with significant growth observed over the past two decades. In terms of the global research landscape, the United States and China dominate the field. Shanghai Jiao Tong University, the University of Pittsburgh, and Ming Ho University are the top three institutions contributing to research in this area. Biomaterials is identified as the central journal in terms of cocitation analysis. Our analysis also reveals new areas of development, such as 3D printing, platelet lysate, and clinical practice, as well as current trends in hydrogels, nanomaterials, and extracellular vesicles. These findings point to exciting prospects for future ASCTE research. Unlike previous subjective reviews, our bibliometric analysis provides an objective assessment of the current state and emerging trends in ASCTE research, allowing researchers to identify popular research areas and explore new directions in this dynamic field.

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 μg for all nine antibodies, compared with ∼10 μg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.