Tissue engineering. Part C, Methods最新文献

The articles and reviews in the field of decellularized extracellular matrix (dECM) from 2001 to 2021 were retrieved and extracted from the Web of Science Core Collection. The R package Bibliometrix, CiteSpace, VOSviewer, and the online BIBLIOMETRC platform were utilized for bibliometric analysis, including specific characteristics of annual publications, influential countries/regions, core journals, leading institutions, keywords, key references, cocited authors, journals and institutions, cooperation, and historical direct citations. Our study concluded core references that fueled the development of dECM and highlighted current research directions, hotpots, and trends. From 2001 to 2021, 3,046 publications were retrieved in total, including 2,700 articles and 349 reviews. The United States (n = 895) produced the majority of publications, and the University of Pittsburgh (n = 318) published most productions. Biomaterials were identified as the most productive and influential journal in the dECM field considering the number of publications (n = 194), and total citations (n = 15,694). Immunomodulation, bioreactors, aging, three-dimensional (3D) bioprinting, bone tissue engineering, bioink, hydrogel, biomaterials, and regeneration were the latest high-frequency keywords, indicating the emerging frontiers of dECM. In the field, decellularization techniques lay the foundation. Orthotopic transplantation of recellularized dECM and induction of specific cell differentiation promoted the bursts of research. The 3D bioprinting and hydrogel based on dECM were extensively studied in recent years. The present study provided developmental trajectories, current research status, global collaboration patterns, hotpots, and trending topics of dECM. Decellularization techniques, tissue engineering to regenerate organs, and improvements in application are the major themes over the past two decades. Impact Statement The review article is significant because decellularized extracellular matrix (dECM), which derived from biological tissues and removal of immunogenic cells, is characterized by safety, biocompatibility, and low in toxicity. Showing great application prospects, dECM has been applied in multiple scenarios of tissue repairment and reconstruction, among the most popular topics in tissue engineering. Thus, analyzing and concluding the development, current condition and future trends are of great significance. Comparing to conventional review, this review article systemically and comprehensively concluded the historical development, current status, and research trending topics. Thus, it allows scholars to get a rapid overview of the dECM field, and plan research directions.

Cathepsins are a family of cysteine proteases responsible for a variety of homeostatic functions throughout the body, including extracellular matrix remodeling, and have been implicated in a variety of degenerative diseases. However, clinical trials using systemic administration of cathepsin inhibitors have been abandoned due to side effects, so local delivery of cathepsin inhibitors may be advantageous. In these experiments, a novel microfluidic device platform was developed that can synthesize uniform, hydrolytically degradable microparticles from a combination of poly(ethylene glycol) diacrylate (PEGDA) and dithiothreitol (DTT). Of the formulations examined, the 10-polymer weight percentage 10 mM DTT formulation degraded after 77 days in vitro. A modified assay using the DQ Gelatin Fluorogenic Substrate was used to demonstrate sustained release and bioactivity of a cathepsin inhibitor (E-64) released from hydrogel microparticles over 2 weeks in vitro (up to ∼13 μg/mL released with up to ∼40% original level of inhibition remaining at day 14). Altogether, the technologies developed in this study will allow a small-molecule, broad cathepsin inhibitor E-64 to be released in a sustained manner for localized inhibition of cathepsins for a wide variety of diseases.

Isolated individual myofibers are valuable experimental models that can be used in various conditions to understand skeletal muscle physiology and pathophysiology at the tissue and cellular level. This report details a time- and cost-effective method for isolation of single myofibers from the flexor digitorum brevis (FDB) muscle in both young and aged mice. The FDB muscle was chosen for its documented history in single myofiber experiments. By modifying published methods for FDB myofiber isolation, we have optimized the protocol by first separating FDB muscle into individual bundles before the digestion, followed by optimizing the subsequent digestion medium conditions to ensure reproducibility. Morphological and functional assessments demonstrate a high yield of isolated FDB myofibers with sarcolemma integrity achieved in a shorter time frame than previous published procedures. This method could be also adapted to other types of skeletal muscle. Additionally, this highly reproducible method can greatly reduce the number of animals needed to yield adequate numbers of myofibers for experiments. Thus, this advanced method for myofiber isolation has the potential to accelerate research in skeletal muscle physiology and screening potential therapeutics "ex vivo" for muscle diseases and regeneration.

The heart contains diverse endothelial cell types. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of these cells, we reported our protocol for isolating EECs from porcine hearts and for establishing an EEC population through cell sorting. In addition, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs stained positively for classic phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs at 48 h (1310 ± 251 cells vs. 597 ± 130 cells, p = 0.0361) and at 96 h (2873 ± 257 cells vs. 1714 ± 342 cells, p = 0.0002). Yet EECs migrated more slowly than HUVECs to cover a scratch wound at 4 h (5% ± 1% wound closure vs. 25% ± 3% wound closure, p < 0.0001), 8 h (15% ± 4% wound closure vs. 51% ± 12% wound closure, p < 0.0001), and 24 h (70% ± 11% wound closure vs. 90% ± 3% wound closure, p < 0.0001). Finally, the EECs maintained their endothelial phenotype by positive expression of CD31 through more than a dozen passages (three populations of EECs showing 97% ± 1% CD31⁺ cells in over 14 passages). In contrast, the HUVECs showed significantly reduced CD31 expression over high passages (80% ± 11% CD31⁺ cells over 14 passages). These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell types when studying or modeling diseases of interest.

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG), while the axons are extended into the three divisions, including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we used a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this method, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies.

Mesenchymal stem cell and 3D printing-based bone tissue engineering present a promising technique to repair large-volume bone defects. Its success is highly dependent on cell attachment, spreading, osteogenic differentiation, and in vivo survival of stem cells on 3D-printed scaffolds. In this study, we applied human salivary histatin-1 (Hst1) to enhance the interactions of human adipose-derived stem cells (hASCs) on 3D-printed β-tricalcium phosphate (β-TCP) bioceramic scaffolds. Fluorescent images showed that Hst1 significantly enhanced the adhesion of hASCs to both bioinert glass and 3D-printed β-TCP scaffold. In addition, Hst1 was associated with significantly higher proliferation and osteogenic differentiation of hASCs on 3D-printed β-TCP scaffolds. Moreover, coating 3D-printed β-TCP scaffolds with histatin significantly promotes the survival of hASCs in vivo. The ERK and p38 but not JNK signaling was found to be involved in the superior adhesion of hASCs to β-TCP scaffolds with the aid of Hst1. In conclusion, Hst1 could significantly promote the adhesion, spreading, osteogenic differentiation, and in vivo survival of hASCs on 3D-printed β-TCP scaffolds, bearing a promising application in stem cell/3D printing-based constructs for bone tissue engineering.

This cross-sectional pilot study explored extracellular vesicle (EV)-derived gene expression of markers for bone turnover and pro-inflammatory cytokines in periodontal disease. Whole unstimulated saliva was collected from 52 participants (18 healthy, 13 gingivitis, and 21 stages III/IV periodontitis), from which salivary small extracellular vesicles (sEVs) were enriched using the size-exclusion chromatography method, and characterized by morphology, EV-protein, and size distribution, using transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), and Nanoparticle Tracking Analysis (NTA), respectively. Bone turnover markers and pro-inflammatory cytokines in salivary sEVs were evaluated using reverse transcription PCR. Salivary sEVs morphology, mode, size distribution, and particle concentration were comparable between healthy, gingivitis, and periodontitis patients. The CD9+ subpopulation was significantly higher in periodontitis-derived salivary sEVs compared with healthy. The detection of sEVs mRNA for osterix and tumor necrosis factor-alpha was significantly decreased and increased, respectively, in periodontitis compared with healthy controls, with good discriminatory power for periodontitis diagnosis (area under the curve >0.72). This pilot study demonstrated that salivary sEVs mRNAs may serve as a potential noninvasive biomarker source for periodontitis diagnosis.