Tissue engineering. Part C, Methods最新文献

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes^®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Considerable research is being undertaken to develop novel biomaterials-based approaches for surgical reconstruction of bone defects. This extends to three-dimensional (3D) printed materials that provide stable, structural, and functional support in vivo. However, few preclinical models can simulate in vivo human biological conditions for clinically relevant testing. In this study we describe a novel ovine model that allows evaluation of in vivo osteogenesis via contact with bone and/or periosteum interfaced with printed polymer bioreactors loaded with biomaterial bone substitutes. The infraspinous scapular region of 14 Dorset cross sheep was exposed. Vascularized periosteum was elevated either attached to the infraspinatus muscle or separately. In both cases, the periosteum was supplied by the periosteal branch of the circumflex scapular vessels. In eight sheep, a 3D printed 4-chambered polyetheretherketone bioreactor was wrapped circumferentially in vascularized periosteum. In 6 sheep, 12 double-sided 3D printed 2-chambered polyetherketone bioreactors were secured to the underlying bone allowing direct contact with the bone on one side and periosteum on the other. Our model enabled simultaneous testing of up to 24 (12 double-sided) 10 × 10 × 5 mm bioreactors per scapula in the flat contact approach or a single 40 × 10 mm four-chambered bioreactor per scapula using the periosteal wrap. De novo bone growth was evaluated using histological and radiological analysis. Of importance, the experimental model was well tolerated by the animals and provides a versatile approach for comparing the osteogenic potential of cambium on the bone surface and elevated with periosteum. Furthermore, the periosteal flaps were sufficiently large for encasing bioreactors containing biomaterial bone substitutes for applications such as segmental mandibular reconstruction.

To learn about advances in skeletal muscle tissue engineering (SMTE) in recent years, we used VOSviewer and Citespace software to quantitatively analyze and visualize relevant literature in the Web of Science database during the period 2012-2022. By mapping high-frequency keyword relationship networks, keyword time zones, and journal article cocitations, we clarified the areas of great interest, evolutionary paths, and developmental trends in research on SMTE. We conducted an in-depth analysis of highly cited and representative articles at various stages to summarize the mainstream research areas of great interest in SMTE and discussed the future development and challenges in this field, intending to provide a reference for the clinical treatment of skeletal muscle injury repair. We found that a collaborative network of authors has formed in this field; the journals publishing SMTE articles belong to the fields of biomaterials and tissue engineering, and open-access journals have played a key role in the promotion of the development of SMTE; and in the past decade, there has been rapid progress in SMTE research in terms of both depth and breadth. Impact statement Compared with the literature review method, bibliometrics can provide a comprehensive knowledge of a knowledge area based on a huge amount of literature. In this article, based on the Web of Science database, CiteSpace, and Vosviewer visualization tools were used to measure and analyze the literature reports in the field of skeletal muscle tissue engineering (SMTE). The research hotspots and cutting-edge information on SMTE were mined in terms of the number of publications, the number of citations, the keywords, the authors, and the publishing institutions to understand the current status of the research on SMTE in the world, to provide a reference for related researchers, engineering research in the field of SMTE, to comprehensively understand the current status of global research in the field of SMTE, and to provide a reference for related researchers.

Angiogenesis induced by growth factor administration, which can augment the blood supply in regenerative applications, has drawn wide attention in medical research. Longitudinal monitoring of vascular structure and development in vivo is important for understanding and evaluating the dynamics of involved biological processes. In this work, a dual-modality imaging system consisting of photoacoustic microscopy (PAM) and optical coherence tomography (OCT) was applied for noninvasive in vivo imaging of angiogenesis in a murine model. Fibrin scaffolds, with and without basic fibroblast growth factor (bFGF), were implanted in a flexible imaging window and longitudinally observed over 9 days. Imaging was conducted at 3, 5, 7, and 9 days after implantation to monitor vascularization in and around the scaffold. Several morphometric parameters were derived from the PAM images, including vessel area density (VAD), total vessel length (TVL), and vessel mean diameter (VMD). On days 7 and 9, mice receiving bFGF-laden fibrin gels exhibited significantly larger VAD and TVL compared to mice with fibrin-only gels. In addition, VMD significantly decreased in +bFGF mice versus fibrin-only mice on days 7 and 9. Blood vessel density, evaluated using immunohistochemical staining of explanted gels and underlying tissue on day 9, corroborated the findings from the PAM images. Overall, the experimental results highlight the utility of a dual-modality imaging system in longitudinally monitoring of vasculature in vivo with high resolution and sensitivity, thereby providing an effective tool to study angiogenesis.

The aim of this study was to assess the bone regeneration potential of a polydioxanone (PDO) scaffold together with recombinant human bone morphogenetic protein-2 (rhBMP-2) for the reconstruction of large bone defect. In total, 24 male rats (6 months old) were subjected to bilateral femoral stabilization using titanium plates to create a 2 mm gap, and reconstruction using rhBMP-2 (Infuse^®; 3.25 μg). The bone defects were covered with PDO (PDO group), or with titanium mesh (Ti group). Animals were euthanized on days 14 and 60. Simultaneously, 16 rats received PDO and Ti in their dorsum for the purpose of biocompatibility analysis at 3, 5, 7, and 10 days postoperatively. X-ray densitometry showed a higher density in the PDO group on day 14. On day 60, coverage of the bone defect with PDO showed a larger quantity of newly formed bone than that found for the Ti group, a lower inflammatory infiltrate value, and a more significant number of blood vessels on day 14. By immunohistochemical assessment, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) showed higher labeling on day 14 in the PDO group. On day 60, bone morphogenetic protein-2 (BMP-2) showed higher labeling in the PDO group, whereas Ti showed higher labeling for osteoprotegerin, nuclear factor kappa B ligand-activating receptor, RUNX2, and OCN. Furthermore, biocompatibility analysis showed a higher inflammatory response in the Ti group. The PDO scaffold enhanced bone regeneration when associated with rhBMP-2 in rat femur reconstruction. Impact statement Regeneration of segmental bone defects is a difficult task, and several techniques and materials have been used. Recent advances in the production of synthetic polymers, such as polydioxanone (PDO), produced by three-dimensional printing, have shown distinct characteristics that could improve tissue regeneration even in an important bone defect. The present preclinical study showed that PDO membranes used as scaffolds to carry recombinant human bone morphogenetic protein-2 (rhBMP-2) improved bone tissue regeneration by more than 8-fold when compared with titanium mesh, suggesting that PDO membranes could be a feasible and useful material for use in guided bone regeneration. (In English, viable is only used for living creatures capable of sustaining life.

The decellularized extracellular matrix (ECM) of cartilage is a widely used natural bioscaffold for constructing tissue-engineered cartilage due to its good biocompatibility and regeneration properties. However, current decellularization methods for accessing decellularized cartilaginous tissues require multiple steps and a relatively long duration to produce decellularized cartilage. In addition, most decellularization strategies lead to damage of the microstructure and loss of functional components of the cartilaginous matrix. In this study, a novel decellularization strategy based on a hydrostatic pressure (HP) bioreactor was introduced, which aimed to improve the efficiency of producing integral decellularized cartilage pieces by combining physical and chemical decellularization methods in a perfusing manner. Two types of cartilaginous tissues, auricular cartilage (AC) and nucleus pulposus (NP) fibrocartilage, were selected for comparison of the effects of ordinary, positive, and negative HP-based decellularization according to the cell clearance ratio, microstructural changes, ECM components, and mechanical properties. The results indicated that applying positive HP improved the efficiency of producing decellularized AC, but no significant differences in decellularization efficiency were found between the ordinary and negative HP-treated groups. However, compared with the ordinary HP treatment, the application of the positive or negative HP did not affect the efficiency of decellularized NP productions. Moreover, neither positive nor negative HP influenced the preservation of the microstructure and components of the AC matrix. However, applying negative HP disarranged the fibril distribution of the NP matrix and reduced glycosaminoglycans and collagen type II contents, two essential ECM components. In addition, the positive HP was beneficial for maintaining the mechanical properties of decellularized cartilage. The recellularization experiments also verified the good biocompatibility of the decellularized cartilage produced by the present bioreactor-based decellularization method under positive HP. Overall, applying positive HP-based decellularization resulted in a superior effect on the production of close-to-natural scaffolds for cartilage tissue engineering. Impact statement In this study, we successfully constructed a novel hydrostatic pressure (HP) bioreactor and used this equipment to produce decellularized cartilage by combining physical and chemical decellularization methods in a perfusing manner. We found that positive HP-based decellularization could improve the production efficiency of integral decellularized cartilage pieces and promote the maintenance of matrix components and mechanical properties. This new decellularization strategy exhibited a superior effect in the production of close-to-natural scaffolds and positively impacts cartilage tissue engineering.

Intervertebral disc degeneration (IVDD) is a major cause of low back pain, and several studies have evaluated the efficacy of extracellular vesicles (EVs) in the treatment of IVDD. The databases PubMed, Embase, and Cochrane Library were systematically searched from inception to the end of 2022 to identify studies investigating the therapeutic potential of cell-derived EVs for IVDD treatment. The following outcome measures were utilized: magnetic resonance imaging (MRI) Pfirrmann grading system, disc height index (DHI), histological grading, and apoptosis rate. A comprehensive meta-analysis was conducted, including a total of 13 articles comprising 19 studies involving 218 experimental animals. Comparative analysis between normal cell-derived EVs and placebo revealed significant reductions in MRI grade, increased DHI values, decreased nucleus pulposus cell apoptosis rates, and improved tissue grades. These findings collectively demonstrate the effective inhibition of IVDD through the application of EVs derived from cells. In conclusion, this study provides an updated synthesis of evidence supporting the efficacy of EVs as a promising therapeutic approach for IVDD treatment.

A major obstacle to the implantation of ex vivo engineered tissues is the incorporation of functional vascular supply to support the growth of new tissue and to minimize ischemic injury. Existing prevascularization systems, such as arteriovenous (AV) loop-based systems, require microsurgery, limiting their use to larger animals. We aimed to develop an implantable device that can be prevascularized to enable vascularization of tissues in small rodents, and test its application on the vascularization of embryonic kidneys. Implanting the chamber between the abdominal aorta and the inferior vena cava, we detected endothelial cells and vascular networks after 48 h of implantation. Loading the chamber with collagen I (C), Matrigel (M), or Matrigel + vascular endothelial growth factor) (MV) had a strong influence on vascularization speed: Chambers loaded with C took 7 days to vascularize, 4 days for chambers with M, and 2 days for chambers with MV. Implantation of E12.5 mouse embryonic kidneys into prevascularized chambers (C, MV) was followed with significant growth and ureteric branching over 22 days. In contrast, the growth of kidneys in non-prevascularized chambers was stunted. We concluded that our prevascularized chamber is a valuable tool for vascularizing implanted tissues and tissue-engineered constructs. Further optimization will be necessary to control the directional growth of vascular endothelial cells within the chamber and the vascularization grade. Impact Statement Vascularization of engineered tissue, or organoids, constructs is a major hurdle in tissue engineering. Failure of vascularization is associated with prolonged ischemia time and potential tissue damage due to hypoxic effects. The method presented, demonstrates the use of a novel chamber that allows rapid vascularization of native and engineered tissues. We hope that this technology helps to stimulate research in the field of tissue vascularization and enables researchers to generate larger engineered vascularized tissues.

In recent years the need for in vitro skin models as a replacement for animal studies has resulted in significant progress in the development of skin-on-a-chip models. These devices allow the fine control of the microenvironment of the model and the incorporation of chemical and physical stimuli. In this study, we describe the development of an easy and low-budget open-top dynamic microfluidic device for skin-on-a-chip experiments using polydimethylsiloxane and a porous polyethylene terephthalate membrane. The chip allows the incorporation of compressive stimuli during the cultivation period by the use of syringe pumps. Proof-of-concept results show the successful differentiation of the cells and establishment of the skin structure in the chip. The microfluidic skin-on-a-chip models presented in this study can serve as a platform for future drug and feasibility studies.

The effect and mechanism of type III recombinant humanized collagen (hCOLIII) on human vascular endothelial EA.hy926 cells at the cellular and molecular levels were investigated. The impact of hCOLIII on the proliferation of EA.hy926 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assay, the effect of hCOLIII on cell migration was investigated by scratch assay, the impact of hCOLIII on cell cycle and apoptosis was detected by flow cytometry, the ability of hCOLIII to induce angiogenesis of EA.hy926 cells was evaluated by angiogenesis assay, and the effect of hCOLIII on vascular endothelial growth factor (VEGF) expression was detected by real-time reverse transcription-polymerase chain reaction analysis. The hCOLIII at concentrations of 0.5, 0.25, and 0.125 mg/mL all showed specific effects on the proliferation and migration of human vascular endothelial cells. It could also affect the cell cycle, increase the proliferation index, and increase the expression level of VEGF in human vascular endothelial cells. In the meantime, hCOLIII at the concentration of 0.5 mg/mL also showed a promoting effect on vessel formation. hCOLIII can potentially promote the endothelization process of blood vessels, mainly by affecting the proliferation, migration, and vascular-like structure of human endothelial cells. At the same time, hCOLIII can promote the expression of VEGF. This collagen demonstrated its potential as a raw material for cardiovascular implants.