Tissue engineering. Part C, Methods最新文献

The nasal cavity is covered with respiratory epithelia, including ciliated cells that eliminate foreign substances trapped in the mucus. In hereditary diseases such as primary ciliary dyskinesia and cystic fibrosis, respiratory epithelial functions are irreversibly impaired; however, no radical treatment has been established yet. Thus, we considered that the transplantation of normal airway epithelia (AE) into the nasal epithelia is one of the strategies that could lead to radical treatment in the future. In our previous study, human induced pluripotent stem cell-derived AE (hiPSC-AE) on the vitrigel membrane were transplanted into the scraped area of the nasal septal mucosa of nude rats. Although human-derived ciliated cells, club cells, and basal cells were observed, they were located in the cysts within the submucosal granulation tissue but not in the nasal mucosal epithelia and the transplanted cells may not contribute to the function of the nasal mucosa with this condition. Therefore, to achieve more functional transplantation, we prepared the graft differently in this study by wrapping the collagen sponge in hiPSC-AE on the vitrigel membrane. As a result, we found the transplanted cells surviving in the nasal mucosal epithelia. These results suggest that hiPSC-AE transplanted into the nasal cavity could be viable in the nasal mucosa. In addition, our method leads to the establishment of nasal mucosa-humanized rats that are used for the development of the drugs and therapeutic methods for hereditary diseases of nasal respiratory epithelia.

Decellularized porcine myocardium is commonly used as scaffolding for engineered heart tissues (EHTs). However, structural and mechanical heterogeneity in the myocardium complicate production of mechanically consistent tissues. In this study, we evaluate the porcine psoas major muscle (tenderloin) as an alternative scaffold material. Head-to-head comparison of decellularized tenderloin and ventricular scaffolds showed only minor differences in mean biomechanical characteristics, but tenderloin scaffolds were less variable and less dependent on the region of origin than ventricular samples. The active contractile behavior of EHTs made by seeding tenderloin versus ventricular scaffolds with human-induced pluripotent stem cell-derived cardiomyocytes was also comparable, with only minor differences observed. Collectively, the data reveal that the behavior of EHTs produced from decellularized porcine psoas muscle is almost identical to those made from porcine left ventricular myocardium, with the advantages of being more homogeneous, biomechanically consistent, and readily obtainable.

To date, islet transplantation to treat type 1 diabetes mellitus remains unsuccessful in long-term follow-up, mainly due to failed engraftment and reconstruction of the islet niche. Alternative approaches, such as islet embedding structures (IESs) based on 3D printing have been developed. However, most of them have been implanted subcutaneously and only a few are intended for direct integration into the vascular system through anastomosis. In this study, we 3D printed a proof-of-concept IES using gelatin methacrylate biocompatible ink. This structure consisted of a branched vascular system surrounding both sides of a central cavity dedicated to islets of Langerhans. Furthermore, we designed a bioreactor optimized for these biological structures. This bioreactor allows seeding and perfusion experiments under sterile and physiological conditions. Preliminary experiments aimed to analyze if the vascular channel could successfully be seeded with mature endothelial cells and the central cavity with rat islets. Subsequently, the structures were used for a humanized model seeding human endothelial progenitor cells (huEPC) within the vascular architecture and human islets co-cultured with huEPC within the central cavity. The constructs were tested for hemocompatibility, suture strength, and anastomosability. The 3D printed IES appeared to be hemocompatible and anastomosable using an alternative cuff anastomosis in a simple ex vivo perfusion model. While rat islets alone could not successfully be embedded within the 3D printed structure for 3 days, human islets co-cultivated with huEPC successfully engrafted within the same time. This result emphasizes the importance of co-culture, nursing cells, and islet niche. In conclusion, we constructed a proof-of-concept 3D printed islet embedding device consisting of a vascular channel that is hemocompatible and perspectively anastomosable to clinical scale blood vessels. However, there are numerous limitations in this model that need to be overcome to transfer this technology to the bedside.

Hepatocyte transplantation has been utilized as a therapy for congenital metabolic liver diseases such as hemophilia and for liver function support in acute liver failure. Hepatocyte sheet technology using a thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted dish is expected to provide an efficient cell transplantation method because the resulting hepatocyte sheet possesses extracellular matrix (ECM) on the basal surface, which enhances attachment to the target sites. However, the cultured hepatocytes consume large amounts of oxygen, leading to the loss of a few hepatocytes within the confluent culture sheet owing to a lack of oxygen. To circumvent this problem, this work demonstrates the shortening of diffusion distance, that is, the medium depth, to accelerate oxygen supply from the gas phase/medium interface to the cultured hepatocytes, allowing them to form a monolayer hepatocyte sheet. Incubation of hepatocytes with medium at a depth of 1.3 mm facilitates confluent culture of hepatocytes for 72 h, whereas viable hepatocytes decreased at 2.6 mm depth. Hepatocyte sheets are formed on a 0.5 μg/cm² fibronectin-physisorbed PIPAAm-grafted dish during 72 h incubation at 37°C. Detachment of the cultured hepatocyte sheet from the PIPAAm-grafted dish where the surface becomes hydrophilic at 20°C is accomplished by scraping the periphery of the sheet using a cell scraper. Furthermore, the apical side of the hepatocyte sheet can be physically grabbed using a gelatin-coated membrane, and the sheet with ECM on the basal surface can be readily transferred to the target site after melting the coated gelatin at 37°C. Both methods are beneficial for creating tissue models by layering with another type of cell sheets, and for quick transplantation, such as into the subcutaneous space and orthotopic transplantation on the surface of the liver.

Muscle tissue engineering has been the focus of extensive research due to its potential for numerous medical applications, including ex vivo actuator development and clinical treatments. In this study, we developed a method for harvesting muscle fiber in a floatable and translocatable manner utilizing thermally expandable hydrogels with a chemically patterned polydopamine (PD) layer generated by microcontact printing (μCP). The μCP of PD on the hydrogel facilitated the formation of stripe patterns with varying widths of printed/nonprinted area (50/50, 100/100, and 200/200 μm). The spatially controlled adhesion of C2C12 myoblasts on the PD patterns produced clearly distinguishable muscle fibers, and translocated muscle fibers exhibited preserved extracellular matrix and junction proteins. Furthermore, the development of anisotropic arrangements and mature myotubes within the fibers suggests the potential for functional control of engineered muscle tissues. Overall, the muscle fiber harvesting method developed herein is suitable for both translocation and floating and is a promising technique for muscle tissue engineering as it mimics the structure-function relationship of natural tissue.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

An animal model of osteoarthritis (OA) induced by monosodium iodoacetate (MIA) can be effectively adjusted based on the concentration of MIA to control the onset, progression, and severity of OA as required. The rat temporomandibular joint osteoarthritis (TMJOA) model using MIA is a useful tool for studying the effectiveness of disease-modifying OA drugs in TMJOA research. However, the intricate and complex anatomy of the rat TMJ often poses challenges in achieving consistent TMJOA induction during experiments. In the previous article, a reference point was established by drawing parallel lines based on the line connecting the external ear and the zygomatic arch. However, this is not suitable for the anatomical characteristics of the rat. We used the zygomatic arch as a reference, which is a technical protocol that considers it. In our protocol, we designated a point ∼1 mm away from the point where the zygomatic arch bends toward the ear as the injection site. To ensure precise injection of MIA and increase the likelihood of inducing OA, it is recommended to insert the needle at a 45° angle so that the needle tip contacts the joint projection. To confirm TMJOA induction, we identified changes in the condyle using in vivo microcomputed tomography (CT) in a rat model of MIA-induced OA and measured the degree of pain-related inflammation using head withdrawal threshold (HWT) measurements. Micro-CT scanning revealed typical OA-like lesions, including degenerative changes and subchondral bone remodeling induced by MIA in the TMJ. Pain, a major clinical feature of OA, showed an appropriate response corresponding to the structural changes shown in micro-CT scanning. In addition, the MIA concentration suitable for long-term observation of lesions was determined through ex vivo micro-CT imaging and HWT measurements. The 8 mg concentration exhibited a significant difference compared with others, confirming the sustained presence of lesions, particularly through changes in subchondral bone over an extended period. Consequently, we have successfully established a reliable rat TMJOA induction model and identified the MIA concentration suitable for long-term observation of subchondral bone research, which will greatly contribute to the study of TMJOA-an incurable disease lacking specific treatment options. The Clinical Trial Registration number is 2021-12-208.

Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum^®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 10⁶ cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 10⁶ cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.

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.