Tissue engineering. Part C, Methods最新文献

Tissue-engineered oral epithelium (ΤΕΟΕ) was developed after comparing various culture conditions, including submerged (SUB) and air-liquid interface (ALI) human cell expansion options. Barrier formation was evaluated via transepithelial electrical resistance (TEER) and calcein permeation via spectrofluorometry. TEOE was further assessed for long-term viability via live/dead staining and development of intercellular connections via transmission electron microscopy. Tissue architecture was evaluated via histochemistry and the expression of pancytokeratin (pCK) via immunohistochemistry. The effect of two commonly used dental resinous monomers on TEOE was evaluated for alterations in cell viability and barrier permeability. ALI/keratinocyte growth factor-supplemented (ALI-KGS) culture conditions led to the formation of an 8-20-layer thick, intercellularly connected epithelial barrier. TEER values of ALI-KGS-developed TEOE decreased compared with all other tested conditions, and the established epithelium intensively expressed pCK. Exposure to dental monomers affected the integrity and architecture of TEOE and induced cellular vacuolation, implicating hydropic degeneration. Despite structural modifications, the permeability of TEOE was not substantially affected after exposure to the monomers. In conclusion, the biological properties of the TEOE mimicking the physiological functional conditions and its value as biocompatibility assessment tool for dental materials were characterized.

Transforming growth factor beta (TGF-β) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-β activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-β activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-β's autoinduction response, whereby active TGF-β (aTGF-β) signaling in cells induces their biosynthesis and secretion of new TGF-β in its latent form (LTGF-β). As such, cell-secreted LTGF-β can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-β in live cartilage tissues. Here, we detect LTGF-β1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-β3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-β secretion levels increase proportionally to aTGF-β exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-β exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-β is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-β secretion and exposed TGF-β activity. Accordingly, we perform quantifications of TGF-β activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-β activity in aTGF-β-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-β activity as a result of a nominal 3 μg/mL loading dose. Overall, cell-secreted LTGF-β can serve as a robust biomarker to quantify in situ activity of TGF-β in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.

This study evaluated the efficacy of synthetic bone blocks, composed of hydroxyapatite (HA) or β-tricalcium phosphate (B-TCP), which were produced by additive manufacturing and used for the repair of critical size bone defects (CSDs) in rat calvaria. Sixty rats were divided into five groups (n = 12): blood clot (CONTROL), 3D-printed HA (HA), 3D-printed β-TCP (B-TCP), 3D-printed HA + autologous micrograft (HA+RIG), and 3D-printed β-TCP + autologous micrograft (B-TCP+RIG). CSDs were surgically created in the parietal bone and treated with the respective biomaterials. The animals were euthanized at 30 and 60 days postsurgery for microcomputed tomography (micro-CT) histomorphometric, and immunohistochemical analysis to assess new bone formation. Micro-CT analysis showed that both biomaterials were incorporated into the animals' calvaria. The HA+RIG group, especially at 60 days, exhibited a significant increase in bone formation compared with the control. The use of 3D-printed bioceramics resulted in thinner trabeculae but a higher number of trabeculae compared with the control. Histomorphometric analysis showed bone islands in close contact with the B-TCP and HA blocks at 30 days. The HA blocks (HA and HA+RIG groups) showed statistically higher new bone formation values with further improvement when autologous micrografts were included. Immunohistochemical analysis showed the expression of bone repair proteins. At 30 days, the HA+RIG group had moderate Osteopontin (OPN) staining, indicating that the repair process had started, whereas other groups showed no staining. At 60 days, the HA+RIG group showed slight staining, similar to that of the control. Osteocalcin (OCN) staining, indicating osteoblastic activity, showed moderate expression in the HA and HA+RIG groups at 30 days, with slight expression in the B-TCP and B-TCP+RIG groups. The combination of HA blocks with autologous micrografts significantly enhanced bone repair, suggesting that the presence of progenitor cells and growth factors in the micrografts contributed to the improved outcomes. It was concluded that 3D-printed bone substitute blocks, associated with autologous micrografts, are highly effective in promoting bone repair in CSDs in rat calvaria.

Biomaterials derived from biological matrices have been widely investigated due to their great therapeutic potential in regenerative medicine, since they are able to induce cell proliferation, tissue remodeling, and angiogenesis in situ. In this context, highly vascularized and proliferative tissues, such as the uterine wall, present an interesting source to produce acellular matrices that can be used as bioactive materials to induce tissue regeneration. Therefore, this study aimed to establish an optimized protocol to generate decellularized uterine scaffolds (dUT), characterizing their structural, compositional, and biomechanical properties. In addition, in vitro performance and in vivo biocompatibility were also evaluated to verify their potential applications for tissue repair. Results showed that the protocol was efficient to promote cell removal, and dUT general structure and extracellular matrix composition remained preserved compared with native tissue. In addition, the scaffolds were cytocompatible, allowing cell growth and survival. In terms of biocompatibility, the matrices did not induce any signs of immune rejection in vivo in a model of subcutaneous implantation in immunocompetent rats, demonstrating an indication of tissue integration after 30 days of implantation. In summary, these findings suggest that dUT scaffolds could be explored as a biomaterial for regenerative purposes, which is beyond the studies in the reproductive field.

Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.

Tissue engineering research fundamentally relies on experiments to advance knowledge, utilizing various models for research on both humans and animals. With scientific progress, experimental models have become increasingly complex over time. This complexity sometimes blurs the distinction between categories, making terminology less consistent. In biomedical research, three overarching terms are commonly used to characterize experimental environments: in vitro, ex vivo, and in vivo. While in vitro translates from Latin as "in glass," referring historically to experimental conditions in a test tube or petri dish, in vivo experiments occur within a living organism's natural environment. Conversely, ex vivo originates from living tissue outside its host environment while striving to maintain conditions as close to the host surroundings as possible. In the tissue engineering and regenerative medicine (TE&RM) community, there needs to be more clarity between in vitro and ex vivo terminology, with historical definitions sometimes disregarded and new terms often introduced without rigorous scientific justification. At this juncture, the question arises of when to refer to experiments as in vitro or ex vivo or whether the terms may be used synonymously in some instances. Moreover, what criteria must ex vivo experiments meet to be legitimately defined as such? This perspective is intended to address questions that would assist the TE&RM community in better understanding the differences between in vitro and ex vivo models. Impact Statement In the tissue engineering & regenerative medicine literature, the terms "in vitro" and "ex vivo" are often used interchangeably to describe experiments. This interchangeable usage can lead to a compromised interpretation of research results and, consequently, misleading scientific conclusions and teachings. This perspective aims to provide clarity on the various definitions of experimental designs. It also highlights the issue of using terms with inconsistent meanings that have origins dating back to the distant past. It's important to note that scientific definitions constantly evolve, and there is a scientifically rooted responsibility to evaluate and rethink the current state of affairs critically.

Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behavior, presented in illustrations and microscopy image examples intended to serve as a reference "atlas." To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored versus nonscored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Most important, significant differences in cell morphology and cytoskeletal protein content between scored versus nonscored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts versus only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, nonscoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.

Hypertrophic scarring is a common complication in severely burned patients who undergo autologous skin grafting. Meshed skin grafts tend to contract during wound healing, increasing the risk of pathological scarring. Although various technologies have been used to study cellular contraction, current methods for measuring contractile forces at the tissue level are limited and do not replicate the complexity of native tissues. Self-assembled skin substitutes (SASSs) were developed at the "Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX" (LOEX) and are used as permanent full-thickness skin grafts. The autologous skin substitutes are produced using the self-assembly method, allowing the cultured cells to produce their own extracellular matrix (ECM) leading to a tissue-engineered substitute resembling the native skin. The level of contraction of the SASSs during the fabrication process is patient-dependent. Thus, because of its architecture and composition, SASS is an interesting model to study skin contraction in vitro. Unfortunately, standard measurement methods are unsuited for SASS contraction assessment, mainly due to incompatibilities between the SASS manufacturing process and the current contraction force measurement methods. Here, we present an innovative contraction measurement method specifically designed to quantify the contractile behavior of tissue-engineered substitutes, without disrupting the protocol of production. The method uses C-shape anchoring frames that closes at different speed and magnitude according to the tissue contractile behavior. A finite element analysis model is then used to associate the frame deformation to a contractile force amplitude. This paper shows that the method can be used to measure the contraction force of tissues produced with cells displaying different contractile properties, such as primary skin fibroblasts and myofibroblasts. It can also be used to study the effects of cell culture conditions on tissue contraction, such as serum concentration. This protocol can be easily and affordably applied and tuned to many regenerative medicine applications or contraction-related pathological studies.