Tissue engineering. Part C, Methods最新文献

Mouse embryonic fibroblasts (MEFs) have been widely used as feeder cells in embryonic stem cell cultures because they can mimic the embryonic microenvironment. Milk fat globule-epidermal growth factor 8 (MFGE8) is expressed during mouse gonadal development, 10.5-13.5 embryonic, and is also found in MEF-conditioned medium (MEF-CM). Feeder-less culture of human-induced pluripotent stem cells (iPSCs) with MEF-CM significantly decreased the number of adherent cells when an inhibitory antibody against MFGE8 was used. The concentration of mouse MFGE8 in MEF-CM, as measured by an ELISA (Enzyme-Linked Immunosorbent Assay), was 0.16-1.24 μg/mL. Mouse MFGE8 and human MFGE8 have partially different molecular structures. Both the recombinant mouse MFGE8 and human MFGE8 significantly promoted cell adhesion of human iPSCs at medium-added concentrations of 2 μg/mL. This cell adhesion was also strongly inhibited by Arginylglycylaspartic acid (RGD) inhibitors, suggesting that it is dependent on the RGD sequence. The integrin αVβ5 expressed in iPSCs was thought to be involved in binding to the RGD sequence. MEF-CMs have long been an essential bio-derived material for the feeder culture method of iPSC culture. This study demonstrates that MFGE8 in MEF-CM is a functional factor in the promoting of cell adhesion of human iPSCs. Furthermore, the use of MFGE8-containing media demonstrates that iPSCs can be established and cultured while maintaining pluripotency and inducing three germ layer differentiation. The results of this study suggest the possibility of using MFGE8 as a scaffold material suitable for inducing differentiation when reproducing in vivo maturation in vitro.

Scaffold-free tissue engineering strategies using cellular aggregates, microtissues, or organoids as "biological building blocks" could potentially be used for the engineering of scaled-up articular cartilage or endochondral bone-forming grafts. Such approaches require large numbers of cells; however, little is known about how different chondrogenic growth factor stimulation regimes during cellular expansion and differentiation influence the capacity of cellular aggregates or microtissues to fuse and generate hyaline cartilage. In this study, human bone marrow mesenchymal stem/stromal cells (MSCs) were additionally stimulated with bone morphogenetic protein 2 (BMP-2) and/or transforming growth factor (TGF)-β1 during both monolayer expansion and subsequent chondrogenic differentiation in a microtissue format. MSCs displayed a higher proliferative potential when expanded in the presence of TGF-β1 or TGF-β1 and BMP-2. Next, the chondrogenic potential of these human MSCs was explored in a medium-high throughput microtissue system. After 3 weeks of culture, MSCs stimulated with BMP-2 during expansion and differentiation deposited higher levels of glycosaminoglycans (GAGs) and collagen, while staining negative for calcium deposits. The fusion capacity of the microtissues was not impacted by these different growth factor stimulation regimes. After 3 weeks of fusion, it was observed that MSCs stimulated with TGF-β1 during expansion and additionally with BMP-2 during chondrogenic differentiation deposited the highest levels of sulfated GAGs. No increase in type X collagen deposition was observed with additional growth factor stimulation. This study demonstrates the importance of carefully optimizing MSC expansion and differentiation conditions when developing modular tissue engineering strategies (e.g., cellular aggregates and microtissues) for cartilage tissue engineering applications.

The failure of orthopedic implants can significantly impact patients physiologically, psychologically, and economically. A bibliometric study of the field of surface modification for antimicrobial purposes in orthopedic implants provides insights into its developmental trajectory and offers valuable predictions for future advancements, thus playing a pivotal role in guiding research in this domain. Relevant publications on surface modification for antimicrobial purposes in orthopedic implants published between 2014 and 2024 were selected from the Web of Science (Core Collection) dataset and analyzed using VOSviewer and Citespace. The analysis encompassed 725 articles. Over the past decade, there has been a steady increase in the number of publications related to surface modification for antimicrobial purposes in orthopedic implants, with China emerging as the primary contributor. Novel antimicrobial materials development, osteogenesis, and angiogenesis have become focal areas of research interest in this domain. Surface modification for antimicrobial purposes in orthopedic implants garners increasing attention. Research in this field is anticipated to expand, with future focus likely to revolve around novel material applications, repair outcomes, and underlying mechanisms.

Plant-derived hydrocolloids offer promising prospects in biomedical applications. Among these, Flaxseed hydrocolloid (FSH) can form a soft, elastic, and biocompatible hydrocolloid with tunable viscosity and superior swelling capacity, making it an attractive scaffold. This study introduces a green extraction method for FSH, employing a single-step aqueous extraction process and fabrication of FSH scaffold. Despite growing interest, the pristine form of FSH has not been investigated for sustainable long-term three-dimensional (3D) cell culture. Here, FSH scaffolds were thoroughly characterized for their morphological, chemical, mechanical, and biological properties. 3D cell culture experiments were conducted using NIH-3T3 mouse fibroblast cells, and cell viability was assessed using live/dead and Alamar Blue assays. High cell viability was sustained for long term compared with 2D cell culture. Cell adhesion and 3D cellular morphology on FSH scaffold for 30 days were monitored by scanning electron microscopy analysis. Also, collagen type-I and F-actin expressions were analyzed by immunostaining after 30 days of culture, resulting in 5- and 4-fold increments of fluorescence intensity, respectively. Results indicate sustained cell viability in the long term and favorable cell-material interaction, demonstrating the potential of FSH as a scaffold. This study emphasizes the importance of the green extraction approach, improving the biocompatibility and functionality of FSH tissue engineering applications.

As society advances, an increasing number of people are focusing on the antiaging process of the body and seeking ways to maintain youthful facial features. Intradermal injection has been used to effectively improve the rough and wrinkled skin, playing a role in skin rejuvenation. However, the main component of intradermal injection products is cross-linked sodium hyaluronate (SHA), which has biological toxicity and potential carcinogenicity. In this study, amino acids were used as hyaluronidase inhibitors and combined with non-cross-linked SHA to prepare a synergically stable SHA composite hydrogel. The effects of amino acids on the viscosity and enzyme activity of the hydrogel were investigated. To determine the stability and antioxidant properties of the composite hydrogel, the effects of the introduction of stabilizer and antioxidant on the hydrogel properties were systematically studied. The results of the in vitro study showed that the introduction of amino acids effectively reduced the activity of hyaluronidase, addressing the problem of rapid hydrolysis and the short half-life of SHA hydrogel in vivo. In addition, the results revealed that NaCl stabilizer, niacinamide, and vitamin B12 antioxidants effectively maintained the stability and antioxidant properties of the hydrogels. In vivo results showed that SHA composite hydrogels had no irritating effect on the skin, and the subcutaneous experiments of mice showed that SHA composite hydrogel still retained a high content after 4 weeks. Therefore, the SHA composite hydrogels have promising applications in the field of medical cosmetology.

Owing to the high occurrence of tissue detachment during the sample preparation process, the application of multiplex immunohistochemistry (mIHC) technology is limited in the field of fragile tissue samples, such as tendons, ligaments, and bones. To optimize a method for preparing sections for mIHC on fragile tissue samples, taking the human anterior cruciate ligament as an example, paraffin-embedded continuous sections with a thickness of 4 μm were divided into two groups: baking groups underwent routine section processing, and after being mounted on glass slides, they were baked at 65°C for 4 h, 8 h, or 24 h; ultraviolet (UV) photosensitive cross-linking groups used adhesive-coated slides for mounting and were directly subjected to UV light-induced cross-linking, with the cross-linking time set at 0 s, 20 s, 40 s, 1 min, 2 min, 3 min, 4 min, and 5 min, respectively. After deparaffinization and rehydration, we simulated the microwave step, which was most likely to cause tissue detachment during the mIHC experimental procedure, and then, the sections were stained with eosin. Finally, using the optimal cross-linking time selected from the UV cross-linking groups, mIHC staining of tendon and bone tissues was performed. After deparaffinization and rehydration, both groups were able to maintain the integrity of the tissue structure, except for the slides from the UV-sensitive cross-linking 0 s group, which showed complete tissue detachment. Following the seventh microwave treatment, the baking groups presented significant tissue detachment. The UV cross-linking groups were affected by the cross-linking time, and severe tissue detachment occurred with cross-linking times of 20 s, 40 s, and 5 min, whereas the tissues cross-linked for 1 min, 2 min, 3 min, and 4 min all maintained complete tissue morphology and structure. Finally, after 2 min of cross-linking, the results of spectral imaging revealed that the tissue morphology and structure were intact. During the process of mIHC staining, photocrosslinking with UV irradiation for 1-4 min effectively preserves the integrity of the tissue morphological structure.

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.

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" 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 extracellular matrix 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 close at different speeds and magnitudes 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 article 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. Impact Statement The protocol presented in this article is a new and simple method to quantify contraction forces present in tissue-engineered substitutes. Using finite element analysis, it allows for the measurement of a contraction force rather than a surface reduction as usually provided by other tissue contraction measurement methods. The results shown are in correlation with the current literature relevant to tissue contraction. It can be easily implemented, and hence, this method will open up new avenues to study tissue contraction of living substitutes engineered with various cell types and to optimize culture conditions.

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.

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.