Tissue Engineering Part A最新文献

Keratin as an abundantly available natural protein from sources such as hair, wool, and feathers possesses excellent biocompatibility, biodegradability, and bioactivity that support cell growth. Recent advances in extracting, purifying, and characterizing keratin have led to the development of various keratin-based biomaterials, such as fibers, gels, films, and nanoparticles via conventional fabrication methods. However, these biomaterials are often limited by simple geometries, weak mechanical strength, and limited reproducibility. Emerging 3D printing technologies offer a promising alternative, allowing the creation of keratin-based scaffolds with precise architecture, tunable mechanical strength, and reproducible geometries. Despite keratin's abundance and biological advantages, the use of keratin in 3D printing remains relatively underexplored. This review provides a comprehensive overview of keratin's molecular structure and biochemistry, its diverse natural sources, extraction and purification methodologies, and the cross-linking mechanisms (chemical, UV, and enzymatic) used to formulate printable keratin-based inks. Furthermore, it discusses the biomedical applications of keratin-derived bioinks in tissue engineering and additive biomanufacturing, with emphasis on skin and bone regeneration. Combining keratin's biological functionality with the design flexibility of 3D printing offers a sustainable and cost-effective pathway toward next-generation biomaterials for regenerative medicine.

Frontalis suspension surgery is the preferred treatment option for patients with poor levator function ptosis. This procedure connects the affected eyelid to the brow using sling material, harnessing the action of the frontalis muscle to elevate the upper eyelid. Various sling materials have been used, most commonly silicone rods and fascia lata. However, both have notable limitations: silicone rods carry a relatively high risk of postoperative inflammation and ptosis recurrence, while fascia lata, due to its low elasticity, may cause blinking dysfunction and exposure keratopathy. Additionally, fascia lata harvesting poses challenges in young children. Therefore, there is a need for an alternative human tissue sling material that is both readily available and capable of overcoming the limitations of established sling materials. This study aimed to evaluate the viability of human umbilical cord grafts as a novel sling material for frontalis suspension surgery in ptosis patients. We developed a new method for dissecting and dehydrating umbilical cord tissue and assessed its mechanical and histological properties using uniaxial tensile testing and histological analysis. Untreated umbilical cord grafts exhibited mechanical strength (15.9546 ± 2.6117 N) and strain (96.8674 ± 3.6707%) values intermediate between those of silicone rod and fascia lata. Alcohol dehydration significantly increased ultimate tensile strength and maximum strain, ultimate strength values exceeding those of silicone rod. These grafts withstood forces exceeding those generated during forced blinking, outperforming silicone rod in strength and exhibiting greater elasticity than fascia lata. Histological analysis revealed abundant collagen and glycosaminoglycans within Wharton's jelly, alongside elastic fiber-rich regions in vessel walls. The presence of these extracellular matrix components likely underlies the grafts' favorable mechanical properties. Overall, umbilical cord grafts may emerge as a promising alternative to conventional sling materials in ptosis surgery, potentially addressing limitations in material availability. Impact Statement This study introduces human umbilical cord grafts as a novel sling material for frontalis suspension surgery in patients with ptosis. We developed a new method for dissecting and dehydrating umbilical cord tissue. Our results suggest that umbilical cord graft may offer sufficient tensile strength and strain, potentially reducing recurrence rates and minimizing postoperative complications. This work lays the groundwork for future studies exploring the clinical application of umbilical cord-derived biomaterials in surgical procedures.

Background: Bile duct jejunal anastomosis is a standard reconstruction method following bile duct resection. Nevertheless, this procedure is technically intricate and carries significant postoperative risks. This study evaluated bile duct regeneration in pigs using artificial bile ducts (ABDs) made of gelatin hydrogel nonwoven fabric (GHNF). Experiment: An ABD composed of polyglycolic acid (PGA) as the inner layer and GHNF as the outer layer was implanted in the defect of the bile duct in pigs. After a 105-day implantation period, tissue samples were analyzed via histology, immunohistochemistry, and RNA sequencing. Results: The implantation of the ABD promoted fibroblast infiltration, extracellular matrix (ECM) formation, and bile duct epithelial regeneration in the site of the bile duct defect by postoperative day 105. Histological analysis revealed complete absorption and replacement of GHNF by collagen-rich ECM. Immunohistochemistry studies indicated the presence of CK19-positive bile duct epithelial cells in the ABD area, suggesting the successful regeneration of the entire bile duct structure. Furthermore, RNA sequencing revealed gene expression patterns analogous to those observed in native bile ducts, showing a similarity with a significant correlation coefficient between the regenerated and the native bile ducts. Differentially expressed genes related to ECM formation, such as COL3A1, SPARC, and COL1A1, were highly expressed, along with growth factors such as FGF1, FGF7, FGF18, FGF22, TGFβ1, and TGFβ3. Conclusions: The experimental findings demonstrated the successful regeneration of bile duct tissue by the ABD made of GHNF implanted in pigs, thereby signifying its potential for future clinical applications.

Human organoid model systems have changed the landscape of developmental biology and basic science. They serve as a great tool for human-specific interrogation. In order to advance our organoid technology, we aimed to test the compatibility of a piezoelectric material with organoid generation, because it will create a new platform with the potential for sensing and actuating organoids in physiologically relevant ways. We differentiated human pluripotent stem cells into spheroids following the traditional human intestinal organoid (HIO) protocol atop a piezoelectric nanofiber scaffold. We observed that exposure to the biocompatible piezoelectric nanofibers promoted spheroid morphology 3 days sooner than with the conventional methodology. At day 28 of culture, HIOs grown on the scaffold appeared similar. Both groups were readily transplantable and developed well-organized laminated structures. Graft sizes between groups were similar. Upon characterizing the tissue further, we found no detrimental effects of the piezoelectric nanofibers on intestinal patterning or maturation. Furthermore, to test the practical feasibility of the material, HIOs were also matured on the nanofiber scaffolds and treated with ultrasound, which lead to increased cellular proliferation which is critical for organoid development and tissue maintenance. This study establishes a proof of concept for integrating piezoelectric materials as a customizable platform for on-demand electrical stimulation of cells using remote ultrasonic waveforms in regenerative medicine.

Transplantation of stem cell-derived β cells is a promising treatment for type-1 diabetes, increasing the supply of insulin-producing cells beyond that of cadaveric islet transplantation. Transplant success is limited by cell death after transplantation, with insufficient oxygen and nutrient accessibility strongly contributing to apoptosis and de-differentiation. Herein, we investigate cotransplantation of endothelial cells and fibroblasts with stem cell-derived β cells to enhance survival and function posttransplantation. A microporous poly (lactide coglycolide) scaffold was used for culture and transplantation of stem cell-derived β cells. Coculture of the stem cell-derived β cells with endothelial cells and fibroblasts generated vascular networks during in vitro culture, which persisted through transplantation and enhanced in vivo vascularization. 7-days of in vitro culture supported enhanced survival of transplanted cells, though function in terms of insulin secretion and reduction of hyperglycemia was compromised. However, 3-days of preculture led to both improved survival and function of the stem cell-derived β cells, with transplant recipients demonstrating reduced fasting blood glucose levels. These studies demonstrate the potential and some constraints on the application of vascularization strategies to enhance function of stem cell-derived β cells with transplantation to extrahepatic sites.

Micronized collagen-based bioscaffolds are increasingly used in clinical applications for wound repair and soft tissue regeneration. This study compared the structural properties of four different commercially available micronized products derived from either reconstituted collagen (pRC), urinary bladder matrix (pUBM), or ovine forestomach matrix (mOFM, mOFMµ). The test articles were characterized by laser diffraction analysis, scanning electron microscopy (SEM), micro-computed tomography (micro-CT), packing density, differential scanning calorimetry, rheometry, proteolytic stability, agarose gel electrophoresis, and blood clotting index. Particle size and surface morphology, assessed by laser diffraction, SEM, and micro-CT, revealed marked differences in particle size, shape, and aggregation. Packing density ranged from 80.3 ± 2.7 mg/cm³ (mOFM) to 484.7 ± 17.8 mg/cm³ (pRC). Thermal analysis demonstrated the native structure of the OFM-based test articles (T_m, 59.80 ± 0.11°C and 58.15 ± 0.15°C) relative to pUBM and pRC (T_m, 41.06 ± 0.06°C and 40.59 ± 0.23°C). Rheological testing revealed that mOFM and mOFMµ had increased cohesive energy, indicating better mechanical resilience when the micronized materials were rehydrated to form a paste. The OFM-based test articles exhibited the greatest resistance to proteolytic digestion (T_1/2, 12.730 ± 1.232 and 5.759 ± 0.1296). All the test articles, except for the reconstituted collagen product, demonstrated hemostasis in whole blood. Micronized reconstituted collagen showed immediate dissolution and no fluid absorption, hemostasis, or resistance to proteolytic digestion, whereas micronized OFM showed the greatest proteolytic stability and packing density. Substantial differences among the micronized bioscaffolds were revealed from the analysis, most likely due to their different source materials and manufacturing processes. Careful consideration of these parameters is warranted when selecting a micronized product for soft tissue applications.

Retinal organoids (ROs) are currently used to study retinal development and diseases but cannot model glaucoma because they fail to form a nerve fiber layer (NFL) and optic nerve (ON). Utilizing three-dimensional bioprinting, ON head astrocytes (ONHAs) and vascular endothelial cells, both of which contribute to NFL development in vivo but are absent in ROs, were positioned at the center of scaffolds seeded with retinal ganglion cells (RGCs). In experiments using ONHAs isolated from developing retinas, polarization of RGC neurite growth increased by 43% while ONHA from adult retinas or astrocytes from the developing peripheral retina or developing cortex did not increase polarization above controls. Furthermore, RGC-seeded scaffolds increased both the number and rate of ONHAs migrating out from the printed center compared to scaffolds lacking RGCs, mimicking the migration pattern observed during retinal development. Finally, in scaffolds containing both ONHAs and endothelial cells, the endothelial cells preferentially migrate on and only form vascular tube structures on scaffolds also containing RGCs. These results suggest that recreating the developmental organization of the retina can recapitulate the mechanism of NFL development and retinal vascularization in vitro. This step is not only necessary for the development of retinal models of glaucoma but has the potential for translation to other parts of the central nervous system.

The periosteum serves as a local source of osteoprogenitor cells and vasculature, therefore influencing the key processes of osteogenesis and neovascularization during bone healing. However, it is often not considered in traditional bone tissue engineering strategies. The periosteum consists of two stratified cell layers, including an inner cambium layer, which serves as a local source of osteoblasts (OBs) and osteoprogenitor cells, and an outer fibrous layer, which hosts vasculature, collagen fibers, and support cells. While several studies have investigated different methodologies to produce tissue-engineered periosteum (TEP) substitutes, few have evaluated the roles of specific cell types within the inner cambium layer and their patterning in 3D environments on underlying bone tissue development. Therefore, we sought to investigate whether mesenchymal stem cells (MSCs) alone, OBs alone, or a 1:1 mixture of the two would result in increased osteogenic differentiation of bone layer MSCs in a 3D bioprinted periosteum-bone coculture model in vitro. We first evaluated these effects in a 2D transwell model, demonstrating that OB-containing cultures, either alone or in a mixed population with MSCs, upregulated alkaline phosphatase activity and runt-related transcription factor 2 (RUNX2) expression. In the 3D bioprinted model, the mixed population showed higher levels of RUNX2 expression and calcium deposition, indicating increased osteogenic differentiation within the bone layer. Results obtained from this study provide evidence that a mixed population of MSCs and OBs within the inner cambium layer of TEP can increase bone regeneration.