Tissue Engineering Part A最新文献

Cell morphology is not only integral to its function within the body but also plays a critical role in cellular behavior and fate. In tissue engineering, cell-scaffold interactions play a critical role because scaffold physical and biochemical characteristics, such as pore size, fiber alignment, and surface architecture, directly influence cellular morphology and behavior. These interactions impact key biological processes, including adhesion, proliferation, migration, and differentiation of the cells, ultimately influencing tissue formation and regeneration. This study investigated how scaffold topography and culture time influence fibroblast morphology and behavior in a bilayer scaffold consisting of randomly oriented fiber layer and aligned fiber layer. Fibroblasts were seeded onto the scaffolds and cultured for 1, 3, 6, or 9 days, and nuclear and cytoskeletal morphologies were quantified using shape descriptors, including nuclear and cellular roundness, eccentricity, aspect ratio, and area ratio. The results demonstrate that scaffold fiber alignment significantly modulates cellular morphology, with aligned fibers promoting elongated, aligned morphologies and randomly oriented fibers favoring branched, multidirectional spreading. Culture time emerged as a key factor, as cells on both surfaces exhibited more rounded, stabilized morphologies by day 6, suggesting time-dependent remodeling and interaction with the scaffold microarchitecture. Specifically, aligned fiber-like scaffold surfaces may benefit regeneration of uniaxially aligned tissues, such as tendon, ligament, or nerve, whereas random fiber-like scaffold surfaces may support stromal or bone environments requiring isotropic spreading. Furthermore, the bilayer scaffold architecture holds promise for complex tissue interfaces, such as the periodontium or osteochondral units, where region-specific topographical cues are essential for functional tissue integration.

Traumatic injuries lead to volumetric muscle loss (VML) and nerve damage that cause chronic functional deficits. Due to the inability of mammalian skeletal muscle to regenerate after VML damage, engineered scaffolds have been explored to address this challenge, but with limited success in functional restoration. We introduce novel bioactive amorphous silicon oxynitride (SiONx) biomaterials with surface properties and Si ion release to accelerate muscle and nerve cell differentiation for functional tissue regeneration. Micropatterned scaffolds were designed and developed on Si-wafer to test the effect of SiONx on myogenesis and neurogenesis. The scaffolds were created using UV photolithography to first pattern their surface, followed by the deposition of SiONx through plasma enhanced chemical vapor deposition (PECVD). X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) confirmed the uniform chemical structure of an amorphous SiONx film on the patterned surfaces. Atomic force microscopy and scanning electron microscopy (SEM) elucidated the surface morphology with a uniform 2 μm grating microstructure. The 2 µm pattern size is within the range of cellular dimensions, allowing for effective cell-surface interactions. Further, 2 µm features provide sufficient contact points for cell adhesion without overwhelming the cell's ability to interact with the surface. Two separate studies were conducted with SiONx biomaterials and Si ions alone. This was done to understand how Si ions impact cell response separate from the surfaces. C2C12 mouse myoblasts and NG108 neuronal cells were cultured on SiONx biomaterials. In separate studies, we tested the effect of Si ion treatments with these cells (cultured on tissue culture plastic). Cell culture studies demonstrated enhanced C2C12 myoblast attachment and proliferation on SiONx surfaces. High-resolution SEM and fluorescence images revealed highly aligned myotubes (from C2C12 cells) and axons (from NG108 cells) in a parallel direction to the micropatterned SiONx scaffolds. GAP43 expression, neurite outgrowth, and alignment were significantly increased with the Si-ions and SiONx biomaterials. These findings suggest that SiONx scaffolds enhance muscle and nerve cell adhesion and growth and promote the formation of aligned myotubes and axons on the pattern surfaces.

Primary open-angle glaucoma is a prevalent type of degenerative eye disease that results in lifelong blindness, and its critical pathogenic cause is trabecular meshwork (TM) dysfunction or decreased TM cellularity. Considering that TM develops from neural crest cells (NCCs), we investigate the potential of human embryonic stem cell (hESC)-derived NCCs transplantation for TM regeneration. We used a chemically defined method to induce the differentiation of NCCs and injected 1.0 × 10⁶ hESC-derived NCCs combined with 100 μmol/L Y-27632 into the anterior chamber of rabbit. Intraocular pressure (IOP), TM, and corneal changes of rabbits with cell transplantation were examined with TonoPEN AVIA, slit lamp microscope, dual-immunofluorescence staining, and optical coherence tomography. The hESC-derived NCCs underwent homogenous differentiation over the course of 5 days' induction, which expressed the typical neural crest markers HNK-1, P75, SOX10, and AP-2α. NOD/SCID mice received injections of hESC-derived NCCs in the groin or axilla. There was no teratoma formation. When intracamerally injected, hESC-derived NCCs integrated into the TM tissue and expressed mature TM cell markers Aqp1, Chi3l1, and Timp3 after 7 days transplantation in rabbit eyes. The IOP and central corneal thickness basically maintained at normal levels within 2 weeks. No significant adverse effects in rabbits with hESC-derived NCC injection were observed after 5 weeks of cell transplantation. Our findings indicate that hESC-derived NCCs could integrate into the TM tissue and differentiate into mature TM cells after being injected intracamerally, showing a potential therapeutic approach to addressing TM dysfunction in the treatment of glaucoma.

Insufficient vascularization is the main barrier to creating engineered bone grafts for treating large and ischemic defects. Modular tissue engineering approaches have promise in this application because of the ability to combine tissue types and localize microenvironmental cues to drive desired cell function. In direct bone formation approaches, it is challenging to maintain sustained osteogenic activity, since vasculogenic cues can inhibit tissue mineralization. This study harnessed the physiological process of endochondral ossification to create multiphase tissues that allowed concomitant mineralization and vessel formation. Mesenchymal stromal cells in pellet culture were differentiated toward a cartilage phenotype, followed by induction to chondrocyte hypertrophy. Hypertrophic pellets (HPs) exhibited increased alkaline phosphatase activity, calcium deposition, and osteogenic gene expression relative to chondrogenic pellets. In addition, HPs secreted and sequestered angiogenic factors, and supported new blood vessel formation by cocultured endothelial cells and undifferentiated stromal cells. Multiphase constructs created by combining HPs and vascularizing microtissues and maintained in an unsupplemented basal culture medium were shown to support robust vascularization and sustained tissue mineralization. These results demonstrate a promising in vitro strategy to produce multiphase-engineered constructs that concomitantly support the generation of mineralized and vascularized tissue in the absence of exogenous osteogenic or vasculogenic medium supplements.

Skin aging involves changes in extracellular matrix components, such as wrinkles and pigmentation. Caviar extract (CE) is a promising compound for skin rejuvenation, but effective topical delivery requires optimized carriers. This study evaluated polyvinyl alcohol/carboxymethyl chitosan (PVA/CMC) hydrogels loaded with CE at concentrations of 2%, 3.5%, and 5% as scaffolds to influence the epithelial differentiation of adipose-derived mesenchymal stem cells (ADMSCs). Hydrogels were synthesized using a freeze-thaw method and characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy, swelling and degradation tests, and mechanical analysis. Biocompatibility and cell migration were assessed using MTT and scratch assays; at the same time, expression of cytokeratin-18 (CK-18) and pan-cytokeratin (pan-CK) was measured via reverse transcription-quantitative polymerase chain reaction and immunocytochemistry (ICC), respectively. FTIR confirmed successful CE incorporation, and SEM revealed a porous structure. Hydrogels with 3.5% and 5% CE demonstrated a good balance between swelling and degradation over 336 h. The biocompatibility tests showed that 5% CE supported enhanced long-term cell growth. The scratch assay indicated improved cell migration, and transcriptional analysis revealed significantly higher CK-18 levels in ADMSCs treated with PVA/CMC/CE 5% (p < 0.001). ICC results showed significantly higher pan-CK expression at 3.5% CE (41.82%) and 5% CE (48.16%), suggesting that CE promotes repair processes. These findings suggest that 5% CE-loaded PVA/CMC hydrogel could be an effective option for skin regeneration and antiaging. Impact Statement Caviar extract (CE) was considered a bioactive ingredient, along with polyvinyl alcohol (PVA) and carboxymethyl chitosan (CMC) polymers, to prepare a functional and practical hydrogel without hazardous components for anti-aging and cosmetic applications. In the present study, the PVA/CMC hydrogel contains various concentrations of CE (3.5% and 5%), is biocompatible, and enhances cellular viability and migration of adipose-derived mesenchymal stem cell. Our results demonstrated that the synergistic effect of CE and CMC could promote the expression of cytokeratin-18 gene and pan-cytokeratin protein and play a critical role in stimulating skin regeneration.

Biological materials composed of extracellular matrix (ECM) or its components have been successfully used for tissue repair and reconstruction. Preclinical studies, along with a cohort study following stage T1A esophageal adenocarcinoma (EAC) resection, have shown that ECM biomaterials can restore esophageal mucosa and submucosa without cancer recurrence. However, the molecular mechanisms underlying these effects remain largely unexplored. The present study investigates the in vitro effects of ECM degradation products from nonmalignant esophageal (eECM) and urinary bladder (ubECM) sources on EAC cell proliferation, migration, and associated signaling pathways. Both eECM and ubECM significantly inhibited OE33 cell proliferation, with eECM exhibiting a stronger effect-reducing proliferation to 25% at 24 h and 7% at 72 h compared with pepsin control (p < 0.001). A high-throughput cell surface marker screen followed by gene and protein expression analysis revealed that both ECM sources downregulated CD164 and CXCR4, reducing CXCR4 protein levels by approximately 50% (p = 0.006 for eECM, p = 0.007 for ubECM). Notably, only eECM significantly suppressed OE33 cell migration (p ≤ 0.0001) and downregulated bone morphogenetic protein 4 BMP4 expression, along with its downstream targets pSMAD1/5/8, ID2, and SNAI2, thereby reducing epithelial-mesenchymal transition. These findings support the concept that biochemical cues from nonmalignant ECM modulate neoplastic cell behavior. Given the involvement of PI3K-Akt and BMP4 signaling in EAC progression, ECM-based strategies may warrant further investigation as potential therapeutic approaches following esophageal cancer resection.

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.