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Cancer-associated cachexia (CAC) is a complex metabolic and behavioral syndrome with multiple manifestations that involve systemic inflammation, weight loss, and adipose lipolysis. It impacts the quality of life of patients and is the direct cause of death in 20–30% of cancer patients. The severity of fat loss and adipose tissue remodeling negatively correlate with patients’ survival outcomes. To address the mechanism of fat loss and design potential approaches to prevent the process, it will be essential to understand CAC pathophysiology through white adipose tissue models. In the present study, an engineered human white adipose tissue (eWAT) model based on three-dimensional (3D) bioprinting was developed and induced with pancreatic cancer cell-conditioned medium (CM) to mimic the status of CAC in vitro. We found that the CM induction significantly increased the lipolysis and accumulation of the extracellular matrix (ECM). The 3D eWATs were further vascularized to study the influence of vascularization on lipolysis and CAC progression, which was largely unknown. Results demonstrated that CM induction improved the angiogenesis of vascularized eWATs (veWATs), and veWATs demonstrated decreased glycerol release but increased UCP1 expression, compared to eWATs. Many unique inflammatory cytokines (IL-8, CXCL-1, GM-CSF, etc) from the CM were detected and supposed to contribute to eWAT lipolysis, UCP1 up-regulation, and ECM development. In response to CM induction, eWATs also secreted inflammatory adipokines related to the metastatic ability of cancer, muscle atrophy, and vascularization (NGAL, CD54, IGFBP-2, etc). Our work demonstrated that the eWAT is a robust model for studying cachectic fat loss and the accompanying remodeling of adipose tissue. It is therefore a useful tool for future research exploring CAC physiologies and developing potential therapies.

In the past decade, cartilage tissue engineering has arisen as a promising therapeutic option for degenerative joint diseases, such as osteoarthritis, in the hope of restoring the structure and physiological functions. Hydrogels are promising biomaterials for developing engineered scaffolds for cartilage regeneration. However, hydrogel-delivered mesenchymal stem cells or chondrocytes could be exposed to elevated levels of reactive oxygen species (ROS) in the inflammatory microenvironment after being implanted into injured joints, which may affect their phenotype and normal functions and thereby hinder the regeneration efficacy. To attenuate ROS induced side effects, a multifunctional hydrogel with an innate anti-oxidative ability was produced in this study. The hydrogel was rapidly formed through a dynamic covalent bond between phenylboronic acid grafted hyaluronic acid (HA-PBA) and poly(vinyl alcohol) and was further stabilized through a secondary crosslinking between the acrylate moiety on HA-PBA and the free thiol group from thiolated gelatin. The hydrogel is cyto-compatible and injectable and can be used as a bioink for 3D bioprinting. The viscoelastic properties of the hydrogels could be modulated through the hydrogel precursor concentration. The presence of dynamic covalent linkages contributed to its shear-thinning property and thus good printability of the hydrogel, resulting in the fabrication of a porous grid construct and a meniscus like scaffold at high structural fidelity. The bioprinted hydrogel promoted cell adhesion and chondrogenic differentiation of encapsulated rabbit adipose derived mesenchymal stem cells. Meanwhile, the hydrogel supported robust deposition of extracellular matrix components, including glycosaminoglycans and type II collagen, by embedded mouse chondrocytes in vitro. Most importantly, the hydrogel could protect encapsulated chondrocytes from ROS induced downregulation of cartilage-specific anabolic genes (ACAN and COL2) and upregulation of a catabolic gene (MMP13) after incubation with H2O2. Furthermore, intra-articular injection of the hydrogel in mice revealed adequate stability and good biocompatibility in vivo. These results demonstrate that this hydrogel can be used as a novel bioink for the generation of 3D bioprinted constructs with anti-ROS ability to potentially enhance cartilage tissue regeneration in a chronic inflammatory and elevated ROS microenvironment.

Cancer-associated cachexia (CAC) is a complex metabolic and behavioral syndrome with multiple manifestations that involve systemic inflammation, weight loss, and adipose lipolysis. It impacts the quality of life of patients and is the direct cause of death in 20%–30% of cancer patients. The severity of fat loss and adipose tissue remodeling negatively correlate with patients’ survival outcomes. To address the mechanism of fat loss and design potential approaches to prevent the process, it will be essential to understand CAC pathophysiology through white adipose tissue models. In the present study, an engineered human white adipose tissue (eWAT) model based on three-dimensional (3D) bioprinting was developed and induced with pancreatic cancer cell-conditioned medium (CM) to mimic the status of CAC in vitro. We found that the CM induction significantly increased the lipolysis and accumulation of the extracellular matrix (ECM). The 3D eWATs were further vascularized to study the influence of vascularization on lipolysis and CAC progression, which was largely unknown. Results demonstrated that CM induction improved the angiogenesis of vascularized eWATs (veWATs), and veWATs demonstrated decreased glycerol release but increased UCP1 expression, compared to eWATs. Many unique inflammatory cytokines (IL-8, CXCL-1, GM-CSF, etc) from the CM were detected and supposed to contribute to eWAT lipolysis, UCP1 up-regulation, and ECM development. In response to CM induction, eWATs also secreted inflammatory adipokines related to the metastatic ability of cancer, muscle atrophy, and vascularization (NGAL, CD54, IGFBP-2, etc). Our work demonstrated that the eWAT is a robust model for studying cachectic fat loss and the accompanying remodeling of adipose tissue. It is therefore a useful tool for future research exploring CAC physiologies and developing potential therapies.

Functional reconstruction of craniomaxillofacial defects is challenging, especially for the patients who suffer from traumatic injury, cranioplasty, and oncologic surgery. Three-dimensional (3D) printing/bioprinting technologies provide a promising tool to fabricate bone tissue engineering constructs with complex architectures and bioactive components. In this study, we implemented multi-material 3D printing to fabricate 3D printed PCL/hydrogel composite scaffolds loaded with dual bioactive small molecules (i.e. resveratrol and strontium ranelate). The incorporated small molecules are expected to target several types of bone cells. We systematically studied the scaffold morphologies and small molecule release profiles. We then investigated the effects of the released small molecules from the drug loaded scaffolds on the behavior and differentiation of mesenchymal stem cells (MSCs), monocyte-derived osteoclasts, and endothelial cells. The 3D printed scaffolds, with and without small molecules, were further implanted into a rat model with a critical-sized mandibular bone defect. We found that the bone scaffolds containing the dual small molecules had combinational advantages in enhancing angiogenesis and inhibiting osteoclast activities, and they synergistically promoted MSC osteogenic differentiation. The dual drug loaded scaffolds also significantly promoted in vivo mandibular bone formation after 8 week implantation. This work presents a 3D printing strategy to fabricate engineered bone constructs, which can likely be used as off-the-shelf products to promote craniomaxillofacial regeneration.

Fiber constructed yarns are the elementary building blocks for the generation of implantable biotextiles, and there are always needs for designing and developing new types of yarns to improve the properties of biotextile implants. In the present study, we aim to develop novel nanofiber yarns by combining nanostructure that more closely mimic the extracellular matrix (ECM) fibrils of native tissues with biodegradability, strong mechanical properties and great textile processibility. A novel electrospinning system which integrates yarn formation with hot drawing process was developed to fabricate poly(L-lactic acid) (PLLA) nanofiber yarns (NYs). Compared to the PLLA NYs without hot drawing, the thermally drawn PLLA NYs presented superbly-orientated fibrous structure and notably enhanced crystallinity. Importantly, they possessed admirable mechanical performances, which matched and even exceeded the commercial PLLA microfiber yarns (MYs). The thermally drawn PLLA NYs were also demonstrated to notably promote the adhesion, alignment, proliferation, and tenogenic differentiation of human adipose derived mesenchymal stem cells (hADMSCs) compared to the PLLA NYs without hot drawing. The thermally drawn PLLA NYs were further processed into various nanofibrous tissue scaffolds with defined structures and adjustable mechanical and biological properties using textile braiding and weaving technologies, demonstrating the feasibility and versatility of thermally drawn PLLA NYs for textile-forming utilization. The hADMSCs cultured on PLLA NY-based textiles presented enhanced attachment and proliferation capacities than those cultured on PLLA MY-based textiles. This work presents a facile technique to manufacture high performance PLLA NYs, which opens up opportunities to generate advanced nanostructured biotextiles for surgical implant applications.

Engineered tendon grafts offer a promising alternative for grafting during the reconstruction of complex tendon tears. The tissue engineered tendon substitutes have the advantage of increased biosafety and the option to customize their biochemical and biophysical properties to promote tendon regeneration. In this study, we developed a novel centrifugal melt electrospinning (CME) technique, with the goal of optimizing the fabrication parameters to generate fibrous scaffolds for tendon tissue engineering. The effects of CME processing parameters, including rotational speed, voltage, and temperature, on fiber properties (i.e. orientation, mean diameter, and productivity) were systematically investigated. By using this solvent-free and environmentally-friendly method, we fabricated both random and aligned poly (L-lactic acid) (PLLA) fibrous scaffolds with controllable mesh thickness. We also investigated and compared their morphology, surface hydrophilicity, and mechanical properties. We seeded human adipose derived mesenchymal stem cells (HADMSC) on various PLLA fibrous scaffolds and conditioned the constructs in tenogenic differentiation medium (TDM) for up to 21 days, to investigate the effects of fiber alignment and scaffold thickness on cell behavior. Aligned fibrous scaffolds induced cell elongation and orientation through a contact guidance phenomenon and promoted HADMSC proliferation and differentiation towards tenocytes. At the early stage, thinner scaffolds were beneficial for HADMSC proliferation, but the scaffold thickness had no significant effects on cell proliferation for longer-term cell culture. We further co-seeded HADMSC and human umbilical vein endothelial cells (HUVEC) on aligned PLLA fibrous mats and determined how the vascularization affected HADMSC tenogenesis. We found that co-cultured HADMSC-HUVEC expressed more tendon related markers on the aligned fibrous scaffold. The co-culture systems promoted in vitro HADMSC differentiation towards tenocytes. These aligned fibrous scaffolds fabricated by CME technique could potentially be utilized to repair and regenerate tendon defects and injuries with cell co-culture and controlled vascularization.

The aortic valve exhibits complex three-dimensional (3D) anatomy and heterogeneity essential for long-term efficient biomechanical function. These are, however, challenging to mimic in de novo engineered living tissue valve strategies. We present a novel simultaneous 3D-printing/photocrosslinking technique for rapidly engineering complex, heterogeneous aortic valve scaffolds. Native anatomic and axisymmetric aortic valve geometries (root wall and tri-leaflets) with 12 to 22 mm inner diameters (ID) were 3D printed with poly-ethylene glycol-diacrylate (PEG-DA) hydrogels (700 or 8000 MW) supplemented with alginate. 3D printing geometric accuracy was quantified and compared using Micro-CT. Porcine aortic valve interstitial cells (PAVIC) seeded scaffolds were cultured for up to 21 days. Results showed that blended PEG-DA scaffolds could achieve over 10-fold range in elastic modulus (5.3±0.9 to 74.6±1.5 kPa). 3D printing times for valve conduits with mechanically contrasting hydrogels were optimized to 14 to 45 minutes, increasing linearly with conduit diameter. Larger printed valves had greater shape fidelity (93.3±2.6, 85.1±2.0, and 73.3±5.2% for 22, 17, and 12 mm ID porcine valves; 89.1±4.0, 84.1±5.6, and 66.6±5.2% for simplified valves). PAVIC seeded scaffolds maintained near 100% viability over 21 days. These results demonstrate that 3D hydrogel printing with controlled photocrosslinking can rapidly fabricate anatomical heterogeneous valve conduits that support cell engraftment.

Biomaterials for scaffolds and scaffold fabrication techniques are two key elements in scaffold-based tissue engineering. Nanocomposites that consist of biodegradable polymers and osteoconductive bioceramic nanoparticles and advanced scaffold manufacturing techniques, such as rapid prototyping (RP) technologies, have attracted much attention for developing new bone tissue engineering strategies. In the current study, poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) microspheres and calcium phosphate (Ca-P)/PHBV nanocomposite microspheres were fabricated using the oil-in-water (O/W) and solid-in-oil-in-water (S/O/W) emulsion solvent evaporation methods. The microspheres with suitable sizes were then used as raw materials for scaffold fabrication via selective laser sintering (SLS), which is a mature RP technique. A three-factor three-level complete factorial design was applied to investigate the effects of the three factors (laser power, scan spacing, and layer thickness) in SLS and to optimize SLS parameters for producing good-quality PHBV polymer scaffolds and Ca-P/PHBV nanocomposite scaffolds. The plots of the main effects of these three factors and the three-dimensional response surface were constructed and discussed. Based on the regression equation, optimized PHBV scaffolds and Ca-P/PHBV scaffolds were fabricated using the optimized values of SLS parameters. Characterization of optimized PHBV scaffolds and Ca-P/PHBV scaffolds verified the optimization process. It has also been demonstrated that SLS has the capability of constructing good-quality, sophisticated porous structures of complex shape, which some tissue engineering applications may require.