Silymarin enriched gelatin methacrylamide bioink imparts hepatoprotectivity to 3D bioprinted liver construct against carbon tetrachloride induced toxicity.

Three-dimensional liver bioprinting is an emerging technology in the field of regenerative medicine that aids in the creation of functional tissue constructs that can be used as transplantable organ substitutes. During transplantation, the bioprinted donor liver must be protected from the oxidative stress environment created by various factors during the transplantation procedure, as well as from drug-induced damage from medications taken as part of the post-surgery medication regimen following the procedure. In this study, Silymarin, a flavonoid with the hepatoprotective properties were introduced into the GelMA bioink formulation to protect the bioprinted liver against hepatotoxicity. The concentration of silymarin to be added in GelMA was optimised, bioink properties were evaluated, and HepG2 cells were used to bioprint liver tissue. Carbon tetrachloride (CCl4) was used to induce hepatotoxicity in bioprinted liver, and the effect of this chemical on the metabolic activities of HepG2 cells was studied. The results showed that Silymarin helps with albumin synthesis and shields liver tissue from the damaging effects of CCl4. According to gene expression analysis, CCl4 treatment increased TNF-α and the antioxidant enzyme SOD expression in HepG2 cells while the presence of silymarin protected the bioprinted construct from CCl4-induced damage. Thus, the outcomes demonstrate that the addition of silymarin in GelMA formulation protects liver function in toxic environments.

Fabrication and evaluation of a bi-layered gelatin based scaffold with arrayed micro-pits for full-thickness skin construct.

There is an unmet need for a reliable and reproducible method for incorporating hair follicle derived stem cells in tissue engineered skin models to reconstitute hair follicles. This study discloses a novel method for introducing hair follicle derived stem cells in microneedle embossed micro-pits of a bilayer skin equivalent fabricated from a gelatin based scaffold. The microneedles are hard and strong enough to penetrate the upper layer of the bilayer gelatin based scaffold that corresponds to the epidermis and permeates down to lower layer that corresponds to dermal layer. This strategic location will mimic the natural niche of hair follicle stem cells for picking up signals from both the epidermis and dermis. Hair follicle stem cells are trapped in to these micro-pits by vacuum assisted cell seeding. The bilayer system consists of two distinct electrospun layers in a single processing step, representing outer epidermal layer and inner dermal layer with hair follicle stem cells in embedded pits, resulting in the formation of a closed representation of a complete skin.

Biocompatibility evaluation of antioxidant cocktail loaded gelatin methacrylamide as bioink for extrusion-based 3D bioprinting.

Three-dimensional (3D) liver bioprinting is a promising technique for creating 3D liver models that can be used forin vitrodrug testing, hepatotoxicity studies, and transplantation. The functional performance of 3D bioprinted liver constructs are limited by the lack of cell-cell interactions, which calls for the creation of bioprinted tissue constructs with high cell densities. This study reports the fabrication of 3D bioprinted liver constructs using a novel photocrosslinkable gelatin methacrylamide (GelMA)-based bioink formulation. However, the formation of excess free radicals during photoinitiation poses a challenge, particularly during photocrosslinking of large constructs with high cell densities. Hence, we designed a bioink formulation comprising the base polymer GelMA loaded with an antioxidant cocktail containing vitamin C (L-ascorbic acid (AA)) and vitamin E (α-tocopherol (α-Toc)). We confirmed that the combination of antioxidants loaded in GelMA enhanced the ability to scavenge intracellular reactive oxygen species formed during photocrosslinking. The GelMA formulation was evaluated for biocompatibilityin vitroandin vivo. These results demonstrated that the bioink had adequate rheological characteristics and was biocompatible. Furthermore, when compared to bioprinted constructs with lower cell density, high-density primary rat hepatocyte constructs demonstrated improved cell-cell interactions and liver-specific functions like albumin and urea secretion, which increased 5-fold and 2.5-fold, respectively.

High-throughput production of liver parenchymal microtissues and enrichment of organ-specific functions in gelatin methacrylamide microenvironment.

Liver parenchymal microtissues (LPMTs) are three-dimensional (3D) aggregates of hepatocytes that recapitulate in vivo-like cellular assembly. They are considered as a valuable model to study drug metabolism, disease biology, and serve as ideal building blocks for liver tissue engineering. However, their integration into the mainstream drug screening process has been hindered due to the lack of simple, rapid techniques to produce a large number of uniform microtissues and preserve their structural-functional integrity over the long term. Here, we present a high-throughput methodology to produce LPMTs in a novel, economic, and reusable Hanging-drop Culture Chamber (HdCC). A drop-on-demand bioprinting approach was optimized to generate droplets of HepG2 cell suspension on a polyethylene terephthalate substrate. The substrates carrying droplets were placed inside a novel HdCC and incubated to obtain 1600 LPMTs having a size of 200-300 µm. Tissue size, cell viability, cellular arrangement and polarity, and insulin-mediated glucose uptake by LPMTs were analyzed. The microtissues were viable and exhibited an active response to insulin stimulation. Cells within the microtissue reorganized to form hepatic plate-like structures and expressed apical (Multidrug Resistance Protein 2 [MRP2]) and epithelial (Zonula Occludens 1 [ZO1]) markers. Further to maintain the structural integrity and enhance the functional capabilities, LPMTs were sandwiched within gelatin methacrylamide (GelMA) hydrogel and the liver-specific functions were monitored for 2 weeks. The results showed that the 3D structure of LPMTs in GelMA sandwich was maintained while the albumin secretion, urea synthesis, and cytochrome P450 activity were enhanced compared with LPMTs in suspension. In conclusion, this study presents a novel culture chamber for mass production of microtissues and a method for enhancing organ-specific functions of LPMTs in vitro.

A Novel, Single Step, Highly Sensitive In-Vitro Cell-Based Metabolic Assay Using Honeycomb Microporous Polymer Membranes.

This study describes a novel, simple and versatile system for cell-based assays at the bench-top. The system consists of Polyurethane (PU) based honeycomb membrane with the active compounds/assay reagents dispensed on its pore linings. Membranes with functionalized pores were thus created and used for conducting cell based assays. As proof-of-concept Flourocein acetate (FDA) and Propidium iodide (PI) were embedded on the pore linings and live/dead assays were performed on L929 and Hacat cell lines. The results proved the sensitivity of the membrane based cell assay. To ensure the capacity of this system for high throughput applications, membrane based live/dead assay was performed on L929 cells with varying levels of viability. The results from this experiment were quantified by microscopic and spectrofluourimetric techniques both of which were found to correlate well. It was concluded that this simple membrane based cell assay is highly versatile and enables multiple compounds to be tested on the same cell/tissue. Furthermore, this method requires low volumes of assay reagents and eliminates many of the wet techniques that are involved in a conventional assay, without compromising on the sensitivity. It is anticipated that this functionalized membrane system could be easily adapted for both manual and automated high content screening experiments including in vitro biomaterial evaluation as well as cytotoxicity of nanomaterials.

Evaluation of dynamic creep properties of surgical mesh prostheses--uniaxial fatigue.

In this study, we examine the dynamic creep behavior of four commonly used commercial hernia meshes (Prolene, Ultrapro, Vypro, and Vypro II). The meshes, differing from each other with respect to composition and architecture, were tested under uniaxial tension at simulated physiological loads and environmental conditions. The changes in percentage strain elongation, secant modulus, and cyclic energy dissipation over 100,000 cycles were compared. All of the meshes evaluated were found to be overengineered compared to physiological-loading criteria and displayed good load-carrying performance. When all meshes were tested at 37 degrees C in physiological saline, they survived 100,000 cycles of sinusoidal loading without fracture, except Ultrapro. Interestingly, irrespective of the differences in structure and composition, all meshes underwent strain-hardening and permanent plastic deformation. Scanning electron micrographs of the meshes showed evidence of yarn thinning, decrimping, and fracture. The results of this study suggest that strain-hardening of the meshes under dynamic loading could be a possible cause for complications related to abdominal mobility during long-term implantations.

The fabrication and characterization of biodegradable HA/PHBV nanoparticle-polymer composite scaffolds.

This study reports the fabrication and characterization of nano-sized hydroxyapatite (HA)/poly(hydroxyabutyrate-co-hydroxyvalerate) (PHBV) polymer composite scaffolds with high porosity and controlled pore architectures. These scaffolds were prepared using a modified thermally induced phase-separation technique. This investigation focuses on the effect of fabrication conditions on the overall pore architecture of the scaffolds and the dispersion of HA nanocrystals within the composite scaffolds. The morphologies, mechanical properties and in vitro bioactivity of the composite scaffolds were investigated. It was noted that the pore architectures could be manipulated by varying phase-separation parameters. The HA particles were dispersed in the pore walls of the scaffolds and were well bonded to the polymer. The introduction of HA greatly increased the stiffness and strength, and improved the in vitro bioactivity of the scaffolds. The results suggest these newly developed nano-HA/PHBV composite scaffolds may serve as an effective three-dimensional substrate in bone tissue engineering.

Biological evaluation of pliable hydroxyapatite-ethylene vinyl acetate co-polymer composites intended for cranioplasty.

Hydroxyapatite (HAP) is undoubtedly a material suitable for repairing the defective bone tissue. However, the brittleness and non-malleability of HAP limit its clinical application as a cranioplastic analogue. To improve these properties, pliable, osteoconductive composites composed of HAP and ethylene vinyl acetate co-polymer (EVA) have been developed. This study reports the biocompatibility evaluation of the newly developed composite material. Composites of two compositions, containing 40 and 50 volume percentage of HAP, were evaluated. In vitro cell culture cytotoxity studies were carried out using L929 cell line. Intracutaneous irritation studies, and intramuscular implantation studies were carried out on rabbits. Cell culture studies showed that the composite was non-cytotoxic to mouse fibroblast cell line. Intracutaneous irritation studies did not show any gross signs of tissue reaction. Histological analysis after six months of implantation in the paravertebral muscles of rabbit showed that all the implants under study were covered with a thin soft tissue capsule. On the basis of these observations, we conclude that the composite materials are biocompatible and hence are a candidate material for implantation in the cranium.

Effect of vinyl acetate content on the sintering behavior of hydroxyapatite-ethylene vinyl acetate copolymer composites.

Ethylene vinyl acetate copolymer (EVA) alone could be used as a binder material for the fabrication of hydroxyapatite (HAP) into intricate shapes for various bone substitute applications. It was observed that as the vinyl acetate content in the polymer was increased from 12 to 28 wt % an increase in the sintered density of the HAP was observed. Retention of the shapes of HAP in the molded form was also observed.