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The limited replicative potential of primary hepatocytes (Hep) is a major hurdle for obtaining sufficient quantity and quality hepatocytes during cell therapy in patients with liver failure. Intrahepatic cholangiocyte organoids (ICOs) derived from intrahepatic bile ducts differentiate into both hepatocytes and cholangiocytes in vitro. Here, we studied in vivo effects of transplanting ICOs and Hep in chronic liver injury mice models. Well characterized primary mouse ICOs and Hep were mixed in decellularized liver (DCL) matrix hydrogels and injected into the subcapsular left lateral liver lobe of CCl4-induced liver injury models whereas mice given DCL alone were in the sham group. Two weeks post-transplantation, transplanted liver lobes were collected and studied by histology and RNA sequencing. Transplanted animals did not exhibit any tumors, mortality or morbidity. Mice livers transplanted with ICOs had increased cellular proliferation and vascularization as compared to Hep transplanted mice or sham. Collagen deposition in the liver was significantly reduced and serum albumin levels were significantly increased in transplanted groups compared to the sham group. Expression of genes associated with hepatocyte differentiation was highest in Hep transplanted livers among the three groups, but they were also upregulated in ICO transplanted livers compared to sham. Our study demonstrates that ICOs encapsulated in DCL hydrogels when transplanted in chronically injured mice livers engraft well and show hepatocyte differentiation and reduction of fibrosis, indicating that hydrogel transplanted cholangiocyte organoids may serve as an efficient cell source and therapy for renewal of hepatocytes, restoration of hepatocyte functions and resolution of liver injury. [ABSTRACT FROM AUTHOR]

Silk is a widely accepted biomaterial for tissue regeneration owing to its tunable biomechanical properties and ease of chemical modification. However, a number of aspects associated with its clinical use are still debated. Indeed, to achieve clinical success, a biomaterial must favorably interact with host tissues without evoking local or systemic immuno-inflammatory responses. The analysis of immune responses associated with silk under in vitro and in vivo conditions provides useful insights, improving the understanding of the functional characteristics of silk biomaterials and further promoting their clinical application. Silk evokes moderate immune responses upon implantation in vivo, depending on the material structure, fabrication method, degradation time, and implantation in soft or hard tissue sites, which rapidly subside within a few days/weeks. In vitro studies indicate that its immune-stimulatory properties are largely due to inherent protein conformation and differential processing parameters. Strategically controlled levels of immune responses in vivo with marginal immunogenicity of silk-based biomaterials may contribute to matrix remodeling and replacement by native tissue matrix around the implanted site. Therefore, immunomodulatory strategies should be developed to promote the use of silk-based biomaterials as promising candidates for numerous clinical applications. [ABSTRACT FROM AUTHOR]

RNA-based therapeutics have exhibited remarkable potential in targeting genetic factors for disease intervention, exemplified by recent mRNA vaccines for COVID-19. Nevertheless, the intrinsic instability of RNA and challenges related to its translational efficiency remain significant obstacles to the development of RNA as therapeutics. This study introduces an innovative RNA delivery approach using a silk fibroin (SF) and positively charged gelatin (Gel) hydrogel matrix to enhance RNA stability for controlled release. As a proof of concept, whole-cell RNA was incorporated into the hydrogel to enhance interactions with RNA molecules. Additionally, molecular modeling studies were conducted to explore the interactions between SF, collagen, chitosan (Chi), and the various RNA species including ribosomal RNAs (28S, 18S, 8.5S, and 5S rRNAs), transfer RNAs (tRNA-ALA, tRNA-GLN, and tRNA-Leu), as well as messenger RNAs (mRNA-GAPDH, mRNA-β actin, and mRNA-Nanog), shedding light on the RNA–polymer interaction and RNA stability; SF exhibits a more robust interaction with RNA compared to collagen/gel and chitosan. We confirmed the molecular interactions of SF and RNA by FTIR and Raman spectroscopy, which were further supported by AFM and contact angle measurement. This research introduces a novel RNA delivery platform and insights into biopolymer–RNA interactions, paving the way for tailored RNA delivery systems in therapeutics and biomedical applications. [ABSTRACT FROM AUTHOR]

The development of transplantable cell sheets of functional keratocytes embedded within an aligned collagen type I matrix is a viable approach for constructing a bioequivalent of corneal stroma. Thermoresponsive materials based on poly(N-isopropylacrylamide) (PolyNIPA) have been utilized to recover carrier-free corneal cell sheets by inducing temperature changes. In this study, we employed direct-write assembly (DWA) to develop microperiodic parallel patterns of silk–PolyNIPA and gelatin–PolyNIPA. Semi-interpenetrating networks of PolyNIPA hybrids (with silk/gelatin) exhibited temperature-responsive nature and thereby have potential use in cell sheet engineering. Silk–PolyNIPA and gelatin–PolyNIPA hybrids demonstrated a hydrophobic surface at 37 °C (i.e. above their lower critical solution temperature) with a contact angle of 59°± 0.3° and 55°± 3°, respectively, whereas the surface roughness of silk–PolyNIPA was double that of gelatin–PolyNIPA. The reduction of temperature to 20 °C resulted in a decrease in the value of surface roughness and water contact angle for both hybrids. All four parallel patterned substrates guided corneal cell alignment along the direction of the patterns. Collagen type-I and aggrecan gene expression was higher when the cells were grown over the gelatin–PolyNIPA matrix after 3 weeks of culture when compared to silk–PolyNIPA. In addition, a significantly higher metabolic activity as well as enhanced vinculin expression of keratocytes on the gelatin–PolyNIPA matrix indicated the improved cytocompability compared to the silk, gelatin and silk–PolyNIPA matrices. Interestingly, the detachment of keratocytes cell sheet was achieved from the silk–PolyNIPA and gelatin–PolyNIPA planar films only within 10 min and 30 min, respectively, but the patterns could not yield intact sheet recovery. Hence, we conclude that while gelatin–PolyNIPA hybrids with parallel patterns fabricated using DWA will benefit from the application of cellular alignment, some optimization in the pattern parameters may be required for rapid sheet recovery from such substrates. Understanding the keratocytes responses to such hybrid biomaterials suggests viable options to develop a corneal stromal bioequivalent. [ABSTRACT FROM AUTHOR]