3D bioprinting of mouse pre-osteoblasts and human MSCs using bioinks consisting of gelatin and decellularized bone particles

One of the key challenges in biofabrication applications is to obtain bioinks that provide a balance between printability, shape fidelity, cell viability, and tissue maturation. Decellularization methods allow the extraction of natural extracellular matrix, preserving tissue-specific matrix proteins. However, the critical challenge in bone decellularization is to preserve both organic (collagen, proteoglycans) and inorganic components (hydroxyapatite) to maintain the natural composition and functionality of bone. Besides, there is a need to investigate the effects of decellularized bone (DB) particles as a tissue-based additive in bioink formulation to develop functional bioinks. Here we evaluated the effect of incorporating DB particles of different sizes (≤45 and ≤100 μm) and concentrations (1%, 5%, 10% (wt %)) into bioink formulations containing gelatin (GEL) and pre-osteoblasts (MC3T3-E1) or human mesenchymal stem cells (hTERT-MSCs). In addition, we propose a minimalistic bioink formulation using GEL, DB particles and cells with an easy preparation process resulting in a high cell viability. The printability properties of the inks were evaluated. Additionally, rheological properties were determined with shear thinning and thixotropy tests. The bioprinted constructs were cultured for 28 days. The viability, proliferation, and osteogenic differentiation capacity of cells were evaluated using biochemical assays and fluorescence microscopy. The incorporation of DB particles enhanced cell proliferation and osteogenic differentiation capacity which might be due to the natural collagen and hydroxyapatite content of DB particles. Alkaline phosphatase activity is increased significantly by using DB particles, notably, without an osteogenic induction of the cells. Moreover, fluorescence images display pronounced cell-material interaction and cell attachment inside the constructs. With these promising results, the present minimalistic bioink formulation is envisioned as a potential candidate for bone tissue engineering as a clinically translatable material with straightforward preparation and high cell activity

3D printed gelatin/decellularized bone composite scaffolds for bone tissue engineering: Fabrication, characterization and cytocompatibility study

Three-dimensional (3D) printing technology enables the design of personalized scaffolds with tunable pore size and composition. Combining decellularization and 3D printing techniques provides the opportunity to fabricate scaffolds with high potential to mimic native tissue. The aim of this study is to produce novel decellularized bone extracellular matrix (dbECM)-reinforced composite-scaffold that can be used as a biomaterial for bone tissue engineering. Decellularized bone particles (dbPTs, ∼100 ​μm diameter) were obtained from rabbit femur and used as a reinforcement agent by mixing with gelatin (GEL) in different concentrations. 3D scaffolds were fabricated by using an extrusion-based bioprinter and crosslinking with microbial transglutaminase (mTG) enzyme, followed by freeze-drying to obtain porous structures. Fabricated 3D scaffolds were characterized morphologically, mechanically, and chemically. Furthermore, MC3T3-E1 mouse pre-osteoblast cells were seeded on the dbPTs reinforced GEL scaffolds (GEL/dbPTs) and cultured for 21 days to assess cytocompatibility and cell attachment. We demonstrate the 3D-printability of dbPTs-reinforced GEL hydrogels and the achievement of homogenous distribution of the dbPTs in the whole scaffold structure, as well as bioactivity and cytocompatibility of GEL/dbPTs scaffolds. It was shown that Young's modulus and degradation rate of scaffolds were enhanced with increasing dbPTs content. Multiphoton microscopy imaging displayed the interaction of cells with dbPTs, indicating attachment and proliferation of cells around the particles as well as into the GEL-particle hydrogels. Our results demonstrate that GEL/dbPTs hydrogel formulations have potential for bone tissue engineering

Physical characterisation of an alginate/lysozyme nano-laminate coating and its evaluation on ‘coalho’ cheese shelf life

This work aimed at the characterisation of a nanolaminate coating produced by the layer-by-layer methodology and its evaluation on the preservation of ‘Coalho’ cheese. Initially, five alternate layers of alginate and lysozyme were assembled in an aminolysed/charged polyethylene terephthalate (A/C PET) and physically characterised by UV/VIS spectroscopy, contact angle, water vapour (WVTR) and oxygen (OTR) transmission rates and scanning electron microscopy. Afterwards, the same methodology was used to apply the nano-laminate coating in ‘Coalho’ cheese and its shelf life was evaluated during 20 days in terms of mass loss, pH, lipid peroxidation, titratable acidity and microbial count. UV/VIS spectroscopy and contact angle analyses confirmed the layers’ deposition and the successful assembly of nano-laminate coating on A/C PET surface. The coating presented WVTR and OTR values of 1.03×10−3 and 1.28× 10−4 g m−2 s−1, respectively. After 20 days, coated cheese showed lower values of mass loss, pH, lipidic peroxidation, microorganisms’ proliferation and higher titratable acidity in comparison with uncoated cheese. These results suggest that gas barrier and antibacterial properties of alginate/lysozyme nanocoating can be used to extend the shelf life of ‘Coalho’ cheese.The author Bartolomeu G. de S. Medeiros is recipient of a scholarship from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES-Brazil). The author Marthyna P. Souza is recipient of a scholarship from Fundacao de Amparo a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE, Brazil) and was recipient of a scholarship from Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES/PDEE-Brazil). The authors Ana C. Pinheiro, Ana I. Bourbon and Miguel A. Cerqueira are recipients of a fellowship (SFRH/BD/48120/2008, SFRH/BD/73178/2010 and SFRH/BPD/72753/2010, respectively), supported by Fundacao para a Ciencia e Tecnologia, POPH-QREN and FSE (FCT, Portugal). Maria G. Carneiro-da-Cunha express is gratitude to the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for research grant. The present work was supported by CAPES/PROCAD/NF/1415/2007. The support of EU Cost Action FA0904 is gratefully acknowledged

Chitosan-hybrid poss nanocomposites for bone regeneration: The effect of poss nanocage on surface, morphology, structure and in vitro bioactivity

WOS: 000510955500064PubMed: 31622724POSS, regarded as the smallest silica particle, is widely used as nanofiller in polymer systems. POSS-based nanocomposites are deduced as novel materials having potency for biomedical applications owing to the enhanced biocompatibility and physicochemical characteristics. The aim of this work was to integrate the beneficial features of chitosan (CS) and OctaTMA-POSS nanoparticle to design nanocomposite for bone tissue regeneration. The nanocomposite scaffolds were fabricated by freeze-drying. The effects of POSS incorporation on morphology and structure of CS matrix were examined. Bioactivity and osteogenic effects of the POSS nanoparticles were investigated with cytocompatibility, cell proliferation, alkaline phosphatase activity, osteocalcin production and biomineralization assays. PUSS incorporation altered the surface morphology by increasing surface roughness. Nanocomposite scaffolds with 82-90% porosity exhibited an increase in compression modulus of scaffolds (78-107 kPa) compared to control CS group (56 kPa). Results indicated that CS-POSS scaffolds were found cytocompatible with 3T3, MG-63 and Saos-2 cell lines. POSS incorporation showed promising effects on osteoblast adhesion and proliferation as well as increasing ALP activity, octeocalcin secretion and biomineralization of cells. (C) 2019 Elsevier B.V. All rights reserved

Bioactive fish scale incorporated chitosan biocomposite scaffolds for bone tissue engineering

Recently, biologically active natural macromolecules have come into prominence to be used as potential materials in scaffold design due to their unique characteristics which can mimic the human tissue structure with their physical and chemical similarity. Among them, fish scale (FS) is a biologically active material with its structural similarity to bone tissue due to including type I collagen and hydroxyapatite and also have distinctive collagen arrangement. In the present study, it is aimed to design a novel composite scaffold with FS incorporation into chitosan (CH) matrix for bone tissue regeneration. Therefore, two biological macromolecules, fish scale and chitosan, were combined to produce bio-composite scaffold. First, FS were decellularized with the chemical method and disrupted physically as microparticles (100 in), followed by dispersal in CH with ultrasonic homogenisation, CH/FS scaffolds were fabricated by lyophilization technique. Scaffolds were characterized physically, chemically, mechanically, and morphologically. SEM and porosity results showed that CH/FS scaffolds have uniform pore structure showing high porosity. Mechanical properties and degradation rate are enhanced with increasing FS content. In vitro cytotoxicity, proliferation and osteogenic activity of the scaffolds were evaluated with SaOS-2 cell line. CH/FS scaffolds did not show any cytotoxicity effect and the cells were gradually proliferated during culture period. Cell viability results showed that, FS microparticles had a proliferative effect on SaOS-2 cells when compared to control group. ALP activity and biomineralization studies indicated that FS micro particle reinforcement increased osteogenic activity during culture period. As a biological macromolecule with unique characteristics, FS was found as cytocompatible and provided promising effects as reinforcement agents for polymeric scaffolds. In conclusion, fabricated CH/FS bio-composites showed potential for bone tissue engineering applications. (C) 2019 Elsevier B.V. All rights reserved