Osteoblast responsive biosilica-enriched gelatin microfibrillar microenvironments.

Engineering of scaffolds for bone regeneration is often inspired by the native extracellular matrix mimicking its composite fibrous structure. In the present study, we used low loadings of diatomite earth (DE) biosilica to improve the bone regeneration potential of gelatin electrospun fibrillar microenvironments. We explored the effect of increasing the DE content from 1 % to 3 % and 5 %, respectively, on the physico-chemical properties of the fibrous scaffolds denoted FG_DE1, FG_DE3, FG_DE5, regarding the aqueous media affinity, stability under simulated physiological conditions, morphology characteristics, and local mechanical properties at the surface. The presence of biosilica generated composite structures with lower swelling degrees and higher stiffness when compared to gelatin fibers. Increasing DE content led to higher Young modulus, while the stability of the protein matrix in PBS, at 37 °C, over 21 was significantly decreased by the presence of diatomite loadings. The best preosteoblast response was obtained for FG_DE3, with enhanced mineralization during the osteogenic differentiation when compared to the control sample without diatomite. 5 % DE in FG_DE5 proved to negatively influence cells' metabolic activity and morphology. Hence, the obtained composite microfibrillar scaffolds might find application as osteoblast-responsive materials for bone tissue engineering.

Inorganic/Biopolymers Hybrid Hydrogels Dual Cross-Linked for Bone Tissue Regeneration.

In tissue engineering, the potential of re-growing new tissue has been considered, however, developments towards such clinical and commercial outcomes have been modest. One of the most important elements here is the selection of a biomaterial that serves as a "scaffold" for the regeneration process. Herein, we designed hydrogels composed of two biocompatible natural polymers, namely gelatin with photopolymerizable functionalities and a pectin derivative amenable to direct protein conjugation. Aiming to design biomimetic hydrogels for bone regeneration, this study proposes double-reinforcement by way of inorganic/biopolymer hybrid filling composed of Si-based compounds and cellulose nanofibers. To attain networks with high flexibility and elastic modulus, a double-crosslinking strategy was envisioned-photochemical and enzyme-mediated conjugation reactions. The dual cross-linked procedure will generate intra- and intermolecular interactions between the protein and polysaccharide and might be a resourceful strategy to develop innovative scaffolding materials.

Development of Biocomposite Alginate-Cuttlebone-Gelatin 3D Printing Inks Designed for Scaffolds with Bone Regeneration Potential.

Fabrication of three-dimensional (3D) scaffolds using natural biomaterials introduces valuable opportunities in bone tissue reconstruction and regeneration. The current study aimed at the development of paste-like 3D printing inks with an extracellular matrix-inspired formulation based on marine materials: sodium alginate (SA), cuttlebone (CB), and fish gelatin (FG). Macroporous scaffolds with microporous biocomposite filaments were obtained by 3D printing combined with post-printing crosslinking. CB fragments were used for their potential to stimulate biomineralization. Alginate enhanced CB embedding within the polymer matrix as confirmed by scanning electron microscopy (ESEM) and micro-computer tomography (micro-CT) and improved the deformation under controlled compression as revealed by micro-CT. SA addition resulted in a modulation of the bulk and surface mechanical behavior, and lead to more elongated cell morphology as imaged by confocal microscopy and ESEM after the adhesion of MC3T3-E1 preosteoblasts at 48 h. Formation of a new mineral phase was detected on the scaffold's surface after cell cultures. All the results were correlated with the scaffolds' compositions. Overall, the study reveals the potential of the marine materials-containing inks to deliver 3D scaffolds with potential for bone regeneration applications.

Nanoengineered biomimetic hydrogels: A major advancement to fabricate 3D-printed constructs for regenerative medicine.

Nanostructured compounds already validated as performant reinforcements for biomedical applications together with different fabrication strategies have been often used to channel the biophysical and biochemical features of hydrogel networks. Ergo, a wide array of nanostructured compounds has been employed as additive materials integrated with hydrophilic networks based on naturally-derived polymers to produce promising scaffolding materials for specific fields of regenerative medicine. To date, nanoengineered hydrogels are extensively explored in (bio)printing formulations, representing the most advanced designs of hydrogel (bio)inks able to fabricate structures with improved mechanical properties and high print fidelity along with a cell-interactive environment. The development of printing inks comprising organic-inorganic hybrid nanocomposites is in full ascent as the impact of a small amount of nanoscale additive does not translate only in improved physicochemical and biomechanical properties of bioink. The biopolymeric nanocomposites may even exhibit additional particular properties engendered by nano-scale reinforcement such as electrical conductivity, magnetic responsiveness, antibacterial or antioxidation properties. The present review focus on hydrogels nanoengineered for 3D printing of biomimetic constructs, with particular emphasis on the impact of the spatial distribution of reinforcing agents (0D, 1D, 2D). Here, a systematic analysis of the naturally-derived nanostructured inks is presented highlighting the relationship between relevant length scales and size effects that influence the final properties of the hydrogels designed for regenerative medicine.

3D Bioprinting of Biosynthetic Nanocellulose-Filled GelMA Inks Highly Reliable for Soft Tissue-Oriented Constructs.

Bioink-formulations based on gelatin methacrylate combined with oxidized cellulose nanofibrils are employed in the present study. The parallel investigation of the printing performance, morphological, swelling, and biological properties of the newly developed hydrogels was performed, with inks prepared using methacrylamide-modified gelatins of fish or bovine origin. Scaffolds with versatile and well-defined internal structure and high shape fidelity were successfully printed due to the high viscosity and shear-thinning behavior of formulated inks and then photo-crosslinked. The biocompatibility of 3D-scaffolds was surveyed using human adipose stem cells (hASCs) and high viability and proliferation rates were obtained when in contact with the biomaterial. Furthermore, bioprinting tests were performed with hASCs embedded in the developed formulations. The results demonstrated that the designed inks are a versatile toolkit for 3D bioprinting and further show the benefits of using fish-derived gelatin for biofabrication.

Cellulose Nanofiber-Based Hydrogels Embedding 5-FU Promote Pyroptosis Activation in Breast Cancer Cells and Support Human Adipose-Derived Stem Cell Proliferation, Opening New Perspectives for Breast Tissue Engineering.

The structure and biocompatibility analysis of a hydrogel based on cellulose nanofibers (CNFs) combined with alginate/pectin (A.CNF or P.CNF) and enriched with 1% or 5% 5-FU revealed more favorable properties for the cellular component when pectin was dispersed within CNFs. 5-Fluorouracil (5-FU) is an antimetabolite fluoropyrimidine used as antineoplastic drug for the treatment of multiple solid tumors. 5-FU activity leads to caspase-1 activation, secretion and maturation of interleukins (IL)-1, IL-18 and reactive oxygen species (ROS) generation. Furthermore, the effects of embedding 5-FU in P.CNF were explored in order to suppress breast tumor cell growth and induce inflammasome complex activation together with extra- and intracellular ROS generation. Exposure of tumor cells to P.CNF/5-FU resulted in a strong cytotoxic effect, an increased level of caspase-1 released in the culture media and ROS production-the latter directly proportional to the concentration of anti-tumor agent embedded in the scaffolds. Simultaneously, 5-FU determined the increase of p53 and caspase-1 expressions, both at gene and protein levels. In conclusion, P.CNF/5-FU scaffolds proved to be efficient against breast tumor cells growth due to pyroptosis induction. Furthermore, biocompatibility and the potential to support human adipose-derived stem cell growth were demonstrated, suggesting that these 3D systems could be used in soft tissue reconstruction post-mastectomy.

Electrospinning Fabrication and Cytocompatibility Investigation of Nanodiamond Particles-Gelatin Fibrous Tubular Scaffolds for Nerve Regeneration.

This paper reports the electrospinning fabrication of flexible nanostructured tubular scaffolds, based on fish gelatin (FG) and nanodiamond nanoparticles (NDs), and their cytocompatibility with murine neural stem cells. The effects of both nanofiller and protein concentration on the scaffold morphology, aqueous affinity, size modification at rehydration, and degradation are assessed. Our findings indicate that nanostructuring with low amounts of NDs may modify the fiber properties, including a certain regional parallel orientation of fiber segments. NE-4C cells form dense clusters that strongly adhere to the surface of FG50-based scaffolds, while also increasing FG concentration and adding NDs favor cellular infiltration into the flexible fibrous FG70_NDs nanocomposite. This research illustrates the potential of nanostructured NDs-FG fibers as scaffolds for nerve repair and regeneration. We also emphasize the importance of further understanding the effect of the nanofiller-protein interphase on the microstructure and properties of electrospun fibers and on cell-interactivity.

Bioinspired 3D printable pectin-nanocellulose ink formulations.

The assessment of several ink formulations for 3D printing based on two natural macromolecular compounds is presented. In the current research we have exploited the fast crosslinking potential of pectin and the remarkable shear-thinning properties of carboxylated cellulose nanofibrils, which is known to induce a desired viscoelastic behavior. Prior to 3D printing, the viscoelastic properties of the polysaccharide inks were evaluated by rheological measurements and injectability tests. The reliance of the printing parameters on the ink composition was established through one-dimensional lines printing, the base units of 3D-structures. The performance of the 3D-printed structures after ionic cross-linking was evaluated in terms of mechanical properties and rehydration behavior. MicroCT was also used to evaluate the morphology of the 3D-printed objects regarding the effect of pectin/nanocellulose ratio on the geometrical features of scaffolds. The proportionality between the two polymers proved to be the determining factor for the firmness and strength of the printed objects.

Nanocomposite particles with improved microstructure for 3D culture systems and bone regeneration.

Nano-apatite and gelatin-alginate hydrogel microparticles have been prepared by a one-step synthesis combined with electrostatic bead generation, for the reconstruction of bone defects. Based on the analysis of bone composition, architecture and embryonic intramembranous ossification, a bio-inspired fabrication has been developed. Accordingly, the mineral phase has been in situ synthesized, calcifying the hydrogel matrix while the latter was crosslinked, finally generating microparticles that can assemble into a bone defect to ensure interconnected pores. Although nano-apatite-biopolymer composites have been widely investigated, microstructural optimization to provide improved distribution and stability of the mineral is rarely achieved. The optimization of the developed method progressively resulted in two types of formulations (15P and 7.5P), with 15 and 7.5 (wt%) phosphate content in the initial precursor. The osteolytic potential was investigated using differentiated macrophages. A commercially available calcium phosphate bone graft substitute (Eurocer 400) was incorporated into the hydrogel, and the obtained composites were in vitro tested for comparison. The cytocompatibility of the microparticles was studied with mouse osteoblast-like cell line MC3T3-E1. Results indicated the best in vitro performance have been obtained for the sample loaded with 7.5P. Preliminary evaluation of biocompatibility into a critical size (3 mm) defect in rabbits showed that 7.5P nanocomposite is associated with newly formed bone in the proximity of the microparticles, after 28 days.

Hybrid networks based on epoxidized camelina oil.

Lately, renewable resources received great attention in the macromolecular compounds area, regarding the design of the monomers and polymers with different applications. In this study the capacity of several modified vegetable oil-based monomers to build competitive hybrid networks was investigate, taking into account thermal and mechanical behavior of the designed materials. In order to synthesize such competitive nanocomposites, the selected renewable raw material, camelina oil, was employed due to the non-toxicity and biodegradability behavior. General properties of epoxidized camelina oil-based materials were improved by loading of different types of organic-inorganic hybrid compounds - polyhedral oligomeric silsesquioxane (POSS) bearing one (POSS1Ep) or eight (POSS8Ep) epoxy rings on the cages. In order to identify the chemical changes occurring after the thermal curing reactions, FT-IR spectrometry was employed. The new synthesized nanocomposites based on epoxidized camelina oil (ECO) were characterized by dynamic mechanical analyze and thermogravimetric analyze. The morphology of the ECO-based materials was investigate by scanning electron microscopy and supplementary information regarding the presence of the POSS compounds were establish by energy dispersive X-ray analysis and X-ray photoelectron spectroscopy. The smooth materials without any separation phase indicates a well dispersion of the Si-O-Si cages within the organic matrix and the incorporation of this hybrid compounds into the ECO network demonstrates to be a well strategy to improve the thermal and mechanical properties, simultaneously.

A 3D Porous Gelatin-Alginate-Based-IPN Acts as an Efficient Promoter of Chondrogenesis from Human Adipose-Derived Stem Cells.

Cartilage has limited regeneration potential. Thus, there is an imperative need to develop new strategies for cartilage tissue engineering (CTE) amenable for clinical use. Recent CTE approaches rely on optimal cell-scaffold interactions, which require a great deal of optimization. In this study we attempt to build a novel gelatin- (G-) alginate- (A-) polyacrylamide (PAA) 3D interpenetrating network (IPN) with superior performance in promoting chondrogenesis from human adipose-derived stem cells (hADSCs). We show that our G-A-PAA scaffold is capable of supporting hADSCs proliferation and survival, with no apparent cytotoxic effect. Moreover, we find that after exposure to prochondrogenic conditions a key transcription factor known to induce chondrogenesis, namely, Sox9, is highly expressed in our hADSCs/G-A-PAA bioconstruct, along with cartilage specific markers such as collagen type II, CEP68, and COMP extracellular matrix (ECM) components. These data suggest that our G-A-PAA structural properties and formulation might enable hADSCs conversion towards functional chondrocytes. We conclude that our novel G-A-PAA biomatrix is a good candidate for prospective in vivo CTE applications.

Perilipin expression reveals adipogenic potential of hADSCs inside superporous polymeric cellular delivery systems.

Recent progress in tissue engineering and regenerative medicine envisages the use of cell-scaffold bioconstructs to best mimic the natural in vivo microenvironment. Our aim was not only to develop novel 3D porous scaffolds for regenerative applications by the association of gelatin (G), alginate (A), and polyacrylamide (PAA) major assets but also to evaluate their in vitro potential to support human adipose-derived stem cells (hADSCs) adipogenesis. G-A-PAA biomatrix investigated in this work is an interesting substrate combining the advantages of the three individual constituents, namely, biodegradability of G, hydrophilicity of A and PAA, superior elasticity at compression with respect to the G-A and PAA controls, and the capacity to generate porous scaffolds. hADSCs inside these novel interpenetrating polymer networks (IPNs) were able to populate the entire scaffold structure and to display their characteristic spindle-like shape as a consequence of a good interaction with G component of the matrices. Additionally, hADSCs proved to display the capacity to differentiate towards mature adipocytes, to accumulate lipids inside their cytoplasm, and to express perilipin late adipogenic marker inside novel IPNs described in this study. On long term, this newly designed biomatrix aims to represent a stem cell delivery system product dedicated for modern regenerative strategies.

Biocompatibility assessment of novel collagen-sericin scaffolds improved with hyaluronic Acid and chondroitin sulfate for cartilage regeneration.

Cartilage tissue engineering (CTE) applications are focused towards the use of implantable biohybrids consisting of biodegradable scaffolds combined with in vitro cultured cells. Hyaluronic acid (HA) and chondroitin sulfate (CS) were identified as the most potent prochondrogenic factors used to design new biomaterials for CTE, while human adipose-derived stem cells (ASCs) were proved to display high chondrogenic potential. In this context, our aim was not only to build novel 3D porous scaffolds based on natural compounds but also to evaluate their in vitro biological performances. Therefore, for prospective CTE, collagen-sericin (Coll-SS) scaffolds improved with HA (5% or 10%) and CS (5% or 10%) were used as temporary physical supports for ASCs and were analyzed in terms of structural, thermal, morphological, and swelling properties and cytotoxic potential. To complete biocompatibility data, ASCs viability and proliferation potential were also assessed. Our studies revealed that Coll-SS hydrogels improved with 10% HA and 5% CS displayed the best biological performances in terms of cell viability, proliferation, morphology, and distribution. Thus, further work will address a novel 3D system including both HA 10% and CS 5% glycoproteins, which will probably be exposed to prochondrogenic conditions in order to assess its potential use in CTE applications.

Hybrid polymeric hydrogels for ocular drug delivery: nanoparticulate systems from copolymers of acrylic acid-functionalized chitosan and N-isopropylacrylamide or 2-hydroxyethyl methacrylate.

Nanoparticulate hybrid polymeric hydrogels (10-70 nm) have been obtained via the radical-induced co-polymerization of acrylic acid-functionalized chitosan with either N-isopropylacrylamide or 2-hydroxyethyl methacrylate, and the materials have been investigated for their ability to act as controlled release vehicles in ophthalmic drug delivery. Studies on the effects of network structure upon swelling properties, adhesiveness to substrates that mimic mucosal surfaces and biodegradability, coupled with in vitro drug release investigations employing ophthalmic drugs with differing aqueous solubilities, have identified nanoparticle compositions for each of the candidate drug molecules. The hybrid nanoparticles combine the temperature sensitivity of N-isopropylacrylamide or the good swelling characteristics of 2-hydroxyethyl methacrylate with the susceptibility of chitosan to lysozyme-induced biodegradation.