Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

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.

Bioinspired 3D scaffolds with antimicrobial, drug delivery, and osteogenic functions for bone regeneration.

A major clinical challenge today is the large number of bone defects caused by diseases or trauma. The development of three-dimensional (3D) scaffolds with adequate properties is crucial for successful bone repair. In this study, we prepared biomimetic mesoporous bioactive glass (MBG)-based scaffolds with and without ceria addition (up to 3 mol %) to explore the biological structure and chemical composition of the marine sponge Spongia Agaricina (SA) as a sacrificial template. Micro-CT examination revealed that all scaffolds exhibited a highly porous structure with pore diameters primarily ranging from 143.5 µm to 213.5 µm, facilitating bone ingrowth. Additionally, smaller pores (< 75 µm), which are known to enhance osteogenesis, were observed. The undoped scaffold displayed the highest open porosity value of 90.83%. Cytotoxicity assessments demonstrated that all scaffolds were noncytotoxic and nongenotoxic toward osteoblast cells. Moreover, scaffolds with higher CeO2 content promoted osteogenic differentiation of dental pulp stem cells, stimulating calcium and osteocalcin secretion. The scaffolds also exhibited antimicrobial and antibiofilm effects against Staphylococcus aureus (S. aureus) as well as drug delivery ability. Our research findings indicated that the combination of MBG, natural biological structure, and the addition of Ce exhibited a synergistic effect on the structure and biological properties of scaffolds for applications in bone tissue engineering.

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.

Osteoblast-like cell behavior on porous scaffolds based on poly(styrene) fibers.

Scaffolds of nonresorbable biomaterials can represent an interesting alternative for replacing large bone defects in some particular clinical cases with massive bone loss. Poly(styrene) microfibers were prepared by a dry spinning method. They were partially melted to provide 3D porous scaffolds. The quality of the material was assessed by Raman spectroscopy. Surface roughness was determined by atomic force microscopy and vertical interference microscopy. Saos-2 osteoblast-like cells were seeded on the surface of the fibers and left to proliferate. Cell morphology, evaluated by scanning electron microscopy, revealed that they can spread and elongate on the rough microfiber surface. Porous 3D scaffolds made of nonresorbable poly(styrene) fibers are cytocompatible biomaterials mimicking allogenic bone trabeculae and allowing the growth and development of osteoblast-like cells in vitro.