Clinical potential of plasma-functionalized graphene oxide ultrathin sheets for bone and blood vessel regeneration: Insights from cellular and animal models.

Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.

Fibronectin/thermo-responsive polymer scaffold as a dynamic ex vivo niche for mesenchymal stem cells.

In this paper, we created a dynamic adhesive environment (DAE) for adipose tissue-derived mesenchymal stem cells (ADMSCs) cultured on smart thermo-responsive substrates, i.e., poly (N-isopropyl acrylamide) (PNIPAM), via introducing periodic changes in the culture temperature. We further explored the particular role of adsorbed fibronectin (FN), an important cell adhesive protein that was recently attributed to the recruitment of stem cells in the niche. The engineered FN/PNIPAM DAE system significantly increased the symmetric renewal of ADMSCs, particularly between passages 7 and 9 (p7-p9), before it dropped down to the level of the control (FN-coated TC polystyrene). This decline in the growth curve was consistent with the increased number of senescent cells, the augmented average cell size and the suppressed FN matrix secretion at late passages (p10-p12), all of them characteristic for stem cells ageing, which equivocally tended to slow down at our DAE system. FN supported also the osteogenic response of ADMSCs (apart from the previous observations with plain PNIPAM substrata) indicated by the significant increase of alkaline phosphatase (ALP) activity at days 7 and 14. The minimal changes in the Ca deposition, however, suggest a restricted effect of DAE on the early osteogenic response of ADMSCs only. Thus, the engineering of niche-like DAE involving FN uncovers a new tissue engineering strategy for gaining larger amounts of functionally active stem cells for clinical application.

Establishing multiple osteogenic differentiation pathways of mesenchymal stem cells through different scaffold configurations.

Mimicking the complex organization of the extracellular matrix (ECM), especially its structure and dimensionality, is necessary to produce living tissues from stem cells. In compliance with a previously established role of nanofiber organization for the osteogenic differentiation of stem cells, here we used hybrid fibrinogen/poly(l-lactide-ε-caprolactone) (FBG/PLCL) nanofibers arranged in aligned and honeycomb configurations, to recapitulate the highly oriented ECM of the cortical bone and the sponge-like (i.e., honeycomb) environment of the cancellous one, respectively. Using special bilayered constructs, we demonstrate that the dimensionality (i.e., 2D vs. 3D) of the nanofibers as well as their architecture (i.e., honeycomb vs. aligned) affects differently the overall morphology and the expression of multiple osteogenic genes of human adipose-derived mesenchymal stem cells (ADMSCs). The cells had elongated shape with markedly increased cell mobility when seeded on aligned nanofibers. Conversely, on honeycomb-shaped nanofibers, ADMSCs initially concentrated inside the honeycomb curvatures adopting rounded morphology, but late, they formed network-like structures overlaying the honeycomb curvatures. By employing quantitative polymerase chain reaction (qPCR), we further show that a 3D environment generally supports the multiple osteogenic response of ADMSCs, but honeycomb and aligned architectures promote rather different differentiation pathways.

Three Dimensional Honeycomb Patterned Fibrinogen Based Nanofibers Induce Substantial Osteogenic Response of Mesenchymal Stem Cells.

Stem cells therapy offers a viable alternative for treatment of bone disorders to the conventional bone grafting. However clinical therapies are still hindered by the insufficient knowledge on the conditions that maximize stem cells differentiation. Hereby, we introduce a novel 3D honeycomb architecture scaffold that strongly support osteogenic differentiation of human adipose derived mesenchymal stem cells (ADMSCs). The scaffold is based on electrospun hybrid nanofibers consisting of poly (L-lactide ε-caprolactone) and fibrinogen (PLCL/FBG). Classical fibers orientations, random or aligned were also produced and studied for comparison. The overall morphology of ADMSC's generally followed the nanofibers orientation and dimensionality developing regular focal adhesions and direction-dependent actin cytoskeleton bundles. However, there was an initial tendency for cells rounding on honeycomb scaffolds before ADMSCs formed a distinct bridging network. This specific cells organization appeared to have significant impact on the differentiation potential of ADMSCs towards osteogenic lineage, as indicated by the alkaline phosphatase production, calcium deposition and specific genes expression. Collectively, it was observed synergistic effect of nanofibers with honeycomb architecture on the behavior of ADMSCs entering osteogenic path of differentiation which outlines the potential benefits from insertion of such bioinspired geometrical cues within scaffolds for bone tissue engineering.

Electrospun honeycomb as nests for controlled osteoblast spatial organization.

Honeycomb nanofibrous scaffolds were elaborated by electrospinning onto micro-patterned collectors either with poly(ϵ-caprolactone) (PCL) or poly(D, L-lactic acid) (PLA). The unimodal distribution of fiber diameters, observed for PLA, led to relatively flat scaffolds; on the other hand, the bimodal distribution of PCL fiber diameters significantly increased the relief of the scaffolds' patterns due to the preferential deposition of the thick fiber portions on the walls of the collector's patterns via preferential electrostatic interaction. Finally, a biological evaluation demonstrated the effect of the scaffolds' relief on the spatial organization of MG63 osteoblast-like cells. Mimicking hemi-osteons, cell gathering was observed inside PCL honeycomb nests with a size ranging from 80 to 360 µm.