Fabrication of Customizable and Reproducible 3D Chondrocyte-Laden Nanofibrous Architectures: Effect of Specific Fiber Alignments and Porosities on Chondrocyte Response under Cyclic Compression.

Electrospinning has been widely employed to fabricate complex extracellular matrix-like microenvironments for tissue engineering due to its ability to replicate structurally biomimetic micro- and nanotopographic cues. Nevertheless, these nanofibrous structures are typically either confined to bidimensional systems or confined to three-dimensional ones that are unable to provide controlled multiscale patterns. Thus, an electrospinning modality was used in this work to fabricate chondrocyte-laden nanofibrous scaffolds with highly customizable three-dimensional (3D) architectures in an automated manner, with the ultimate goal of recreating a suitable 3D scaffold for articular cartilage tissue engineering. Three distinct architectures were designed and fabricated by combining multiple nanofibrous and chondrocyte-laden hydrogel layers and tested in vitro in a compression bioreactor system. Results demonstrated that it was possible to precisely control the placement and alignment of electrospun polycaprolactone and gelatin nanofibers, generating three unique architectures with distinctive macroscale porosity, water absorption capacity, and mechanical properties. The architecture organized in a lattice-like fashion was highly porous with substantial pore interconnectivity, resulting in a high-water absorption capacity but a poor compression modulus and relatively weaker energy dissipation capacity. The donut-like 3D geometry was the densest, with lower swelling, but the highest compression modulus and improved energy dissipation ability. The third architecture combined a lattice and donut-like fibrous arrangement, exhibiting intermediary behavior in terms of porosity, water absorption, compression modulus, and energy dissipation capacity. The properties of the donut-like 3D architecture demonstrated great potential for articular cartilage tissue engineering, as it mimicked key topographic, chemical, and mechanical characteristics of chondrocytes' surrounding environment. In fact, the combination of these architectural features with a dynamically compressive mechanical stimulus triggered the best in vitro results in terms of viability and biosynthetic production.

Customizing 3D Structures of Vertically Aligned Carbon Nanotubes to Direct Neural Stem Cell Differentiation.

Neural tissue-related illnesses have a high incidence and prevalence in society. Despite intensive research efforts to enhance the regeneration of neural cells into functional tissue, effective treatments are still unavailable. Here, a novel therapeutic approach based on vertically aligned carbon nanotube forests (VA-CNT forests) and periodic VA-CNT micropillars produced by thermal chemical vapor deposition is explored. In addition, honeycomb-like and flower-like morphologies are created. Initial viability testing reveals that NE-4C neural stem cells seeded on all morphologies survive and proliferate. In addition, free-standing VA-CNT forests and capillary-driven VA-CNT forests are created, with the latter demonstrating enhanced capacity to stimulate neuritogenesis and network formation under minimal differentiation medium conditions. This is attributed to the interaction between surface roughness and 3D-like morphology that mimics the native extracellular matrix, thus enhancing cellular attachment and communication. These findings provide a new avenue for the construction of electroresponsive scaffolds based on CNTs for neural tissue engineering.

Three-dimensional nanofibrous and porous scaffolds of poly(ε-caprolactone)-chitosan blends for musculoskeletal tissue engineering.

One of the established tissue engineering strategies relies on the fabrication of appropriate materials architectures (scaffolds) that mimic the extracellular matrix (ECM) and assist the regeneration of living tissues. Fibrous structures produced by electrospinning have been widely used as reliable ECM templates but their two-dimensional structure restricts, in part, cell infiltration and proliferation. A recent technique called thermally-induced self-agglomeration (TISA) allowed to alleviate this drawback by rearranging the 2D electrospun membranes into highly functional 3D porous-fibrous systems. Following this trend, the present research focused on preparing polycaprolactone/chitosan blends by electrospinning, to then convert them into 3D structures by TISA. By adding different amounts of chitosan, it was possible to accurately modulate the physicochemical properties of the obtained 3D nanofibrous scaffolds, leading to highly porous constructs with distinct morphologic and mechanical features. Viability and proliferation studies using adult human chondrocytes also revealed that the biocompatibility of the scaffolds was not impaired after 28 days of cell culture, highlighting their potential to be included into musculoskeletal tissue engineering applications, particularly cartilage repair.

Bio-electrospraying assessment toward in situ chondrocyte-laden electrospun scaffold fabrication.

Electrospinning has been widely used to fabricate fibrous scaffolds for cartilage tissue engineering, but their small pores severely restrict cell infiltration, resulting in an uneven distribution of cells across the scaffold, particularly in three-dimensional designs. If bio-electrospraying is applied, direct chondrocyte incorporation into the fibers during electrospinning may be a solution. However, before this approach can be effectively employed, it is critical to identify whether chondrocytes are adversely affected. Several electrospraying operating settings were tested to determine their effect on the survival and function of an immortalized human chondrocyte cell line. These chondrocytes survived through an electric field formed by low needle-to-collector distances and low voltage. No differences in chondrocyte viability, morphology, gene expression, or proliferation were found. Preliminary data of the combination of electrospraying and polymer electrospinning disclosed that chondrocyte integration was feasible using an alternated approach. The overall increase in chondrocyte viability over time indicated that the embedded cells retained their proliferative capacity. Besides the cell line, primary chondrocytes were also electrosprayed under the previously optimized operational conditions, revealing the higher sensitivity degree of these cells. Still, their post-electrosprayed viability remained considerably high. The data reported here further suggest that bio-electrospraying under the optimal operational conditions might be a promising alternative to the existent cell seeding techniques, promoting not only cells safe delivery to the scaffold, but also the development of cellularized cartilage tissue constructs.

Biomimetic Graphene/Spongin Scaffolds for Improved Osteoblasts Bioactivity via Dynamic Mechanical Stimulation.

Biomimetics offers excellent prospects for design a novel generation of improved biomaterials. Here the controlled integration of graphene oxide (GO) derivatives with a 3D marine spongin (MS) network is explored to nanoengineer novel smart bio-based constructs for bone tissue engineering. The results point out that 3D MS surfaces can be homogeneously coated by layer-by-layer (LbL) assembly of oppositely charged polyethyleneimine (PEI) and GO. Notably, the GOPEI@MS bionanocomposites present a high structural and mechanical stability under compression tests in wet conditions (shape memory). Dynamic mechanically (2 h of sinusoidal compression cyclic interval (0.5 Hz, 0-10% strain)/14 d) stimulates GOPEI@MS seeded with osteoblast (MC3T3-E1), shows a significant improvement in bioactivity, with cell proliferation being two times higher than under static conditions. Besides, the dynamic assays show that GOPEI@MS bionanocomposites are able to act as mechanical stimulus-responsive scaffolds able to resemble physiological bone extracellular matrix (ECM) requirements by strongly triggering mineralization of the bone matrix. These results prove that the environment created by the system cell-GOPEI@MS is suitable for controlling the mechanisms regulating mechanical stimulation-induced cell proliferation for potential in vivo experimentation.

Multi-layered electrospinning and electrospraying approach: Effect of polymeric supplements on chondrocyte suspension.

Articular cartilage was expected to be one of the first tissues to be successfully engineered, but replicating the complex fibril architecture and the cellular distribution of the native cartilage has proven difficult. While electrospinning has been widely used to reproduce the depth-dependent fibre architecture in 3D scaffolds, the chondrocyte-controlled distribution remains an unsolved problem. To incorporate cells homogeneously through the depth of scaffolds, a combination of polymer electrospinning and cell seeding is necessary. A multi-layer approach alternating between polymer electrospinning with chondrocyte electrospraying can be a solution. Still, the success of this process is related to the survival rate of the electrosprayed chondrocytes embedded within the electrospun mesh. In this regard, the present study investigated the impact of the multi-layered process and the supplementation of the electrospray chondrocyte suspension with different concentrations of Gelatin and Alginate on the viability of electrosprayed chondrocytes embedded within a Polycaprolactone/Gelatin electrospun mesh and on the mechanical properties of the resulting meshes. The addition of Gelatin in the chondrocyte suspension did not increase significantly (p > 0.05) the percentage of viable electrosprayed chondrocytes (25%), while 3 wt% Alginate addition led to a significant (p < 0.05) increase in chondrocyte viability (50%) relative to the case without polymer supplement (15%). Furthermore, the addition of both polymer supplements increased the mechanical properties of the multi-layer construct. These findings imply that this multi-layered approach can be applied to cartilage TE allowing for automated chondrocyte integration during scaffolds creation.

Boosting in vitro cartilage tissue engineering through the fabrication of polycaprolactone-gelatin 3D scaffolds with specific depth-dependent fiber alignments and mechanical stimulation.

Due to the limited self-healing ability of natural cartilage, several tissue engineering strategies have been explored to develop functional replacements. Still, most of these approaches do not attempt to recreate in vitro the anisotropic organization of its extracellular matrix, which is essential for a suitable load-bearing function. In this work, different depth-dependent alignments of polycaprolactone-gelatin electrospun fibers were assembled into three-dimensional scaffold architectures to assess variations on chondrocyte response under static, unconfined compressed and perfused culture conditions. The in vitro results confirmed that not only the 3D scaffolds specific depth-dependent fiber alignments potentiated chondrocyte proliferation and migration towards the fibrous systems, but also the mechanical stimulation protocols applied were able to enhance significantly cell metabolic activity and extracellular matrix deposition, respectively.

Electrospinning of bioactive polycaprolactone-gelatin nanofibres with increased pore size for cartilage tissue engineering applications.

Polycaprolactone (PCL) electrospun scaffolds have been widely investigated for cartilage repair application. However, their hydrophobicity and small pore size has been known to prevent cell attachment, proliferation and migration. Here, PCL was blended with gelatin (GEL) combining the favorable biological properties of GEL with the good mechanical performance of the former. Also, polyethylene glycol (PEG) particles were introduced during the electrospinning of the polymers blend by simultaneous electrospraying. These particles were subsequently removed resulting in fibrous scaffolds with enlarged pore size. PCL, GEL and PEG scaffolds formulations were developed and extensively structural and biologically characterized. GEL incorporation on the PCL scaffolds led to a considerably improved cell attachment and proliferation. A substantial pore size and interconnectivity increase was obtained, allowing cell infiltration through the porogenic scaffolds. All together these results suggest that this combined approach may provide a potentially clinically viable strategy for cartilage regeneration.

Microfabrication of a biomimetic arcade-like electrospun scaffold for cartilage tissue engineering applications.

In recent years, the engineering of biomimetic cellular microenvironments has emerged as a top priority for regenerative medicine, being the in vitro recreation of the arcade-like cartilaginous tissue one of the most critical challenges due to the notorious absence of cost- and time-efficient microfabrication techniques capable of building 3D fibrous scaffolds with precise anisotropic properties. Taking this into account, we suggest a feasible and accurate methodology that uses a sequential adaptation of an electrospinning-electrospraying set up to construct a hierarchical system comprising both polycaprolactone (PCL) fibres and polyethylene glycol sacrificial microparticles. After porogen leaching, the bi-layered PCL scaffold was capable of presenting not only a depth-dependent fibre orientation similar to natural cartilage, but also mechanical features and porosity proficient to encourage an enhanced cell response. In fact, cell viability studies confirmed the biocompatibility of the scaffold and its ability to guarantee suitable cell adhesion, proliferation and migration throughout the 3D anisotropic fibrous network during 21 days of culture. Additionally, likewise the hierarchical relationship between chondrocytes and their extracellular matrix, the reported PCL scaffold was able to induce depth-dependent cell-material interactions responsible for promoting a spatial modulation of the morphology, alignment and density of the cells in vitro.

Understanding the composition-structure-bioactivity relationships in diopside (CaO·MgO·2SiO2)-tricalcium phosphate (3CaO·P2O5) glass system.

The present work is an amalgamation of computation and experimental approach to gain an insight into composition-structure-bioactivity relationships of alkali-free bioactive glasses in the CaO-MgO-SiO2-P2O5 system. The glasses have been designed in the diopside (CaO·MgO·2SiO2; Di)-tricalcium phosphate (3CaO·P2O5; TCP) binary join by varying the Di/TCP ratio. The melt-quenched glasses have been investigated for their structure by molecular dynamic (MD) simulations as well as by nuclear magnetic resonance spectroscopy (NMR). In all the investigated glasses silicate and phosphate components are dominated by Q(2) (Si) and Q(0) (P) species, respectively. The apatite forming ability of the glasses was investigated using X-ray diffraction (XRD), infrared spectroscopy after immersion of glass powders in simulated body fluid (SBF) for time durations varying between 1 h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the investigated glasses showed good bioactivity without any substantial variation. A significant statistical increase in metabolic activity of human mesenchymal stem cells (hMSCs) when compared to the control was observed for Di-60 and Di-70 glass compositions under both basal and osteogenic conditions.