Cell seeding process optimization on polycaprolactone-gelatin-arcade-like-architecture-scaffolds

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Development of a DoE with a new electrospinning system for cartilage tissue engineering

Electrospinning is currently one of the most used techniques to produce fibrous synthetic tissues such as cartilage and bone. To replicate cartilage tissue engineering functionality, one of the most important characteristics is the alignment of the resulting fibre meshes in a three-dimensional (3D) fashion. Here, a newly developed electrospinning collector system is tested in order to understand how the process parameters affected the obtained fibre meshes topography. For that, a polymer consisting of PCL/Gelatin was electrospun using the electrostatic potential to create a fibre mesh. A Design of the Experiments (DoE) approach was implemented, to determine whether the variation of the main process parameters led to significant effects on the mesh dimensional characteristics. The process parameters analyzed were the velocity of the collecting bands, the linear velocity of the fibre deposition table and the flow rate. The analyzed mesh characteristics were the fibre diameter, the distance between the fibres and pore size. The effect of each of the three factors was statistically analyzed using ANOVA, as well as the interaction between them. Complementary an ANOVA linear regression approach was developed to predict the distance between the fibres. This statistical regression was then compared with a predictive theoretical model and with the experimental results. The results obtained indicate the presence of interactions between the three process parameters analyzed. The three process parameters showed statistical significance in the distance between the fibres, however, the velocity of the deposition table was the process parameter that presented the highest effect.publishe

Chondrocyte incorporation onto electrospun scaffolds for cartilage tissue engineering

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Electrosprayed cells proliferative behaviour in a 3d microporous scaffold

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Electrospraying of primary chondrocytes for cartilage repair

Electrospun scaffolds have long been used for cartilage repair, due to the topographic similarity between the electrospun fibers and the collagen fibers of the extracellular matrix (ECM) in the native cartilage. Still, while their nanotophography can be beneficial for the cell proliferative and spreading behavior, it greatly reduces the inter-fiber pore size, hindering cell migration and relegating tissue formation to the surface of the scaffold [1]. A possible solution for this structura l limitation would be the direct incorporation of cells into the fibers during electrospinning of the fibrous scaffold, overcoming the challenges of cell infiltration into small pore sizes by literally surrounding cells with the fiber matrix as it is produced [1]. This can be achieved using cell electrospraying, a concept first introduced in 2005 by Jayasinghe, enables the deposition of living cells onto specific targets by exposing the cell suspension to an external high intensity electric field [2]. Cell exposure to the electric field, as well as the shear stress of passing through the cell electrospraying apparatus may affect cell viability and function, so several types of cells have been electrosprayed, and no significant influence was observed on a genetic, genomic and physiological level [4]. In fact, our previous work has demonstrated this inertness from a chondrocyte cell line (C28-I2) [5]. Still, these immortalized cells are genetically modified, and might not not accurately replicate the physiological conditions. Primary chondrocytes possess little proliferative ability, showing considerable dedifferentiation from a chondrocyte-like to a more fibroblast-like phenotype over time, particularly if growth factors are not used [5]. In this regard, electrospraying experiments were performed with primary chondrocytes to assess the process influence on chondrocyte viability. After 24 hour-incubation, chondrocyte metabolic activity was measured, and these electrosprayed (E) cells were then slip and cultured in well plates and in threedimensional anisotropic fibrous/porous scaffolds under static and perfused conditions. Non-electrosprayed (NE) cells were considered for comparison. The obtained results confirmed that the behaviour of primary chondrocytes upon electric field exposure was significantly different from that obtained for the chondrocyte cell line, which can be attributed to the lower recovery ability of these cells. Nonetheless, an increasing proliferation rate was observed over time. The proliferation performance of NE and E primary chondrocytes on 3D environment followed a similar trend, with E primary chondrocytes possessing a significantly lower viability than the NE primary chondrocytes. The application of perfused conditions to the E chondrocyte-seeded scaffolds greatly increased the chondrocyte viability to values similar to the ones obtained for NE chondrocyte-seeded scaffolds. Even though the electrosprayed primary chondrocytes suffered a substantial proliferative delay, they were able to recover, particularly under perfused conditions, suggesting that these conditions should be implemented after the electrospraying process, so that this technology might become an effective approach to uniformly incorporate primary chondrocytes into electrospun scaffolds.publishe

Fabrication of electrospun scaffolds with cell laden hydrogel for cartilage tissue engineering

Tissue engineering strategies create artificial substitutes for the regeneration of damaged tissues, beginning with the fabrication of scaffolds moving then to cell incorporation onto those scaffolds and subsequent tissue growth in vitro. Cell seeding techniques, unfortunately, are usually ineffective to develop scaffolds with homogenous cell distribution, resulting in non-functional tissue formation [1]. With electrospun scaffolds, cell incorporation becomes even more challenging. Electrospun scaffolds are a very tightly packed layer of fibers with small pores, that makes difficult the migration of cells onto the scaffolds, as well as, the diffusion of nutrients and wastes. To overcome this drawback, the direct incorporation of cells, using electrospraying technique, onto the scaffolds during the electrospinning process has been reported. Cell electrospraying is a jet-based technique that allows the spray of living cells onto the materials by applying an electric charge in a cellular suspension [2]. Several studies have proved that cells can survive and proliferate after electrospraying process [3], [4]. Still, previous work has shown that while uniformly distributed cell-laden scaffolds can be fabricated using this technique, some issues remain. Cell desiccation on top of the fibers due to longer duration of the experiment and inadequate cell environment – low temperature and CO2 concentration – and solvent toxicity are the main limitations for the optimal efficiency of cell electrospray process onto electrospun fibers. In this regard, in this work, the production of electrospun scaffolds was combined with the electrospray of chondrocyte laden hydrogel creating a shield/protection around the cells during and after the electrospray process, preventing its dehydration. For that, a polymeric solution of polycaprolactone (PCL) and gelatin was electrospun alternately with a chondrocyte-laden sodium alginate hydrogel electrospray. Sodium alginate is a natural polymer widely used in biomedical engineering due to its biocompatibility, biodegradability and ability to form hydrogels [5]. The prepared scaffolds were then cultured for 7 days and the respective cell viability assessed. The percentage of viability was calculated as a ratio of the metabolic activity of the electrosprayed chondrocytes and the metabolic activity of chondrocytes that did not underwent any process. The chondrocyte distribution was also evaluated. On the first day of culture, the results showed that the cellular viability was higher than the one previous reported, demonstrating that the alginate hydrogel allowed the cells to survive and helps in its attachment. After 7 days of culture, cells continue alive with considerable viability increasing. It was also shown that it was possible to incorporate cells homogenously distributed by electrospraying process using the chondrocyte laden hydrogel. These results emphasize the potential value that the hydrogels can have on the electrospraying process with the electrospun scaffolds.publishe

Bilayered arcade-like scaffolds for articular cartilage repair

Articular cartilage is a highly organized tissue that it is adapted to the complex mechanical loading in joints. Given the limited self-healing of this tissue, tissue engineering (TE) strategies have explored the development of 3D anisotropic fibrous scaffolds with the implementation of specific mechanical stimulus. However, this functionally is dependent on the ability to recreate the depth-dependent collagen fibre alignment on the 3D fibrous scaffolds, which has been considerably challenging. In this work, bilayered structures with different fibre orientations were fabricated via polycaprolactone and Gelatin electrospinning and polyethylene glycol (PEG) particles electrospraying. After PEG removal, large interfibre spaces were created, that were compatible with chondrocyte migration The in vitro studies confirmed the biocompatibility of the scaffolds and their ability to guarantee cell attachment and migration through the scaffold. The mechanical stimulation applied through unconfined compression substantially improved chondrocyte response. These results confirmed the potential of the developed 3D bilayered scaffolds for articular cartilage TE.publishe

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.publishe

Automated fabrication of 3D chondrocyte-laden anisotropic scaffolds for articular cartilage

Tissue engineering (TE) strategies for repairing and regenerating articular car-tilage face critical challenges to approximate the biochemical and biomechanical microen-vironment of native tissue, particularly regarding collagen fibril depth-orientation and chondrocyte distribution. Here, a recently developed electromechanically 3D electrospin-ning platform was employed to develop three-dimensional (3D) anisotropic electrospun scaffolds in a fully automated manner with simultaneous chondrocyte incorporation. As expected, the 3D scaffolds possessed an arcade-like fibrous configuration with a uniform chondrocyte distribution. Overall, the results suggest that this combined approach has potential for cartilage TE.publishe

Bio-electrospraying of chondrocytes for cartilage tissue enginnering applications

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