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.

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.

Novel bioengineering strategies for the ex vivo fabrication of native-like tissue-engineered cartilage are crucial for the translation of these approaches to clinically manage highly prevalent and debilitating joint diseases. Bioreactors that provide different biophysical stimuli have been used in tissue engineering approaches aimed at enhancing the quality of the cartilage tissue generated. However, such systems are often highly complex, expensive, and not very versatile. In the current study, a novel, cost-effective, and customizable perfusion bioreactor totally fabricated by additive manufacturing (AM) is proposed for the study of the effect of fluid flow on the chondrogenic differentiation of human bone-marrow mesenchymal stem/stromal cells (hBMSCs) in 3D porous poly(ɛ-caprolactone) (PCL) scaffolds. hBMSCs are first seeded and grown on PCL scaffolds and hBMSC–PCL constructs are then transferred to 3D-extruded bioreactors for continuous perfusion culture under chondrogenic inductive conditions. Perfused constructs show similar cell metabolic activity and significantly higher sulfated glycosaminoglycan production (≈1.8-fold) in comparison to their non-perfused counterparts. Importantly, perfusion bioreactor culture significantly promoted the expression of chondrogenic marker genes while downregulating hypertrophy. This work highlights the potential of customizable AM platforms for the development of novel personalized repair strategies and more reliable in vitro models with a wide range of applications.

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.

Designing and fabricating hierarchical geometries for tissue engineering (TE) applications is the major challenge and also the biggest opportunity of regenerative medicine in recent years, being the in vitro recreation of the arcade-like cartilaginous tissue one of the most critical examples due to the current inefficient standard medical procedures and the lack of fabrication techniques capable of building scaffolds with the required architecture in a cost and time effective way. 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 system comprising both fibres and sacrificial microparticles. Polycaprolactone (PCL) and polyethylene glycol were respectively used as bulk and sacrificial biomaterials, leading to a bi-layered PCL scaffold which presented not only a depth-dependent fibre orientation similar to natural cartilage, but also mechanical features and porosity compatible with cartilage TE approaches. 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. Additionally, likewise the natural anisotropic cartilage, the PCL scaffold was capable of inducing oriented cell-material interactions since the morphology, alignment and density of the chondrocytes changed relatively to the specific topographic cues of each electrospun layer.

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.

Polycaprolactone (PCL) electrospun scaffolds have long been used for cartilage tissue engineering applications due to their biocompatibility, biodegradability, good mechanical properties and easy processability. However, their inherent hydrophobicity prevents cell adhesion and cell proliferation. On the other hand, natural polymers, such as gelatin, have been reported to support cell adhesion due to its hydrophilic character and the presence of cell recognition sites. Another common limitation of PCL electrospun scaffolds is their inherent small pores, which can hinder cell migration. The introduction of a sacrificial agent on the scaffolds, such as polyethylene glycol (PEG), which can be co-electrospun with the polymer of interest, has been reported to overcome this limitation. The sacrificial polymer is then dissolved away in water, resulting in an electrospun scaffolds with increased porosity. The present work combines these approaches to improve the surface properties and the scaffolds’ porosity that will benefit cell adhesion, migration and proliferation. Thus, a new series of electrospun scaffolds composed of PCL, gelatin and PEG sacrificial particles were fabricated and characterized on their chemical composition, wettability, topography and biocompatibility using an articular cartilage progenitor cell line. According to the results obtained, the addition of gelatin led to an increased hydrophilicity of the scaffolds, which resulted in better cell adhesion and proliferation. The introduction of PEG sacrificial particles enlarged the pore size of the scaffolds to values comparable to the cell diameter and allowed cell migration through the scaffold.