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

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Improving surface properties and porosity of electrospun scaffolds for cartilage tissue engineering

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.publishe

Chondrocyte incorporation onto electrospun scaffolds for cartilage tissue engineering

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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

Electrosprayed cells proliferative behaviour in a 3d microporous scaffold

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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

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

Desenvolvimento de scaffolds anisotrópicos de PCL e gelatina para a regeneração de cartilagem

Um dos maiores desafios da engenharia de tecidos de cartilagem é a dificuldade de imitar o ambiente bioquímico e biomecânico da cartilagem nativa. Até à data, várias estratégias de engenharia de tecidos de cartilagem conseguiram desenvolver cartilagem artificial com propriedades bioquímicas semelhantes às do tecido nativo [1]. No entanto as propriedades mecânicas da cartilagem in-vitro permanecem inferiores às da nativa. Uma das principais limitações da cartilagem artificial é que esta não exibe as variações zonais da cartilagem nativa [2-3]. A organização das fibras de colagénio em forma de arcada ao longo da profundidade da cartilagem nativa é importante e deve ser replicada na cartilagem artificial para tornar se mecanicamente funcional [2-3]. As condições de cultura que têm impacto sobre a síntese de colagénio e sua organização fibrilar incluem os scaffolds e a estimulação mecânica. Alguns investigadores sugerem o uso de scaffolds fibrosos anisotrópicos, a fim de proporcionar um arquétipo para organizar a nova matriz extracelular depositada. A utilização da técnica de eletrofiação para o desenvolvimento de scaffolds fibrosos para a engenharia de tecidos da cartilagem já foi reportada, visto que as matrizes de nanofibras poliméricas alinhadas produzidas mimetizam a topografia da matriz extracelular da cartilagem nativa e funcionam como suporte para organizar a deposição de nova matriz extracelular produzida por células nelas semeadas [4]. Alguns investigadores sugerem que a aplicação de estímulos mecânicos variáveis em profundidade que estimulem de forma diferenciada a síntese de matriz extracelular e logo uma diferente orientação fibrilar em profundidade [5]. A policaprolactona (PCL) é um poliéster sintético, biocompatível e biodegradável que apresenta elevada resistência mecânica e é facilmente processável. As matrizes de nanofibras de PCL mimetizam topograficamente a matriz extracelular no tecido cartilagíneo. No entanto, a hidrofobicidade inerente deste material pode prevenir a adesão, migração, proliferação e diferenciação celular. A combinação de PCL com polímeros naturais tem sido utilizada para obter propriedades mecânicas e biológicas complementares, uma vez que os polímeros naturais possuem uma superfície hidrofílica e recetores reconhecíveis pelas células. A gelatina é um polímero natural derivado do colagénio, que constitui maioritariamente a matriz extracelular da cartilagem [6]. Assim sendo, neste trabalho foi explorada a combinação de estimulação mecânica com a utilização de scaffolds fibrosos anisotrópicos de PCL e gelatina produzidos por electrofiação, envolvidos numa estrutura porosa de óxido de grafeno (GO) e colagénio, para estimular a proliferação celular e produção de matriz extracelular cartilagínea. Várias arquiteturas foram desenvolvidas. As propriedades topográficas, mecânicas e a capacidade de absorção de água dos scaffolds foram analisadas e, posteriormente a biocompatibilidade dos mesmos foi investigada utilizando células progenitoras da cartilagem articular. A estimulação mecânica das células semeadas nos scaffolds por compressão cíclica foi efetuada com recurso um biorreator desenvolvido e patenteado pela equipa [7]. Os resultados obtidos demonstraram que estas estruturas permitem não só a adesão, mas também a proliferação celular. A estimulação mecânica aplicada gerou uma resposta positiva das células, através da produção de elementos da matriz extracelular da cartilagem.publishe

Bio-electrospraying of chondrocytes for cartilage tissue enginnering applications

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