Decellularized extracellular matrices (ECMs) are able to provide the necessary and specific cues for remodeling and maturation of tissue-specific cells. Nevertheless, their use for typical biofabrication applications requires chemical modification or mixing with other polymers, mainly due to the limited viscoelastic properties. In this study, we hypothesize that a bioink exclusively based on decellularized kidney ECM (dKECM) could be used to bioprint renal progenitor cells. To address these aims, porcine kidneys were decellularized, lyophilized and digested to yield a viscous solution. Then, the bioprinting process was optimized using an agarose microparticle support bath containing transglutaminase for enzymatic crosslinking of the dKECM. This methodology was highly effective to obtain constructs with good printing resolution and high structural integrity. Moreover, the encapsulation of primary renal progenitor cells resulted in high cell viability, with creation of 3D complex structures over time. More importantly, this tissue-specific matrix was also able to influence cellular growth and differentiation over time. Taken together, these results demonstrate that unmodified dKECM bioinks have great potential for bioengineering renal tissue analogs with promising translational applications and/or for in vitro model systems. Ultimately, this strategy may have greater implications on the biomedical field for the development of bioengineered substitutes using decellularized matrices from other tissues.
Three-dimensional (3D)-printed polycaprolactone (PCL)-based scaffolds have been extensively proposed for Tissue Engineering (TE) applications. Currently, the majority of the scaffolds produced are not representative of the complex arrangement of natural structures, since the internal morphologies follow an orthogonal and regular pattern. In order to produce scaffolds that more closely replicate the structure of the extracellular matrix (ECM) of tissues, herein both circular and sinusoidal scaffolds were fabricated and compared to their conventional orthogonal counterparts. This is an innovative, versatile and efficient strategy to 3D print PCL scaffolds with unique curved geometries. The morphology and the mechanical behavior of the scaffolds were assessed. The biological response was analyzed by evaluating the cell seeding efficiency, cell adhesion, proliferation, and osteogenic activity of an osteoblastic-like cell line seeded in these scaffolds. The scaffolds were designed and produced to have a similar porosity of about 56%. The non-orthogonal structures demonstrated lead to higher values of Young's modulus, both under dry conditions and when immersed in PBS. Moreover, the biological data corroborate that non-orthogonal scaffolds influence the cellular responses in a positive manner, namely in the osteogenic activity when compared with the orthogonal controls. These results suggest that introducing less orthogonal elements, which better mimic the tissue ECM and architecture, may have a positive influence on the cellular behavior, being a potential strategy to address bone tissue engineering applications.
The aim of this work was to determine the influence of the biomaterial environment on human mesenchymal stem cell (hMSC) fate when cultured in supports with varying topography. Poly(vinylidene fluoride) (PVDF) culture supports were prepared with structures ranging between 2D and 3D, based on PVDF films on which PVDF microspheres were deposited with varying surface density. Maintenance of multipotentiality when cultured in expansion medium was studied by flow cytometry monitoring the expression of characteristic hMSCs markers, and revealed that cells were losing their characteristic surface markers on these supports. Cell morphology was assessed by scanning electron microscopy (SEM). Alkaline phosphatase activity was also assessed after seven days of culture on expansion medium. On the other hand, osteoblastic differentiation was monitored while culturing in osteogenic medium after cells reached confluence. Osteocalcin immunocytochemistry and alizarin red assays were performed. We show that flow cytometry is a suitable technique for the study of the differentiation of hMSC seeded onto biomaterials, giving a quantitative reliable analysis of hMSC-associated markers. We also show that electrosprayed piezoelectric poly(vinylidene fluoride) is a suitable support for tissue engineering purposes, as hMSCs can proliferate, be viable and undergo osteogenic differentiation when chemically stimulated.
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