Despite the great advances that the tissue engineering field has experienced over the last two decades, the amount of in vitro engineered tissues that have reached a stage of clinical trial is limited. While many challenges are still to be overcome, the lack of vascularization represents a major milestone if tissues bigger than approximately 200 µm are to be transplanted. Cell survival and homeostasis is to a large extent conditioned by the oxygen and nutrient transport (as well as waste removal) by blood vessels on their proximity and spontaneous vascularization in vivo is a relatively slow process, leading all together to necrosis of implanted tissues. Thus, in vitro vascularization appears to be a requirement for the advancement of the field. One of the main approaches to this end is the formation of vascular templates that will develop in vitro together with the targeted engineered tissue. Bioprinting, a fast and reliable method for the deposition of cells and materials on a precise manner, appears as an excellent fabrication technique. In this review, we provide a comprehensive background to the fields of vascularization and bioprinting, providing details on the current strategies, cell sources, materials and outcomes of these studies.
Nowadays, there is an ever-increasing interest in the development of systems able to guide and influence cell activities for bone regeneration. In this context, we have explored for the first time the combination of type-I collagen and superparamagnetic iron oxide nanoparticles (SPIONs) to design magnetic and biocompatible electrospun scaffolds. For this purpose, SPIONs with a size of 12 nm were obtained by thermal decomposition and transferred to an aqueous medium via ligand exchange with dimercaptosuccinic acid (DMSA). The SPIONs were subsequently incorporated into type-I collagen solutions to prove the processability of the resulting hybrid formulation by means of electrospinning. The optimized method led to the fabrication of nanostructured scaffolds composed of randomly oriented collagen fibers ranging between 100 and 200 nm, where SPIONs resulted distributed and embedded into the collagen fibers. The SPIONs-containing electrospun structures proved to preserve the magnetic properties of the nanoparticles alone, making these matrices excellent candidates to explore the magnetic stimuli for biomedical applications. Furthermore, the biological assessment of these collagen scaffolds confirmed high viability, adhesion, and proliferation of both pre-osteoblastic MC3T3-E1 cells and human bone marrow-derived mesenchymal stem cells (hBM-MSCs).
The use of biomaterials and scaffolds to boost bone regeneration is increasingly gaining interest as a complementary method to the standard surgical and pharmacological treatments in case of severe injuries and pathological conditions. In this frame, the selection of biomaterials and the accurate assessment of the manufacturing procedures are considered key factors in the design of constructs able to resemble the features of the native tissue and effectively induce specific cell responses. Accordingly, composite scaffolds based on type-I-collagen can mimic the composition of bone extracellular matrix (ECM), while electrospinning technologies can be exploited to produce nanofibrous matrices to resemble its architectural organization. However, the combination of collagen and electrospinning reported several complications due to the frequent denaturation of the protein and the variability of results according to collagen origin, concentration, and solvent. In this context, the strategies optimized in this study enabled the preparation of collagen-based electrospun scaffolds characterized by about 100 nm fibers, preserving the physico-chemical properties of the protein thanks to the use of an acetic acid-based solvent. Moreover, nanoparticles of mesoporous bioactive glasses were combined with the optimized collagen formulation, proving the successful design of composite scaffolds resembling the morphological features of bone ECM at the nanoscale.
Human mesenchymal stem/stromal cells (hMSCs) present a great opportunity for tissue regeneration due to their multipotent capacity. However, when cultured on 2D tissue culture polystyrene (TCPS) plates, hMSCs lose their differentiation capacity and clinical potential. It has been reported that cells need a more physiologically relevant micro-environment that allows them to maintain their phenotype. Here, we have developed a 3D alginate hydrogel functionalized with the Arg-Gly-Asp (RGD) sequence and having low mechanical stiffness that mimics the mechanical properties (>5 KPa) of bone marrow. hMSCs cultured in these hydrogels appeared to be halted in G1 phase of the cell cycle and to be non-proliferative, as shown by flow cytometry and 5-Ethynyl-2'-deoxyuridine (EdU) staining, respectively. Their quiescent state was characterized by an upregulation of enhancer of zeste homolog 1 (EZH1) at the gene level, forkhead box O3 (FoxO3) and cyclin-dependent kinase inhibitor 1B (p27) at the gene and protein levels compared to hMSCs grown in 2D TCPS. Comparative studies in 3D hydrogels of alginate-RGD presenting higher concentration of the peptide or in collagen hydrogels revealed that independently of the concentration of RGD or the chemistry of the adhesion motives, hMSCs cultured in 3D presented a similar phenotype. This quiescent phenotype was exclusive of 3D cultures. In 2D, even when cells were starved of fetal bovine serum (FBS) and became also non-proliferative, the expression of these markers was not observed. We propose that this difference may be the result of mammalian target of rapamycin complex 1 (mTORC1) being downregulated in hMSCs cultured in 3D hydrogels, which induces cells to be in deep quiescence and be kept alive ex vivo for a long period of time. Our results represent a step forward towards understanding hMSCs quiescence and its molecular pathways, providing more insight for hMSCs cell therapies.
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