Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.

The capacity of bone grafts to repair critical size defects can be greatly enhanced by the delivery of mesenchymal stem cells (MSCs). Adipose tissue is considered the most effective source of MSCs (ADSCs); however, the efficiency of bone regeneration using undifferentiated ADSCs is low. Therefore, this study proposes scaffolds based on polycaprolactone (PCL), which is widely considered a suitable MSC delivery system, were used as a three-dimensional (3D) culture environment promoting osteogenic differentiation of ADSCs. PCL scaffolds enriched with 5% tricalcium phosphate (TCP) were used. Human ADSCs were cultured in osteogenic medium both on the scaffolds and in 2D culture. Cell viability and osteogenic differentiation were tested at various time points for 42 days. The expression of RUNX2, collagen I, alkaline phosphatase, osteonectin and osteocalcin, measured by real-time polymerase chain reaction was significantly upregulated in 3D culture. Production of osteocalcin, a specific marker of terminally differentiated osteoblasts, was significantly higher in 3D cultures than in 2D cultures, as confirmed by western blot and immunostaining, and accompanied by earlier and enhanced mineralization. Subcutaneous implantation into immunodeficient mice was used for in vivo observations. Immunohistological and micro-computed tomography analysis revealed ADSC survival and activity toward extracellular production after 4 and 12 weeks, although heterotopic osteogenesis was not confirmed -probably resulting from insufficient availability of Ca/P ions. Additionally, TCP did not contribute to the upregulation of differentiation on the scaffolds in culture, and we postulate that the 3D architecture is a critical factor and provides a useful environment for prior-to-implantation osteogenic differentiation of ADSCs.

Fused deposition modeling has been used to fabricate three-dimensional (3D) scaffolds for tissue engineering applications, because it allows to tailor their pore network. Despite the proven flexibility in doing so, a limited amount of studies have been performed to evaluate whether specific pore shapes have an influence on cell activity and tissue formation. Our study aimed at investigating the influence of internal pore architecture on the biological and mechanical properties of 3D scaffolds seeded with mesenchymal stromal cells. Polycaprolactone scaffolds with six different geometries were fabricated. The 3D samples were manufactured with different lay-down pattern of the fibers by varying the layer deposition angle from 0°/15°/30°, to 0°/30°/60°, 0°/45°/90°, 0°/60°/120°, 0°/75°/150°, and 0°/90°/180°. The scaffolds were investigated by scanning electron microscopy and micro computed tomographical analysis and displayed a fully interconnected pore network. Cell proliferation and differentiation toward the osteogenic lineage were evaluated by DNA, alkaline phosphatase activity, and polymerase chain reaction. The obtained scaffolds had structures with open porosity (50%-60%) and interconnected pores ranging from 380 to 400 µm. Changing the angle deposition affected significantly the mechanical properties of the scaffolds. With increasing the angle deposition between successive layers, the elastic modulus increased as well. Cellular studies also showed influence of the internal architecture on cell adhesion and proliferation within the 3D construct, yet limited influence on cell differentiation was observed.

This paper is concerned with reliable and physically sound elasticity determination of rapid-prototyped tissue engineering scaffolds made of poly-L-lactide (PLLA), with and without small portions of tricalcium phosphate (TCP) inclusions. At the level of overall scaffolds, that is, that of several millimeters, multiple uniaxial loading-unloading (quasistatic) tests were performed, giving access to the scaffolds' Young's moduli, through stress-strain characteristics during unloading. In addition, acoustic tests with 0.05 MHz frequency delivered an independent access to elastic properties, in terms of the normal components of the scaffolds' stiffness tensors. The latter strongly correlate, in a linear fashion, with the Young's moduli from the unloading tests, revealing porosity independence of Poisson's ratio. The magnitude of the latter is in full agreement with literature data on polymers. Both of these facts underline that both ultrasound tests and quasistatic unloading tests reliably provide the elastic properties of tissue engineering scaffolds.

Background: Vascularization is important for the clinical application of tissue engineered products. Both adiposederived stem cells (ASCs) and surgical prefabrication can be used to induce angiogenesis in scaffolds. Our aim was to compare the angiogenic potential of ASC-seeded scaffolds combined with scaffold prefabrication with that of non-seeded, non-prefabricated scaffolds. Methods: For prefabrication, functional blood vessels were introduced into the scaffold using a flow-through pedicle system. ASCs were isolated from rat fat deposits. Three-dimensional-printed cylindrical poly-ε-caprolactone scaffolds were fabricated by fused deposition modelling. Three groups, each containing six rats, were investigated by using non-seeded, ASC-seeded, and osteogenic induced ASC-seeded scaffolds. In each group, one rat was implanted with two scaffolds in the inguinal region. On the right side, a scaffold was implanted subcutaneously around the inferior epigastric vessels (classic prefabrication group). On the left side, the inferior epigastric vessels were placed inside the prefabricated scaffold in the flow-through pedicle system (flow-through prefabrication group). The vessel density and vascular architecture were examined histopathologically and by μCT imaging, respectively, at 2 months after implantation. Results: The mean vessel densities were 10-and 5-fold higher in the ASC-seeded and osteogenic induced ASCseeded scaffolds with flow-through prefabrication, respectively, than in the non-seeded classic prefabricated group (p < 0.001). μCT imaging revealed functional vessels within the scaffold. Conclusion: ASC-seeded scaffolds with prefabrication showed significantly improved scaffold vasculogenesis and could be useful for application to tissue engineering products in the clinical settings.

Background/Aim: Most melanomas develop in hypoxic conditions. Since hypoxia via HIF-1 induces glycolysis, a process essential for malignant melanoma growth/survival, the goal of this study was to analyze the influence of hypoxia on the expression of HIF-1 target genes involved in glucose metabolism. Materials and Methods: The response of melanoma cell lines to hypoxic conditions was analyzed by RT-PCR and western blotting. A Kaplan-Meier survival analysis for patients with high and low expression level of PFKFB4 was performed. Further analysis of patients' data was performed using the R/Bioconductor environment. Results: Induction of PFKFB4 gene expression can be considered a crucial mechanism behind glycolysis enhancement in hypoxic melanoma cells. Analysis of a publicly available database revealed that high PFKFB4 expression contributes to poor prognosis of melanoma patients. Conclusion: Currently available anti-melanoma therapeutic strategies may significantly benefit from agents targeting PFKFB4 activity.

The purpose of the current study was to evaluate the usefulness of adipose-derived stem cells (ASCs) for bone injury therapy. Lipoaspirates were collected from the abdomen regions of 17 healthy female donors (mean age 49 ± 6 years) using Coleman technique or Body-jet liposuction. In the present study, the primary objective was the in vitro characteristics of human ASCs. The secondary objective was the optimization of the cell seeding process on 3D-printed scaffolds using polycaprolactone (PCL) or polycaprolactone covered with tricalcium phosphate (PCL + 5% TCP). Biological evaluation of human ASC showed high efficiency of isolation obtaining a satisfying amount of homogeneous cell populations. Results suggest that ASCs can be cultured in vitro for a long time without impairing their proliferative capacity. Growth kinetics shows that the highest number of cells can be achieved in passage 5 and after the 16th passage; there is a significant decrease of cell numbers and their proliferative potential. The percentage of colony forming units from the adipose stem cells is 8% ± 0.63% (p<0.05). It was observed that the accumulation of calcium phosphate in the cells in vitro, marked with Alizarin Red S, was increased along with the next passage. Analysis of key parameters critically related to the cell seeding process shows that volume of cell suspension and propagation time greatly improve the efficiency of seeding both in PCL and PCL + 5% TCP scaffolds. The cell seeding efficiency did differ significantly between scaffold materials and cell seeding methods (p<0.001). Increased seeding efficiency was observed when using the saturation of cell suspension into scaffolds with additional incubation. Alkaline phosphatase level production in PCL + 5% TCP scaffold was better than in PCL-only scaffold. The study results can be used for the optimization of the seeding process and quantification methods determining the successful implementation of the preclinical model study in the future tissue engineering strategies.

Abstract. Fabrication of scaffolds for tissue engineering (TE) applications becomes a very important research topic in present days. The aim of the study was to create and evaluate a hybrid polymeric 3D scaffold consisted of nano and microfibers, which could be used for bone tissue engineering. Hybrid structures were fabricated using rapid prototyping (RP) and electrospinning (ES) methods. Electrospun nanofibrous mats were incorporated between the microfibrous layers produced by RP technology. The nanofibers were made of poly(L-lactid) and polycaprolactone was used to fabricate microfibers. The micro-and nanostructures of the hybrid scaffolds were examined using scanning electron microscopy (SEM). X-ray microtomographical (µCT) analysis and the mechanical testing of the porous hybrid structures were performed using SkyScan 1172 machine, equipped with a material testing stage. The scanning electron microscopy and micro-tomography analyses showed that obtained scaffolds are hybrid nanofibers/microfibers structures with high porosity and interconnected pores ranging from 10 to 500um. Although, connection between microfibrous layers and electrospun mats remained consistent under compression tests, addition of the nanofibrous mats affected the mechanical properties of the scaffold, particularly its elastic modulus. The results of the biocompatibility tests didn't show any cytotoxic effects and no fibroblast after contact with the scaffold showed any damage of the cell body, the cells had proper morphologies and showed good proliferation. Summarizing, using RP technology and electrospinning method it is possible to fabricate biocompatible scaffolds with controllable geometrical parameters and good mechanical properties.