A hybrid construct with tailored 3D structure for directing pre-vascularization in engineered tissues.

Hybrid 3D constructs combining different structural components afford unique opportunities to engineer functional tissues. Creating functional microvascular networks within these constructs is crucial for promoting integration with host vessels and ensuring successful engraftment. Here, we present a hybrid 3D system in which poly (ethylene oxide terephthalate)/poly (butylene terephthalate) fibrous scaffolds are combined with pectin hydrogels to provide internal topography and guide the formation of microvascular beds. The sequence/method of seeding human endothelial cells (EC) and mesenchymal stromal cells (MSC) into the system had a significant impact on microvessel formation. Optimal results were obtained when EC were directly seeded onto the fibrous scaffold, followed by the addition of hydrogel-embedded MSC. This approach facilitated the development of highly oriented microvascular networks along the fibers. These networks were lumenized, supported by a basement membrane, and stabilized by pericyte-like cells, persisting for at least 28 days in vitro. Furthermore, culture under pro-angiogenic and osteoinductive conditions induced MSC osteogenic differentiation without impairing microvessel formation. Upon subcutaneous implantation in mice, the pre-vascularized constructs were infiltrated by host vessels, and human microvessels were still present after 2 weeks. Overall, the proposed hybrid 3D system, combined with an optimized cell-seeding protocol, offers an effective approach for directing the formation of robust and geometrically oriented microvessels, making it promising for tissue engineering applications.

Incorporation of graphene oxide into poly(ɛ-caprolactone) 3D printed fibrous scaffolds improves their antimicrobial properties.

Implantable medical devices infection and consequent failure is a severe health issue, which can result from bacterial adhesion, growth, and subsequent biofilm formation at the implantation site. Graphene-based materials, namely graphene oxide (GO), have been described as potential antibacterial agents when immobilized and exposed in polymeric matrices. This work focuses on the development of antibacterial and biocompatible 3D fibrous scaffolds incorporating GO. Poly(ε-caprolactone) scaffolds were produced, with and without GO, using wet-spinning combined with additive manufacturing. Scaffolds with different GO loadings were evaluated regarding physical-chemical characterization, namely GO surface exposure, antibacterial properties, and ability to promote human cells adhesion. Antimicrobial properties were evaluated through live/dead assays performed with Gram-positive and Gram-negative bacteria. 2 h and 24 h adhesion assays revealed a time-dependent bactericidal effect in the presence of GO, with death rates of adherent S. epidermidis and E. coli reaching ~80% after 24 h of contact with scaffolds with the highest GO concentration. Human fibroblasts cultured for up to 14 days were able to adhere and spread over the fibers, independently of the presence of GO. Overall, this work demonstrates the potential of GO-containing fibrous scaffolds to be used as biomaterials that hinder bacterial infection, while allowing human cells adhesion.

Leveling Up Hydrogels: Hybrid Systems in Tissue Engineering.

Hydrogels can mimic several features of the cell native microenvironment and have been widely used as synthetic extracellular matrices (ECMs) in tissue engineering and regenerative medicine (TERM). However, some applications have specifications that hydrogels cannot efficiently fulfill on their own. Incorporating reinforcing structures like fibrous scaffolds or particles into hydrogels, as hybrid systems, is a promising strategy to improve their functionality. We describe recent advances in the fabrication and application of these hybrid systems, where structural properties and stimuli responsiveness of hydrogels are enhanced while their ECM-like features are preserved. Furthermore, we discuss how these systems can contribute to the development of more complex tissue engineered structures in the rapidly evolving field of TERM.

Cyanobacterium-Derived Extracellular Carbohydrate Polymer for the Controlled Delivery of Functional Proteins.

The unicellular cyanobacterium Cyanothece sp. CCY 0110 is a highly efficient producer of extracellular polymeric substances (EPS), releasing up to 75% of the polymer to the culture medium. The carbohydrate polymer released to the medium (RPS) was previously isolated and characterized; it is composed of nine different monosaccharides including two uronic acids, and also containing peptides and sulfate groups. Here it is shown that the RPS spontaneously assembles with proteins at high concentrations leading to a phase transition. The proteins are released progressively and structurally intact near physiological conditions, primarily through the swelling of the polymer-protein matrix. The releasing kinetics of the proteins can be modulated through the addition of divalent cations, such as calcium. Notably, the polymer is not toxic to human dermal neonatal fibroblasts in vitro at RPS concentrations bellow 0.1 mg mL-1 . The results show that this polymer is a good candidate for the delivery of therapeutic macromolecules.

Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 µm to 1.950 ± 0.553 µm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 µm and 396.1 ± 32.3 µm, in the z-axis between 36.5 ± 5.3 µm and 70.7 ± 8.8 µm, average fiber diameters between 69.4 ± 6.1 µm and 99.0 ± 9.4 µm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 µm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 µm).

Finite element analysis of pectus carinatum surgical correction via a minimally invasive approach.

Pectus carinatum (PC) is a chest deformity caused by a disproportionate growth of the costal cartilages compared to the bony thoracic skeleton, pulling the sternum towards, which leads to its protrusion. There has been a growing interest on using the 'reversed Nuss' technique as a minimally invasive procedure for PC surgical correction. A corrective bar is introduced between the skin and the thoracic cage and positioned on top of the sternum highest protrusion area for continuous pressure. Then, it is fixed to the ribs and kept implanted for about 2-3 years. The purpose of this work was to (a) assess the stresses distribution on the thoracic cage that arise from the procedure, and (b) investigate the impact of different positioning of the corrective bar along the sternum. The higher stresses were generated on the 4th, 5th and 6th ribs backend, supporting the hypothesis of pectus deformities correction-induced scoliosis. The different bar positioning originated different stresses on the ribs' backend. The bar position that led to lower stresses generated on the ribs backend was the one that also led to the smallest sternum displacement. However, this may be preferred, as the risk of induced scoliosis is lowered.

Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

In situ-forming hydrogels of pectin, a polysaccharide present in the cell wall of higher plants, were prepared using an internal ionotropic gelation strategy based on calcium carbonate/d-glucono-δ-lactone, and explored for the first time as cell delivery vehicles. Since no ultrapure pectins are commercially available yet, a simple and efficient purification method was established, effectively reducing the levels of proteins, polyphenols and endotoxins of the raw pectin. The purified pectin was then functionalized by carbodiimide chemistry with a cell-adhesive peptide (RGD). Its gelation was analyzed by rheometry and optimized. Human mesenchymal stem cells embedded within unmodified and RGD-pectin hydrogels of different viscoelasticities (1.5 and 2.5 wt%) remained viable and metabolically active for up to 14 days. On unmodified pectin hydrogels, cells remained isolated and round-shaped. In contrast, within RGD-pectin hydrogels they elongated, spread, established cell-to-cell contacts, produced extracellular matrix, and migrated outwards the hydrogels. After 7 days of subcutaneous implantation in mice, acellular pectin hydrogels were considerably degraded, particularly the 1.5 wt% hydrogels. Altogether, these findings show the great potential of pectin-based hydrogels, which combine an interesting set of easily tunable properties, including the in vivo degradation profile, for tissue engineering and regenerative medicine.

Correction: Biofunctionalized pectin hydrogels as 3D cellular microenvironments.

Correction for 'Biofunctionalized pectin hydrogels as 3D cellular microenvironments' by Sara C. Neves et al., J. Mater. Chem. B, 2015, 3, 2096-2108.

Chitosan/poly(epsilon-caprolactone) blend scaffolds for cartilage repair.

Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. µCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation.