Hybrid spheroid microscaffolds as modular tissue units to build macro-tissue assemblies for tissue engineering.

Since its inception, tissue engineering and regenerative medicine (TERM) has been relying on either scaffold-based or scaffold-free strategies. Recent reports outlined the possibility of a synergistic, convergence approach, referred to as the third TERM strategy, which could alleviate bottlenecks of the two previous options. This strategy requires the fabrication of highly porous microscaffolds, allowing to create single spheroids within each of them. The resulting tissue units can then be combined and used as modular building blocks for creating tissue constructs through a bottom-up self-assembly. Such strategy can have a significant impact for the future of TERM, but so far, no reports have assessed its feasibility in detail. This work reports a first systematic study, which includes a comparison of the in vitro behavior of tissue units based on adipose derived stem cell spheroids cultured within microscaffolds versus conventional spheroids. We first proved that the presence of the microscaffold neither impairs the cells 'ability to form spheroids nor impacts their viability. Importantly, the fusiogenic and the differentiation potential (i.e. chondrogenesis and osteogenesis), which are important features for cellularized building blocks to be used in TERM, are preserved when spheroids are cultured within microscaffolds. Significant benefits of microscaffold-based tissue units include the enhanced cell retention, the decreased compaction and the better control over the size observed when larger tissue constructs are formed through self-assembly. The proof of concept study presented here demonstrates the great potential offered by those microsize tissue units to be used as building blocks for directed tissue self-assembly. STATEMENT OF SIGNIFICANCE: One of the most exciting and recent advances in tissue engineering and regenerative medicine (TERM) is to combine together multiple micro-size cellularized units, which are able to self-assemble altogether to recreate larger tissue constructs. In this work, we produce such modules by forming single spheroids within highly porous microscaffolds, and study how this new microenvironment impacts on the spheroid's behavior and stemness potential. This work highlights as well that such novel route is enabled by two-photon polymerization, which is an additive manufacturing technique offering high spatial resolution down to 100 nm. These findings provide a first scientific evidence about the utilization of hybrid spheroid microscaffold-based tissue units with great perspective as a modular tool for TERM.

Increasing the Microfabrication Performance of Synthetic Hydrogel Precursors through Molecular Design.

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (<0.7) and higher stiffness (>36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures.

Technological advancements for the development of stem cell-based models for hepatotoxicity testing.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Enhanced osteogenic activity with boron-doped nanohydroxyapatite-loaded poly(butylene adipate-co-terephthalate) fibrous 3D matrix.

In this study, three dimensional (3D) poly(butylene adipate-co-terephthalate) (PBAT) fibrous scaffolds with more than 90% porosity were fabricated via wet electrospinning method. Amorphous hydroxyapatite (HAp) and boron (B) doped hydroxyapatite (B-HAp) nanoparticles were produced by microwave-assisted biomimetic precipitation and encapsulated into PBAT fibres with the ratio of 5% (w/w) in order to enhance osteogenic activity of the scaffolds. Cell culture studies were carried out with human bone marrow derived stem cells (hBMSCs) and they showed that alkaline phosphatase (ALP) activity and the amounts of collagen and calcium were higher on B containing PBAT (B-HAp-PBAT) scaffolds during the 28-day culture period than that of the PBAT scaffolds. Moreover, hBMSCs cultivated on B-HAp-PBAT scaffolds showed significantly higher expression levels of both early and late stage osteogenic genes e.g. ALP, collagen I (COL-I), osteocalcin (OCN) and osteopontin (OPN) at day 28 than that of the PBAT scaffolds. Scanning electron microscope (SEM) photographs and energy dispersive X-ray (EDX) analysis indicated that hBMSCs produced high amounts of mineralized extracellular matrix (ECM) mainly on the surface of the 3 D matrices. This study demonstrates that boron-containing 3 D nanofibrous PBAT scaffolds with their osteoinductive and osteoconductive properties can be used as alternative constructs for bone tissue engineering.

Poly(butylene adipate-co-terephthalate) scaffolds: processing, structural characteristics and cellular responses.

The aliphatic-aromatic copolyester, poly(butylene adipate-co-terephthalate) (PBAT) combines good mechanical and thermal properties with biodegradation ability. However, until now, researches in its potential medical use remain limited. Only in a few studies blends of PBAT with routinely used biocompatible polymers had been prepared and investigated regarding tissue engineering applications. Therefore, in this study, we decided to determine processability of neat PBAT as a scaffold material for bone tissue by using different fabrication methods i.e. solvent evaporation, electrospinning, solvent casting-particulate leaching (SCPL) and melt molding-particulate leaching. The results of physicochemical characterizations and cell culture studies with MC3T3-E1 preosteoblasts confirmed that neat PBAT has favorable characteristics for bone tissue engineering, however, fabrication method strongly affects the cellular responses. Regarding to the characterizations and cell cultures, PBAT scaffolds produced by SCPL and electrospinning are proposed to be used for bone tissue engineering.

Honey-based PET or PET/chitosan fibrous wound dressings: effect of honey on electrospinning process.

In this study, fibrous mats were fabricated via electrospinning from solutions of polyethylene terephthalate (PET), PET/chitosan, and PET/honey at different concentrations. The effect of honey and chitosan on electrospinning process was investigated and compared. Fibers containing chitosan had a beaded or ribbon-like/branched morphology, but this morphology improved in the presence of honey. The diameter of electrospun fibers decreased with an increased ratio of honey in PET solution. In addition, fiber deposition area in the collector increased by increasing the honey content. PET/chitosan and PET/honey fibrous mats reached an equilibrium water content in 15 min and their water uptake capacities, which are important for exudating wounds, were found in the range of 280-430% on dry basis. Cytotoxicity evaluation demonstrated that fibers exhibited no cytotoxic activity. This study discloses that PET fibrous mats especially electrospun in the presence of honey could be proposed as potential wound dressing materials owing to their improved processing abilities besides their suitable structural properties.