Multiscale-Engineered Muscle Constructs: PEG Hydrogel Micro-Patterning on an Electrospun PCL Mat Functionalized with Gold Nanoparticles

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes’ alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle,as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle’s physiological functions.

Bioprinting of biomimetic self-organised cartilage with a supporting joint fixation device

Despite sustained efforts, engineering truly biomimetic articular cartilage (AC) via traditional top-down approaches remains challenging. Emerging biofabrication strategies, from 3D bioprinting to scaffold-free approaches that leverage principles of cellular self-organisation, are generating significant interest in the field of cartilage tissue engineering as a means of developing biomimetic tissue analogues in vitro. Although such strategies have advanced the quality of engineered cartilage, recapitulation of many key structural features of native AC, in particular a collagen network mimicking the tissue’s ‘Benninghoff arcade’, remains elusive. Additionally, a complete solution to fixating engineered cartilages in situ within damaged synovial joints has yet to be identified. This study sought to address both of these key challenges by engineering biomimetic AC within a device designed to anchor the tissue within a synovial joint defect. We first designed and fabricated a fixation device capable of anchoring engineered cartilage into the subchondral bone. Next, we developed a strategy for inkjet printing porcine mesenchymal stem/stromal cells (MSCs) into this supporting fixation device, which was also designed to provide instructive cues to direct the self-organisation of MSC condensations towards a stratified engineered AC. We found that a higher starting cell-density supported the development of a more zonally defined collagen network within the engineered tissue. Dynamic culture was implemented to further enhance the quality of this engineered tissue, resulting in an approximate 3 fold increase in glycosaminoglycan and collagen accumulation. Ultimately this strategy supported the development of AC that exhibited near-native levels of glycosaminoglycan accumulation (>5% WW), as well as a biomimetic collagen network organisation with a perpendicular to a parallel fibre arrangement (relative to the tissue surface) from the deep to superficial zones via arcading fibres within the middle zone of the engineered tissue. Collectively, this work demonstrates the successful convergence of novel biofabrication methods, bioprinting strategies and culture regimes to engineer a hybrid implant suited to resurfacing AC defects.

The Effects of Macrophages Condition Media on the Vessel-Like Structure Formation of Pre-Vascularized Bone Marrow Mesenchymal Stem Cell Sheets

Background With the development of tissue engineering and regenerative medicine, engineered tissue constructs have become more prevalent and are now used as substitutions of patients’ damaged organs or tissues. To date, a scaffold-free cell transplantation technique based on cell sheets have been widely used in clinic. While poor vascularization and adverse immune reactions to biomedical devices remain crucial challenges in tissue engineering. The inflammatory response represents one of mainly and important regulators of vascularization and engineered tissue function restoration. This study investigated the effect of macrophage secretion on the formation of microvascular-like structures of human umbilical vein endothelial cells (HUVECs) in the bone marrow mesenchymal stem cell (BMSC) sheets. Methods Researchers differentiated the human monocytic leukemia cell line (THP-1) cells into the resting state of macrophage (M0) by phorbol 12-myristate 13-acetate (PMA) and further activated them into pro-inflammation and pro-healing states with the use of various cytokines. Condition media derived from kinds of macrophages were used to investigate the impact of macrophage on the viability and arrangement of HUVECs on BMSC sheets. Cytokines related to angiogenesis were detected in the culture supernatant of HUVECs, BMSC sheets, and pre-vascularized sheets. Results Results indicated that macrophages may guide the arrangement of endothelial cell through the paracrine pathway. The cell sheets that were cultured in the microenvironment with pro-inflammatory macrophages had fewer cell layers compared to those generated in other media. Furthermore, after experiencing high levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-α) in the first 3 days while high level of platelet-derived growth factor (PDGF)-BB but low TNF-α and VEGF concentration in the next 4 days, pre-vascularized sheets had the most significant micro-vessel network. Conclusions The fates of pre-vascularized sheets regulated by macrophages secreted factors in microenvironment and sequential released cytokines could even promote neovascularization and angiogenesis.

Integrating melt-electrowriting and inkjet bioprinting for engineering structurally organized articular cartilage

Successful cartilage engineering requires the generation of biological grafts mimicking the structure, composition and mechanical behaviour of the native tissue. Here melt-electrowriting (MEW) was used to produce arrays of polymeric structures whose function was to orient the growth of cellular aggregates spontaneously generated within these structures, and to provide tensile reinforcement to the resulting tissues. Inkjeting was used to deposit defined numbers of cells into MEW structures, which self-assembled into an organized array of spheroids within hours, ultimately generating a hybrid tissue that was hyaline-like in composition. Structurally, the engineered cartilage mimicked the histotypical organization observed in skeletally immature synovial joints. This biofabrication framework was then used to generate scaled-up (50mm x 50mm) cartilage implants containing over 3,500 cellular aggregates in under 15 minutes. After 8 weeks in culture, a 50-fold increase in the compressive properties of these MEW reinforced tissues were observed, while the tensile properties were still dominated by the polymer network, resulting in a composite construct demonstrating tension-compression nonlinearity mimetic of the native tissue. Helium ion microscopy further demonstrated the development of an arcading collagen network within the engineered tissue. This hybrid bioprinting strategy provides a versatile and scalable approach to engineer cartilage biomimetic grafts for biological joint resurfacing.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold’s mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.

Electrospun natural rubber latex biocomposite for scaffolds in tissue engineering

Fibrous scaffold along with seed cells are essential players for engineered tissue regeneration. Recently, PLGA/epoxidized poly(isoprene) dense membranes have been evaluated for cell growth and have shown satisfactory results. However, porous and fibrous structures suitable for obtaining 3D supports have not yet been evaluated for the PLGA/epoxidized poly(isoprene). The present work aimed to establish the electrospinning conditions for obtaining electrospun membranes with a smaller diameter of fibers and adequate morphology, which were characterized in vitro by their physical, chemical and biological properties. The best electrospun fibers were obtained from the following conditions: an applied voltage of 15 kV, a needle-collector distance of 20 cm and, a flow rate of 5 mL/h. The functional groups of the polymers involved in the blend did not show any changes. The mechanical properties of the electrospun membranes are within the lower limits known to human skin and some soft tissues. The in vitro degradation test showed a loss of mass of approximately 20% in 28 days. Significant adhesion and proliferation of human adipose–derived mesenchymal stem cells were demonstrated, indicating that there was cellular penetration into the scaffold and proliferation. Therefore, the preliminary results suggest that the electrospun PLGA/epoxidized poly(isoprene) membranes have high potential for application as a 3D tissue engineering scaffold.