We present a new strategy for the fabrication of artificial skeletal muscle tissue with functional morphologies based on an innovative 3D bioprinting approach. The methodology is based on a microfluidic printing head coupled to a co-axial needle extruder for high-resolution 3D bioprinting of hydrogel fibers laden with muscle precursor cells (C2C12). To promote myogenic differentiation, we formulated a tailored bioink with a photocurable semi-synthetic biopolymer (PEG-Fibrinogen) encapsulating cells into 3D constructs composed of aligned hydrogel fibers. After 3-5 days of culture, the encapsulated myoblasts started migrating and fusing, forming multinucleated myotubes within the 3D bioprinted fibers. The obtained myotubes showed high degree of alignment along the direction of hydrogel fiber deposition, further revealing maturation, sarcomerogenesis, and functionality. Following subcutaneous implantation in the back of immunocompromised mice, bioprinted constructs generated organized artificial muscle tissue in vivo. Finally, we demonstrate that our microfluidic printing head allows to design three dimensional multi-cellular assemblies with an exquisite compartmentalization of the encapsulated cells. Our results demonstrate an enhanced myogenic differentiation with the formation of parallel aligned long-range myotubes. The approach that we report here represents a robust and valid candidate for the fabrication of macroscopic artificial muscle to scale up skeletal muscle tissue engineering for human clinical application.

In this work we demonstrate how to print 3D biomimetic hydrogel scaffolds for cartilage tissue engineering with high cell density (>10(7) cells ml(-1)), high cell viability (85 ÷ 90%) and high printing resolution (≈100 μm) through a two coaxial-needles system. The scaffolds were composed of modified biopolymers present in the extracellular matrix (ECM) of cartilage, namely gelatin methacrylamide (GelMA), chondroitin sulfate amino ethyl methacrylate (CS-AEMA) and hyaluronic acid methacrylate (HAMA). The polymers were used to prepare three photocurable bioinks with increasing degree of biomimicry: (i) GelMA, (ii) GelMA + CS-AEMA and (iii) GelMA + CS-AEMA + HAMA. Alginate was added to the bioinks as templating agent to form stable fibers during 3D printing. In all cases, bioink solutions were loaded with bone marrow-derived human mesenchymal stem cells (BM-MSCs). After printing, the samples were cultured in expansion (negative control) and chondrogenic media to evaluate the possible differentiating effect exerted by the biomimetic matrix or the synergistic effect of the matrix and chondrogenic supplements. After 7, 14, and 21 days, gene expression of the chondrogenic markers (COL2A1 and aggrecan), marker of osteogenesis (COL1A1) and marker of hypertrophy (COL10A1) were evaluated qualitatively by means of fluorescence immunocytochemistry and quantitatively by means of RT-qPCR. The observed enhanced viability and chondrogenic differentiation of BM-MSCs, as well as high robustness and accuracy of the employed deposition method, make the presented approach a valid candidate for advanced engineering of cartilage tissue.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

On several paintings of James Ensor (1860-1949), a gradual fading of originally bright yellow areas, painted with the pigment cadmium yellow (CdS), is observed. Additionally, in some areas exposed to light, the formation of small white-colored globules on top of the original paint surface is observed. In this paper the chemical transformation leading to the color change and to the formation of the globules is elucidated. Microscopic X-ray absorption near-edge spectroscopy (mu-XANES) experiments show that sulfur, originally present in sulfidic form (S(2-)), is oxidized during the transformation to the sulfate form (S(6+)). Upon formation (at or immediately below the surface), the highly soluble cadmium sulfate is assumed to be transported to the surface in solution and reprecipitates there, forming the whitish globules. The presence of cadmium sulfate (CdSO(4).2H(2)O) and ammonium cadmium sulfate [(NH(4))(2)Cd(SO(4))(2)] at the surface is confirmed by microscopic X-ray diffraction measurements, where the latter salt is suspected to result from a secondary reaction of cadmium sulfate with ammonia. Measurements performed on cross sections reveal that the oxidation front has penetrated into the yellow paint down to ca. 1-2 microm. The morphology and elemental distribution of the paint and degradation product were examined by means of scanning electron microscopy equipped with an energy-dispersive spectrometer (SEM-EDS) and synchrotron radiation based micro-X-ray fluorescence spectrometry (SR micro-XRF). In addition, ultraviolet-induced visible fluorescence photography (UIVFP) revealed itself to be a straightforward technique for documenting the occurrence of this specific kind of degradation on a macroscale by painting conservators.

Gelatin methacrylate (GelMA) is an inexpensive, photocrosslinkable, cell-responsive hydrogel which has drawn attention for a wide range of tissue engineering applications. The potential of GelMA scaffolds was demonstrated to be tunable for different tissue engineering (TE) applications through modifying the polymer concentration, methacrylation degree, or UV light intensity. Despite the promising results of GelMA hydrogels in tissue engineering, the influence of polymer concentration for bone tissue engineering (BTE) scaffolds was not established yet. Thus, in this study, we have demonstrated the effect of polymer concentration in GelMA scaffolds on osteogenic differentiation. We prepared GelMA scaffolds with 5 and 10% polymer concentrations and characterized the scaffolds in terms of porosity, pore size, swelling characteristics, and mechanical properties. Subsequent to the scaffolds characterization, the scaffolds were seeded with bone marrow derived rat mesenchymal stem cells and cultured in osteogenic media to evaluate the possible osteogenic differentiation effect exerted by the polymer concentration. After 7, 14, 21, and 28 days, DNA content, calcium deposition, and alkaline phosphatase (ALP) activity of scaffolds were evaluated quantitatively by colorimetric bioassays. Furthermore, the distribution of the calcium deposition within the scaffolds was attained qualitatively and quantitatively by microcomputer tomography (mCT). Our data suggest that GelMA hydrogels prepared with 5% polymer concentration has promoted homogeneous extracellular matrix calcification and it is a great candidate for BTE applications.