Vivek Mudera

The physiological O₂ microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O₂, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O₂. At 21% O₂, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O₂ hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

There are several three-dimensional (3D) skeletal muscle (SkM) tissue engineered models reported in the literature. 3D SkM tissue engineering (TE) aims to recapitulate the structure and function of native (in vivo) tissue, within an in vitro environment. This requires the differentiation of myoblasts into aligned multinucleated myotubes surrounded by a biologically representative extracellular matrix (ECM). In the present work, a new commercially available 3D SkM TE culture chamber manufactured from polyether ether ketone (PEEK) that facilitates suitable development of these myotubes is presented. To assess the outcomes of the myotubes within these constructs, morphological, gene expression, and ECM remodeling parameters were compared against a previously published custom-built model. No significant differences were observed in the morphological and gene expression measures between the newly introduced and the established construct configuration, suggesting biological reproducibility irrespective of manufacturing process. However, TE SkM fabricated using the commercially available PEEK chambers displayed reduced variability in both construct attachment and matrix deformation, likely due to increased reproducibility within the manufacturing process. The mechanical differences between systems may also have contributed to such differences, however, investigation of these variables was beyond the scope of the investigation. Though more expensive than the custom-built models, these PEEK chambers are also suitable for multiple use after autoclaving. As such this would support its use over the previously published handmade culture chamber system, particularly when seeking to develop higher-throughput systems or when experimental cost is not a factor.

Biomineralization is a dynamic, complex, lifelong process by which living organisms control precipitations of inorganic nanocrystals within organic matrices to form unique hybrid biological tissues, for example, enamel, dentin, cementum, and bone. Understanding the process of mineral deposition is important for the development of treatments for mineralization-related diseases and also for the innovation and development of scaffolds. This review provides a thorough overview of the up-to-date information on the theories describing the possible mechanisms and the factors implicated as agonists and antagonists of mineralization. Then, the role of calcium and phosphate ions in the maintenance of teeth and bone health is described. Throughout the life, teeth and bone are at risk of demineralization, with particular emphasis on teeth, due to their anatomical arrangement and location. Teeth are exposed to food, drink, and the microbiota of the mouth; therefore, they have developed a high resistance to localized demineralization that is unmatched by bone. The mechanisms by which demineralization–remineralization process occurs in both teeth and bone and the new therapies/technologies that reverse demineralization or boost remineralization are also scrupulously discussed. Technologies discussed include composites with nano- and micron-sized inorganic minerals that can mimic mechanical properties of the tooth and bone in addition to promoting more natural repair of surrounding tissues. Turning these new technologies to products and practices would improve health care worldwide.

Neuromuscular and neurodegenerative diseases are conditions that affect both motor neurons and the underlying skeletal muscle tissue. At present, the majority of neuromuscular research utilizes animal models and there is a growing need to develop novel methodologies that can be used to help understand and develop treatments for these diseases. Skeletal muscle tissue-engineered constructs exhibit many of the characteristics of the native tissue such as accurate fascicular structure and generation of active contractions. However, to date, there has been little consideration toward the integration of engineered skeletal muscle with motor neurons with the aim of neuromuscular junction (NMJ) formation, which would provide a model to investigate neuromuscular diseases and basic biology. In the present work we isolated primary embryonic motor neurons and neonatal myoblasts from Sprague-Dawley rats, and cocultured the two cell types in three-dimensional tissue-engineered fibrin hydrogels with the aim of NMJ formation. Immunohistochemistry revealed myotube formation in a fascicular arrangement and neurite outgrowth from motor neuron cell bodies toward the aligned myotubes. Furthermore, colocalization of pre- and postsynaptic proteins and chemical inhibition of spontaneous myotube twitch indicated the presence of NMJs in the innervated constructs. When electrical field stimulation was employed to evoke isometric contractions, maximal twitch and tetanic force were higher in the constructs cocultured with motor neurons, which may, in part, be explained by improved myotube cytoskeletal organization in these constructs. The fabrication of such constructs may be useful tools for investigating neuromuscular pharmaceuticals and improving the understanding of neuromuscular pathologies.

Skeletal muscle (SkM) is a tissue that responds to mechanical load following both physiological (exercise) or pathophysiological (bed rest) conditions. The heterogeneity of human samples and the experimental and ethical limitations of animal studies provide a rationale for the study of SkM plasticity in vitro. Many current in vitro approaches of mechanical loading of SkM disregard the three-dimensional (3D) structure in vivo. Tissue engineered 3D SkM, that displays highly aligned and differentiated myotubes, was used to investigate mechano-regulated gene transcription of genes implicated in hypertrophy/atrophy. Static loading (STL) and ramp loading (RPL) at 10 % strain for 60 min were used as mechano-stimulation with constructs sampled immediately for RNA extraction. STL increased IGF-I mRNA compared to both RPL and CON (control, p = 0.003 and 0.011 respectively) whilst MMP-9 mRNA increased in STL and RPL compared to CON (both p < 0.05). IGFBP-2 mRNA was differentially regulated in RPL and STL compared to CON (p = 0.057), whilst a reduction in IGFBP-5 mRNA was found for STL and RPL compared to CON (both p < 0.05). There was no effect in the expression of putative atrophic genes, myostatin, MuRF-1 and MAFBx (all p > 0.05). These data demonstrate a transcriptional signature associated with SkM hypertrophy within a tissue-engineered model that more greatly recapitulates the in vivo SkM structure compared previously published studies.