3D Printing of Anisotropic Bone-Mimetic Structure with Controlled Fluid Flow Stimuli for Osteocytes: Flow Orientation Determines the Elongation of Dendrites

Although three-dimensional (3D) bioprinting techniques enable the construction of various living tissues and organs, the generation of bone-like oriented microstructures with anisotropic texture remains a challenge. Inside the mineralized bone matrix, osteocytes play mechanosensing roles in an ordered manner with a well-developed lacunar-canaliculi system. Therefore, control of cellular arrangement and dendritic processes is indispensable for construction of artificially controlled 3D bone-mimetic architecture. Herein, we propose an innovative methodology to induce controlled arrangement of osteocyte dendritic processes using the laminated layer method of oriented collagen sheets, combined with a custom-made fluid flow stimuli system. Osteocyte dendritic processes showed elongation depending on the competitive directional relationship between flow and substrate. To the best of our knowledge, this study is the first to report the successful construction of the anisotropic bone-mimetic microstructure and further demonstrate that the dendritic process formation in osteocytes can be controlled with selective fluid flow stimuli, specifically by regulating focal adhesion. Our results demonstrate how osteocytes adapt to mechanical stimuli by optimizing the anisotropic maturation of dendritic cell processes.

Imaging endogenous synaptic proteins in primary neurons at single-cell resolution using CRISPR/Cas9

Fluorescence imaging at single-cell resolution is a crucial approach to analyzing the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a nonviral strategy that enables the tagging of endogenous loci by CRISPR/Cas9-mediated genome editing combined with a nucleofection technique. The method allowed expression of fluorescently tagged proteins at endogenous levels, and we successfully achieved tagging of a presynaptic protein, synaptophysin (Syp), and a postsynaptic protein, PSD-95, in cultured postmitotic neurons. Superresolution fluorescence microscopy of fixed neurons confirmed the identical localization patterns of the tagged proteins to those of endogenous ones verified by immunohistochemistry. The system is also applicable for multiplexed labeling and live-cell imaging. Live imaging with total internal reflection fluorescence microscopy of a single dendritic process of a neuron double-labeled with Syp-mCherry and PSD-95-EGFP revealed the previously undescribed dynamic localization of the proteins synchronously moving along dendritic shafts. Our convenient and versatile strategy is potent for analysis of proteins whose ectopic expressions perturb cellular functions.

Studies on the pigmented and unpigmented photoreceptors of the cercaria of Cryptocotyle lingua (Creplin) from Littorina littorea (L.)

The cercaria of Cryptocotyle lingua possesses a pair of pigmented rhabdomeric photoreceptors situated antero-dorsally, and a median unpigmented rhabdomeric photoreceptor lying slightly ventral to the middle region of the body immediately anterior to the transverse commissure. Each pigmented photoreceptor consists of two asymmetrical pigment cells containing two symmetrical back-to-back pigment cups facing respectively antero-laterally and postero-medianly, with one retinular cell associated with each pigment cup. The microvilli of the rhabdomere in each cup are radially arranged around a triradiate dendritic process; in the centre of each microvillus is a strand presumably the visual pigment. The retinular cell and dendritic process contain a large number of mitochondria. The rhabdomere of the median unpigmented photoreceptor is enclosed in a thin-walled unpigmented cup opening posteriorly and giving off processes, some of which contain mitochondria, into the surrounding parenchyma. This rhabdomere resembles the equivalent of two rhabdomeres of the paired photoreceptors. Axonal processes from the retinular cells of the paired and unpaired photoreceptors enter the neuropile of the cerebral ganglia and transverse commissure respectively. The cercaria is markedly photopositive and responds also to shadow by active bursts of swimming. It is suggested that the paired dorsal photoreceptors may be sensitive to light of higher intensity and the median more ventral photoreceptor to light of lower intensity and to shadow.

Interreceptor Contacts in Olfactory Mucosa of Prog and Turtle

Besides the mechanical role of supporting elements, supporting cells in the vertebrates olfactory mucosa have been attributed the role of secretion and isolation of olfactory receptors. The role of isolating elements has been presently investigated and the data obtained prove this role should be revised. With the transmission electron microscope sections perpendicular and parallel to the surface of the olfactory mucosa were studied. In perpendicular sections it has been observed that receptors (r) are on occasion in mutual direct contact at the level of the junctional complex (Fig. 1). Furthermore, along their dendritic process they come close together both along large areas or through specific armlike (a) processes. (Fig. 2). In sections parallel to the surface the receptors arrange themselves in annular interconnected patterns. Each receptor, moreover, extends short protrusions (the same armlike processes (arrow) as seen in perpendicular sections) to the neighboring neurons (Fig. 3).

The development of the body wall of Polymorphus minutus (Acanthocephala) in its intermediate host Gammarus pulex

The ultrastructure and development of the body wall of Polymorphus minutus is described during the acanthella stages in the intermediate host Gammarus pulex.The cortex of the early acanthella is simple in structure, consisting of a syncytium containing giant nuclei, mitochondria, Golgi bodies, endoplasmic reticulum and a few lipid bodies. The cuticle is penetrated by pores which open into vesicles in a vesicular region.The cortex gives rise to the body wall, neck and lemnisci of the adult, and may be considered as divided initially into two and later three functionally different regions: an outer absorptive region, an inner storage region and a layer between which develops to provide firm, resistant skeletal protection.Absorption is facilitated by the increase in surface area due to the pores which penetrate the surface. This region is functional throughout development and the pores increase in number, possibly by division, as the animal grows. Beneath the pores a pore canal layer develops towards the end of the late acanthella stage.During development supporting fibres are deposited throughout the cortex. Beneath the vesicular layer a concentration of mitochondria, Golgi bodies and endoplasmic reticulum deposit the fibres of the felt layer. In the anterior meta-soma no felt layer is found, but the body spines originate from this region.The inner region of the cortex, or radial layer, contains the giant nuclei, which are arranged in the early acanthella in 5 rings of 4, 6, 5 or 6, 6 and 5 nuclei respectively. During development they produce dendritic process and finally fragment. They are considered here to be concerned with the formation of the felt layer and lemnisci rather than the ‘lacunar channels’. The inner cortical region becomes ensely packed with lipid bodies, and it is suggested that these may be responsible, owing to fixation artifact, for the so-called ‘lacunar channels’ previously described in these regions, the extensive cavities seen in some light microscope preparations being filled with a fluid lipid rather than an aqueous solution.The lemnisci are similar in appearance to the cortex except for regions of concentrated endoplasmic reticulum.The mitochondria in the cortex of the acanthella appear to be aerobic rather than anaerobic, as they possess many cristae and are larger than the mitochondria of the adults.My thanks are due to Dr D. W. T. Crompton for advice and encouragement, and to Dr D. H. Northcote and Dr F. B. P. Wooding for time on the Phillips Electron Microscope 100. This work was carried out during the tenure of an S.R.C. studentship.