Growth plate and articular cartilage constitute a single anatomical entity early in development but later separate into two distinct structures by the secondary ossification center (SOC). The reason for such separation remains unknown. We found that evolutionarily SOC appears in animals conquering the land - amniotes. Analysis of the ossification pattern in mammals with specialized extremities (whales, bats, jerboa) revealed that SOC development correlates with the extent of mechanical loads. Mathematical modeling revealed that SOC reduces mechanical stress within the growth plate. Functional experiments revealed the high vulnerability of hypertrophic chondrocytes to mechanical stress and showed that SOC protects these cells from apoptosis caused by extensive loading. Atomic force microscopy showed that hypertrophic chondrocytes are the least mechanically stiff cells within the growth plate. Altogether, these findings suggest that SOC has evolved to protect the hypertrophic chondrocytes from the high mechanical stress encountered in the terrestrial environment.

46Growth plates are narrow discs of cartilage, ultimately required for longitudinal growth 47 of all mammals including humans. However, originally the growth plate and articular 48 cartilage were a single anatomical entity, an epiphyseal cartilage, as appeared in early 49 tetrapods and in mammalian development. The reason, why the growth plates evolved 50 as spatially separate organs, remains unknown. 51Here, we demonstrate that the epiphyseal growth plate first appeared as an 52individual organ in amniotes due to the formation of a novel bony structure, the 53 secondary ossification center (SOC), which spatially separates articular cartilage and 54 the growth plate. Since amniotes translocate their entire growth period on land, we next 55 explored the role of mechanical demands faced by bones growing under weight-bearing 56conditions. Comparison of mammals whose limbs are subjected to greater or lesser 57 mechanical demands (i.e., Chiropterans (bats), Cetaceans (whales) and Dipodidae 58(jerboa)) revealed that the presence of an SOC is correlated to the extent of these 59 demands. Mathematical modelling in combination with physical and biological 60 experiments showed that the SOC reduces shear and normal stresses within the growth 61 plate, allowing epiphyseal chondrocytes to withstand a six-fold higher load before 62 undergoing caspase-dependent apoptosis via the YAP-p73 pathway. Furthermore, the 63 hypertrophic chondrocytes, the cells primarily responsible for bone elongation were 64 least mechanically stiff and most sensitive to weight bearing. 65Our results demonstrate that evolution of the epiphyseal cartilage into a 66 separate organ allows epiphyseal chondrocytes to withstand the high mechanical stress 67 placed on them by the terrestrial environment. 68 69 70Main text 71 72The skeleton articulates via articular cartilage and grow in length via the 73 epiphyseal cartilage, often presented as growth plates, tiny discs of cartilage located to 74 the end of long bones and containing epiphyseal chondrocytes. These chondrocytes 75 proliferate, align in the longitudinal direction and then undergo several-fold of 76 enlargement (hypertrophy). Thereafter hypertrophic chondrocytes undergo apoptosis 77 or trans-differentiation 1 leaving their calcified extracellular matrix as a scaffold for 78invading blood vessels and osteoblasts to form new bone tissue. The process of bone 79 growth on cartilage template is called endochondral bone formation. Recent 3D 80 microanatomical characterization of the 380-million-year-old lobe-finned fish 81Eusthenopteron 2 revealed longitudinally-oriented trabeculae within the shaft of their 82 humeri ( Fig.

Summary Regenerative medicine opens new opportunities in the repair of cicatricial lesions of the vocal folds. Here, we present a thorough morphological study, with the focus on the collagen structures in the mucosa of the vocal folds, dedicated to the effects of stem cells on the vocal folds repair after cicatricial lesions. We used a conventional experimental model of a mature scar of the rabbit vocal folds, which was surgically excised with a simultaneous implantation of autologous bone marrow‐derived mesenchymal stem cells (MSC) into the defect. The restoration of the vocal folds was studied 3 months postimplantation of stem cells and 6 months after the first surgery. The collagen structure assessment included histology, immunohistochemistry and atomic force microscopy (AFM) studies. According to the data of optical microscopy and AFM, as well as to immunohistochemical analysis, MSC implantation into the vocal fold defect leads not only to the general reduction of scarring, normal ratio of collagens type I and type III, but also to a more complete restoration of architecture and ultrastructure of collagen fibres in the mucosa, as compared to the control. The collagen structures in the scar tissue in the vocal folds with implanted MSC are more similar to those in the normal mucosa of the vocal folds than to those of the untreated scars. AFM has proven to be an instrumental technique in the assessment of the ultrastructure restoration in such studies. Lay Description Regenerative medicine opens new opportunities in the repair of the vocal fold scars. Because collagen is a main component in the vocal fold mucosa responsible for the scar formation and repair, we focus on the collagen structures in the mucosa of the vocal folds, using a thorough morphological study based on histology and atomic force microscopy (AFM). Atomic force microscopy is a scanning microscopic technique which allows revealing the internal structure of a tissue with a resolution up to nanometres. We used a conventional experimental model of a mature scar of the rabbit vocal folds, surgically excised and treated with a mesenchymal stem cells transplant. Our morphological study, primarily AFM, explicitly shows that the collagen structures in the scarred vocal folds almost completely restore after the stem cell treatment. Thus, the modern microscopic methods, and especially AFM are instrumental tools for monitoring the repair of the vocal folds scars.

The results of laser-induced formation of luminescent structures on the surface of poly-2.2’-n-oxydiphenylene-5.5’-bis-benzimidazole films, obtained by the coating method from a formic acid solution, are presented. The structures are formed using cw 405-nm laser radiation with intensities of 102 – 104 W cm–2. It is shown (using methods of optical, atomic-force, and electron microscopy) that the structures formed on the surface are microbubble foam-like aggregations. Their formation is modelled as a sequence of the following processes: release of formic acid molecules, their condensation on matrix defects in the surface layer, and explosive boiling as a result of heating this layer of polymer film by the laser beam. The luminescence of these structures is due to the weakening of the concentration quenching from closely spaced luminescence centres in benzimidazole cycles during their emergence to the surface and the increase in the distance between them due to the extension on bubble aggregates on the surface.