Non-uniform strain distribution within rat cartilaginous growth plate under uniaxial compression

Growth plates are highly inhomogeneous in morphology and composition. Mechanical loading can modulate longitudinal bone growth, though the mechanisms underlying this mechanobiology are poorly understood. The proximal tibial growth plates of six rats were tested in vitro under uniaxial compression to 5% strain, and confocal microscopy was used to track and capture images of fluorescently labeled cell nuclei with increasing applied strains. The local strain patterns through the growth plate thickness were quantified using texture correlation analysis. The technique of texture correlation analysis was first validated by comparing theoretical simulated strain maps generated from numerically distorted images. The texture correlation algorithm was sensitive to the grid size superimposed on the original image, but remained insensitive to parameters related to the size of the final image mask, which was searched by the correlation algorithm for each grid point of the original image. Within the growth plate, experimental strain distributions were non-uniform in all six specimens. Growth plates were mostly under compression strains. The strain distributions differed among the histomorphological zones of the growth plate, which was most obvious in specimens with regular growth plate shape: higher compressive strains (4-8 times higher than the applied 5% strain) were located mainly in regions overlapping the reserve and hypertrophic zones with lower compressive strains in the proliferative zone. This study documents the non-uniform mechanical behavior of growth plate across its three histological zones when exposed to compression. Further investigation is required to establish the significance of non-uniform strain fields during growth in vivo.     

Electrically regulated cellular morphological and cytoskeletal changes on an optically transparent electrode

Electrically regulated morphological and cytoskeletal changes of HeLa cells were studied on an optically transparent electrode (OTE), on which potential-applied surface cellular behavior and morphogenesis were easily observed. Upon application of a potential, HeLa cells in an OTE exhibited remarkable morphological changes above +0.5 V (vs. Ag/AgCl) and below 0 V. At each potential in this potential range, change in F-actin distribution was observed using a fluorescent probe (rhodamine phalloidin). These results suggest that an electrical field induces a subcellular cytoskeletal change. Electrostimulation of cells with OTE can be a valuable strategy for the manipulation of cultured human cells.     

Physiological and growth changes in micropropagated Citrus macrophylla explants due to salinity

Salinity is one of the major abiotic stresses affecting arable crops worldwide, and is the most stringent factor limiting plant distribution and productivity. In the present study, the possible use of in vitro culture to evaluate the growth and physiological responses to salt-induced stress in cultivated explants of Citrus macrophylla was analyzed. For this purpose, micropropagated adult explants were grown in proliferation and rooting media supplemented with different concentrations of NaCl. All growth parameters were decreased significantly by these NaCl treatments; this was accompanied by visible symptoms of salt injury in the proliferated shoots from 60 mM NaCl and in the rooted shoots from 40 mM NaCl. Malondialdehyde (MDA) increased with increasing salinity in proliferated shoots, indicating a rising degree of membrane damage. The concentration of total chlorophyll significantly decreased in the presence of NaCl, and this effect was more pronounced in the rooted explants. The Na⁺ and Cl⁻ concentrations in the explants increased significantly with the salinity level, but Cl⁻ levels were higher in the proliferated explants than in the rooted explants. For osmotic adjustment, high concentrations of compatible solutes (proline and quaternary ammonium compounds—QAC) accumulated in salt-stressed plants in proliferation, but differences were not observed in rooted explants. In proliferation, proline and QAC were highly correlated with the sodium and chloride concentrations in the explants, indicating a possible role of these compounds in osmotic adjustment. The plant concentrations of NO₃⁻, K⁺, Mg²⁺, Ca⁺ and Fe were also affected by the NaCl concentration of the medium. We suggest that the important deleterious effects in the in vitro explants of Citrus macrophylla grown at increasing NaCl concentrations were due mainly to toxic effects of saline ions, particularly Cl⁻, at the cellular level.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE₂) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE₂. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE₂ production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE₂ synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE₂ synthesis after 18 hours, suggesting that PGE₂ synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Feeding, morphological changes and allometric growth during starvation in miiuy croaker larvae

We investigated the effects of the timing of first feeding (larvae in F0, F1, F2, F3 and S were first fed on day 3, 4, 5, 6 days after hatching (DAH) and unfed, respectively) on feeding, morphological changes, survival and growth in miiuy croaker larvae at 24°C. The fed larvae initiated feeding on 3 DAH and reached point of no return (PNR) on 6 DAH. Larvae in F0 and F1 groups survived apparently better than F2 group at the end of the experiment on 36 DAH. High larval mortality occurred from 3 to 7 DAH in all feeding groups, accounting for 40% (F0, F1 and F2 groups) to 90% (F3 and S groups) of the total mortality. Larvae in F0 and F1 groups grew better than F2 group throughout the experiment. Eye diameter, body height, head height and mouth gape of the first feeding larvae were more sensitive to starvation than other morphometrics and could be used as indicators for evaluating their nutritional status. Results indicated that delayed first feeding over 1 day after yolk exhaustion could lead to poor larval survival and growth. To avoid starvation and obtain good growth in culturing, larvae feeding should be initiated within 1 day after yolk exhaustion at 24°C.     

Elemental changes associated with chondrocyte differentiation in rat rib growth plate

Summary Quantitative X-ray microanalysis was under-taken to follow the elemental changes that occur in the process of chondrocyte differentiation. For analysis at the cellular level, semi-thick freeze-dried cryosections of rat rib growth plate cartilage were used. For evaluation of the elemental concentrations at the subcellular level, thin sections of freeze-dried and low temperature vacuum embedded cartilage were analyzed. Levels of Na, P, S, Cl, K, and Ca were determined in the cells and extracellular matrix in different zones of the cartilage — resting, proliferative, and hypertrophic. Proliferative cells had a sodium concentration that was twice that of resting cells, suggesting that Na may play an important role in the regulation of DNA- and protein-synthesis in chondrocytes. A concomitant rise in Na and S concentration occurred between resting zone and proliferative zone cartilage matrix. The high concentrations of Na and K in the matrix are probably due to the high amount of sulfate in proteoglycans which may bind these cations.     

Elemental changes associated with chondrocyte differentiation in rat rib growth plate

Quantitative X-ray microanalysis was under-taken to follow the elemental changes that occur in the process of chondrocyte differentiation. For analysis at the cellular level, semi-thick freeze-dried cryosections of rat rib growth plate cartilage were used. For evaluation of the elemental concentrations at the subcellular level, thin sections of freeze-dried and low temperature vacuum embedded cartilage were analyzed. Levels of Na, P, S, Cl, K, and Ca were determined in the cells and extracellular matrix in different zones of the cartilage--resting, proliferative, and hypertrophic. Proliferative cells had a sodium concentration that was twice that of resting cells, suggesting that Na may play an important role in the regulation of DNA- and protein-synthesis in chondrocytes, A concomitant rise in Na and S concentration occurred between resting zone and proliferative zone cartilage matrix. The high concentrations of Na and K in the matrix are probably due to the high amount of sulfate in proteoglycans which may bind these cations.     

Effects of serum, fibroblast growth factor, and platelet-derived growth factor on explants of rat tail tendon: a morphological study

Eighteen tail tendon fascicles were explanted from a 40-day postpartum rat and maintained in both serum-supplemented and serum-free Eagle's minimal essential medium for 2 weeks. Epitendon and paratendon connective tissues were excluded from these explants. Tendon fibroblasts maintained in serum-supplemented medium proliferated and synthesized collagen. Tendon fibroblasts explanted in serum-free medium remained viable but did not proliferate. Fibroblast growth factor and platelet-derived growth factor were shown to stimulate proliferation of mature tendon fibroblasts in serum-free medium.     

Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilage

The pericellular matrix (PCM) is a narrow region of tissue that completely surrounds chondrocytes in articular cartilage. Previous theoretical models of the "chondron" (the PCM with enclosed cells) suggest that the structure and properties of the PCM may significantly influence the mechanical environment of the chondrocyte. The objective of this study was to quantify changes in the three-dimensional (3D) morphology of the chondron in situ at different magnitudes of compression applied to the cartilage extracellular matrix. Fluorescence immunolabeling for type-VI collagen was used to identify the boundaries of the cell and PCM, and confocal microscopy was used to form 3D images of chondrons from superficial, middle, and deep zone cartilage in explants compressed to 0%, 10%, 30%, and 50% surface-to-surface strain. Lagrangian tissue strain, determined locally using texture correlation, was highly inhomogeneous and revealed depth-dependent compressive stiffness and Poisson's ratio of the extracellular matrix. Compression significantly decreased cell and chondron height and volume, depending on the zone and magnitude of compression. In the superficial zone, cellular-level strains were always lower than tissue-level strains. In the middle and deep zones, however, tissue strains below 25% were amplified at the cellular level, while tissue strains above 25% were decreased at the cellular level. These findings are consistent with previous theoretical models of the chondron, suggesting that the PCM can serve as either a protective layer for the chondrocyte or a transducer that amplifies strain, such that cellular-level strains are more homogenous throughout the tissue depth despite large inhomogeneities in local ECM strains.     

Grazing-induced morphological and growth rate changes in Anarthrophyllum rigidum,a Patagonian leguminous shrub

Grazing influences the morphology and growth rate of shrubs, and consequently, their population dynamics. It has been shown that grazing directly affects the growth of shrubs. On the other hand, the reduction of grass biomass by herbivores reduces soil–water competition between grasses and shrubs, and indirectly, could enhance the growth of shrubs. However, the assessment of the long-term effects of grazing on the growth of shrubs in the arid Patagonia has been hampered by the lack of long and homogeneous records of plant population dynamics and primary production. In this study, we combined growth-ring and allometric analyses to assess the long-term effect of grazing on individuals of Anarthrophyllum rigidum, a leguminous shrub widely distributed across the Patagonian steppe. A. rigidum has evergreen leaves rich in proteins that constitute an important complement to the diet of sheep, particularly in winter when the abundance of grasses is reduced. Our observations indicate that individuals of A. rigidum nearby the water source used by livestock were smaller in size (35.5 cm vs. 67.39 cm), presented a larger number of basal branches (23 vs. 12), and showed slower rates of growth (8.2 mm year^?1 vs. 14.3 mm year^?1) than individuals located far from the water source. This first quantification of the long-term effects of grazing on A. rigidum in the dry Patagonian steppe suggests that beneficial effects of grazing through the reduction of grasses that compete with shrubs for soil–water should be more obvious for livestock non-preferred than preferred shrubs     

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.     

Mechanical properties of the porcine growth plate and its three zones from unconfined compression tests

The aim of the study was to determine intrinsic mechanical properties of the complete growth plate and its reserve, proliferative and hypertrophic zones. Growth plate disk samples from newborn swine's ulnae were tested using stress relaxation tests under unconfined compression. The Transversely Isotropic Biphasic Model (TIBPE) derived by [Cohen, B., Lai, W. M., Mow, V. C., 1998. A transversely isotropic biphasic model for unconfined compression of growth plate and chondroepiphysis. Journal of Biomechanical Engineering, 120, pp. 491–496] was used to extract intrinsic mechanical properties using a four-parameter optimization procedure. Significant differences were found for the transverse permeability k₁, the Poisson's ratio in the transverse plane ν₂₁, the out-of-plane Poisson's ratio ν₃₁ and the out-of-plane Young's modulus E₃ between the reserve zone and the proliferative zone as well as between the reserve zone and the hypertrophic zone. The same trends were obtained for the Young's modulus in the transverse plane E₁, but significant differences were also found between the reserve zone and the complete growth plate. The proliferative and hypertrophic zones are half as stiff as the reserve zone along the compression axis and about three times less stiff than the reserve zone in the transverse plane. These two zones are also three times as permeable as the reserve zone in the radial direction. The mechanical behavior of the newborn porcine distal ulna growth plate is non-uniform along its thickness. The reserve zone, with its greater zonal component at that development stage, has noteworthy effects on the complete growth plate intrinsic mechanical properties. This study provides, for the very first time, an investigation of the intrinsic mechanical properties of the reserve, proliferative and hypertrophic zones of the growth plate.     

Expression of basement membrane components through morphological changes in the hair growth cycle

The amount and distribution of fibronectin associated with hair follicles was found to vary during the hair growth cycle in the rat. Immunocytochemical staining of follicles in mid-late anagen (the growth stage) revealed the presence of fibronectin in the dermal papilla matrix, in the basement membrane separating this from the epithelial cells of the hair bulb, and in the basement membrane and connective tissue sheath which underly the cells of the outer root sheath. Early in catagen, the transitional stage, staining of the dermal papilla matrix disappeared. Fibronectin persisted in the basement membrane and connective tissue sheath, which undergo corrugation and apparent thickening in catagen. After follicle shortening, the telogen (resting) stage is reached, at which point fibronectin staining was found to be minimal, being restricted to the basement membrane around the secondary germ. The onset of anagen, involving cell division and follicle elongation, was associated with a great increase in the amount of fibronectin in this zone and in and around the dermal papilla. Analysis of entry into anagen by [3H]thymidine incorporation and autoradiography revealed that growth could be detected before the increase in fibronectin expression. However, growing cells, even in a suprabasal position, always had some fibronectin at their surface. Immunoelectron microscopy of early anagen follicles confirmed the light microscopic findings and also showed that fibronectin was present in small vesicles close to the surface of dermal papilla and some epithelial cells. Increased deposition of laminin and type IV collagen in early anagen follicles was also noted, emphasizing the importance of basement membrane components during morphogenetic events in vivo.     

不同光照条件下三七幼苗形态及生长指标的变化

用遮阳网设置不同透光率(自然全光照的1%、3%、8%、12%和22%)处理,对不同光照条件下三七〔Panax notoginseng(Burk.)F.H.Chen〕幼苗形态指标(株高、冠幅、块根长、主根长、块根直径、茎基径、单株须根数和单株须根长)、干物质积累(不同器官干质量)和分配以及叶片性状(单株叶面积、比叶面积和叶绿素相对含量SPAD值)的变化进行了研究。结果表明:在透光率不同的条件下三七幼苗的形态指标、不同器官干质量及分配以及叶片性状均有明显变化。其中,块根直径、单株须根长、单株须根数、不同器官(块根、须根、根、叶片和茎)干质量和植株总干质量均随透光率增大逐渐提高;株高在透光率22%条件下最高;冠幅和单株叶面积在透光率3%条件下最大;主根长、茎基径、根冠比、根质比及SPAD值均在透光率8%条件下最高;茎质比和叶质比在透光率3%和1%条件下较大;比叶面积随透光率增大逐渐降低。综合分析结果揭示:三七是一种典型的喜阴植物,种植过程中适当遮阳有利于其生长和干物质积累,其中透光率8%对三七幼苗生长较为适宜。