Neutron diffraction measurements of skeletal muscle using the contrast variation technique: analysis of the equatorial diffraction patterns.

Among various methods for structural studies of biological macromolecules, neutron scattering and diffraction have a unique feature in that the contrast between the scattering length density of the molecules and that of the solvent can be varied easily by changing the D2O content in the solvent. This "contrast variation" technique enables one to obtain information on variations of scattering length density of the molecules of interest. Here, in order to explore the possibilities of the contrast variation technique in neutron fiber diffraction, neutron diffraction measurements of skeletal muscles were performed. The neutron fiber diffraction patterns from frog sartorius muscles were measured in various D2O concentrations in the relaxed state where no tension of muscle is produced, and in the rigor state where all myosin heads of the thick filaments bind tightly to actin in the thin filaments. It was shown that in both states, there were reflections having distinct contrast matching points, indicating a variation in the scattering length density of the protein regions in the unit cell of the muscle structure. Analysis of the equatorial reflections by the two-dimensional projected scattering length density map calculations by Fourier synthesis and model calculations provided the phase information of the equatorial reflections, with which the projected scattering length density maps of the unit cell of the hexagonal filament array in both states were calculated. The analysis showed that the scattering length density of the thick filament region was higher than that of the thin filament region, and that the scattering length density of the thick filament backbone changed as muscle went from the relaxed state into the rigor state.

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels.

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.

Quality control of cultured tissues requires tools for quantitative analyses of heterogeneous features developed in manufacturing process.

Tissue engineering and related technology have attracted a great deal of medical attention as promising fields for curing defective tissues in vivo. Nowadays, many companies have been established for supplying the reconstructed grafts of cultured tissues for transplantation. The manufacturing processes generally deals with the handlings of starter cells offered by patients (or donors) as raw materials to cultured tissues as products, requiring the construction of novel ex vivo methodologies based on principles different from conventional processes for chemical and pharmaceutical productions. In addition, the raw materials have heterogeneity depending on the state of patients and location of cell harvests, and the products possess spatial cell distribution in the three dimensional structure. These features request a unique strategy in manufacturing process accompanied with the quality control for raw materials and products. This review article describes the contribution of tissue bankers and biochemical engineers to the quality control of cultured tissues during manufacturing, introducing the advances in methodologies to evaluate spatial heterogeneity of cells (or aggregates) and matrices in cultured tissues.

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction.

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by approximately 0.1% upon activation relative to the relaxing state and increased by approximately 0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca(2+)-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca(2+)-binding and the second induced by actomyosin interaction.

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction.

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.

Morphological regulation of rabbit chondrocytes on glucose-displayed surface.

A culture surface was designed to regulate morphology of rabbit chondrocytes by changing the ratio of D- and L-glucose isomers displayed on a glass plate. With increasing ratio of d-glucose displayed on the surfaces, the efficiency of cell attachment improved, meaning that the attachment exclusively occurred via mediation of an affinity between D-glucose displayed and glucose transporter on cell membrane. At 0% and 100% D-glucose display, the round-shaped cells appeared dominantly, and most of cells became stretched in shape at 50% d-glucose display, indicating that the frequency of round-shaped cells depicted a concave profile against the ratio of D-glucose displayed. From the cytoskeletal staining of F-actin and vinculin, the immature stress fibers with fewer focal contacts were recognized in both the round shaped cells and those stretched in shape on 100% D-glucose-displayed surface. The time-lapse observation revealed that the cells on 100% D-glucose-displayed surface conducted active migration and aggregation with formation of collagen type II. These results suggest that 100% D-glucose-displayed surface can offer culture environment to maintain the chondrocytic phenotype of cells, similarly to the conditions achieved in three-dimensional (3-D) culture.

Structural alterations of thin actin filaments in muscle contraction by synchrotron X-ray fiber diffraction.

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Modeling analysis of myosin-based meridional X-ray reflections from frog skeletal muscles in relaxed and contracting states.

Analysis of the myosin-based meridional intensity data in the X-ray diffraction patterns of live frog skeletal muscles was performed to propose a more precise model for a myosin crown periodicity and an axial disposition of two-headed crossbridges along the thick filament in a sarcomere. Modeling studies revealed that the thick filament has a mixed structure of two different periodicities of the myosin crossbridge crown arrangement and that the crown periodicity and the axial disposition of crossbridges are altered when muscle goes from the relaxed state to the contracting state. Factors that primarily affect the meridional intensities were examined.

Network formation through active migration of human vascular endothelial cells in a multilayered skeletal myoblast sheet.

Autologous transplantation of myoblast sheet has attracted attention as a new technique for curing myocardial infarction. Myoblast sheet has the ability to secret cytokines that improve heart function via the facilitation of angiogenesis on affected part. To mimic the in vivo angiogenesis in the myoblast sheet after transplantation, a five-layered cell sheet of human skeletal muscle myoblasts (HSMMs) was overlaid on human umbilical vein endothelial cells (HUVECs) which enables evaluation of dynamic HUVEC behavior. HUVECs existing initially at the bottom of the sheet changed to be a stretched shape and migrated upward compared with the surrounding HSMMs in the sheet. Prolonged incubation resulted in network formation of HUVECs in the middle of the sheet, although non-networked HUVECs continued to migrate to the top of the sheet, which meant the spatial habitation of HUVECs in the cell sheet. Image processing was performed to determine the variation in the extent of network formation at different HUVEC densities. It was found that the extent of formed network depended on the frequency of encounters among HUVECs in the middle of the sheet. The present system, which can evaluate network formation, is considered to be a promising in vitro angiogenesis model.

Evaluation of vertical cell fluidity in a multilayered sheet of skeletal myoblasts.

The procedure for fabricating a multilayered cell sheet has been developed by combining multiple sheets using a thermo-responsive surface and stamp system. Confocal laser scanning microscopy revealed that the fluidity of a multilayered sheet of skeletal myoblasts could be estimated as vertical diffusivity and changed upon addition of dermal fibroblasts.

X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles.

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

Growth and differentiation potentials in confluent state of culture of human skeletal muscle myoblasts.

The transitional behaviors of myoblasts toward differentiation were investigated in the cultures at the low and high seeding densities (respectively, X(0)=1.0x10(3) and 2.0x10(5) cells/cm(2)). In the culture at the low seeding density, an increase in confluence degree accompanied a decrease in growth potential (R(p)), being R(p)=0.85 and 0.11 at t=48 and 672 h, respectively. Myoblasts seeded at the high density resulted in the immediate cessation of growth with keeping the low range of R(p)=0.02-0.09 throughout the culture. The reduction of R(p) led to the generation of three subpopulations of cells in proliferative, quiescent and differentiated states. Close cell contacts in the confluent state of high seeding culture induced cell quiescence to a higher extent with suppressing differentiation.

Morphological evaluation of chondrogenic potency in passaged cell populations.

The present study describes the morphological assessment of chondrogenic potency during a cell expanding process through serial subculturing of rabbit chondrocytes at different levels of population doublings (PD) in a T-flask with a conventional polystyrene surface. The passaged populations were seeded on a high-density collagen surface (CL surface) and in a collagen gel (CL gel) scaffold to evaluate the planar and spatial morphologies of the chondrocytes, respectively, as well as the gene expressions of mRNA for collagen types I and II. The planar morphological estimation was based on roundness (R(c)) of chondrocyte cells at different PD values after 1 day incubation on the CL surface. The frequency of round-shaped cells with R(c)>0.9 (f(R)) decreased with increasing PD values, accompanied by an increase in collagen type I mRNA level. At PD=17.8, the frequency reached f(R)=0.12, which was less than one-sixth of that at PD=0. A similar trend was found with respect to the passaged chondrocytes embedded in the CL gels by estimating the spatial morphology in terms of sphericity (S(c)) determined 4 days after seeding. With an increase in PD value, the frequency in spherical-shaped cells with S(c)>0.9 (f(S)) decreased and the mRNA expression of collagen type I increased, giving f(S)=0.28 at PD=17.8 which was less than a quarter of that at PD=0. From these results, the cell morphologies on the CL surface and in the CL gel were proposed as indicators for understanding chondrogenic potentials concerning the phenotypes and differentiated states in the population during cell expansion, ultimately leading to quality control of tissue-engineered cartilage.

Synergic stimulation of laminin and epidermal growth factor facilitates the myoblast growth through promoting migration.

The dynamic behaviors of human skeletal muscle myoblasts were investigated in the culture on a laminin-coated surface in the presence of 100 ng/ml epidermal growth factor (EGF) in medium. The coexistence of laminin and EGF caused the enhancement of myoblast migration, giving an average migration rate of 62.0 microm/h, which was 2.7 times that on a plain surface. This encouraged migration could be a driving force to separate the dividing cells from each other, accompanied by shortened disjunction time of daughter cells to complete cytokinesis. In addition, the synergic effect of laminin and EGF led to the promotion of myoblast growth with keeping a relatively high fraction of proliferative cells during the culture for 150 h, which is considered to arise from the reduced frequency of cell-cell contacts during cytokinesis and thereby suppressing the process towards myotube formation after cell division.

Effect of transforming growth factor-beta1 on morphological characteristics relating to migration and differentiation of rabbit chondrocytes cultured in collagen gels.

The influence of transforming growth factor-beta1 (TGFbeta1) on the behavior of rabbit chondrocytes embedded in collagen gels was examined in terms of cell migration and consequent architecture of cell aggregation. In a low-seeding density culture (X(0)=2.0 x 10(5) cells/cm(3)) TGFbeta1 (0-10.0 ng/ml) was added and observed during a 14-d culture period. Stereoscopic observation was performed on 5 d employing the morphology-related parameter of sphericity (S(c)) for individual cells in the gels. The frequency of migrating cells with S(c) less than 0.95 increased in a dose-dependent manner in response to TGFbeta1. Moreover, the frequency of migrating cells in the culture with 10.0 ng/ml TGFbeta1 was 0.32, two times higher than that in the reference culture without TGFbeta1, while the frequency of dividing cells in the same culture was less than half of that in the reference culture. The histological observation of cultured gels on 14 d revealed that the starburst and loose aggregates with the spindle-shaped cells emerged in the TGFbeta1-free culture, accompanying the poor production of collagen type II by the cells. On the other hand, the spherical-shaped cells were observed in the starburst aggregates with rich excretion of collagen type II in the culture with 5.0 ng/ml TGFbeta1. Moreover, the mRNA levels of differentiation-marker genes (collagen types I and II) were regulated in accordance with the morphological analyses concerning the cell migration and aggregation in the cultures with and without TGFbeta1. From these results, it was concluded that TGFbeta1 had a culture time-dependent effect on the morphological characteristics relating to the migration and differentiation of the chondrocytes in the collagen gel-embedded cultures seeded at low density, that is, the growth factor promotes cell migration with deteriorated proliferation in the early culture phase, and accelerates the transformation of spindle-shaped cells to spherical-shaped ones in the prolonged culture.

Rigor-force producing cross-bridges in skeletal muscle fibers activated by a substoichiometric amount of ATP.

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of approximately 100 microM ATP, approximately 2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (approximately 50% of the maximal active force), which gradually recovered to the original force level with a time constant of approximately 4 s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1-1.9 s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.