Effects of Loading Orientation on the Morphology of the Predicted Yielded Regions in Trabecular Bone

While the effects of bone mineral density and architecture in osteoporotic bone have been studied extensively, the micromechanics of yielding and failure have received less attention. However, understanding architectural features associated with failure should provide insight into assessing bone quality. In this study, microstructural finite element models were used to compute regions of tissue level yielding in ten bovine tibial trabecular bone samples. The morphology, number, and mean volume of the yielded regions were quantified for four apparent strains under two loading conditions. For on-axis loading, the mean aspect ratio of the tissue that yielded due to compressive strain increased with increasing apparent strain, expanding along the principal trabecular orientation. This suggests that tissue level yielding progresses along vertical trabeculae when a specimen is loaded on-axis. The number, but not the volume, of the regions yielded due to tensile strain increased with increasing applied load, consistent with relaxation and redistribution of stresses around the yielded regions. When the specimens were compressed perpendicular to the principal axis, the aspect ratio of the yielded regions was close to one, while the number, mean volume, and mean thickness of the yielded regions increased. This indicates that localized high strains consistent with bending rather than axial deformation of struts occur at the tissue level. Overall, the results provide new insight into trabecular bone failure, which is relevant to assessing diagnostic tests for fracture risk or evaluating osteoporosis treatments.     

Reorientation of myofilaments during contraction of a vertebrate smooth muscle.

The purpose of the investigation was to determine whether filaments within smooth muscle cells changed their orientation (with respect to the main axis of the cell) during contraction. The stomach muscle of Bufo marinus was used, since its cells may be easily isolated, enabling direct observation in living cells. In addition to still micrography, cinemicrography was used to record continuously during contraction. Polarization microscopy revealed a change in birefringence after contraction, with relaxed cells exhibiting uniform birefringence while contracted cells displayed a discontinuous pattern. Movies revealed a progressive change in orientation of birefringent elements from nearly parallel to the cell's main axis in relaxed cells to increasingly larger angles to the cell's axis as contraction progressed. Phase-contrast microscopy revealed a change in filamentous components, from being parallel to the cell's axis in relaxed cells to being in an undulating or helical pattern during concentration. Cell shape tended to follow the configuration of the filamentous component. Electron microscopy of muscle strips corroborated the observations of living cells and substantiated the conclusion that filaments change their orientation from parallel to oblique (with respect to the cell's axis) during shortening with an undulating or helical pattern of filaments in shortened muscles.     

Influence of an initiating microsplit on the resistance to compression-induced rupture of the articular surface

Cartilage-on-bone samples from bovine patellae containing a defined stellar or linear initiating split in the articular surface were incrementally loaded in direct compression with intervening rehydration, until articular surface rupture occurred. All patellae were either normal or exhibited a mild level of surface fibrillation. In all cases the actual loading site was free of disruption. The average rupture stress of the healthy cartilage was significantly higher than that of the mildly degenerate cartilage, and in both tissue categories average rupture stresses were lower for the linear split morphology than for the stellar. We propose that this contrasting rupture behavior is explained by differences in both secondary lineal surface strains associated with the depth of compressive indentation and in the ability of the fibrillar network within the surface layer to re-arrange itself in the localized regions of stress concentration around the initiating split.     

Measuring integrated cellular mechanical stress response at focal adhesions by optical tweezers

The ability of cells to sustain mechanical stress is largely modulated by the cytoskeleton. We present a new application of optical tweezers to study cell's mechanical properties. We trap a fibronectin-coated bead attached to an adherent H4II-EC3 rat hepatoma cell in order to apply the force to the cell surface membrane. The bead position corresponding to the cell's local mechanical response at focal adhesions is measured with a quadrant detector. We assessed the cell response by tracking the evolution of the equilibrium force for 40 cells selected at random and selected a temporal window to assess the cell initial force expression at focal adhesions. The mean value of the force within this time window over 40 randomly selected bead∕cell bounds was 52.3 pN. Then, we assessed the responses of the cells with modulation of the cytoskeletons, namely the ubiquitous actin-microfilaments and microtubules, plus the differentiation-dependent keratin intermediate filaments. Notably, a destabilization of the first two networks led to around 50 and 30% reductions in the mean equilibrium forces, respectively, relative to untreated cells, whereas a loss of the third one yielded a 25% increase. The differences in the forces from untreated and treated cells are resolved by the optical tweezers experiment.     

Influence of an Initiating Microsplit on the Resistance to Compression-Induced Rupture of the Articular Surface

Cartilage-on-bone samples from bovine patellae containing a defined stellar or linear initiating split in the articular surface were incrementally loaded in direct compression with intervening rehydration, until articular surface rupture occurred. All patellae were either normal or exhibited a mild level of surface fibrillation. In all cases the actual loading site was free of disruption. The average rupture stress of the healthy cartilage was significantly higher than that of the mildly degenerate cartilage, and in both tissue categories average rupture stresses were lower for the linear split morphology than for the stellar. We propose that this contrasting rupture behavior is explained by differences in both secondary lineal surface strains associated with the depth of compressive indentation and in the ability of the fibrillar network within the surface layer to re-arrange itself in the localized regions of stress concentration around the initiating split.     

Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix.

Lapine and canine marrow stromal cells were found to contain a contractile actin isoform, alpha-smooth muscle actin (SMA), by immunohistochemistry and Western blot analysis. The SMA was found to be incorporated into stress fibers that were prominently displayed by the cells in monolayer culture. The cell content of this actin isoform increased with passage number. The contractility of SMA-expressing stromal cells was demonstrated by their contraction of collagen-glycosaminoglycan analogs of extracellular matrix into which they were seeded. The demonstration that marrow-derived stromal cells express the SMA gene may explain recent findings of this expression in musculoskeletal connective tissue cells including osteoblasts, chondrocytes, and fibrochondrocytes that may be derived from this mesenchymal stem cell. The implications of these findings for tissue engineering strategies employing marrow stromal cells are also discussed.     

Fibrocartilage tissue engineering: the role of the stress environment on cell morphology and matrix expression

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development.     

Histochemical Analyses of Tissue-Engineered Human Menisci

The field of tissue engineering remains one of the least explored areas of current meniscal research but holds great promise. In this investigation, meniscal fibrochondrocytes were isolated from fresh human meniscal tissue and seeded onto synthetic polyglycolic acid (PGA) scaffolds. Constructs were implanted into the dorsal subcutaneous space of athymic nude mice. Control scaffolds, devoid of meniscal cells, were simultaneously implanted in additional mice. Constructs were harvested over 12 weeks and treated with a variety of histochemical stains to analyze general specimen morphology, cellular viability and proliferation, and collagen secretion. Results indicate that meniscal fibrochondrocyte proliferation increased over the time of implantation with cellular consolidation occurring as the PGA scaffolding was progressively hydrolyzed. Collagen production also increased over time. There were favorable similarities between constructs and human meniscal controls in terms of cellular morphology, phenotypic expression, and collagen production. These initial findings demonstrate procedures supporting proliferation of meniscal fibrochondrocytes, expression of fibrochondral phenotype, and the formation of putative meniscal tissue.     

Platelet derived growth factor-AB enhances knee meniscal cell activity in vitro

Meniscal healing especially in the inner avascular region has always been a major challenge. In this study we investigated the potential for platelet derived growth factor-AB (PDGF-AB) to promote meniscal tissue regeneration in the inner (avascular), middle, and outer (vascular) zones of the meniscus. Various concentrations of PDGF-AB were tested on sheep meniscal cell cultures. We used the radioactive thymidine uptake assay to assess cell proliferation, and the radioactive sulphur and proline uptake assays and Blyscan assay to assess matrix formation. In general, PDGF-AB stimulated both cell proliferation and matrix formation by cells from all meniscal zones. PDGF-AB at a concentration of 100 ng/ml increased cell proliferation and matrix formation by eight and four fold respectively, by fibrochondrocytes cultured from all meniscal zones (p<0.001). These results indicate that fibrochondrocytes present within the avascular region of the meniscus have the ability to proliferate and form new matrix when exposed to anabolic cytokines such as PDGF-AB.     

Porcine mitral valve interstitial cells in culture

There are connective tissue cells present within the interstitium of the heart valves. This study was designed to isolate and characterize mitral valve interstitial cells from the anterior leaflet of the mitral valve. Explants obtained from the distal part of the leaflet, having been scraped free of surface endocardial cells, were incubated in medium 199 supplemented with 10% fetal bovine serum. Cells grew out of the explant after 3 to 5 days and by 3 weeks these cells were harvested and passaged. Passages 1 to 22 were characterized in several explant sets. The cells showed a growth pattern reminiscent of fibroblasts. Growth was dependent on serum concentration. Cytoskeletal localization of actin and myosin showed prominent stress fibers. Ultrastructural studies showed many elongated cells with prominent stress fibers and some gap junctions and few adherens junctions. There were as well cells with fewer stress fibers containing prominent Golgi complex and dilated endoplasmic reticulum. In the multilayered superconfluent cultures, the former cells tended to be on the substratum of the dish or surface of the multilayered culture, whereas the latter was generally located within the layer of cells. Extracellular matrix was prominent in superconfluent cultures, often within the layers as well. Labeling of the cells with antibody HHF 35 (Tsukada T, Tippens D, Gordon D, Ross R, Gown AM: Am J Pathol 126:51, 1987), which recognizes smooth muscle cell actin, showed prominent staining of the elongated stress fiber-containing cells and much less in the secretory type cells. These studies show that interstitial mitral valve cells can be grown in culture and that either two different cell types or one cell type with two phenotypic expressions is present in culture.     

An MRI-based leg model used to simulate biomechanical phenomena during cuff algometry: a finite element study

Cuff pressure stimulation is applicable for assessing deep-tissue pain sensitivity by exciting a variety of deep-tissue nociceptors. In this study, the relative transfer of biomechanical stresses and strains from the cuff via the skin to the muscle and the somatic tissue layers around bones were investigated. Cuff pressure was applied on the lower leg at three different stimulation intensities (mild pressure to pain). Three-dimensional finite element models including bones and three different layers of deep tissues were developed based on magnetic resonance images (MRI). The skin indentation maps at mild pressure, pain threshold, and intense painful stimulations were extracted from MRI and applied to the model. The mean stress under the cuff position around tibia was 4.6, 4.9 and around fibula 14.8, 16.4 times greater than mean stress of muscle surface in the same section at pain threshold and intense painful stimulations, respectively. At the same stimulation intensities, the mean strains around tibia were 36.4, 42.3 % and around fibula 32.9, 35.0 %, respectively, of mean strain on the muscle surface. Assuming strain as the ideal stimulus for nociceptors the results suggest that cuff algometry is less capable to challenge the nociceptors of tissues around bones as compared to more superficially located muscles.     

Cat retinal ganglion cells: size and shape of receptive field centres

1. Receptive field centres of 144 sustained and transient retinal ganglion cells were mapped in cats under light pentobarbitone anaesthesia.2. Sustained on-centre, sustained off-centre, transient on-centre and transient off-centre cells had different mean sizes of receptive field centre, with some overlap between their distributions.3. For each class of cell, central fields had the smallest field-centres; progressively larger field-centres were encountered more peripherally.4. All classes of ganglion cells tended to have slightly elliptical receptive field centres. Major axes of over half of all receptive fields were oriented within 20 degrees of horizontal. These trends were independent of pupil dimensions, or of receptive field eccentricity or position in the visual field. The results almost certainly reflect asymmetry in retinal wiring.5. Two cells of thirty-nine tested were sensitive to axis of motion; in both cases the preferred and major axis were horizontal. A further cell was orientation specific.     

组织块法与酶消化法培养大鼠毛囊干细胞的比较

背景：研究证实毛囊干细胞比毛囊间表皮干细胞更有增生能力,近年来受到广泛关注,成为种子细胞的研究热点。 目的：比较组织块法和两步酶法培养大鼠毛囊干细胞的生物学特性。 方法：体式显微镜下分离大鼠触须部的毛囊,分别用组织块法和两步酶法培养毛囊干细胞,利用反复差速贴壁法纯化细胞,定期观察细胞生长状况及形态,流式细胞仪检测第3代毛囊干细胞CD34、β1整合素的表达。 结果与结论：两步酶法获得的细胞生长速度快,获得的细胞量多,而组织块法获得的细胞生长速度较慢,获得的细胞量也少。流式细胞仪分析显示酶消化法培养组PE-CD34、FITC-β1整合素的表达分别为(39.52±19.57)%和(93.46±4.73)%,组织块法培养组相应为(19.20±11.53)%和(363.57±14.42)%,两组间差异有显著性意义(P < 0.05)。总的来说,两种方法均能培养出实验所需毛囊干细胞,可根据不同实验需求选择恰当的培养方法。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞的分化

背景：颞下关节盘软骨缺损修复在口腔临床上仍然是较大的挑战,具有多向分化潜能的脂肪间充质干细胞为成纤维软骨类组织修复带来了希望。目前使用转化生长因子β3诱导脂肪间充质干细胞向纤维软骨细胞分化的研究很少。 目的：观察转化生长因子β3对脂肪间充质干细胞生长形貌及向成纤维软骨分化的影响。 方法：采用转化生长因子β3诱导SD大鼠脂肪间充质干细胞,观察成纤维软骨细胞分化的细胞形态,组织学和免疫荧光染色等方法检测脂肪间充质干细胞产生的细胞外基质Ⅰ,Ⅱ型胶原和蛋白多糖表达情况,评价脂肪间充质干细胞作为纤维软骨组织工程种子细胞的可行性。 结果与结论：倒置荧光显微镜观察结果显示脂经转化生长因子β3生长因子诱导之后,细胞有明显的聚集生长现象,形态呈多角形、多边形,细胞外基质分泌增多。阿利新蓝染色结果表明,经转化生长因子β3诱导脂肪间充质干细胞显示明显的深蓝色,表明脂肪间充质干细胞合成了大量的糖胺聚糖。免疫染色结果表明,在转化生长因子β3 诱导下,脂肪间充质干细胞合成Ⅰ,Ⅱ型胶原细胞外基质。提示转化生长因子β3可诱导脂肪间充质干细胞向成纤维软骨样细胞分化,也意味着脂肪间充质干细胞具有作为工程化纤维软骨种子细胞的潜能。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

In vitro study of the effect of cyclic strains on the dermal fibroblast (GM3384) morphology--mapping of cell responses to strain field

Cells can respond to mechanical forces and actively interact with mechanical stimulations in vitro. Understanding the effect of mechanical loading on cell morphology signifies a critical biomechanics issue in tissue engineering. In this study, human dermal fibroblasts (GM3384) underwent cyclic strain. This was done by culturing a monolayer of the cells onto a transparent membrane and applying a cyclic stress using a computer controlled bioreactor. The cells were mechanically stimulated at around 7% strain with 1 cycle per minute for 2 days. Finite element analysis (FEA) was then employed to characterize the strain field across the substrate membrane in the bioreactor. The results showed that strain distribution were non-uniform in the substrate membrane. The mapping of cell morphology with the strain field revealed that the cells exposed to the equibiaxial strain exhibited the classical spindle morphology while the cells subjected to uniaxial strain changed to a polygonal morphology. It is concluded that the nature of the strain has significant impact on the final cell morphology.     

The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(L-lactide-co-epsilon-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm(2). The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm(2)) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm(2)) to a high shear stress (19.6 dyn/cm(2)) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.     

Mechanical Interactions of Mouse Mammary Gland Cells with Collagen in a Three-Dimensional Construct

An effort to understand the development of breast cancer motivates the study of mammary gland cells and their interactions with the extracellular matrix. A mixture of mammary gland epithelial cells (normal murine mammary gland), collagen, and fluorescent beads was loaded into microchannels and observed via four-dimensional imaging. Collagen concentrations of 1.3, 2, and 3 mg/mL were used. The displacements of the beads were used to calculate strains in the 3D matrix. To ensure physiologically relevant materials properties for analysis, the collagen was characterized using independent tensile testing with strain rates in the range of those measured in the cell–gel constructs. 3D elastic theory for an isotropic material was employed to calculate the stress. The technique presented adds to the field of measuring cell-generated stresses by providing the capability of measuring 3D stresses locally around a single cell and using physiologically relevant materials properties for analysis. The highest strains were observed in the most compliant matrix. Additionally, the stresses fluctuated over time due to the cells’ interaction with the collagen matrix.     

组织块法培养SD大鼠的成骨细胞及鉴定

背景：获得足够量高纯度的成骨细胞比较困难,因此掌握简单而快速提取成骨细胞并进行培养鉴定势在必行。 目的：应用组织块法对SD大鼠成骨细胞的体外培养与鉴定。 方法：取新生(<24 h)SD大鼠颅骨,去除周围多余的组织,将剔净颅骨剪成1 mm3大小的碎块,分别用常规方法和组织块法(改良)方法进行成骨细胞培养,从形态学、碱性磷酸酶和茜苏红染色等方法鉴定。 结果与结论：利用改良方法培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶呈阳性染色,茜素红染色后有矿化结节的形成。组织块法培养的成骨细胞具有典型的成骨细胞成分单一、细胞培养时间短、数量、纯度、密度均一致,从而的得到稳定的成骨细胞系,为进行体外实验建立了良好的平台。     

The alveolar macrophage delivery system. Kinetic studies in cultured explants of murine lung.

The production of alveolar macrophages in a blood-free organ culture system has been studied to determine whether free macrophages undergo mitosis or whether their ongoing production is dependent upon continuing division of interstitial cells. Explants of murine long were attached to cellophane or glass; after 6 days of culture, a population of cells identified morphologically and functionally as macrophages appeared around the central tissue. These cells did not divide, and they disappeared 4 days after removal of the central lung explant. 3H-thymidine labeling of these peripheral macrophages was observed only when the central tissue was present and when the thymidine pulse was 24 hours in duration. Actual cell division was observed only in the interstitial cells of the explant. It is concluded that the interstitial cell population provides a continuing pool of precursor cells that divide and migrate outwards, creating a "steady state" system in which macrophage loss at the periphery is balanced by cell production at the center.     

The CNS-PNS transitional zone of the rat. Morphometric studies at cranial and spinal levels.

The transitional zone is that length of rootlet containing both central and peripheral nervous tissue. The CNS-PNS interface may be defined as the basal lamina covering the intricately interwoven layer of astrocyte processes which forms the CNS surface and which is pierced by axons passing between the CNS and PNS. Study of transitional zone development defines morphologically the growth, relative movement and interaction of central and peripheral nervous tissues as they establish their mutually exclusive territories on either side of the CNS-PNS boundary, and helps to explain the wide variations in the form of the mature transitional zone. Nerve rootlets at first consist of bundles of bare axons. These become segregated by matrices of fine Schwann cell processes peripherally and of astrocyte processes centrally. The latter may prevent Schwann cell invasion of the CNS. Astrocyte processes branch profusely and come to form the principal central nervous tissue component of the transitional zone. Developmental changes in the transitional zone vary markedly between nerves, reflecting differences in its final morphology. Widespread relative movements and migration of CNS and PNS tissues take place during development, so that the central-peripheral interface changes shape and position, commonly oscillating along the proximodistal axis of the rootlet. For example, developing cervical ventral rootlets contain a transient central tissue projection, while that of lumbar ventral rootlets and to a lesser extent that of cervical dorsal rootlets alternately increase and decrease in length. In the developing cochlear nerve, a central tissue projection is present before birth, but regresses somewhat before a marked outgrowth of central nervous tissue along the nerve takes place, which reaches into the modiolus during the first week postnatum. During development, some astrocytic tissue may even break off and migrate distally into the root, giving rise to one or more glial islands within it. During the period immediately preceding birth, Schwann cells come to be present in very large numbers in that part of the rootlet immediately distal to the CNS-PNS interface, the proximal rootlet segment. Here they form prominent sleeves or clusters of closely packed cells which intertwine with and encapsulate one another on the rootlet surface. Such Schwann cell overcrowding in the proximal rootlet segment could result in part from distal overgrowth of the rapidly expanding CNS around axon bundles, which might strip the Schwann cells distally off the bundle segments so engulfed.(ABSTRACT TRUNCATED AT 400 WORDS)