An image-processing technique for measuring the dynamic movement of cell membranes

An automated method has been developed to measure and compare the dynamic movement of cell membranes. Using the red blood cell as a common example the method locates the edge of the cell, with sub-pixel precision at multiple points on the periphery. This method is a different implementation to a technique used for giant unilamellar vesicles and addresses issues relating specifically to biological cells, in particular relating to finding a local minima, calculating equi-spaced measuring points for arbitrary shapes and using the perpendicular direction to the edge for position measurement. Parameters have been defined to characterise the cell's membrane behaviour and the analysis program allows the automatic compilation of multiple tests under varying conditions, and statistical comparison of identical populations of cells.     

Influence of different intensities of vibration on proliferation and differentiation of human periodontal ligament stem cells

Introduction

To understand the effects of low-magnitude, high-frequency (LMHF) mechanical vibration at different intensities on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation.

Material and methods

The effect of vibration on hPDLSC proliferation, osteogenic differentiation, tenogenic differentiation and cytoskeleton was assessed at the cellular, genetic and protein level.

Results

The PDLSC proliferation was decreased after different magnitudes of mechanical vibration; however, there were no obvious senescent cells in the experimental and the static control group. Expression of osteogenesis markers was increased. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA was up-regulated at 0.1 g, 0.3 g, 0.6 g and 0.9 g magnitude, with the peak at 0.3 g. The type I collagen (Col-I) level was increased after vibration exposure at 0.1 g, 0.3 g, and 0.6 g, peaking at 0.3 g. The expression levels of both mRNA and protein of Runx2 and osterix (OSX) significantly increased at a magnitude of 0.1 g to 0.9 g, reached a peak at 0.3 g and then decreased slowly. The scleraxis, tenogenic markers, and mRNA expression decreased at 0.05 g, 0.1 g, and 0.3 g, and significantly increased at 0.6 g and 0.9 g. Compared with the static group, the F-actin stress fibers of hPDLSCs became thicker and clearer following vibration.

Conclusions

The LMHF mechanical vibration promotes PDLSC osteogenic differentiation and implies the existence of a magnitude-dependent effect of vibration on determining PDLSC commitment to the osteoblast lineage. Changes in the cytoskeleton of hPDLSCs after vibration may be one of the mechanisms of the biological effects.     

Evaluation of an experimental periodontal ligament for dental implants.

To improve the devices used in dental implantology, a new experimental supporting element has been developed. The device, made of titanium, poly(methylmethacrylate) and Dacron tissue, has been tested for the osteointegrability of its outer Dacron coat by implants in mandibular bone of rabbits. The Dacron filamentous tissue became incorporated by the bone at 3 month post-implantation. This might provide a reliable mechanical anchorage of the device and a barrier against epithelial proliferation and microbial contamination. Preliminary results relating to in vitro evaluation of the poly(methyl methacrylate)-Dacron fixation strength predict favourable mechanical behaviour at this interface and in the complete implanted device.     

In vivo measurement of the elastic modulus of the human periodontal ligament.

The present study was designated to determine the elastic properties of the periodontal ligament (PDL) in human subjects. A maxillary central incisor was experimentally translated so that stress or strain could be uniformly distributed in the PDL by applying a single force passing through the center of resistance. Displacements were measured under different magnitudes of load using a magnet-magnetic sensing system. From the load-displacement relations, Young's modulus of the PDL was calculated. The values determined were approximately 0.12 MPa under load ranging from 0 to 0.5 N, 0.25 MPa within the range of 0.5-1.0 N, 0.44 MPa under load 1.0-1.5 N, and between 0.69 and 0.96 MPa with 1.5-2.0 N. The values of Young's moduli increased almost exponentially with the increment of load due to a non-linear elasticity of the PDL.     

Innervation of the periodontal ligament in the dog with special reference to the morphology of Ruffini endings.

The distribution and terminal formation of nerves in the periodontal ligament of dog incisors and canines were investigated by immunohistochemistry for neurofilament protein (NFP) and by electron microscopy. The NFP-immunoreactive nerve fibers were found to be densely distributed in the apical third of the periodontal ligament, while they were sparse in the coronal two thirds. Most of the nerve endings in the periodontal ligament showed a tree-like appearance and resembled those nerve endings demonstrated in the periodontal ligament of human and monkey under the category of free nerve endings. Presumable axon terminals of these were slightly thicker than preterminal portions, running along periodontal collagen fibers and tapering within them. In light microscopic images, at least, they differed from the Ruffini endings which are commonly seen in rodents, displaying a glove-like configuration with extremely expanded tips. Under the electron microscope, however, the tree-like endings of the dog appeared similar to the Ruffini endings of rodents: their terminals were filled with mitochondria, covered with a cytoplasmic process of a Schwann cell, and surrounded by collagen fibers. These ultrastructural findings, combined with the results of previous physiological studies suggest that the nerve endings demonstrated in the present study can be identified as Ruffini endings. It is even stressed that the dog-type of Ruffini ending can be regarded as a representative of the sensory receptors in the mammalian periodontal ligament. In addition to these endings, knobbed endings, corpuscular (lamellated and glomerular) endings, and free nerve endings were rarely encountered in the periodontal ligament of incisors and canines of the dog.     

新型壳聚糖-胶原支架与牙周膜细胞的生物相容性

背景：目前胶原作为牙周组织工程支架材料仍具有机械强度差、降解速度快等缺点,将其与壳聚糖复合可改善上述问题。 目的：评估新型壳聚糖-胶原支架材料的体外生物相容性。 方法：通过MTT法评估100%,75%,50%,25%壳聚糖-胶原支架材料浸提液对人牙周膜细胞的毒性。选择第4-6代生长状态良好的人牙周膜细胞与壳聚糖-胶原支架共培养,观察细胞在支架上的生长情况,并检测与壳聚糖-胶原支架复合培养前后人牙周膜细胞碱性磷酸酶活性的变化。 结果与结论：新型壳聚糖-胶原支架具有双层结构,一侧表面致密,一侧表面疏松多孔。MTT法检测不同浓度材料浸提液毒性评级为0或1级。扫描电子显微镜及组织学观察可见细胞在壳聚糖-胶原支架上增殖良好,且致密层可起屏障膜作用,阻挡细胞进入支架内部;复合培养24 h后,人牙周膜细胞的碱性磷酸酶活性与复合培养前无明显差异(P > 0.05),复合培养48,72 h后人牙周膜细胞的碱性磷酸酶活性高于复合培养前(P < 0.05)。以上结果提示新型壳聚糖-胶原支架具有良好的生物相容性及屏障功能,可进一步应用于牙周组织工程的研究。     

Response of periodontal ligament cells to orthodontic force: ultrastructural identification of proliferating fibroblasts.

The morphologic response of periodontal ligament (PDL) cells in an area of tension created by orthodontic force has been assessed by transmission electron microscopy. Young adult male rats were sacrificed at 24, 48, 72, 96 and 120 hours following orthodontic stimulation. The earliest detectable response was the appearance of increased numbers of mitotic cells in the PDL at 24 hours post-stimulation. The most significant ultrastructural feature of these cells was the presence of intracellular vesicles containing collagen microfibrils. These vesicles were identical to profiles present in interphase PDL fibroblasts involved in collagen phagocytosis associated with turnover of the ligament. Between 48 and 120 hours the alveolar bone surface in the region examined was characterized by the presence of newly generated osteoblasts and active bone formation. Intracellular collagen was never observed in osteoblasts. These observations suggest that at least a portio- of the population of PDL cells which proliferate in response to orthodontic force represent functional ligament fibroblasts.     

Progenitor cell populations in the periodontal ligament of mice

Stem cells in a variety of renewal tissues exhibit a slow rate of cell proliferation (Potten et al., 1979). The periodontal ligament of mouse molars was examined for the presence of slowly cycling progenitor cells to provide evidence for the existence of stem cells in this tissue. A pulse injection of 3H-thymidine was administered and mice were sacrificed between 1 hour and 14 days after injection. Analysis of radioautographs using percentage of labeled cells and grain counts demonstrated that a population of label-retaining cells within 10 micron of blood vessels traversed the cell cycle more slowly than proliferating cells located greater than 10 micron from blood vessels. These data suggest that there is a slowly dividing population of progenitor cells in paravascular sites in mouse molar periodontal ligament which may be stem cells.     

Graft placement after the anterior cruciate ligament reconstruction: a new method to evaluate the femoral and tibial placements of the graft.

In order to evaluate graft placement of the anterior cruciate ligament (ACL) reconstruction and its relationship to the clinical outcome of the patient, 100 patients were examined 7 years after an ACL reconstruction. Clinical assessment was performed using the standard knee ligament evaluation form of the International Knee Documentation Committee (IKDC), and the Lysholm and the Marshall knee scores. We developed a new evaluation method of graft placement after an ACL reconstruction, 'the sum score of the graft placement', which takes into account both the femoral and the tibial positions of the graft simultaneously, and our study showed that 'the sum score of the graft placement' has an association to clinical outcome of patients and that it can better explain the long-term osteoarthritic changes at the injured knee than the separate measurements of the femoral and tibial tunnel placements.     

Optimizing the dynamic characteristics of a pneumotachometer's measuring unit

Conclusions 1. A study has been made on the DC of pneumotachometers in order to decrease their dynamic error for which the relative value is particularly large when measuring the maximal volume velocity of forced expiration by healthy people.2. The condition for minimizing the dynamic error of a pneumotachometer is to optimize (according to a criterion of minimal duration for the transient response) the degree of damping for the movable system in the instrument's measuring unit, i.e., make it equal to unity.3. A selection in accord with the procedure developed of the optimal DC and the calculation of the elements that determine their parameters in the design of the measuring unit makes it possible, based on a given value of the relative dynamic measuring error, to plan an optimal design for a pneumotachometer.Kiev Production Union Medapparatura. Translated from Meditsinskaya Tekhnika, No. 2, pp. 39–44, March–April, 1984.     

3H-mannose utilization by fibroblasts of the periodontal ligament

The synthesis, intracellular translocation, and secretion of mannose-containing glycoproteins(s) by periodontal ligament fibroblasts have been investigated by means of electron microscopic radioautography. Tritiated mannose was administered to young mice via jugular vein, and radioautographs were prepared at 5, 10, 20, and 35 minutes, 4 and 8 hours after injection. Analysis of electron microscopic radioautographs revealed a maximum labeling (94%) with 3H-mannose of the rough endoplasmic reticulum at 5 minutes. Labeling of the Golgi components started to increase from 10 minutes (14%) and reached a maximum level at 20 minutes (31.2%). At 35 minutes, secretion granules, dense bodies, profiles of intracellular collagen, and the cell surface were labeled. At 8 hours, most labelling (79.2%) was extracellular, and associated either with the collagenous matrix (43.7%) or the cell surface (35.5%). Cytoplasmic vesicles containing dense materials around collagen fibrils were also labeled at 8 hours. It is concluded that mannose is directly incorporated into the rough endoplasmic reticulum (RER), and that mannose-containing glycoprotein(s) are packaged in the Golgi apparatus into secretory granules. Mannose-containing glycoprotein(s) become distributed on the periodontal ligament (PDL) fibroblast cell surface, cytoplasmic dense bodies, and the extracellular matrix.     

A simple fluorescence labeling method to visualize the three-dimensional arrangement of collagen fibers in the equine periodontal ligament.

In order to display the collagen-fiber arrangement in the equine periodontal ligament an inexpensive and easy staining procedure with fluorescein was applied to paraffin sections. After fluorescein labeling a section was suitable for successful examination with three special microscopical systems: a) fluorescence microscopy b) phase contrast microscopy and c) polarized light microscopy. Collagen fibers were clearly displayed as compact structures in the fluorescence microscope. This distinct feature of the fluorescent image generated an almost three-dimensional impression of the fiber arrangement. Phase contrast microscopy and polarized light microscopical investigations of the same section supplemented the findings with further structural details. This contributed to demonstration of the complex architecture of the PDL, i. e. the varying sizes of the fiber bundles, their specific spatial alignment, and the entheses to the dental cementum.     

A measurement technique to evaluate the macroscopic permeability of the vertebral end-plate

The remodelling response of vertebral segments in idiopathic scoliosis or disc degeneration, shows a modification of the disc hydration. The investigation of mass transport between the disc and the vertebral body is relevant to understanding the normal and pathological behaviour of the spine. The measurement method we adopted, to derive the macroscopic permeability of the vertebral end-plate, used the relaxation pressure due to a transient-flow rate into the biological structure. Our approach mimicked the in vivo conditions of mass transfer between the disc and the vertebral bodies. It minimized the flow-induced matrix compaction and it allowed the boundary conditions of the specimen to be controlled. We proposed a specific theoretical method and an associated device according to poroelastic theory. A preliminary evaluation with a controlled porous medium, preceded a pilot study in an animal model (lumbar L(4)-L(5) segment of a 4 months old female pig). The macroscopic permeability of multilayered cylindrical specimens including the trabecular bone, subchondral bone and cartilage end-plate was measured, using a 'cartilage-towards-bone' fluid flow direction. A histologic evaluation completed the biomechanical approach. Results showed that the central zone was more permeable than the periphery and this concurred with qualitative studies from the literature.     

Attachment of human epithelial cells and periodontal ligament fibroblasts to tooth dentin

A goal of treatment in periodontal therapy is to regenerate a new fibroblastic attachment rather than to repair lost attachment with a long junctional epithelium. To date, there is no evidence that fibroblastic attachment formed during regeneration is stronger or less susceptible to periodontal breakdown than a long junctional epithelial attachment. We measured the rate and strength of attachment of epithelial cells (NHEK) and periodontal ligament fibroblasts (PDLF) cultured individually and cocultured to dentin surfaces to determine which cell type has a faster attachment rate and greater adhesive strength to human dentin, and whether the cell types attach independently. Longitudinal dentin slices were seeded with either PDLF or NHEK for 2 or 24 h. The specimens were placed into a parallel plate flow chamber and defined laminar shear stresses were applied. Shear stress was created by step increases in fluid flow rate. Effluent fluid was collected and cell numbers (detached) were counted using a hemocytometer. Cocultures of PDLF and NHEK at three seeding ratios (10:1, 1:1, 1:10) were also tested. Each cell type attached equally well to polystyrene or dentin. PDLF showed a stronger attachment to polystyrene and dentin at 24 versus 2 h. NHEK attached to polystyrene or dentin equally well at 2 and 24 h. NHEK were more strongly attached after 2 h when compared to PDLF. PDLF were more strongly attached after 24 h versus NHEK. When NHEK and PDLF were seeded together on dentin at a 1:1 ratio, PDLF appeared to be more strongly attached than NHEK at 2 but not 24 h. At a ratio of 10 PDLF:1 NHEK, PDLF appeared to be more strongly attached at 2 and 24 h. At a ratio of 1 PDLF:10 NHEK, NHEK appeared to be more strongly attached at 2 h, but PDLF showed a trend of stronger attachment at 24 h. We conclude that epithelial cells attach more quickly to dentin surfaces than PDLF, but do not demonstrate increased attachment strength over time (PDLF do show increased attachment strength overtime). The purported advantages of periodontal regeneration over periodontal repair are supported by our results. Furthermore, our results support the concept of guided tissue regeneration. On the basis of on cellular competition experiments, epithelial cells and PDLF do not act independently, because epithelial cells enhanced the attachment rate of PDLF.     

Viscoelastic response of the periodontal ligament: an experimental-numerical analysis

A viscoelastic constitutive model for the periodontal ligament (PDL) capable of accounting for large strains, anisotropy, and inelastic time-dependent effects was developed. Anisotropy characteristics are determined by the composite nature of the tissue and, in particular, by the distribution of collagen fibres. Time-dependent viscous phenomena are due to microstructural modifications during loading, such as fluid fluxes moving through the solid matrix and the internal rearrangement of fibers and constitutive adaptation. The viscoelastic model presented here was implemented in a general purpose finite element code. In vitro experimental tests were carried out on the PDL specimens of adult pigs to obtain stress-relaxation and cyclic stress-strain curves. The comparison of experimental and numerical results revealed good correspondence and confirmed the capability of the formulation assumed to properly interpret the viscoelastic behavior of the PDL.     

Multi-scale techniques of measuring the dynamic properties of biological tissues.

The overall aim of this study is to establish relationships between the histology of biological tissues and their mechanical properties, especially their dynamic modulus. The emphasis is on the physical scale of the probe used in relation to the size of the histological features in the material of interest. The selection of a model system (connective tissue) is described along with a corresponding gelatin-based system in which the make-up of the material can be "engineered" reproducibly. Next three experimental rigs are described with regard to their suitability for making measurements at a variety of scales; the micro-scale, the meso-scale and the macro-scale. Finally, some preliminary measurements on the micro-scale rig are presented and preliminary conclusions drawn on the methodology.     

A three-dimensional cell culture model to study the mechano-biological behavior in periodontal ligament regeneration

Periodontitis is a disease affecting the supporting structures of the teeth, which can eventually result in tooth loss. A three-dimensional (3D) tissue culture model was developed that may serve to grow a 3D construct that not only transplants into defective periodontal sites, but also allows to examine the effect of mechanical load in vitro. In the current in vitro study, green fluorescent protein labeled periodontal ligament (PDL) cells form rat incisors were embedded in a 3D matrix and exposed to mechanical loading alone, to a chemical stimulus (Emdogain; enamel matrix derivative [EMD]) alone, or a combination of both. Loading consisted of unilateral stretching (8%, 1?Hz) and was applied for 1, 3, or 5 days. Results showed that PDL cells were distributed and randomly oriented within the artificial PDL space in static culture. On mechanical loading, the cells showed higher cell numbers. Moreover, cells realigned perpendicular to the stretching force depending on time and position, with great analogy to natural PDL tissue. EMD application gave a significant effect on growth and upregulated bone sialoprotein (BSP) and collagen type-I (Col-I), whereas Runx-2 was downregulated. This implies that PDL cells under loading might tend to act similar to bone-like cells (BSP and Col-I) but at the same time, react tendon like (Runx-2). The combination of chemical and mechanical stimulation seems possible, but does not show synergistic effects. In this study, a new model was successfully introduced in the field of PDL-related regenerative research. Besides validating the 3D model to mimic an authentic PDL space, it also provided a useful and well-controlled approach to study cell response to mechanical loading and other stimuli.