High-voltage galvanic stimulation on wound healing in guinea pigs: longer-term effects.

The purpose of this investigation was to determine the effects of high-voltage stimulation (HVS) on wound tensile strength properties and wound closure (histology). Eighteen mature guinea pigs with full-thickness incisions were treated with HVS for 45 minutes daily for 2 weeks; 9 animals were studied after the 14 days of treatment and the remaining 9 were studied 2 weeks later. Five animals (10 wounds) served as controls at each time period. After 2 or 4 weeks, treated and untreated skin was harvested, tested to failure, and prepared for histological examination. Two-week-treated and control wounds had comparable values for peak force to failure, elongation, and energy absorbed to failure. Epithelialization was more advanced in treated animals at 14 days (p < .05). There was a trend (p = .068) toward stronger wounds in 4-week-treated animals (maximum load to failure), but not differences were observed between controls and treated groups for elongation or energy absorbed to failure. Dermal healing appeared to be more advanced in treated animals at 30 days. Although peak force to failure was almost 500g higher for treated guinea pigs after 2 weeks of treatment and more than 700g higher than controls after 4 weeks, mean data were highly variable, so the hypothesis that HVS augments wound strength could not be accepted. It is difficult, however, not to assign clinical significance to the findings.

A method for interactive medical instruction utilizing the World Wide Web.

We describe the implementation of interactive medical teaching programs in radiology and histology which utilize the Internet's World Wide Web (WWW). The WWW standard hypertext interface allows for simple navigation between related documents but does not provide a method for student tracking or question queries. Electronic forms, a recent feature of the WWW, provide the means to present question documents to remote clients and track student performance. A feature of our approach is dynamic creation of HTML documents based upon interaction with database applications. The approach allows multiple simultaneous, yet asynchronous interactions by geographically dispersed students upon the same instructional database and is scalable, providing the capability for multiple image/document servers. The security of the database is assured given that it is not accessible through the Internet.

Periosteal attachment fibers in the rat calvarium.

The anatomical relationships between the fiber tracts and bone lining cells within the rat calvarial periosteum have been studied by electron microscopy. Classical Sharpey's fibers were not observed in this location. Rather, thin unmineralized fibers originating from the periosteum traversed the cambial layer and passed to the bone surface between individual osteoblasts or groups of osteoblasts. The organizational relationship suggests that the osteogenic calvarial cell populations are compartmentalized into domains that might be particularly sensitive and responsive to the biomechanical forces of masticatory muscles.

Toadfish saccular hair cell bundle has a preferred orientation in the otolithic membrane.

The macula of the saccule of the toadfish, Opsanus tau, is covered by an otolithic membrane containing sockets into which the stereocilia and kinocilia of the hair cells project. We have found that the hair cell bundle has a distinct eccentric orientation within this space of the otolithic socket. Although the sockets of the otolithic membrane are irregular in shape, all kinocilia are located closet to the same border of the sockets. Since the socket is a fluid or gel filled space through which the hair cell bundle moves, this orientation may have some significance for transduction since it leaves a larger space in the on direction for stereociliary movement.

Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells.

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.

Polarity effects on wound healing using electric stimulation in rabbits.

The aims of this study were to examine effects of high-voltage stimulation (HVS) on wound closure, tensile properties, and mitotic activity. Thirty-six rabbits were assigned to experimental (n = 18) or control groups. Each animal was anesthetized and a full-thickness incision, 3.5 cm long, was made on its back. After 24 hours, experimental rabbits received HVS for two hours, twice daily, for seven days. Polarity was negative the first three days and positive thereafter. Wound closure for HVS treated rabbits was 100%, which was significantly better than that of control rabbits (87%). Tensile strength values and mitotic activity for experimental and control rabbits were comparable. Histologic examination of the wounds suggested a more rapid rate of epithelialization in treated rabbits. Results suggest that electric stimulation, as given, augments wound closure.

Bone cell populations and histomorphometric correlates to function.

A circadian context has been used to develop information about the proliferative and functional behavior of the cell populations that function to model the long bones of growing rats. We asked: Are the proliferating cells in the growth cartilages and diaphyseal bone of young adult growing rats distributed within single or multiple populations? Can cytomorphometry (TEM-C) be used to determine ultrastructural correlates to the well-defined circadian rhythm of matrix formation displayed by functionally synchronous populations of metaphyseal osteoblasts? Can TEM-C reveal changes in osteoclast ultrastructure that could index a biological rhythm for osteoclastic bone mineralysis/resorption? Kinetic results derived from multiple radiothymidine labeling (DNA synthesis) support the single population model for chondrocytes and diaphyseal osteoprogenitor cells. TEM-C studies at the midpoints of the daily light and dark spans show that osteoblast RER-membrane development and cysternal volumes are maximal at the recorded daytime peak of net collagen synthesis. The extent of metaphyseal osteoclast surface ruffling (mineralysis) is also twofold greater during the day than the night--an observation supporting the concept that bone formation and resorption activities are coupled.

Electrical stimulation effects on cutaneous wound healing in rabbits. A follow-up study.

The purpose of this study was to determine the effects of high voltage monophasic pulsed electrical stimulation on wound healing using positive polarity. Forty-four rabbits were assigned to experimental or control groups and followed for four or seven days. We classified the groups as Exp4, Con4, Exp7, and Con7, respectively. Each animal was anesthetized, and a full-thickness incision, 3.5-cm long, was made on its back. After 24 hours, the Exp4 and Exp7 rabbits received high voltage electrical stimulation for two hours twice daily. Wound closure for the Exp4 rabbits (50%) was significantly less than that of the Con4 rabbits (78%). After seven days, however, the Exp7 and Con7 rabbits had similar wound-closure values (80% and 82%, respectively). Tensile-strength values for the control and experimental animals were comparable at both time periods. Histologic examination of the wounds suggested a more rapid rate of epithelization between the Exp4 and Exp7 rabbits compared with the Con4 and Con7 rabbits. The results of this study are inconclusive, but may indicate that positive-polarity stimulation enhanced wound closure between four and seven days of treatment.

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells.

In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: dramatic shortening and rarefaction of microvilli within 1 min; and recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormone-induced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.

From bone lining cell to osteocyte--an SEM study.

We describe the SEM appearance of the rat endosteal bone lining cell ( BLC ) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.

A cellular investment of bone marrow.

We describe a method for clearly separating the cell layers at the bone-marrow interface, which reveals that the myeloid tissue is invested by an epithelial-like layer of specialized squamous cells we call the marrow sac. The scanning electron microscope showed that the sac was fenestrated and that some of its cells pass as perivascular elements with the marrow capillaries that penetrate the bony cortex. The transmission electron microscope (TEM) showed that the cells comprising the marrow sac are less than 0.1 micrometer thick, overlap at their margins without specialized cell junctions, and are more electron dense than the reticular or fibroblastic cells of the marrow stroma. The fenestrations in the sac were intercellular and were usually occupied by cells having an ultrastructure compatible with an osteoprogenitor cell (OPC) lineage. The observation of a close proximity between the cells of the marrow sac and the osteogenic cells that line the endosteal surfaces of bone suggest that the sac cells, along with the OPCs of the superficial marrow stroma, should be included in any morphological or functional definition of an endosteum.

The ultrastructure of collagen in the dermis of tight-skin (Tsk) mutant mice.

A recently discovered dominant mutation in C57/B10 mice called tight-skin (Tsk) results in hypertrophy of certain collagenous tissues including the dermis and hypodermis. The skin of heterozygotes (Tsk/+) is indurated and substantially stiffer than that of the normal animals (+/+). In this study, an electron microscopical comparison of the skin of these animals revealed that the fibrous architecture of the hypertrophic reticular dermis of Tsk/+ mice is more disorganized than that of the +/+ mice and in many areas, the collagen fibrils are more densely packed. The abundance of fibroblasts with distended endoplasmic reticulum in both the dermis and hypodermis of Tsk/+ mice is consistent with increased collagen synthesis. Several of the changes in the dermis and hypodermis of the Tsk/+ mice are similar to changes reported in sclerodermatous skin of man. Surprisingly, an apparent abnormality in the morphology of some of collagen fibrils in the skin of Tsk/+ mice was found to be at least as prevalent in the "normal" +/+ mice. The reticular dermis of both animals contain scattered fibrils which are much larger in diameter than normal and often have a twisted appearance resulting from either helical grooves in the surface of the fibril or discrete branches which twist about one another.

A liquid film model of tetrakaidecahedral packing to account for the establishment of epidermal cell columns.

The possiblity that the organization of cells into columns in the mammalian epidermis may be a result of the close packing of these cells has been investigated in a model system involving the association of randomly produced soap bubbles into a stable froth. Upon floating to the surface of a liquid, soap bubbles have been found to spontaneously assemble into precise columns of interdigitating bubbles. The tetrakaidecahedral shape and the spatial configuration of these bubbles closely resemble those of stacked epidermal cells, although the columns of a froth were oriented at a 60degrees angle to their substratum rather than at right angles as occurs in the epidermal cell columns. These observations lend support to the theory that the organization of the cells in the epidermis into columns is due to the assumption of the keratocytes of a minimum surface-close packing array. Such an organizing mechanism would be independent of both positional control of the underlying mitoses and active guidance of the cells as they become superficially displaced within the epidermis. The observation that a high rate of cell turnover is incompatible with the epidermal column structure may be related to the finding that rapidly produced soap bubbles do not, at least initially, assemble into a columnar array.

A minimum-surface mechanism to account for the organization of cells into columns in the mammalian epidermis.

It has recently been discovered that the stratum corneum and superficial living epidermis of mammalian skin are organized into neat vertical columns of interdigitating cells. The mechanism for this organization has not yet been determined. This study shows that the stacked organization is not unique to the epidermis but also occurs in cork cambium and the pith of woody plant stems. The structural and spatial organization of the stacked cells in these tissues were compared and found to approximate closely the shape of Kelvin's minimum-surface polygon, the tetrakaidecahedron. Paper models of flattened tetrakaidecahedra were constructed and found to stack columns of interdigitating units which are consistent with all of the structural details seen in stacked cells observed under the light and the scanning electron microscopes. Indeed, only polygons of this type are capable of aggregating without interstices in the manner of the stacked cells. A study of stacked arrays of these models has revealed a mechanism by which the epidermis might become organized in columns of stacked and interdigitating cells. The proposed mechanism assumes that cells within an array seek the smallest possible surface-to-volume ratio and, under appropriate conditions, form stacked tetrakaidecahedra.

Scanning electron microscopy of human placental villi from early and late in gestation.

Human placental villi from term placentas and from 10 to 14 week placentas were examined in the scanning electron microscope (SEM) after critical point drying. When prepared by this method, the syncytial surface appears covered with slender microvilli similar to those seen in transmission electron micrographs. We find no support in our observations for the suggestion that the trophoblastic surface is a mosaic of microvillous and nonmicrovillous areas or that substantial areas lack microvilli. In addition, the villi often show circumferentially oriented furrows. SEM's of the early placentas showed numerous syncytial sprouts, most of which represent stages in the formation of new free or terminal villi.