Effects of calcium antagonists and calcium-buffered salines on wound healing in Xenopus early embryos

The role of calcium in the process of wound closure in Xenopus early embryos was studied. Embryos were wounded in the presence of the calcium antagonists D-600 and TMB-8 or in calcium-buffered salines, and the effects on wound healing were observed by scanning electron microscopy. D-600 and TMB-8 inhibit wound closure and these antagonists appear to act synergistically since their combined effect is greater than their individual effects. Experiments with calcium-buffered salines suggest that wound closure can proceed in the presence of low extracellular calcium. In all conditions there is a correlation between the degree of wound closure and the shapes of the cells at the wound margin; closing wounds are accompanied by cells elongated radial to the wound, gaping (non-closing) wounds are accompanied by cells stretched tangential to the wound. Thus the results suggest that calcium influx may not be a requirement for the changes in cell shape which accompany, and probably effect, wound closure in Xenopus early embryos.     

Effects of concurrent infections on growth, development, distribution, and infectivity of adult Philophthalmus megalurus and Philophthalmus gralli

Chickens were infected concurrently with 10 and 20 metacercariae/eye of Philophthalmus megalurus and Philophthalmus gralli. After 14 and 34 days of growth, the adults were removed and worm lengths, return rates, stage of development, and distribution within the host recorded. In a second experiment, chickens infected on day 1 with 10 metacercariae/eye of P. megalurus were concurrently infected with a similar dose of P. gralli on day 14. At day 34, the worms were removed and evaluated for the same parameters as in the first study. The effect of concurrent infections on worm length of P. gralli but not P. megalurus was significant when compared to single species control groups. In all cases recovery rates of P. megalurus in concurrent infections were significantly lower than controls, whereas P. gralli adults were recovered at lower rates only in 20-metacercariae--14-day infections. Maturation of eggs was delayed in both species in concurrent infections at the higher infection levels. Normal distribution was disrupted more for P. gralli at the higher infection levels and longer growth periods. Philophthalmus megalurus adults rarely left their normal habitat in the conjunctival sac. A delayed infective dose of P. gralli affected both species by disrupting normal distribution patterns, delaying egg development, and, in the case of P. megalurus, reducing the recovery rate. The possible role of crowding and antagonistic effects is discussed.     

Recurrent pilonidal cyst and sinus; a plan of preoperative preparation,operation and postoperative care

Evaluation ten years following radical excision and primary closure of recurrent pilonidal cysts led to the conclusion that the method of preoperative and postoperative care and the surgical technique employed gave satisfactory results. In 50 patients operated upon, the duration of symptoms varied from ten days to six years. Primary healing was achieved in all but one case in which there was slight skin overlapping. Thirty-three of the 50 patients were located for appraisal at the end of ten years. Three had had recurrences. The procedure involved eradication of acute infection preoperatively, wide, en bloc radical excision, with primary closure reattaching flaps centrally to the presacral fascia, and drainage of the depths of the wound.     

Effects of pulsed extremely-low-frequency magnetic fields on skin wounds in the rat

Rats with skin-wounds surgically created on their backs were exposed immediately after surgery and every 12 h thereafter to pulsed, extremely-low-frequency magnetic fields. The shape of the pulse was a positive triangle (50 Hz, 8 mT peak). The rate of healing of skin wounds was evaluated macroscopically and by light and electron microscopy at 6, 12, 21, and 42 days after the operation. A significant increase in the rate of wound contraction was found in rats treated with magnetic fields. Forty-two days after surgery all treated animals show fully closed wounds, while control rats at the same time intervals still lacked a final 6% of the wound surface to be covered. Treated rats showed earlier cellular organization, collagen formation and maturation, and a very early appearance of newly formed vascular network.     

A retrospective analysis of 48 infected sternal wound closures: delayed closure decreases wound complications

Forty-eight patients who suffered sternal wound infections following coronary artery bypass grafting were retrospectively reviewed over a 5-year period. All patients in this study had clinical signs of major infection including redness, pain, and purulence at the time of mediastinal drainage and debridement. One patient died 11 days postoperatively because of heart failure, leaving 47 patients available for long-term follow-up. All muscle flaps (pectoralis and rectus abdominis) survived completely. All wound complications were related to chest wall skin flap dehiscence or continued infection. Seventeen of 22 patients (77 percent) undergoing flap closure 4 days or less after sternal debridement and irrigation suffered wound complications. Five of these 22 patients (23 percent) had major wound complications, meaning that the wound required more than 2 months of care before healing was complete. No major wound complications and only three minor complications (12 percent) occurred in 25 patients undergoing sternal flap closure 5 days or more after mediastinal debridement and irrigation. The frequency and severity of wound complications were significantly decreased in the group of patients undergoing sternal flap closure 5 or more days after sternal drainage and debridement (p < 0.00005). In the majority of cases [29 of 47 (62 percent)], secure sternal wound closure was obtained with a single, split, medially based, right pectoralis major muscle flap.     

The longevity and hatchability of Philophthalmus megalurus and P. gralli miracidia in different environmental conditions.

The effects of salinity, pH, and temperature on the longevity and hatchability of miracidia of Philophthalmus megalurus and P. gralli were determined. Miracidia of both species are able to hatch and survive at saline concentrations much above physiological levels, although these processes are reduced in 2.0--2.4% saline and completely inhibited at 2.6%. The greatest hatching rates for both species were found near neutrality (pH 6--8) but some miracidia hatched at the extreme pH levels of 3 and 12. Philophthalmus megalurus miracidia exhibited longer half-lives under acid conditions (pH 2--6) than P. gralli miracidia; conversely, P. gralli miracidia showed longer half-lives in alkaline conditions (pH 8--11). Hatching and longevity were much greater below room temperature (5--20 C) than above (30--50 C) for miracidia of both species. Temperatures above 50 C proved lethal for eyefluke eggs. Except in acid pH, P. gralli miracidia showed longer half-lives than miracidia of P. megalurus. Comparison to studies on schistosomes revealed that the inhibitory effects of physiological saline and host body temperature on the hatching process of schistosome eggs does not occur in these 2 species of eyeflukes.     

RECURRENT PILONIDAL CYST AND SINUS—A Plan of Preoperative Preparation,Operation and Postoperative Care

Evaluation ten years following radical excision and primary closure of recurrent pilonidal cysts led to the conclusion that the method of preoperative and postoperative care and the surgical technique employed gave satisfactory results. In 50 patients operated upon, the duration of symptoms varied from ten days to six years. Primary healing was achieved in all but one case in which there was slight skin overlapping. Thirty-three of the 50 patients were located for appraisal at the end of ten years. Three had had recurrences.The procedure involved eradication of acute infection preoperatively, wide, en bloc radical excision, with primary closure reattaching flaps centrally to the presacral fascia, and drainage of the depths of the wound.     

A comparison of Texan and Hawaiian strains of the avian eyefluke, philophthalmus gralli, with a cautionary note on the importation of exotic animals

Philophthalmus gralli (Mathis and Leger, 1910) was introduced into the San Antonio, Texas area within the last 25-30 years from an unknown foreign source. Strains of P. gralli originally from Texas and Hawaii were compared for differences in morphology, growth patterns, mating compatibility, and isozyme mobilities. Metacercarial cysts of the 2 strains were compared for viability after storage at room temperature. Adult stages of the 2 strains were indistinguishable based on sucker ratios, vitellaria, and egg sizes. In single- and multiple-worm infections, both strains exhibited similar growth patterns when reared in chickens. Adults of the 2 strains, when transplanted in concurrent infections, readily cross-inseminated. Isozyme mobilities of 5 enzymes were identical in all adult worms examined from both strains. After 3 days, metacercarial cysts from both strains rapidly lost their ability to excyst. The cysts of the Texan strain remained viable for 4 days longer than the Hawaiian strain, although excystment rates were low during that period. The evolutionary implications of this imported species are discussed.     

Photoperiodic modulation of cephalic melatonin in planarians

Endogenous melatonin was detected by high-performance liquid chromatography (HPLC) with electrochemical detection (EC) in the head of the planarian Dugesia dorotocephala. The identity of this elution peak was further confirmed by radioimmunoassay. In groups of planarians adapted to either normal or reversed photoperiods, the melatonin levels were always higher in those heads collected in the dark period than in those collected in the light period. This indicates that primitive animals such as planarians have already evolved a melatonin-metabolizing system that is photically driven in a manner suggestive of the way melatonin synthesis is influenced by light and dark cycles in vertebrates.     

Outcome of scalp donor sites in 450 consecutive pediatric burn patients

The scalp is a useful and reliable donor site in pediatric burn patients that can be multiply harvested with minimal morbidity. Healing complications, however, may include alopecia and chronic folliculitis. To investigate scalp donor-site morbidity, a consecutive series of 2478 pediatric burn patients treated over a 10-year period were reviewed. A total of 450 of these patients had scalp donor sites for wound closure. Percent of total body surface area burned was 46+/-23 percent (mean+/-standard deviation), and the mean number of sequential scalp donor-site harvests was 2.2+/-2 (range, 1 to 10) with mean intervals between harvesting of 6+/-0.6 days. Ten patients (2.2 percent) had related complications. Eight patients developed scalp folliculitis, with Staphylococcus sp as the predominant organism (80 percent). Two patients were managed successfully with wound care alone; the other six patients required surgical debridement and split-thickness skin grafting to achieve wound healing. These eight patients developed varying degrees of alopecia. Two patients developed alopecia without previous folliculitis. Six patients required reconstructive surgery, which consisted of primary closure (3), staged excision (1), and tissue expansion (2). A number of variables were examined to determine any differences in the group that had complications compared with the group of patients that did not. No differences in age, sex, race, burn type, burn size, septic episodes, time to wound closure, or number of times the scalp was harvested were detected. Healed second-degree burns to the scalp that were subsequently taken as donor sites seemed to be a risk factor (p < 0.05) for folliculitis and alopecia. Our study confirms that scalp donor sites are reliable with low morbidity. Complications include alopecia and chronic folliculitis that can be avoided by meticulous technique and avoidance of previously burned areas.     

Comparative study of eye defective worm 'menashi' and regenerating wild-type in planarian, Dugesia ryukyuensis

Inbreeding of the sexualized planarian, Dugesia ryukyuensis, produces eye-defective worms, menashi, in the F1 population. To study the effects of this mutation on the eye, we observed the eye-region of menashi using electron microscopy and compared it with the regenerating eye in wild-type worms. The intact eye of wild-type planarians consisted of a few pigment cells and a number of visual cells. Pigment cells containing spherically-shaped electron-dense melanosomes contacted each other and enclosed rhabdomes of visual cells. Rhabdomes had numerous tubular microvilli extending radially and touching the pigment cells. However, in menashi, various lengths of tubular microvilli were irregularly distributed near the pigment cells, which contained numerous electron-lucent premelanosomes, and no adhesive structures were found between the pigment cells. The premelanosomes of menashi were equal in size to those seen after 2 days of regeneration in wild-type planarians and were similar in maturation to those found after 3 days of regeneration in wild-type planarian. These results suggest that menashi is defective in the mechanism(s) of developing pigment granules and constructing visual cells. These findings also suggest that pigment cells in menashi are defective in the mechanism(s) involved with cell adhesion.     

Wound closure in foetal rat skin.

Foetal rat skin rapidly closes an open wound in organ culture and in vivo, this possibly being unique to organs still in the morphogenetic stage. In the present study, examination was made of morphological changes in foetal rat skin during closure of open wounds inflicted at day 16 of gestation. Phase-contrast microscopy of open-wounded skin cultured in vitro indicated inward spreading of the peripheral skin to be responsible for wound closure. Wound closure in vitro was inhibited by cytochalasin B (10 micrograms/ml), not by hydroxyurea (2 mM), indicating prenatal wound closure to be mediated by regulation of the microfilament system rather than cell proliferation. During wound closure in vitro and in vivo, light and scanning electron microscopy of the peripheral skin showed cells in the periderm, the outermost layer of the foetal epidermis, to elongate centripetally and en masse, whereas the shape of underlying epidermal cells not to change. Numerous spindle-shaped cells and fibrous matrices in the mesenchyme were redistributed, becoming oriented along the wound edge. Following isolation of the mesenchyme and epidermis by treatment with Dispase and separate culturing, the capacity for wound closure in vitro was found to be retained only by the mesenchyme. Cellular activity within the mesenchyme, rather than in the epidermis, would thus appear essential to wound closure in foetal rat.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

Neoplastic transformation in the planarian: I. Cocarcinogenesis and histopathology

Although several investigators have reported that exposure to mammalian carcinogens induces abnormal tumorlike growths and teratogenic remodeling in planarians, there is no general agreement that these, or comparable responses in any other invertebrates, model mammalian carcinogenesis. To investigate this question, freshwater planarians of the species Dugesia dorotocephala were exposed to culture water containing an initiator and a promoter, either alone or in combination. Cadmium, a potent carcinogen, was used as an initiator in the protocol. Treatment with sublethal concentrations of cadmium sulfate produced a benign, but persistent, tumor in a small percentage of the planarians. The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester and well-known promoter, to the cadmium-containing solutions resulted in the induction of a progressive, potentially lethal, transplantable tumor in a large proportion of the treated flatworms. Light and electron microscopy revealed this particular tumor to be composed both of immature cells and of a single mature cell type: newly differentiated, but transformed, reticular cells. Further examination of the infiltrating tissue formations elucidated the profile of differentiation, from a population of mitotically active transformed stem cells through the transitional stages in the associated reticuloma. These results suggest that 1) the freshwater planarian displays the major phenomenology of mammalian cocarcinogenesis and that 2) the planarian reticuloma models several important features of a neoplastic stem cell disease.     

Neoplastic transformation in the planarian: II. Ultrastructure of malignant reticuloma

Cadmium and phorbol ester induced tumorigenesis in the planarian, Dugesia dorotocephala, develops as a cocarcinogenic process involving initiation and promotion in the progression of neoplastic disease. Treatment of intact planarians with sublethal concentrations of cadmium sulfate and 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a type of infiltrating tumor that proved to be potentially lethal. Surgical transplantation of such tumorous tissues into otherwise healthy planarians resulted in the same histopathological progression to lethality, which confirmed the metastatic nature of the neoplasia. Electron microscopic studies revealed that both the chemically-induced and the transplantation-based tumors involved, exclusively, the proliferation and differentiation of abnormal reticular cells, referred to as reticuloma cells. Reticular cells normally are ameboid, phagocytic, and are thought to provide the planarian with a phylogenetic predecessor of an immune surveillance system. A considerable incidence of mitosis was observed within the tumor areas; and the sequence of differentiation, from transformed stem cells to mature but nonfunctional reticuloma cells, was elucidated. This profile of differentiation supports the concept of cellular derivation via stem cell dynamics as opposed to dedifferentiation. A variety of ultrastructural abnormalities were characterized: several of which tend to substantiate the anaplastic quality of the reticuloma, while others are more specifically diagnostic for malignancy. These findings further extend the potential usefulness of the planarian malignant reticuloma as a model system for the study of neoplastic stem cell diseases.     

Implantation of a strip of fascia in the venous wall

In 12 adult rabbits, according to the pull through technique a strip of muscular fascia was implanted in the wall of the femoral vein. Of 11 of these animals, sacrificed at intervals varying from 0 days to 3 months after the operation, the area of implantation was removed and histologically studied. Two cases showed wound infection and one case, moreover, venous thrombosis. Among the other 9 cases, in 2 specimens the end of the strip was found to lie within the venous wall, while in the remaining 7 specimens the strip protruded into the venous lumen. In the latter cases, the strip proved to be firmly attached to the venous wall within 4 days; within the same time the protruding part of the strip became completely covered by venous endothelial cells. Loose connective and fatty tissue adherent to the strip only disappeared, when the end of the strip freely floated in the streaming blood.     

Wound healing ability of Xenopus laevis embryos. II. Morphological analysis of wound marginal epidermis

We previously showed that bisectional wounds made in Xenopus laevis embryos at the primary eye vesicle stage were rapidly closed. In this study, microscopic analyses, including scanning electron microscopy, on the morphology of the epidermis were conducted during wound closure in the half embryos. Bright fluorescence of Texas red-phalloidin showing actin filaments started to be visualized at the cut edge 10 min after wounding. It increased with time, forming a distinguished, though discontinuous, bundle along the wound margin. The wound closure was completely inhibited by 20 microm cytochalasin B, and almost completely by 50 mm 2,3-butanedione 2-monoxime, an inhibitor to myosin ATPase activity. Scanning electron microscopy revealed that the outer epidermal cells became extensively elongated in the radial direction, and the contour of the closing wound edge did not become smoother but remained ragged. Thus, a representative embryonic type of wound closure may be driven in Xenopus embryos by a complex mechanism, involving not only the actin 'purse-string' but also an inward movement of individual cells. Anyhow, the wound closure is a movement of the epidermal sheet maintaining cell-cell contact, and not involving locomotion of single cells separated from the wound edge.     

Two populations of pluripotent stem cells in planarians <Emphasis Type="Italic">Girardia tigrina</Emphasis>

The positional differences in the regenerative capability of individual body parts of the planarian Girardia (Dugesia) tigrina were analyzed. The paper shows the significance of the size and positional differences of individual fragments of planarians for their regenerative capabilities, as well as the fundamental difference in the mechanisms of the head and tail blastema formation. A scheme of regeneration that includes two populations of pluripotent stem cells called neoblasts is suggested. The two populations of neoblasts differ in their role and distribution along the planarian body. Specifically, the population of neoblasts involved in the formation of any blastema migrates to the nearest blastema, and the population participating only in the creation of the head blastema migrates along the planarian body, following the gradient of biomass of the damaged axons arising after the amputation of the head end. The maximal size of the head blastema was found in the fragment obtained after cutting off the head fragment at the eye level, and the maximal portion of all pluripotent stem cells migrating into two blastemas was found in the fragment obtained by cutting the planarian above the mouth, followed by cutting off the head fragment at the eye level.     

Vacuum assisted closure for the treatment of sternal wounds: the bridge between débridement and definitive closure

A method to refine the treatment of sternal wounds using Vacuum Assisted Closure (V.A.C.) therapy as the bridge between débridement and delayed definitive closure is described. A retrospective review of 35 consecutive patients with sternal wound complications over a 2-year period (March of 1999 to March of 2001) was performed. The treatment of sternal wounds with traditional twice-a-day dressing changes was compared with the treatment with the wound V.A.C. device. An analysis of the number of days between initial débridement and closure, number of dressing changes, number and types of flaps needed for reconstruction, and complications was performed. Eighteen patients were treated with traditional twice-a-day dressing changes and 17 patients were treated with V.A.C. therapy alone. The two groups were similar regarding age, sex, type of cardiac procedure, and type of sternal wound. The V.A.C. therapy group had a trend toward a shorter interval between débridement and closure, with a mean of 6.2 days, whereas the dressing change group had mean of 8.5 days. The V.A.C. therapy group had a significantly lower number of dressing changes, with a mean of three, whereas the twice-a-day dressing change group had a mean of 17 (p < 0.05). Reconstruction required an average of 1.5 soft-tissue flaps per patient treated with traditional dressing changes versus 0.9 soft-tissue flaps per patient for those treated with V.A.C. therapy (p < 0.05). Before closure, there was one death among patients undergoing dressing changes and three in the V.A.C. therapy group, all of which were unrelated to the management of the sternal wound. Patients with sternal wounds who have benefited from V.A.C. therapy alone have a significant decrease in the number of dressing changes and number of soft-tissue flaps needed for closure. Finally, the V.A.C. therapy group had a trend toward a decreased number of days between débridement and closure.