Repair and reorganization of minced cardiac muscle in the adult newt (Notophthalmus viridescens)

Studies of the response of adult mammalian and amphibian ventricle to injury have indicated the formation of a connective tissue scar in the place of the wounded or amputated muscle. It has been demonstrated that amphibian myocytes adjacent to a wound surface, unlike mammalian myocytes, have a proliferative capacity. In the present study, a minced cardiac muscle graft was placed into the adult newt ventricle in order to increase the number of myocytes near a wound surface. With such an increased number of reactive myocytes, it was thought a new wall consisting primarily of muscle might be formed. One-sixteenth to one-eighth of the ventricular apex was removed, minced and returned to the amputation surface of the ventricle. General histological and autoradiographic studies were conducted on two sham-operated animals and on five experimental animals which were killed at 5, 10, 20, 30, 50 and 70 days after surgery. Major events of the repair and reorganization of minced cardiac muscle included blood clot formation followed by necrosis of the blood clot and much of the muscle graft. By ten days, an apparent coalescence of muscle fragments and continuity of ventricular and graft lumina were observed, although the graft area never formed an integrated unit with the wounded ventricular wall. The peak of mitotic activity (3.19%) and thymidine labeling (28.1%) of graft cells, including many cells which resembled cardiac myocytes, was observed at 20 days. At 30 days, the graft was observed as a continuous wall composed primarily of muscle fibers. Several 30-, 50- and 70-day grafts had rhythmic contractions. These results suggest that amphibian cardiac muscle has histogenetic and proliferative capacities not attributable to mammalian cardiac muscle.     

Comparison of the effect of bacterial inoculation in musculocutaneous and fasciocutaneous flaps

The skin fascial flap is now recognized as a reliable flap for use in reconstructive surgery. The fasciocutaneous flap has been advocated for coverage of chronic infected wounds after debridement as an alternative to the musculocutaneous flap. Previous experimental and clinical studies have demonstrated the superior resistance of the musculocutaneous flap as compared to the random-pattern flap to bacterial inoculation. A canine model is presented for comparison of the effect of bacterial inoculation in fasciocutaneous and musculocutaneous flaps of similar dimensions. The area of skin necrosis secondary to bacterial inoculation was similar in these two flap types despite greater blood flow and skin oxygen in the fasciocutaneous flap. In a study of closed wound spaces formed by the deep surface of these two flap types, a greater degree of inhibition and elimination of bacterial growth and more collagen deposition are observed in the musculocutaneous wound space than in the fasciocutaneous flap.     

Expression of the WE3 antigen in newt epithelium originating from subcutaneous grafts of skin

mAb WE3 recognizes an antigen that is developmentally regulated in the wound epithelium of regenerating newt limbs. The antigen is precociously expressed when pieces of WE3-negative wound epithelium are grafted subcutaneously (Tassava et al.: Recent Trends in Regeneration Research. New York: Plenum Publishing Co., pp. 37-49, 1989). In the present study, we investigated whether the WE3 antigen is expressed in epidermis of subcutaneous grafts of skin. Small pieces of limb skin were grafted into small tunnels in the lower jaw, limb, and tail, oriented either the same as (epidermis facing out) or opposite to (epidermis facing in) the orientation of the host skin. In most cases, the epithelium migrated from the graft along the wounded surface of the tunnel, closed onto itself, and formed a multilayered "emigrant" epithelium. Infrequently, the migrating epithelium combined with the wound epithelium of the insertion wound. In no case did the epithelium migrate over the cut edge of the grafted dermis. Reactivity to mAb WE3 was first seen at 4 days after grafting, when the migrating epithelium had almost closed over onto itself. By 6 days and thereafter, the entire emigrant epithelium was reactive to mAb WE3. While initially restricted to the emigrant epithelium, at 10 days after grafting and thereafter, reactivity was also seen in the epidermis that remained in contact with the dermis. Expression of the WE3 antigen was not influenced by the orientation of the graft nor by the graft site. The results show that, compared to amputated limbs, the epithelium originating from these grafts precociously expresses the WE3 antigen. Also, epidermis of grafted skin is capable of expressing the WE3 antigen.     

Integration of Acoustic Radiation Force and Optical Imaging for Blood Plasma Clot Stiffness Measurement

Despite the life-preserving function blood clotting serves in the body, inadequate or excessive blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between blood clot stiffness and vascular diseases underscores the importance of quantifying the magnitude and kinetics of blood’s transformation from a fluid to a viscoelastic solid. To measure blood plasma clot stiffness, we have developed a method that uses ultrasound acoustic radiation force (ARF) to induce micron-scaled displacements (1-500 μm) on microbeads suspended in blood plasma. The displacements were detected by optical microscopy and took place within a micro-liter sized clot region formed within a larger volume (2 mL sample) to minimize container surface effects. Modulation of the ultrasound generated acoustic radiation force allowed stiffness measurements to be made in blood plasma from before its gel point to the stage where it was a fully developed viscoelastic solid. A 0.5 wt % agarose hydrogel was 9.8-fold stiffer than the plasma (platelet-rich) clot at 1 h post-kaolin stimulus. The acoustic radiation force microbead method was sensitive to the presence of platelets and strength of coagulation stimulus. Platelet depletion reduced clot stiffness 6.9 fold relative to platelet rich plasma. The sensitivity of acoustic radiation force based stiffness assessment may allow for studying platelet regulation of both incipient and mature clot mechanical properties.     

Wound keratins in the regenerating epidermis of lizard suggest that the wound reaction is similar in the tail and limb

The keratin cytoskeleton of the wound epidermis of lizard limb (which does not regenerate) and tail (which regenerates) hase been studied by qualitative ultrastructural, immunocytochemical, and immunoblotting methods. The process of re-epithelialization is much shorter in the tail than in the limb. In the latter, a massive tissue destruction of bones, and the shrinkage of the old skin over the stump surface, delay wound closure, maintain inflammation, reduce blastemal cell population, resulting in inhibition of regeneration. The expression of special wound keratins found in the newt epidermis (W6) or mammalian epidermis (K6, K16, and K17) is present in the epidermis of both tail and limb of the lizard. These keratins are not immunolocalized in the migrating epithelium or normal (resting) epidermis but only after it has formed the thick wound epithelium, made of lacunar cells. The latter are proliferating keratinocytes produced during the cyclical renewal or regeneration of lizard epidermis. W6-immunolabeled proteic bands mainly at 45-47 kDa are detected by immunoblotting in normal, regenerating, and scarring epidermis of the tail and limb. Immunolabeled proteic bands at 52, 62-67 kDa (with K6), at 44-47, 60, 65 kDa (with K16), and at 44-47 kDa (with K17) were detected in normal and regenerating epidermis. It is suggested that: (1) these keratins constitute normal epidermis, especially where the lacunar layer is still differentiating; (2) the wound epidermis is similar in the limb and tail in terms of morphology and keratin content; (3) the W6 antigen is similar to that of the newt, and is associated with tonofilaments; (4) lizard K6 and K17 have molecular weights similar to mammalian keratins; (5) K16 shows some isoforms or degradative products with different molecular weight from those of mammals; (6) K17 increases in wound keratinocytes and localizes over sparse filaments or small bundles of short filaments, not over tonofilaments joined to desmosomes; and (7) failure of limb regeneration in lizards may not depend on the wound reaction of keratinocytes.     

Hemostasis in larvae of Manduca sexta: formation of a fibrous coagulum by hemolymph proteins

Little is known of the participation of insect hemolymph proteins in wound healing and clot formation. We describe the assembly of purified hemolymph protein from the tobacco hornworm into an extended fibrous coagulum in the absence of hemocytes. This coagulum resembles the clot formed from bovine fibrinogen and thrombin. Structural components of the coagulum are present in hemolymph, however, spontaneous assembly occurs only in hemolymph collected through a wound. The fibrous coagulum assembles from purified structural protein(s) following addition of a non-protein factor from hemolymph, which is also present in Grace's insect cell culture medium.     

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.     

Microfluidic and computational study of structural properties and resistance to flow of blood clots under arterial shear

The ability of a blood clot to modulate blood flow is determined by the clot’s resistance, which depends on its structural features. For a flow with arterial shear, we investigated the characteristic patterns relating to clot shape, size, and composition on the one hand, and its viscous resistance, intraclot axial flow velocity, and shear distributions on the other. We used microfluidic technology to measure the kinetics of platelet, thrombin, and fibrin accumulation at a thrombogenic surface coated with collagen and tissue factor (TF), the key clot-formation trigger. We subsequently utilized the obtained data to perform additional calibration and validation of a detailed computational fluid dynamics model of spatial clot growth under flow. We then ran model simulations to gain insights into the resistance of clots formed under our experimental conditions. We found that increased thrombogenic surface length and TF surface density enhanced the bulk thrombin and fibrin generation in a nonadditive, synergistic way. The height of the platelet deposition domain—and, therefore, clot occlusivity—was rather robust to thrombogenic surface length and TF density variations, but consistently increased with time. Clot viscous resistance was non-uniform and tended to be higher in the fibrin-rich, inner “core” region of the clot. Interestingly, despite intraclot structure and viscous resistance variations, intraclot flow velocity variations were minor compared to the abrupt decrease in flow velocity around the platelet deposition region. Our results shed new light on the connection between the structure of clots under arterial shear and spatiotemporal variations in their resistance to flow.

     

Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage

Full-thickness excisional wounds were made in the dorsal skin of rat fetuses at day 16 and day 18 of gestation. A small patch of skin surrounding the open wound was cut out, mounted on a plastic ring and incubated in an organ culture system. In the presence of serum, the open wound in the day-16 fetal skin closed within three days of culture. During the wound-closure process, no new structures were formed in the wound space, and no conspicuous changes were noted in the histological architecture of the surrounding skin during culture, indicating that the wound closure may result from a centripetal movement of the surrounding skin only. In contrast, the size of the open wound in the day-18 fetal skin remained almost unchanged for one week, but a thin acellular network spread over the wound space within one day of culture. The predominant component of the network was cross-linked fibrin, as disclosed by scanning electron microscopy and sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. The network served as a scaffold for the ingrowth of fibroblast-like cells. These stage-dependent differences in fetal wound healing were consistent with an in vivo study showing that the day-16 wound was covered with the surrounding skin itself, whereas the day-18 wound was covered with newly formed epidermis and invaded by inflammatory cells. The present investigation strongly indicates the prenatal occurrence of a fetal-to-adult transition in the wound-healing pattern of rat skin.     

Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface.

The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.     

The healing skin wound: a novel site of action of the chemokine C10

To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. For this purpose we generated a subtractive cDNA library from normal mouse back skin and 1-day full-thickness excisional wounds. One of the differentially expressed genes encodes the chemokine C10. Using Northern blotting, RNase protection assay and Western blotting, we confirmed the injury-induced expression of C10 at the mRNA and protein level. Maximal levels of C10 mRNA and protein were seen at day 1 after wounding, and expression levels subsequently declined. In situ hybridization and immunohistochemistry revealed expression of C10 in macrophages of the clot and the granulation tissue as well as in keratinocytes of the epidermis and the hair follicles at the wound edge. Since C10 is a potent chemoattractant for macrophages, our results suggest that this chemokine contributes to the strong macrophage influx observed in the healing skin wound.     

Promotive effects of far-infrared ray on full-thickness skin wound healing in rats

The biological effects of far-infrared ray (FIR) on whole organisms remain poorly understood. The aim of our study was to investigate not only the hyperthermic effect of the FIR irradiation, but also the biological effects of FIR on wound healing. To evaluate the effect of FIR on a skin wound site, the speed of full-thickness skin wound healing was compared among groups with and without FIR using a rat model. We measured the skin wound area, skin blood flow, and skin temperature before and during FIR irradiation, and we performed histological inspection. Wound healing was significantly more rapid with than without FIR. Skin blood flow and skin temperature did not change significantly before or during FIR irradiation. Histological findings revealed greater collagen regeneration and infiltration of fibroblasts that expressed transforming growth factor-beta1 (TGF-beta1) in wounds in the FIR group than in the group without FIR. Stimulation of the secretion of TGF-beta1 or the activation of fibroblasts may be considered as a possible mechanisms for the promotive effect of FIR on wound healing independent of skin blood flow and skin temperature.     

Cytoplasmic filaments and gap junctions in epithelial cells and myofibroblasts during wound healing

During the healing of an experimental skin wound, epidermal cells and granulation tissue fibroblasts (myofibroblasts) develop an extensive cytoplasmic contactile apparatus. Concurrently, the proportion of epidermal cell surface occupied by gap junctions increases when compared to normal skin, and newly formed gap junctions appear between myofibroblasts; this suggests that epidermal cell migration and granulation tissue contraction are synchronized phenomena.     

Structure and function of plasminogen/plasmin system

The main physiological function of plasmin is blood clot fibrinolysis and restoration of normal blood flow. To date, however, it became apparent that in addition to thrombolysis, the plasminogen/plasmin system plays an important physiological and pathological role in a number of other essential processes: degradation of the extracellular matrix, embryogenesis, cell migration, tissue remodeling, wound healing, angiogenesis, inflammation, and tumor cell migration. This review focuses on structural features of plasminogen, regulation of its activation by physiological plasminogen activators, inhibitors of plasmin, and plasminogen activators, and the role of plasminogen binding to fibrin, cellular receptors, and extracellular ligands in various functions performed by plasmin thus formed.     

Impact of Tissue Factor Localization on Blood Clot Structure and Resistance under Venous Shear

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ～0.1 and ～2 molecules/μm². Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot’s structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot’s occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow.     

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.     

Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair

The ability to heal wounds is vital to all organisms. In mammalian tissues, alterations in intermediate filament (IF) gene expression represent an early reaction of cells surviving injury. We investigated the role of keratin IFs during the epithelialization of skin wounds using a keratin 6alpha and 6beta (K6alpha/K6beta)-null mouse model. In skin explant culture, null keratinocytes exhibit an enhanced epithelialization potential due to increased migration. The extent of the phenotype is strain dependent, and is accompanied by alterations in keratin IF and F-actin organization. However, in wounded skin in vivo, null keratinocytes rupture as they attempt to migrate under the blood clot. Fragility of the K6alpha/K6beta-null epidermis is confirmed when applying trauma to chemically treated skin. We propose that the alterations in IF gene expression after tissue injury foster a compromise between the need to display the cellular pliability necessary for timely migration and the requirement for resilience sufficient to withstand the rigors of a wound site.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Functional morphology and evolution of tail autotomy in salamanders

Basal tail constriction occurs in about two-thirds of the species of plethodontid salamanders. The constriction, which marks the site of tail autotomy, is a result of a reduction in length and diameter of the first caudal segment. Gross and microscopic anatomical studies reveal that many structural specializations are associated with basal constriction, and these are considered in detail. Areas of weakness in the skin at the posterior end of the first caudal segment, at the attachment of the musculature to the intermyotomal septum at the anterior end of the same segment, and between the last caudosacral and first caudal vertebrae precisely define the route of tail breakage. During autotomy the entire tail is shed, and a cylinder of skin one segment long closes over the wound at the end of the body. It is suggested that specializations described in this paper have evolved independently in three different groups of salamanders. Experiments and field observations reveal that, contrary to expectations, frequency of tail breakage is less in species with apparent provisions for tail autotomy than in less specialized species. The tail is a very important, highly functional organ in salamanders and it is suggested that selection has been for behavioral and structural adaptations for control of tail loss, rather than for tail loss per se.     

Cultured epithelial autografts for giant congenital nevi

Eight pediatric patients with giant congenital nevi confluent over 21 to 51 percent body surface area were treated by excision and grafting. The nevus was excised to the muscle fascia, and the open wound was grafted with cultured epithelial autografts and split-thickness skin grafts. The patients have been followed from 17 to 56 months. Seventeen operations were performed in the eight patients, excising a mean of 6.9 percent body surface area at each procedure. The mean duration of anesthesia was 3.7 hours, and the mean operative blood loss was 12.3 percent estimated blood volume. The mean "take" for the cultured epithelial autografts was 68 percent, and for the split-thickness skin grafts, 84 percent. Epithelialization of open wound areas adjacent to the grafts was somewhat slower for the cultured epithelial autografts than for the split-thickness skin grafts, but it led to a healed wound in all patients except one. Ten of the 17 areas grafted with cultured epithelial autografts resulted in small open wounds that required regrafting. Wound contraction under the cultured epithelial autografts and under split-thickness skin grafts was similar and depended more on the anatomic site grafted than on the type of graft employed. in 16 of 17 operations, the cultured epithelium remained as a permanent, durable skin coverage. The use of cultured epithelial autografts allowed a larger area of excision than would have been possible with split-thickness skin grafts alone and, therefore, a more rapid removal of nevus. Cultured epithelial autograft are an important new technique in the care of patients with giant congenital nevi.