Actin architecture of cultured human thyroid cancer cells: predictor of differentiation?

The actin cytoskeleton is important for cell structure and motility. A disordered actin architecture has been correlated with a high metastatic potential in melanoma, fibrosarcoma, and colon cancer models. Thyrotropin is known to induce growth and differentiation in cultured thyroid cells, whereas the carcinogenic phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) causes dedifferentiation and malignant transformation in many cell lines. We therefore assessed the effect of thyrotropin and TPA on the actin architecture of FTC-133 human follicular thyroid cancer cells in continuous culture. Staining of filamentous actin with rhodamine phalloidin showed that 1 mU/ml or 30 mU/ml thyrotropin-induced actin polymerization was detectable at 1 hour but more notable at 24 hours. Similarly TPA (0.008 to 10 mumol/L) caused rapid actin fiber disruption and redistribution to the cell periphery. Secondary antibody staining for alpha-actinin, a protein that binds and crosslinks actin, was more prominent after treatment with thyrotropin but decreased after TPA. These findings indicate that the actin cytoskeleton has a dynamic response to trophic factors. Thyrotropin promoted actin polymerization, but TPA caused depolymerization. These effects may correlate with cellular alpha-actinin levels. Actin architecture may therefore reflect the state of differentiation of thyroid tumor cells.     

Covering by a flap induces apoptosis of granulation tissue myofibroblasts and vascular cells

It has recently been shown that during the healing of an open wound, apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Because reduced contraction and a decrease in the number of fibroblastic cells have been described in wounds covered with a successful skin graft, we hypothesized that apoptosis could be responsible for these phenomena. Using in situ labeling of fragmented DNA, immunohistochemistry for alpha-smooth muscle actin, and electron microscopy, we have studied in rats the evolution of 10-day-old wound tissue covered with a total skin flap (containing epidermis, dermis, and the cutaneous muscle). In 10-day-old wound tissue, few apoptotic vascular cells and rare apoptotic myofibroblasts were present; the number of apoptotic cells increased slightly 72 hours later. In wounds covered with total skin flaps, the number of apoptotic vascular and myofibroblastic cells increased drastically 6 hours after flap application with a maximum at 24 and 48 hours, respectively. A decrease of apoptotic cell number was noted at 72 hours; at this time, the size of the granulation tissue was greatly reduced and showed extracellular matrix remodeling. Total flaps were more efficient in the induction of granulation tissue cell apoptosis compared with dermo-epidermal flaps. Moreover, the control application of full-thickness skin autografts, which were not viable 7 days later, did not induce apoptosis 24 hours after implantation. Our results indicate that covering granulation tissue with a skin flap results in a massive apoptotic process, possibly by means of a (some) locally released substance(s).     

Site-specific alteration of actin assembly visualized in living renal epithelial cells during ATP depletion

Disruption of normal actin organization in renal tubular epithelial cells is an important element of renal injury induced by ischemia. Studies of fixed cells indicate that the cytoskeleton is disrupted by both ischemia and ATP depletion in a site-specific manner. However, few studies have examined these effects in living cells, and the relationship between the time course of ATP reduction and alteration of the cytoskeleton remains unclear. Here, time-lapse video images of cultured renal epithelial cells expressing an enhanced green fluorescent protein (EGFP)-actin fusion protein were obtained, and the kinetics of fluorescence actin distribution before and during ATP depletion is quantified and compared with measured ATP levels. This study found that assembly of lamellar actin is inhibited rapidly as cellular ATP levels are reduced, whereas disruption of actin in stress fibers is more gradual and persistent. Actin associated with focal adhesions is largely resistant to ATP depletion in these experiments, and, consistent with previous studies, particulate aggregates of actin were formed within the cytoplasm of ATP-depleted cells. Most surprisingly, time-lapse imaging of EGFP-actin distribution, quantitative fluorescence imaging of phalloidin-stained cells, and ultrastructural studies indicate that assembly of actin filaments occurs at sites of epithelial cell-cell attachment in ATP-depleted cells. This assembly is initiated early during ATP depletion and continues after ATP levels are maximally reduced. Assembly of actin at sites of cell-cell attachment may be an element of the pathology of injury induced by ischemia, or alternatively, could reflect the function of a protective mechanism. These studies directly demonstrate site-specific alteration of actin assembly in living epithelial cells during ATP depletion. The results also reveal that actin reorganization continues after ATP levels are maximally decreased and that epithelial cell-cell attachments are sites of actin assembly in ATP-depleted cells.     

Subcutaneous tissue fibroblasts transfected with muscle and nonmuscle actins: A good in vitro model to study fibroblastic cell plasticity

Cultured fibroblasts develop several biochemical and morphological properties of smooth muscle cells, particularly the expression of alpha-smooth muscle actin, the actin isoform typical of vascular smooth muscle cells. They resemble modified fibroblasts or myofibroblasts observed in granulation tissue during wound repair and in fibrotic situations. We have analysed by immunolabeling the fate of exogenous epitope-tagged actin isoforms by transfection of the corresponding cDNAs into fibroblasts cultured from rat subcutaneous tissue. Tagged muscle actins were efficiently integrated into stress fibers and did not produce obvious changes in cell shape of transfected cells. Transfected nonmuscle actins in contrast changed the morphology and were not or poorly incorporated into stress fibers. These cultured subcutaneous fibroblasts behave similarly to smooth muscle cells when transfected with the same actin encoding cDNAs, indicating another common characteristic of these two cell types in sorting and targeting actin isoforms. Subcutaneous fibroblasts transfected with muscle and nonmuscle actin isoforms provide a good in vitro model to analyze the intracellular sorting of isoactins and to improve our knowledge of myofibroblast characterization and differentiation during tissue repair as well as to understand the relationships between modifications of actin cytoskeleton, adhesion and extracellular matrix proteins.     

Granulation tissue regression induced by musculocutaneous advancement flap coverage

BACKGROUND: Clinical experience suggests that granulation tissue may be inhibited by coverage with a musculocutaneous flap. We hypothesized that coverage of an open wound with a musculocutaneous flap would result in regression and apoptosis of the wound's granulation tissue. METHODS: In the first experiment, 32 rats underwent excisional wounding; 16 underwent musculocutaneous flap coverage of their granulation tissue on postwounding day 8, and then 16 rats (8 controls + 8 flaps) were killed on both postwounding days 10 and 12 (2 and 4 days after the flap procedure, respectively). In the second experiment, 18 rats were wounded, and on postwounding day 5 the rats underwent flap coverage (n = 6), wound edge release/mobilization (the first step of the flap procedure) without flap coverage (n = 6), or dressing change only (n = 6); all rats were killed on postwounding day 6 (24 hours after the secondary intervention). Apoptosis was quantified with the terminal deoxynucleotidyl transferase-mediated nick-end labeling assay. RESULTS: Placement of a musculocutaneous flap over an 8-day-old excisional wound in the first experiment increased the apoptotic rate in the granulation tissue from 0% to 1% (controls) to 5% to 10% at both 2 and 4 days after flap coverage (P <.05). Cell population density decreased 50% in the flap-covered granulation tissue compared with the controls (P <.05). In the second experiment, circumferential release of the granulation tissue resulted in an equivalent increase in granulation tissue apoptosis over controls compared to that induced by the full flap procedure. CONCLUSIONS: Coverage of established granulation tissue with a musculocutaneous flap resulted in histologic regression of the wound's granulation tissue after 2 to 4 days of flap coverage and induced at least a 5-fold increase in the apoptotic rate of the granulation tissue. Releasing the wound edge increased granulation tissue apoptosis to a level equivalent to that produced by the musculocutaneous flap procedure, suggesting that alteration of the wound's mechanical environment is responsible for the acute induction of apoptosis in this model.     

Mechanisms and factors involved in development of hypertrophic scars

Fibroblasts in granulation tissue (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle cells, such as the presence of microfilament bundles and the expression of -smooth muscle actin. The mechanisms leading to the development of such myofibroblastic features remain unclear. Both in vivo and in vitro investigations suggest the participation of growth factors, cytokines, and extracellular matrix in the regulation of -smooth muscle actin expression. During normal wound healing, myofibroblasts disappear when the wound is fully re-epithelialized. In contrast, the expression of -smooth muscle actin is a permanent feature of myofibroblasts present in fibrocontractive diseases. In hypertrophic scars, the presence of these cells may represent an important element in the pathogenesis of retraction. Different therapeutic modalities available for the treatment of hypertrophic scars cause a decrease in the number of -smooth muscle actin expressing myofibroblasts. Further studies regarding the expression of cytoskeleton markers in fibroblastic cells are needed to understand the mechanisms of scarring and fibrosis.     

Delayed wound healing in aged rats is associated with increased collagen gel remodeling and contraction by skin fibroblasts, not with differences in apoptotic or myofibroblast cell populations

Aging has been anecdotally reported to result in prolonged wound healing. Measurement of punch biopsy wound closure in young (4 month old) and old (36 month old) rats indicated there was a significant delay in wound closure by old rats during the early phase of repair, after which closure rates were equivalent. The delay in granulation tissue accumulation in older animals could involve premature programmed cell death (apoptosis); however, apoptotic fibroblasts in sponge granulation tissue and tissue culture were less abundant in samples from old rats relative to young rats. Myofibroblasts express alpha-smooth muscle actin, and they are believed to be important in wound contraction. There were no significant differences in overall abundance or distribution of alpha-smooth muscle actin containing myofibroblasts in granulation tissue and in cultured granulation tissue fibroblasts regardless of the age of the donor rat. The spatial distribution of myofibroblasts and apoptotic cells was distinct. Fibroblasts from granulation tissue and skin explants were placed in a collagen gel contraction assay prior to the 5th passage to determine their in vitro contractility. While granulation tissue fibroblasts from young and old rats showed similar collagen gel contractility, skin fibroblasts from old rats displayed greater collagen gel contractile behavior than young skin fibroblasts. Greater gel contractility of fibroblasts from old rats appeared to result, in large part, from the ability of those cells to cause generalized gel degradation. Gelatin zymography indicated a greater abundance of matrix metalloproteinase-2 in supernatants from gels containing skin fibroblasts from old rats. Taken together, these results suggest that the age-associated healing delay in the rat may not be related to the appearance or abundance of distinct myofibroblast or apoptotic cell populations. Proteolysis may have a significant role in delayed wound healing in aged animals.     

Role of actin cytoskeleton in prostaglandin-induced protection against ethanol in an intestinal epithelial cell line

Prostaglandins (PGs) protect a variety of gastrointestinal cells against injury induced by ethanol and other noxious agents. This investigation attempted to discern the mechanism of cytoprotection as it relates to the relationship between actin and PGs in IEC-6 cells (a rat intestinal epithelial cell line). IEC-6 cells were incubated in Dulbecco's modified Eagle's medium +/- 16,16-dimethyl prostaglandin E(2) (dmPG, 2.6 microM) for 15 min and subsequently incubated in medium containing 1, 2.5, 5, 7.5, and 10% ethanol (EtOH). Cells were then processed for immunocytochemistry using FITC-phalloidin in order to stain the actin cytoskeleton, and cell viability was determined by trypan blue exclusion. Quantitative Western immunoblotting of fractioned G-actin (nonpolymerized; S1) and F-actin (polymerized; S2) was also carried out. EtOH concentrations equal to and greater than 5% led to the collapse of the actin cytoskeleton as depicted by extensive disorganization and fragmentation. In addition, these same EtOH concentrations significantly decreased the S2 fraction and increased the S1 pool of actin. Preincubation with dmPG prevented collapse of the actin cytoskeleton, significantly increased the S2 polymerized fraction as determined by quantitative immunoblotting, and increased cell viability in EtOH-treated cultures. Prior incubation with cytochalasin D, an actin disruptive agent, not only reduced cell viability but also prevented the cytoprotective effects of dmPG. Phalloidin, an actin stabilizing agent, had effects similar to that of dmPG as demonstrated by stability of the actin cytoskeleton and increased cellular viability. Such findings indicate that PGs are important in the organization and stability of actin under in vitro conditions. These effects on actin may play an essential role in the mechanism of PG-induced cytoprotection.     

Cytoskeletal rearrangement and signal transduction in TGF-beta1-stimulated mesangial cell collagen accumulation

TGF-beta1 has been implicated in glomerular extracellular matrix accumulation, although the precise cellular mechanism(s) by which this occurs is not fully understood. The authors have previously shown that the Smad signaling pathway is present and functional in human glomerular mesangial cells and plays a role in activating type I collagen gene expression. It also was determined that TGF-beta1 activates ERK mitogen-activated protein kinase in mesangial cells to enhance Smad activation and collagen expression. Here, it was shown that TGF-beta1 rapidly induces cytoskeletal rearrangement in human mesangial cells, stimulating smooth muscle alpha-actin detection in stress fibers and promoting focal adhesion complex assembly and redistribution. Disrupting the actin cytoskeleton with cytochalasin D (Cyto D) selectively decreased basal and TGF-beta1-induced cell-layer collagen I and IV accumulation. The balance of matrix metalloproteinases (MMP) and inhibitors was altered by Cyto D or TGF-beta1 alone, increasing MMP activity, increasing MMP-1 expression, and decreasing tissue inhibitor of matrix metalloproteinase-2 expression. Cyto D also decreased basal and TGF-beta1-stimulated alpha1(I) collagen mRNA but did not inhibit TGF-beta-stimulated alpha1(IV) mRNA expression. A similar decrease in alpha1(I) mRNA expression caused by the actin polymerization inhibitor latrunculin B was partially blocked by the addition of jasplakinolide, which promotes actin assembly. The Rho-family GTPase inhibitor C. difficile toxin B or the Rho-associated kinase inhibitor Y-27632 also blocked TGF-beta1-stimulated alpha1(I) mRNA expression. Cytoskeletal disruption reduced Smad2 phosphorylation but had little effect on mRNA stability, TGF-beta receptor number, or receptor affinity. Thus, TGF-beta1-mediated collagen I accumulation is associated with cytoskeletal rearrangement and Rho-GTPase signaling.     

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, alpha-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6+/-22.2 cells/mm2, p<0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83+/-1.10%, p<0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1+/-1.3 cells/mm2, p<0.001 when compared with normal skin). Intense alpha-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-beta1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-beta1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.     

Wound splinting regulates granulation tissue survival

PURPOSE: Fibroblast survival within an in vitro collagen matrix is dependent on matrix anchorage to a rigid substratum. The purpose of this study was to determine whether granulation tissue survival in vivo also is dependent on matrix anchorage. We hypothesized that splinting an excisional wound (i.e., anchoring the wound edges) would promote granulation tissue survival and that desplinting a splinted wound would produce granulation tissue apoptosis. METHODS: Eighteen Wistar rats (3 months, 350 g) underwent excisional wounding (2 x 2 cm, dorsal skin) with immediate wound splinting (a metal template affixed with sutures) on day 0. On day 6, rats (n = 6 per group) underwent splint removal (desplinted), splint removal with circumferential incision of the wound edge (desplint/release), or no intervention (splinted); sacrifice of all animals was on day 7. Frozen sections of granulation tissue were stained with TUNEL or H and E; data were analyzed with ANOVA and the unpaired t test. RESULTS: The cross-sectional and surface area of the desplinted and desplint/release granulation tissue both decreased compared to the splinted granulation tissue (*P < 0.05). The nuclear density of the desplint/release granulation tissue was 25% less compared to the splinted granulation tissue (*P < 0.05). The desplinted and desplint/release apoptotic rates were twice and >10x greater than the splinted apoptotic rate, respectively (*P < 0.05). CONCLUSIONS: The rate of cell death in a splinted wound (an in vivo equivalent of an anchored FPCM) is minimal to nil, which is consistent with our hypothesis. Desplinting and releasing the wound edge of a previously splinted wound (the in vivo equivalent of a detached FPCM) results in granulation tissue regression and a large increase in apoptosis. Desplinting a wound alone results in changes somewhat intermediate to the splinted and desplint/release conditions. Loss of wound anchorage acutely promotes granulation tissue apoptosis.     

Impaired wound contraction in stromelysin-1-deficient mice.

OBJECTIVE: To determine whether the deletion of stromelysin-1, a single metalloproteinase gene product, will alter the time course and quality of dermal wound repair in mice. SUMMARY BACKGROUND DATA: After dermal injury, a highly coordinated program of events is initiated by formation of a fibrin clot, followed by migration of keratinocytes, contraction of the dermis, recruitment of inflammatory macrophages, formation of granulation tissue with angiogenesis, and finally tissue remodeling. Matrix metalloproteinases are rapidly induced in the dermis and granulation tissue and at the leading edge of the epidermis in the healing wounds. METHODS: Incisional and circular full-thickness wounds 2 to 10 mm were made in the dermis of stromelysin-1-deficient and wild-type mice. The wounds were analyzed for rate of cellular migration and epithelialization. The wound contraction was examined by immunohistochemical staining for alpha-smooth muscle actin and fluorescent staining for fibrillar actin. RESULTS: Independent of the age of the animal, excisional wounds in stromelysin-1-deficient mice failed to contract and healed more slowly than those in wild-type mice. Cellular migration and epithelialization were unaffected in the stromelysin-1-deficient animals. The functional defect in these mice is failure of contraction during the first phase of healing because of inadequate organization of actin-rich stromal fibroblasts. CONCLUSIONS: Excisional dermal wound healing is impaired in mice with a targeted deletion in the stromelysin-1 gene. Incisional wound healing is not affected. These data implicate stromelysin-1 proteolysis during early wound contraction and indicate that stromelysin-1 is crucial for the organization of a multicellular actin network.     

Transient dynamic actin cytoskeletal change stimulates the osteoblastic differentiation

Dynamic cytoskeletal changes appear to be one of intracellular signals that control cell differentiation. To test this hypothesis, we examined the effects of short-term actin cytoskeletal changes on osteoblastic differentiation. We found an actin polymerization interfering reagent, cytochalasin D, promoted osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells. We also found that these effects were mediated by the protein kinase D (PKD) pathway. Short-term cytochalasin D treatment increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of the extracellular matrix in MC3T3-E1 cells, with temporary changes in actin cytoskeleton. Furthermore, the disruption of actin cytoskeleton induced phosphorylation of 744/748 serine within the activation loop of PKD in a dose-dependent manner. The protein kinase C (PKC)/PKD inhibitor Go6976 suppressed cytochalasin D-induced acceleration of osteoblastic differentiation, whereas Go6983, a specific inhibitor of conventional PKCs, did not. Involvement of PKD signaling was confirmed by using small interfering RNA to knock down PKD. In addition, another actin polymerization interfering reagent, latrunculin B, also stimulated ALP activity and OCN secretion with PKD activation. On the other hand, the present data suggested that transient dynamic actin cytoskeletal reorganization could be a novel cellular signal that directly stimulated osteoblastic differentiation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Topical vanadate optimizes collagen organization within granulation tissue

Systemic ingestion of vanadate, a nonspecific inhibitor of tyrosine phosphatases, doubles wound breaking strength, enhances the packing of collagen fibers, and prevents the appearance of myofibroblasts in granulation tissue. Will the local application of vanadate mimic the systemic effects? Pairs of polyvinyl alcohol sponges, each with a central reservoir and attached injection port, were subcutaneously implanted in rats. Daily, one implant received 0.2 ml of saline and the other received 0.2 ml of 0.03 mM vanadate in saline. On day 7, harvested sponges had equivalent wet weights. The vanadate‐treated sponges had fibroblasts separated by connective tissue, with a more intense birefringence of the collagen fibers. Transmission electron microscopy showed collagen more uniformly packed in the vanadate treated sponges where collagen fibers were equally spaced and had equal diameters. By immunohistology, myofibroblasts, defined by the expression of α‐smooth muscle actin within stress fibers, were absent in vanadate‐treated granulation tissue. The expression of α‐smooth muscle actin was restricted to smooth muscle cells of blood vessels. Controls had densely packed α‐smooth muscle actin staining myofibroblasts, weak birefringence, and randomly spaced collagen fibers with irregular diameters. We conclude that the local application of vanadate prevents the appearance of myofibroblasts and optimizes the organization of collagen fibers in developing granulation tissue. (WOUND REP REG 2003;11:204–212)     

A novel cell-adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide–chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27– and A5G33–chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33–chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and α-smooth muscle actin, showed that keratinocyte delivery by the present peptide–chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.     

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor involved in all aspects of wound healing. TGF‐β accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol‐anchored protein, as a novel TGF‐β coreceptor and inhibitor of TGF‐β signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild‐type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin‐1α and monocyte chemoattractant protein‐1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.     

Hyaluronidase activity during open wound healing in rabbits: A preliminary report

The presence of an enzyme with hyaluronidase activity has been established for open wound granulation tissue in rabbits. Such activity was unrelated to the presence of inflammatory cells and, in fact, was absent in early wounds. Activity increased 7 days after wounding and remained elevated through the 19-day term of the study. The approximate hyaluronic acid content of granulation tissue during healing decreased markedly during this period. The relationship of hyaluronidase to hyaluronic acid in the process of open would healing is discussed.     

封闭负压吸引在创伤性胫骨骨外露的应用

目的探讨封闭负压吸引在创伤性胫骨骨外露中的疗效。方法 45例创伤性胫骨骨外露（Ⅰ～Ⅳ度）患者清创后测量骨外露面积,将VSD材料按创面面积大小和形态剪裁并将其创面与周围正常组织间断缝合固定,引流管连接负压吸引装置,维持负压40 kPa;7 d后对外露骨质表面处理,再次使用负压吸引材料覆盖。结果 22例Ⅰ、Ⅱ度骨外露者7 d后骨外露全部被新鲜肉芽组织覆盖;15例Ⅲ度骨外露者7 d后骨外露存在,面积减小,对外露骨质表面处理后再次使用封闭负压吸引材料覆盖,骨外露全部被新鲜肉芽组织覆盖;8例Ⅳ度骨外露者使用封闭负压吸引治疗后骨外露不能完全被肉芽组织覆盖,但骨外露面积明显缩小,使用组织皮瓣转移覆盖骨外露。结论封闭负压吸引对面积〈5 cm2的胫骨骨外露能够有效促进肉芽组织生长,并将其完全覆盖。对于〉5 cm2的胫骨骨外露使用封闭负压吸引后能够为组织皮瓣移植创造良好的创面。     

PTH-induced actin depolymerization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+ signaling in osteoblasts.

Disruption of the actin cytoskeleton with cytochalasin D enhanced the mechanically induced increase in intracellular Ca(2+) ([Ca(2+)](i)) in osteoblasts in a manner similar to that of PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Patch-clamp analyses show that the MSCC is directly influenced by alterations in actin integrity. INTRODUCTION: PTH significantly enhances the fluid shear-induced increase in [Ca(2+)](i) in osteoblasts, in part, through increased activation of both the mechanosensitive, cation-selective channel (MSCC) and L-type voltage-sensitive Ca(2+) channel (L-VSCC). Both stimuli have been shown to produce dynamic changes in the organization of the actin cytoskeleton. In this study, we examined the effects of alterations in actin polymerization on [Ca(2+)](i) and MSCC activity in MC3T3-E1 and UMR-106.01 osteoblasts in response to shear +/- PTH pretreatment. MATERIALS AND METHODS: MC3T3-E1 or UMR-106.01 cells were plated onto type I collagen-coated quartz slides, allowed to proliferate to 60% confluency, and mounted on a modified parallel plate chamber and subjected to 12 dynes/cm(2). For patch-clamp studies, cells were plated on collagen-coated glass coverslips, mounted on the patch chamber, and subjected to pipette suction. Modulators of actin cytoskeleton polymerization were added 30 minutes before the experiments, whereas channel inhibitors were added 10 minutes before mechanical stimulation. All drugs were maintained in the flow medium for the duration of the experiment. RESULTS AND CONCLUSIONS: Depolymerization of actin with 1-5 microM cytochalasin D (cyto D) augmented the peak [Ca(2+)](i) response and increased the number of cells responding to shear, similar to the increased responses induced by pretreatment with 50 nM PTH. Stabilization of actin with phalloidin prevented the PTH enhanced [Ca(2+)](i) response to shear. Inhibition of the MSCC with Gd(3+) significantly blocked both the peak Ca(2+) response and the number of cells responding to shear in cells pretreated with either PTH or cyto D. Inhibition of the L-VSCC reduced the peak [Ca(2+)](i) response to shear in cells pretreated with PTH, but not with cyto D. Patch-clamp analyses found that addition of PTH or cyto D significantly increased the MSCC open probability in response to mechanical stimulation, whereas phalloidin significantly attenuated the PTH-enhanced MSCC activation. These data indicate that actin reorganization increases MSCC activity in a manner similar to PTH and may be one mechanism through which PTH may reduce the mechanical threshold of osteoblasts.     

Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation

BACKGROUND: This study was designed to determine whether the nitric oxide (NO) pathway is involved in wound granulation tissue formation. METHODS: A section of the pig abdominal wall (excluding the skin) was excised, creating an incisional hernia. The resulting defect was repaired with silicone sheeting in a manner that mimics a temporary abdominal wall closure. During the 14-day experimental period, porcine omentum adhered to the peritoneal edges of the defect and a highly vascularized granulation tissue formed on both sides of the sheeting. Granulation tissue thickness and wound fluid volume were monitored by ultrasonography and epigastric artery flow velocity was monitored by color Doppler flow analysis at days 2, 4, 7, 9, 11, and 14. Fluid was serially harvested from the wound compartment at days 2, 4, 7, 9, 11, and 14 for nitrite/ nitrate (NOx) analysis. Finally, granulation tissue was harvested at day 14 for immunohistochemical and molecular analyses. RESULTS: There was a significant increase in granulation tissue thickness and wound fluid volume during the 14-day study period. Blood flow to the wound increased significantly by day 4 and returned toward baseline by day 14. Wound fluid NOx levels significantly increased from days 7 to 11 and then decreased to near baseline values by day 14. Wound fluid arginine levels significantly decreased when compared with peritoneal fluid and plasma levels at day 14, while wound fluid ornithine levels significantly increased. Immunohistochemical analysis of granulation tissue at day 14 revealed nitric oxide synthase (NOS) 2 was present in the majority of the cells in the granulation tissue. NOS 3 was expressed in endothelial cells only, and NOS 1 expression was not observed in the granulation tissue. CONCLUSIONS: This study suggests that NO, NOS 2, and arginine may play critical roles in granulation tissue formation and wound healing. Arginase and NOS 2 may compete for available arginine as a substrate, thereby limiting later NO production in favor of sustained ornithine synthesis.