Outcome of open abdominal management following military trauma.

BACKGROUND: Temporary abdominal closure (TAC) is increasingly common after military and civilian major trauma. Primary fascial closure cannot be achieved after TAC in 30 per cent of civilian patients; subsequent abdominal wall reconstruction carries significant morbidity. This retrospective review aimed to determine this morbidity in a UK military cohort. METHODS: A prospectively maintained database of all injured personnel from the Iraq and Afghanistan conflicts was searched from 1 January 2003 to 31 December 2014 for all patients who had undergone laparotomy in a deployed military medical treatment facility. This database, the patients' hospital notes and their primary care records were searched. RESULTS: Laparotomy was performed in a total of 155 patients who survived to be repatriated to the UK; records were available for 150 of these patients. Seventy-seven patients (51·3 per cent) had fascial closure at first laparotomy, and 73 (48·7 per cent) had a period of TAC. Of the 73 who had TAC, two died before closure and two had significant abdominal wall loss from blast injury and were excluded from analysis. Of the 69 remaining patients, 65 (94 per cent) were able to undergo delayed primary fascial closure. The median duration of follow-up from injury was 1257 (range 1-4677) days for the whole cohort. Nine (12 per cent) of the 73 patients who underwent TAC subsequently developed an incisional hernia, compared with ten (13 per cent) of the 77 patients whose abdomen was closed at the primary laparotomy (P = 1·000). CONCLUSION: Rates of delayed primary closure of abdominal fascia after temporary abdominal closure appear high. Subsequent rates of incisional hernia formation were similar in patients undergoing delayed primary closure and those who had closure at the primary laparotomy.

The potential of mesothelial cells in tissue engineering and regenerative medicine applications.

Injury to the serosa through injurious agents such as radiation, surgery, infection and disease results in the loss of the protective surface mesothelium and often leads to fibrous adhesion formation. Mechanisms that increase the rate of mesothialisation are therefore actively being investigated in order to reduce the formation of adhesions. These include intraperitoneal delivery of cultured mesothelial cells as well as administration of factors that are known to increase mesothelial proliferation and migration. An exciting alternative that has only recently received attention, is the possible role of mesothelial progenitor cells in the repair and regeneration of denuded serosal areas. Accumulating evidence suggests that such a population exists and under certain conditions is able to form a number of defined cell types indicating a degree of plasticity. Such properties may explain the extensive use of mesothelial cells in various tissue engineering applications including the development of vascular conduits and peripheral nerve replacements. It is likely that with the rapid explosion in the fields of tissue engineering and regenerative medicine, a greater understanding of the potential of mesothelial progenitor cells to repair, replace and possibly regenerate damaged or defective tissue will be uncovered.

The origin of regenerating mesothelium: a historical perspective.

Bichat first described the mesothelium in 1827 but despite its early discovery, it has only been in recent years that its importance both in health and disease has been realised. One area still poorly understood is that of the mechanisms regulating mesothelial repair. Mesothelial cells are derived from the mesoderm but express many epithelial characteristics. However, mesothelium does not heal in the same way as other epithelial-like cells. Epithelium heals by centripetal migration, with cells at the edge of the wound proliferating and migrating into the injured area. Hertzler in 1919 noted that both large and small peritoneal injuries healed within the same time frame, concluding that the mesothelium could not heal solely by centripetal migration. The exact mechanisms involved in mesothelial regeneration following injury are controversial with a number of proposals suggested to explain the origin of the regenerating cells. This review will examine these proposals and give some insights into the likely mechanisms involved.

Venous ulcer fibroblasts compared with normal fibroblasts show differences in collagen but not fibronectin production under both normal and hypoxic conditions.

Previous immunocytochemical analysis showed that the base of venous ulcers was deficient in fibronectin compared with surrounding "normal" dermis. Here, we investigate whether impaired synthetic ability of ulcer fibroblasts could underlie this observation. Ulcer fibroblasts, established in culture from biopsies of the edge of chronic venous leg ulcers, were compared with normal fibroblasts grown from biopsies of site-and age-matched normal skin for their ability to synthesize matrix molecules. Collagen and fibronectin synthesis were measured following metabolic labeling, as collagenase susceptible counts and counts with gelatin affinity, respectively. More collagen was produced by normal fibroblasts than ulcer fibroblasts, both when the cells were cultured on plastic and in collagen gels. In fibronectin synthesis, however, there was no major difference between the two cell types on either substratum. The hypoxic environment to which ulcer fibroblasts are exposed may have caused the intrinsic differences in collagen synthesis by the two fibroblast types. When we tested the effect of culturing cells under hypoxic conditions, both cell types produced less collagen, especially normal fibroblasts grown in a collagen gel, but there was no effect of hypoxia on fibronectin synthesis. We conclude that venous ulcer edge-derived fibroblasts have an impaired ability to synthesize collagen in vitro, but synthesize fibronectin normally. Therefore, the low level of fibronectin found in venous ulcers is not likely to be due to the inability of ulcer cells to produce it or to the response to hypoxic conditions but may be due to the degradation of synthesized fibronectin by proteases present in these ulcers.

Motoric responses to sucrose in postmature and term infants.

Postmature infants calm less when presented with sucrose solutions than term infants. To further assess the influence of postmaturity on sucrose responsivity, several motoric responses were examined in healthy term neonates and chronically stressed postmature (> or = 288 days gestational age (GA) e.g., > 41 weeks) neonates with Clifford's Syndrome tested between 5-91 h of age. Following baseline observations, each subject was presented with 0.1 ml of a 14% sucrose solution for 10 s each min for 5 min, and observations were continued for 5 min following stimulation. During sucrose stimulation and poststimulation phases, postmature infants showed more tremors during quiet state than term infants. Across conditions, postmature infants exhibited increased finger sucking, rhythmic sucking, but less mouth opening and arm movements than healthy, term infants. Sequence analysis revealed that short-latency mouthing, repetitive movements of the upper and lower lips, followed sucrose infusion reliably in postmature infants, but not term infants. These results suggest that postmaturity is associated with: 1. Increased oral behaviors associated with arousal and self soothing; and 2. Increased sucrose reactivity, as indicated by contingent mouthing and increased tremors.

The use of topical human growth hormone on chronic venous ulcers.

An investigation of the effect of biosynthetic human growth hormone on chronic venous ulcer healing rates.

Perivascular cells in a skin graft are rapidly repopulated by host cells.

Survival of grafted tissues is dependent upon revascularisation. This study investigated revascularisation in a murine skin graft model, using two methods. The first involved 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI) labelling of the wound bed, prior to replacing the skin graft, to allow tracking of host cells into the grafts. At time points between day 3 and day 14 post-surgery, DiI-labelled cells which had tracked into the grafts, were found to co-localise with CD31 positive endothelial cells and patent perfused vessels (fluorescein isothiocyanate (FITC)-dextran perfusion), to show possible association with the vasculature. To further differentiate between graft and host-derived cells, C57BL/6 wild-type grafts were placed on enhanced-green fluorescent protein (e-GFP) transgenic mouse hosts, and at set times post-grafting examined using confocal microscopy. Patent vessels were found at all depths of the graft by day 3. Host (DiI- or GFP-positive) cells were predominantly co-localised with graft vessels in grafts from day 3 onwards, with a similar morphology to control skin. Significantly more GFP labelled host cells were visualised in the superficial dermis at day 5 compared to day 3. Initial restoration of circulation appears to be due to linkage between existing graft and bed vessels, followed by an influx of host cells with a definite perivascular distribution. These findings have implications for skin autografts and tissue engineered skin substitutes.

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. OBJECTIVES: To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. METHODS: Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. RESULTS: The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. CONCLUSIONS: These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Role of plasminogen activators in peritoneal adhesion formation.

Intra-abdominal adhesion formation is a major complication of serosal repair following surgery, ischaemia or infection, leading to conditions such as intestinal obstruction and infertility. It has been proposed that the persistence of fibrin, due to impaired plasminogen activator activity, results in the formation of adhesions between damaged serosal surfaces. This study aimed to assess the role of fibrinolysis in adhesion formation using mice deficient in either of the plasminogen activator proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). We hypothesize that, following serosal injury, mice with decreased peritoneal fibrinolytic activity will be more susceptible to adhesion formation. Adhesion formation was induced in tPA- and uPA-deficient and wild-type mice following either surgical trauma to the serosa with haemorrhage and acute or chronic intraperitoneal inflammation. Adhesion formation was assessed from 1 to 4 weeks post-injury. Mice deficient in tPA were more susceptible to adhesion formation following both a surgical insult and a chronic inflammatory episode compared with uPA-deficient and wild-type mice. In addition, the time of maximal adhesion formation varied depending on the nature of the initial insult. It is proposed that the persistence of fibrin due to decreased tPA activity following surgery or chronic inflammation plays a major role in peritoneal adhesion formation.

Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers.

As part of a major clinical trial, sequential biopsies were taken from the margins of venous leg ulcers during their healing. The changing patterns of tissue architecture and extracellular matrix synthesis during healing were documented histologically and immunocytochemically. Initial biopsies were similar in appearance: prominent fibrin cuffs, variable inflammation, hemosiderin, and red blood cell extravasation. So called "fibrin cuffs" were highly organized structures composed of laminin, fibronectin, tenascin, and collagen as well as trapped leukocytes and fibrin. Fibronectin was absent from the ulcer tissue although collagen was abundant. Major histologic changes were observed after 2 weeks' pressure bandage therapy; hemosiderin, acute inflammation, and granulation tissue with the deposition of fibronectin had all increased and epithelial migration had commenced. Complete epithelialization was frequent by the fourth week of treatment, but the basement membrane was incomplete. At this time, hemosiderin and red blood cell extravasation had decreased and "fibrin cuffs" were virtually absent although chronic inflammation remained. The complex organization of the so-called "fibrin cuffs" may inhibit angiogenesis (but offer protection against increased venous pressure) in addition to their previously ascribed role in causing tissue ischemia.

Growth of nerve fibres into murine peritoneal adhesions.

Adhesions in the peritoneal cavity have been implicated in the cause of intestinal obstruction and infertility, but their role in the aetiology of chronic pelvic pain is unclear. Nerves have been demonstrated in human pelvic adhesions, but the presence of pain-conducting fibres has not been established. The purpose of this study was to use an animal model to examine the growth of nerves during adhesion formation at various times following injury and to characterize the types of fibres present. Adhesions were generated in mice by injuring the surface of the caecum and adjacent abdominal wall, with apposition. At 1-8 weeks post-surgery, adhesions were processed and nerve fibres characterized histologically, immunohistochemically, and ultrastructurally. Peritoneal adhesions had consistently formed by 1 week after surgery and from 2 weeks onwards, all adhesions contained some nerve fibres which were synaptophysin, calcitonin gene-related peptide, and substance P-immunoreactive, and were seen to originate from the caecum. By 4 weeks post-surgery, nerve fibres were found to originate from both the caecum and the abdominal wall, and as demonstrated by acetylcholinesterase histochemistry, many traversed the entire adhesion. Ultrastructural analysis showed both myelinated and non-myelinated nerve fibres within the adhesion. This study provides the first direct evidence for the growth of sensory nerve fibres within abdominal visceral adhesions in a murine model and suggests that there may be nerve fibres involved in the conduction of pain stimuli.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans.

Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA.

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo-matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP-1 and -2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post-wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase-polymerase chain reaction (RT-PCR). TIMP-1 and TIMP-2 proteins are up-regulated from 24 h post-wounding, with a decrease in staining intensity by day 7 for TIMP-2 and by day 14 for TIMP-1. Steady-state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP-1 and -2 by day 3 post-wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP-1 and -2 at all time-points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.

Presence and distribution of sensory nerve fibers in human peritoneal adhesions.

OBJECTIVE: To assess the distribution and type of nerve fibers present in human peritoneal adhesions and to relate data on location and size of nerves with estimated age and with clinical parameters such as reports of chronic pelvic pain. SUMMARY BACKGROUND DATA: Peritoneal adhesions are implicated in the cause of chronic abdominopelvic pain, and many patients are relieved of their symptoms after adhesiolysis. Adhesions are thought to cause pain indirectly by restricting organ motion, thus stretching and pulling smooth muscle of adjacent viscera or the abdominal wall. However, in mapping studies using microlaparoscopic techniques, 80% of patients with pelvic adhesions reported tenderness when these structures were probed, an observation suggesting that adhesions themselves are capable of generating pain stimuli. METHODS: Human peritoneal adhesions were collected from 25 patients undergoing laparotomy, 20 of whom reported chronic pelvic pain. Tissue samples were prepared for histologic, immunohistochemical, and ultrastructural analysis. Nerve fibers were characterized using antibodies against several neuronal markers, including those expressed by sensory nerve fibers. In addition, the distribution of nerve fibers, their orientation, and their association with blood vessels were investigated by acetylcholinesterase histochemistry and dual immunolocalization. RESULTS: Nerve fibers, identified histologically, ultrastructurally, and immunohistochemically, were present in all the peritoneal adhesions examined. The location of the adhesion, its size, and its estimated age did not influence the type of nerve fibers found. Further, fibers expressing the sensory neuronal markers calcitonin gene-related protein and substance P were present in all adhesions irrespective of reports of chronic abdominopelvic pain. The nerves comprised both myelinated and nonmyelinated axons and were often, but not invariably, associated with blood vessels. CONCLUSIONS: This study provides the first direct evidence for the presence of sensory nerve fibers in human peritoneal adhesions, suggesting that these structures may be capable of conducting pain after appropriate stimulation.

Epithelial scatter factor and development of the chick embryonic axis.

Scatter factor, a recently characterised protein secreted by certain embryonic fibroblasts, affects cultured epithelial by increasing cell motility, the breakdown of cell junctions and cell scattering. The process of gastrulation in higher vertebrate embryos, during which the primitive streak forms, involves an epithelial-to-mesenchymal transformation resembling the effects of the factor on cultured cells. The factor was applied locally to chick embryos, using both scatter-factor-secreting cell lines and inert carriers. We found that scatter factor can generate local supernumerary axial structures resembling primitive streak and/or neural plate and conclude that it may have primitive-streak and/or neural-inducing activity in chick embryos.

Human peritoneal adhesions are highly cellular, innervated, and vascularized.

Peritoneal adhesions are a major complication of healing following surgery or infection and can lead to conditions such as intestinal obstruction, infertility, and chronic pain. Mature adhesions are the result of aberrant peritoneal healing and historically have been thought to consist of non-functional scar tissue. The aim of the present study was to analyse the cellular composition, vascularity, and extracellular matrix distribution of human peritoneal adhesions, to determine whether adhesions represent redundant scar tissue or are dynamic regenerating structures. Furthermore, the histological appearance of each adhesion was correlated with the clinical history of the patient, to determine whether maturity or intraperitoneal pathology influences adhesion structure. Human peritoneal adhesions were collected from 29 patients undergoing laparotomy for various conditions and were prepared for histology, immunocytochemistry, and transmission electron microscopy. All adhesions were highly vascularized, containing well-developed arterioles, venules, and capillaries. Nerve fibres, with both myelinated and non-myelinated axons, were present in adhesions from nearly two-thirds of the patients, with increased incidence in those with a malignancy. Approximately one-third of the adhesions contained conspicuous smooth muscle cell clusters lined by collagen fibres of heterogeneous size. Adipose tissue was a consistent feature of all the adhesions, with some areas displaying fibrosis. There appeared to be no correlation between the estimated maturity or site of each adhesion and its histological appearance. However, intraperitoneal pathology at the time of surgery did influence the incidence of some histological features, such as the presence of nerve fibres, clusters of smooth muscle cells, and inflammation. This study challenges previous concepts that adhesions represent non-functional scar tissue and clearly demonstrates that established adhesions are highly cellular, vascularized, and innervated, features more consistent with dynamic, regenerating structures.

An integrin-targeted non-viral vector for pulmonary gene therapy.

Gene therapy offers potential for the treatment of severe respiratory diseases. However, the vectors which are currently available have drawbacks limiting their therapeutic application. Here we report on an integrin-targeted, non-viral gene delivery system for pulmonary gene transfer. We demonstrate that this vector can deliver the lacZ reporter gene to the lung, transfecting bronchial epithelium and parenchymal cells with similar efficiency to an adenoviral vector and with greater efficiency than a cationic liposome. In addition, vector administration can be repeated leading to further gene expression without inducing inflammation. The advantages of this novel gene delivery system provide considerable potential for targeted gene therapy in vivo. Gene Therapy (2000) 7, 393-400.

HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways.

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.