Interleukin-8 levels and activity in delayed-healing human thermal wounds.

There are numerous causes for slow or delayed wound healing. Because slowly healing wounds are often inflamed, we quantitated the inflammatory chemokine, interleukin-8, produced by slowly healing human burn wounds and compared this to interleukin-8 from healed wounds and normal intact skin. Interleukin-8 levels were increased significantly in unhealed wounds (19.7 ng/ml) compared to healed wounds (7.7 ng/ml) or normal skin (5.7 ng/ml). Interleukin-8 in these ranges was added to adult human keratinocytes and fibroblasts. Interleukin-8 significantly decreased keratinocyte replication but had no effect on fibroblast replication. The rate or final degree of fibroblast populated collagen lattice contraction was inhibited at interleukin-8 concentrations between 10 and 30 ng/ml, but not altered at concentrations below 10 ng/ml and above 100 ng/ml. The concurrent application of indomethacin at 10 microg/ml reversed this interleukin-8 induced inhibition. Interleukin-8 inhibited myosin ATPase activity, apparently by reducing the phosphorylation of nonmuscle myosin light chain. We conclude that elevated levels of interleukin-8 may be found during delayed healing, and these elevated interleukin-8 levels may directly contribute to retarded wound closure.

Postnatal pulmonary hypertension after repair of congenital diaphragmatic hernia: predicting risk and outcome.

BACKGROUND: Pulmonary hypertension (PH) after congenital diaphragmatic hernia (CDH) repair remains a significant cause of morbidity and mortality. Although treatment advances have improved overall survival, a new cohort of patients is surviving with PH beyond the postnatal period. Because the clinical entity of postnatal persistent pulmonary hypertension (PPHTN) in CDH patients has not been published, the authors undertook a retrospective study of our neonatal CDH experience to characterize this group of infants. METHODS: Charts of all infants with CDH treated at this institution from January 1991 to June 1997 were reviewed (n = 51). Persistent pulmonary hypertension by echocardiographic (Echo) measurements at the time of discharge identified PPHTN patients. Control survivors had normal pulmonary artery pressures at discharge. Physiological parameters and the results of therapeutic interventions were analyzed to predict PPHTN. RESULTS: Seven infants (four boys, three girls) had PPHTN at discharge. Significant differences with the control group were noted in length of stay, duration of intubation, and duration of nitric oxide therapy. Extracorporeal membrane oxygenation (ECMO) duration was not significantly different between the groups. By 12 months of age, PPHTN resolved in six patients (87%), and one died at 13 months. Regardless of therapy, two parameters showed 100% positive predictive value for identifying patients with PPHTN (P < .001): an Echo demonstrating PH at 2 months of age or continued oxygen requirement at 3 months. Oxygen requirement at 2 months had a 67% predictive value of PPHTN. CONCLUSIONS: With current treatment strategies for CDH, infants can survive with persistent pulmonary hypertension beyond the newborn period. The long-term survival rate is excellent, and normalization of pulmonary artery pressures can be expected. PPHTN can be predicted in those infants with Echo-defined pulmonary hypertension at 2 months.

Repeated additions of hyaluronan alters granulation tissue deposition in sponge implants in mice.

The role for the metabolism of hyaluronic acid in the repair process is uncertain. Fetal dermal wounds do not heal by scarring and have sustained high levels of hyaluronic acid. In contrast, adult dermis is repaired by scarring and has less hyaluronic acid. Initially after injury, hyaluronic acid is elevated in both adult and fetal wounds, and although it remains elevated in fetal repair, it is rapidly degraded in adult wounds. The chronic addition of hyaluronic acid or hyaluronidase to polyvinyl alcohol sponge implants in adult mice was investigated in this study. Polyvinyl alcohol sponge implants containing a central reservoir were placed subcutaneously in the dorsum of adult male CD-1 mice. Mice were divided into three groups: a phosphate-buffered saline control, a 20 microgram hyaluronic acid treatment group, and a 10 U hyaluronidase treatment group. The central reservoir of each sponge implant received appropriate compound every 3 days for 2 weeks via transdermal injection and were then evaluated histologically. At 2 weeks, the cellular density and the quantity of granulation tissue deposition were the greatest in the hyaluronidase group and were lowest in the hyaluronic acid group. In addition, the organization of collagen fiber bundles was the most dense in the hyaluronidase group and least in the hyaluronic acid group. In a second experiment, polyvinyl alcohol sponge implants in mice received either phosphate-buffered saline solution or 20 microgram hyaluronic acid every 3 days for 1 week. On day 5, an aliquot of fluorescently tagged native collagen was injected into the sponges. Sponges were harvested at day 7, cryosections made, and the presence of autofluorescent collagen fibers assessed. The autofluorescent collagen fiber bundles in the phosphate-buffered saline solution group were organized in thick parallel bundles, whereas the collagen bundles from hyaluronic acid-treated implants were organized in fine lacelike structures. Chronic addition of hyaluronic acid appears to mimic the fetal dermal connective tissue matrix in which repair proceeds with diminished collagen deposition, organized in finer collagen fiber bundles in granulation tissue. On the other hand, the removal of hyaluronic acid by the chronic administration of hyaluronidase increases the amount of granulation tissue. Elevated levels of hyaluronic acid in granulation tissue appear to modulate the ability of resident fibroblasts to organize collagen fiber bundles.

Hyaluronic acid stimulates human fibroblast proliferation within a collagen matrix.

Human dermal fibroblasts suspended in a collagen matrix exhibit a 4-day delay in cell division, while the same cells in monolayer divided by day 1. The initial rates of 3H-thymidine incorporation by cells in monolayer or suspended in collagen were not significantly different. When suspended in collagen, there was a threefold increase in the proportion of cells in a tetraploidal (4N) DNA state compared to the same cells in monolayer. Flow cytometry analysis and 3H-thymidine incorporation studies identified the delay of cell division as a consequence of a block in the G2/M of the cell cycle and not an inhibition of DNA synthesis. The inclusion of 150 microg/ml of hyaluronic acid (HA) in the manufacture of fibroblast populated collagen lattices (FPCL) caused a stimulation of cell division, as determined by cell counting; increased the expression of tubulin, as determined by Western blot analysis; and reduced the proportion of cells in a 4N state, as determined by flow cytometry. HA added to the same cells growing in monolayer produced a minimal increase in the rate of cell division or DNA synthesis. HA supplementation of FPCLs stimulated cell division as well as tubulin concentrations, but it did not enhance lattice contraction. The introduction of tubulin isolated from pig brain or purchased tubulin into fibroblasts by electroporation prior to their transfer into collagen lattices promoted cell division in the first 24 hours and enhanced FPCL contraction. It is proposed that tubulin protein, the building blocks of microtubules, is limited in human fibroblasts residing within a collagen matrix. When human fibroblasts are suspended in collagen, one effect of added HA may be to stimulate the synthesis of tubulin which assists cells through the cell cycle.

Inhibiting the differentiation of myocardiocytes by hyaluronic acid.

BACKGROUND: In vitro experimentation found that wounded midgestation fetal mouse hearts heal scarlessly. Scarless repair occurs in an environment enriched in hyaluronic acid (HA), while in the absence of HA and the inclusion of hyaluronidase (HAdase), repair by scarring occurs. Excess HA downregulates the expression of the specific HA receptor, RHAMM (receptor for HA-mediated motility). The expression of RHAMM and the migration of cardiac cells from fetal heart explants were investigated in the presence of excess HA and added HAdase. METHOD: Hearts from Gestational Day 15 fetal mice (term = 20) were cut into four fragments, established as explant cultures, and assigned to one of three treatment groups: 400 micrograms/ml HA, 50 U/ml HAdase, or saline. Cellular outgrow was recorded at Day 7. The character of the migrating cells (fibroblast-like or myocardiocyte) was determined by immunostaining for filamentous actin (f-actin, microfilaments) or desmin (intermediate filaments). The expression of RHAMM was documented also. RESULTS: The inclusions of HA stimulated cell migration and proliferation, perpetuated cells as immature myocardiocytes, and blocked the expression of RHAMM. The inclusion of HAdase limited cell migration and proliferation, promoted the differentiation of cells into myocardiocytes, and increased the number of cells expressing RHAMM. CONCLUSION: Increased concentrations of HA promoted the proliferation and migration of an immature population of myocardiocytes. On the other hand the inclusion of HAdase inhibits the migration and proliferation of cells and promotes the appearance of myocardiocytes with a fibroblast-like morphology. The speculation is that excess HA may promote proliferation and migration of immature myocardiocytes into a heart defect, leading to replacement of lost myocardium with contractile tissue rather than dysfunctional scar.

Hyaluronan induces scarless repair in mouse limb organ culture.

BACKGROUND/PURPOSE: Wounded fetal mouse limbs harvested from two distinct time points in gestation heal differently in organ culture. The healing of a gestational day 14 limb is by scarless repair, whereas gestational day 18 (gd 18) limbs heal by scarring. The persistence of elevated levels of hyaluronic acid (HA) is a major difference in the extracellular matrix of scarless repair. The purpose of this study was to demonstrate that chronic additions of HA to incisional wounds of gd 18 limbs induces scarless repair. METHODS: Time-dated pregnant CD-1 mice (term, 20 days) were killed on gestational day 18 and fetuses were harvested via laparotomy. A through and through stab wound was made in each forelimb with a 1-mm microscapel, and the wound was closed with a single 10-0 nylon suture. The forelimbs were amputated at the level of the shoulder and placed in organ culture. Daily medium changes with 1 mL of BGJb (devoid of serum) were made. Half the cultures received 10 microL of HA (4 mg/mL) directly to the wound site with each medium change. The other half of the cultures received 10 microL of phosphate-buffered saline (PBS-control). At day 7, the limbs were harvested, fixed in methyl Carnoys solution, paraffin embedded, and 5-microm serial sections cut. The sections were stained with H&E or Sirius red/fast green. The sections were viewed in a blinded fashion by two observers. Suture defined the wound site, and the sections were graded for healing by scarring. RESULTS: Minimal limb growth occurred in both control and HA-treated limbs. Grossly, both control and treated limbs healed incisional wounds by 7 days in culture. Limbs from both treatment and control groups showed viability by microscopic analysis. The limbs treated with HA had no appreciable scar morphologically in sections in which epithelial dimpling and suture were evident. The orientation of the collagen fiber bundles in the control wounds were in parallel arrays perpendicular to the incision. The orientation of the collagen fiber bundles in the HA-treated limbs had a basket weave pattern that was indistinguishable from unwounded dermis. The direct repeated additions of HA to healing organ cultured limb explants of gestational day 18 fetal mice promoted scarless repair. CONCLUSIONS: This result demonstrates that chronic elevation of HA in the microenvironment of a wound affects healing by promoting the deposition of a more dermal-like connective tissue matrix in the wound site. The maintenance of elevated levels of HA could have utility in the clinical setting to improve the organization of connective tissue, leading to the reduction of scar complications.

Tumor necrosis factor mediates impaired wound healing in chronic abdominal sepsis.

BACKGROUND: The role of systemic tumor necrosis factor (TNF) as a mediator of impaired wound healing in sepsis is unclear. The purpose of this study was to examine the effects of a specific TNF antagonist (TNFbp) on wound healing during chronic abdominal sepsis. METHODS: Male Sprague-Dawley rats were divided into four groups: control, control + TNFbp, sepsis, and sepsis + TNFbp. Saline (1.0 mL) or TNFbp (1 mg/kg, 1.0 mL) was injected subcutaneously daily, polyvinylalcohol (PVA) sponge implants were placed in subcutaneous pockets, and sepsis was induced by creation of a chronic, intra-abdominal abscess. Sponge implants were removed on day 5 and examined histologically. Granulation tissue infiltration and quality (connective tissue, cellularity, vascularity) were scored on a scale from 1 to 4 in a blinded fashion. RESULTS: Septic mortality (19 vs. 25%) was not influenced by TNFbp. Granulation tissue penetration and quality were decreased in septic animals. The administration of TNFbp significantly attenuated the effects of sepsis on granulation tissue histology, but not to control levels. CONCLUSIONS: These studies provide evidence that TNF contributes to the impaired wound healing observed in this model of chronic abdominal sepsis.