Negative-pressure dressings as a bolster for skin grafts.

Contoured wounds needing closure with skin grafts are often located in complex anatomic regions or are in unusual positions, which make conventional skin graft stabilization techniques cumbersome and ineffective. Often after 72 hours, a skin graft covered with a bolstered dressing has poor take secondary to shear stresses, as well as hematoma formation or serum collection, negating the effectiveness of the stabilizing dressing. The Food and Drug Administration has recently approved vacuum-assisted closure (V.A.C.), an innovative technique using negative pressure, for closure of chronic wounds. This reportedly leads to enhanced granulation tissue formation and consequently more rapid reepithelialization of wounds compared with conventional packing with saline-moistened gauze. Experimental studies have demonstrated increased oxygen tension, decreased bacterial counts, and increased granulation formation occurring under negative-pressure systems. Extending the use of this concept, we have coupled skin grafting with negative-pressure dressings for closure of large, complex open wounds. Our results indicate greater than 95% graft take in all patients in this study. This technique is extremely efficacious, with increased graft take due to total immobilization of the graft, thereby limiting shear forces, elimination of fluid collections, bridging of the graft, and decreased bacterial contamination. Moreover we have noted decreased edema in rotated muscle flaps, improved contour conformity, and shortened hospitalizations.

Regenerative healing of incisional wounds in midgestational murine hearts in organ culture.

OBJECTIVE: Using an organ-culture fetal heart repair model, we explored fetal repair in tissues other than dermis. METHODS: Wounded fetal mouse hearts of 14 and 18 days' gestation (term = 20 days), as well as hearts of 22 days' gestation (newborn), were maintained in serum-free medium. Specimens were fixed at 2, 7, and 11 days and then processed for histologic examination. Small fragments of fetal hearts from all time points were cultured as explants. The migration of cells from the periphery of the explants was compared at day 4, and the pattern of microfilaments in these cells was assessed. RESULTS: In 14-day hearts (n = 18), tissue architecture was rapidly reestablished without an inflammatory response or scarring, constituting regenerative repair. In 18-day hearts (n = 18), no reestablishment of muscle fibers or wound closure occurred. In the 22-day explants (n = 12) the wounds closed by scarring. Cell migration from 14-day explants was 4.7 +/- 2.3 ocular units; from 18-day explants, it was 2.6 +/- 1.1 ocular units; and from 22-day explants, it was 0.9 +/- 0.4 ocular units. Microfilaments of 14-day cells were arranged at the periphery of the cell consistent with cardiomyocytes. Microfilaments of 18- and 22-day cells were arranged in parallel arrays (stress fibers) that were consistent with fibroblasts. CONCLUSIONS: We propose that regenerative healing of 14-day fetal hearts is by the migration of cardiomyocytes. At 18 and 22 days, cardiomyocytes are too differentiated and unable to migrate; hence cell migration is limited to resident fibroblasts, which are deficient at 18 days but sufficient at 21 days to be repaired by the scarring process.

Regenerative healing of incisional wounds in murine fetal lungs maintained in organ culture.

Although fetal dermal repair is known to be fundamentally different from adult healing, the response to wounding in other organs is less well characterized. Scarless repair in mid-gestation dermis with a transition to adult-type healing at term has been shown in fetal organ culture. A lung explant culture system was used to investigate whether wound repair in the fetal lung shows characteristics similar to those found in fetal dermis. Lungs from 14-day and 18-day Cd-1 murine fetuses and 2-day-old newborns, (term = 20 days, n = 24) were wounded by linear incision and incubated at 37 degrees C, in a 21% O2, 5% CO2 environment, in BGJb supplemented with vitamin C and antibiotics. Medium was changed daily. Samples were fixed at 7 days and embedded in paraffin. Sections were stained with hematoxalyn-eosin and Masson Trichrome. Additional 14-day and 18-day samples were frozen in freon and immunohistochemical staining for TGF-beta performed. Other frozen tissues from each time point were homogenized and used to assay for endogenous TGF-beta levels by Western blot analysis. Histology showed reconstitution of tissue architecture across the wound in 14-day and 18-day specimens. In representative histological sections, intact bronchial architecture developed across the previous wound site. No cellular inflammatory response was observed, and collagen deposition was undetectable at the site of the wound by Trichrome staining. By 22 days the lung explants showed a much less ordered repair, including disorganized collagen deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

The extracellular matrix of the fetal wound: hyaluronic acid controls lymphocyte adhesion.

Adhesive interactions between lymphocytes and components of the extracellular matrix (ECM) within a wound environment play a crucial role in determining the inflammatory response following tissue injury. In fetal wounds the extracellular matrix is composed predominantly of hyaluronic acid. Within this environment the inflammatory reaction as a result of injury is minimal. We propose that this lack of an inflammatory cell response in the fetal wound is due to the high levels of hyaluronic acid within the ECM and the inability of lymphocytes to adhere to this matrix component. Therefore, we examined the adhesive properties of fetal lymphocytes to fibronectin, vitronectin, collagen types I, III, IV, V, and hyaluronic acid--ECM components involved in fetal and adult wound environments. Fetal lymphocytes from both spleen and thymus demonstrated significant binding capabilities to fibronectin, vitronectin, and collagen types I and III. No intrinsic binding capabilities were detected to hyaluronic acid. Adhesion was not affected by the addition of IL-1, IFN-gamma, or phorbol dibutyrate. The inability of lymphocytes to adhere to hyaluronic acid helps to explain the lack of inflammation found in fetal wounds and serves to demonstrate the importance of ECM-lymphocyte interactions in determining the inflammatory response during fetal wound healing.

Immune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic-suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat.

The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells (7-9 days, n = 6) or inocula (5 X 10(6) cells) depleted of SIg+ cells (10-13 days, n = 5), but additional depletion of cytotoxic T cells and their precursors (OX8+) resulted in marked prolongation of graft survival (n = 4, 4 X 10(6) cells, 45-67 days). Reducing the reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells (OX8-, SIg-) prolonged graft survival to that observed in unreconstituted recipients (generally greater than 100 days). Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate (stimulation index [SI] 1.01, P = NS) on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated (SI 4.25, P less than .001) and generated greater cytotoxic T lymphocyte (CTL) activity (P less than .001) under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency. Further, though PVG-RT1r1 heart grafts were rejected in 10-12 days (n = 3) by (PVG X WF)F1 recipients, (PVG-RT1r1 X WF)F1----(PVG X WF)F1 heart grafts (RT1Aa disparity) survived greater than 100 days despite an equivalent alloimmune response, and this was shown to correlate with a reduced sensitivity of (PVG-RT1r1 X WF)F1 target cells to lysis by CTL. These data, therefore, strongly suggest that the pivotal role of CTL in the rejection of class-I-disparate heart grafts is, in fact, related to their function in direct cell-mediated cytolysis.

Immune mechanisms in organ allograft rejection. VI. Delayed-type hypersensitivity and lymphotoxin in experimental renal allograft rejection.

The cellular requirements for renal allograft rejection have been reassessed in a rat adoptive transfer model, preceding studies having shown that transplanted kidneys may be rejected in the absence of cytotoxic T cells or specific antibody. Unilaterally nephrectomized, sublethally irradiated (780 rads) LEW recipients of renal allografts from irradiated WF donors, were selectively reconstituted with spleen cells from sensitized syngeneic donors and subjected to delayed nephrectomy of the residual native kidney 3 days posttransplantation. In some experiments the reconstituting inocula were depleted of SIg+ cells (anti-Ig column) or additionally depleted of cytotoxic T cells and their precursors reactive with monoclonal OX8 (rosette depletion). Depleting the reconstituting inocula of SIg+ cells as well as cells reactive with monoclonal OX8 failed (n = 4) to alter the tempo of rejection, as demonstrated by a mean serum creatinine +/- SD on day 8 of 5.4 +/- 3.8 vs. 6.4 +/- 4.2 in recipients (n = 8) reconstituted with unfractionated inocula. These data support a link between DTH and graft rejection, so additional studies were performed to characterize rat lymphotoxin (LT), one of the potential mediators of DTH-induced tissue injury, and to demonstrate the presence of LT in rejecting rat renal allografts. Rat LT, generated in vitro by stimulating spleen cells from specifically sensitized rats with keyhole limpet hemocyanin (100 micrograms m/ml), was shown on gel filtration to have an MW of approximately 50,000. In-vitro-generated rat LT was shown to be heat stable (70 degrees C for 15 min) and soluble in 40% (NH4)2SO4. Rat LT eluted as a single peak on DEAE anion exchange chromatography (0-0.15 M, NaCl osmotic gradient), supporting the existence of but a single molecular form. LT was isolated from rejecting renal allografts on day 6 after renal transplant but undetected (less than 1 unit) in residual native kidneys. This study, therefore, provides substantial support for a link between DTH and renal allograft rejection. Lymphotoxin, one of the potential mediators of tissue injury in this model system, has been partially characterized and demonstrated to be present in rejecting rat renal allografts.

Immune reactivity and immunosuppressive intervention (TLI) in experimental nephritis. I. Immunopathologic correlates in the accelerated autologous form of nephrotoxic serum nephritis.

The progression of histologic changes observed in the accelerated autologous form of nephrotoxic serum nephritis (AA-NTSN) in the inbred LEW rat, ranging from minimal endocapillary proliferation to marked endo- and extra-capillary proliferation together with fibrin deposition and necrosis of glomerular tufts, was closely correlated (day 8) with parallel measures of albuminuria (1.6 leads to 280 mg/24 hr). In the induction phase of AA-NTSN (day 0) a direct linear correlation (r = 0.72, p less than 0.05) was obtained between anti-SGG antibody levels and 24-hr urine albumin excretion. Subsequent observations (day 7) yield an inverse correlation between anti-SGG antibody and albuminuria (r = -0.54, p less than 0.01), and this change was shown to be related to the loss of specific antibody in the urine (r = 0.59, p less than 0.025). Although splenic T cells of rats with AA-NTSN manifest a specific proliferative response to the inducing antigen SGG, and although the magnitude of this proliferative response (day 7) correlates directly with parallel measures of urine albumin excretion (r = 0.71, p less than 0.02), delayed-type hypersensitivity (DTH) to SGG measured by radiometric ear assay was inversely correlated with albuminuria in this model of experimental nephritis (r = 0.80, p less than 0.01). Additional data are presented that strongly suggest that persistent albuminuria at 1 mo after induction of AA-NTSN is independent of ongoing immunopathologic processes. Thus, glomerular injury in AA-NTSN, previously linked to prominent glomerular macrophage accumulation, is shown to be primarily dependent on a brisk antibody response to the planted heterologous (sheep) immunoglobulin, whereas cellular immunity, readily demonstrated in the splenic T cell compartment, is likely to be of lesser import in the pathogenesis of this lesion in the intact animal.