Early growth response factor-1: expression in a rabbit flexor tendon scar model.

BACKGROUND: Adhesion formation limits functional recovery after flexor tendon repair. Various growth factors have been implicated in the adhesion scar process. Early growth response factor-1 (EGR-1), a transcription factor associated with synthesis of a variety of key fibrotic growth factors and expression of extracellular matrix genes, has never been identified in a tendon repair model. METHODS: Thirty New Zealand White rabbit forepaws underwent laceration and repair of the middle digit flexor digitorum profundus equivalent in zone II. Sodium morrhuate, a topical sclerosing agent, or phosphate-buffered saline, a standard control, was applied to the repair during closure of the tendon sheath. Tendons were harvested from operated and unoperated forepaws at increasing time intervals (1, 3, 7, 14, and 28 days). Tissues were analyzed by immunohistochemistry and Masson trichrome staining. RESULTS: Immunohistochemistry demonstrated that EGR-1 is expressed at the site of tendon repair, along the epitenon of the tendon, and in the infiltrate of inflammatory cells in the surrounding sheath-scar matrix. Control, unoperated tendons demonstrated baseline EGR-1 expression within epitenon cells. EGR-1 was maximally expressed on postoperative day 7. Sodium morrhuate and phosphate-buffered saline demonstrated no difference in their ability to augment tendon adhesion scar formation. CONCLUSIONS: : Findings demonstrate the following: (1) EGR-1 expression is increased in the tendon wound environment after flexor tendon laceration repair; (2) normal epitenon cells have low, baseline levels of EGR-1 expression; and (3) sodium morrhuate does not augment scar matrix production more than phosphate-buffered saline. The ideal tendon scar model was not generated.

Transgene expression in a model of composite tissue allotransplantation.

BACKGROUND: Composite tissue allografting may be an ideal solution to many problems requiring reconstructive surgery. Unfortunately, complications associated with chronic immunocompromise are major impediments to widespread use of composite tissue allografting. Current immunosuppressive and immunomodulatory paradigms focus on modification of the recipient through global immunosuppression or donor/recipient chimerism. Alternatively, modifying the allograft to block rejection or promote tolerance could confine deleterious immunosuppressive effects to the graft or eliminate graft rejection. However, a technique introducing genetic information into the transplant is needed. The authors demonstrate the first model for expressing a gene of interest locally in a hind-limb transplant. METHODS: Using a rat hind-limb transplant model, the authors tested the ability of naked DNA infusion, cationic polymer/DNA complex transfection, and adenoviral vector transduction to introduce genetic material into the composite tissue allograft. The marker genes luciferase and green fluorescent protein were used to follow gene expression. RESULTS: Recombinant adenovirus showed rapid, high-level expression of marker genes in the graft, with no detectable expression in recipient animals. Expression was detectable at 18 hours and peaked at 7 days. Levels of expression were lower but above baseline at 4 weeks. CONCLUSIONS: Using an adenoviral vector system, the authors have selectively introduced a marker gene (luciferase) into a transplanted hind-limb rat model. Expression was rapid and seen in a variety of cell types. Adenovirus infection had no impact on limb rejection. This method may be a powerful tool for genetically modifying composite tissue allografts and may contribute to immune tolerance and more widespread use of composite tissue allograft surgery.

Volume analysis of preadipocyte injection for vocal cord medialization.

OBJECTIVE: To compare the volume retention of injected preadipocytes with that of standard fat injection in a paralyzed rabbit true vocal cord. STUDY DESIGN: Prospective analysis with blinded data collection. METHODS: Thirteen New Zealand white rabbits were divided into two groups. Group 1 consisted of seven animals undergoing left-side vocal cord paralysis by resection of a 1-cm segment of the left-side recurrent laryngeal nerve and abdominal fat harvest for isolation of preadipocytes. Preadipocytes were cultured under sterile conditions in cell culture media. Animals in group 2 also underwent left-side vocal cord paralysis without fat harvest. After 10 to 14 days, in a second procedure, group 1 underwent injection of 0.1 mL cultured autologous preadipocytes, and group 2 underwent routine injection of 0.1 mL abdominal fat harvested during the same procedure. At 6 and 12 months, volumetric analysis was performed. RESULTS: Volume analysis at 6 months showed a mean volume of 0.029 mL retained fat in group 2 representing a retention of approximately 29% (SD = 0.023) of the original injected volume. Retention in group 1 animals approximated 0.002 mL (SD = 0.0024) or 2% of the injected volume. Analysis at 12 months showed a mean volume of 0.008 mL (SD = 0.0078) in group 2 and of 0.002 mL (SD = 0.0015) in group 1. Group 2 showed significantly higher volumes of the injected fat at 6 and 12 months (P <.033). CONCLUSION: Volumes obtained with standard fat injection were superior to those obtained with preadipocyte injection at both 6 and 12 months.

A tissue-engineering technique for vascularized laryngotracheal reconstruction.

OBJECTIVE: To perform laryngotracheal reconstruction (LTR) using a vascularized neotracheal segment. DESIGN: A neotracheal segment was created within the sternocleidomastoid muscle. An anterior cricoid split procedure was performed using a pedicled, vascularized neotracheal segment. Results were compared with a control group that underwent anterior cricoid split using standard (avascular) autografted cartilage. Cross-sectional area, cartilage viability, extrusion, mucosalization, and wound healing were compared between groups. SUBJECTS: Sixteen female New Zealand white rabbits. INTERVENTIONS: Eight animals underwent placement of a cartilage-wrapped silicone implant into the sternocleidomastoid muscle. After 2 weeks, the silicone implant was removed, leaving a fibrovascular "foreign body" capsule and the interwoven autografted cartilage. The neotracheal segment was trimmed to create an anterior graft for LTR. The remaining animals underwent standard anterior graft LTR using autografted auricular cartilage. The reconstructed segments were harvested for comparison at 2 and 4 weeks. RESULTS: All reconstructed animals survived the postoperative period. No significant differences in stenosis rates or mucosalization were noted between groups. Two animals in the standard LTR group had microabscess formation, and no graft extrusions were encountered. CONCLUSION: A pedicled neotracheal graft can be used for anterior cricoid split procedures in rabbits.

Local hypothermia during early reperfusion protects skeletal muscle from ischemia-reperfusion injury.

Amputated tissue maintained in a hypothermic environment can endure prolonged ischemia and improve replantation success. The authors hypothesized that local tissue hypothermia during the early reperfusion period may provide a protective effect against ischemia-reperfusion injury similar to that seen when hypothermia is provided during the ischemic period. A rat gracilis muscle flap model was used to assess the protective effects of exposing skeletal muscle to local hypothermia during ischemia only (p = 18), reperfusion only (p = 18), and both ischemia and reperfusion (p = 18). Gracilis muscles were isolated and exposed to hypothermia of 10 degrees C during 4 hours of ischemia, the initial 3 hours of reperfusion, or both periods. Ischemia-reperfusion outcome measures used to evaluate muscle flap injury included muscle viability (percent nitroblue tetrazolium staining), local edema (wet-to-dry weight ratio), neutrophil infiltration (intramuscular neutrophil density per high-power field), neutrophil integrin expression (CD11b mean fluorescence intensity), and neutrophil oxidative potential (dihydro-rhodamine oxidation mean fluorescence intensity) after 24 hours of reperfusion. Nitroblue tetrazolium staining demonstrated improved muscle viability in the experimental groups (ischemia-only: 78.8 +/- 3.5 percent, p < 0.001; reperfusion-only: 80.2 +/- 5.2 percent, p < 0.001; and ischemia-reperfusion: 79.6 +/- 7.6 percent, p < 0.001) when compared with the nonhypothermic control group (50.7 +/- 9.3 percent). The experimental groups demonstrated decreased local muscle edema (4.09 +/- 0.30, 4.10 +/- 0.19, and 4.04 +/- 0.31 wet-to-dry weight ratios, respectively) when compared with the nonhypothermic control group (5.24 +/- 0.31 wet-to-dry weight ratio; p < 0.001, p < 0.001, and p < 0.001, respectively). CD11b expression was significantly decreased in the reperfusion-only (32.65 +/- 8.75 mean fluorescence intensity, p < 0.001) and ischemia-reperfusion groups (25.26 +/- 5.32, p < 0.001) compared with the nonhypothermic control group (62.69 +/- 16.93). There was not a significant decrease in neutrophil CD11b expression in the ischemia-only group (50.72 +/- 11.7 mean fluorescence intensity, p = 0.281). Neutrophil infiltration was significantly decreased in the reperfusion-only (20 +/- 11 counts per high-power field, p = 0.025) and ischemia-reperfusion groups (23 +/- 3 counts, p = 0.041) compared with the nonhypothermic control group (51 +/- 28 counts). No decrease in neutrophil density was observed in the ischemia-only group (40 +/- 15 counts per high-power field, p = 0.672) when compared with the nonhypothermic control group (51 +/- 28 counts). Finally, dihydrorhodamine oxidation was significantly decreased in the reperfusion-only group (45.83 +/- 11.89 mean fluorescence intensity, p = 0.021) and ischemia-reperfusion group (44.30 +/- 11.80, p = 0.018) when compared with the nonhypothermic control group (71.74 +/- 20.83), whereas no decrease in dihydrorhodamine oxidation was observed in the ischemia-only group (65.93 +/- 10.3, p = 0.982). The findings suggest a protective effect of local hypothermia during early reperfusion to skeletal muscle after an ischemic insult. Inhibition of CD11b expression and subsequent neutrophil infiltration and depression of neutrophil oxidative potential may represent independent protective mechanisms isolated to local tissue hypothermia during the early reperfusion period (reperfusion-only and ischemia-reperfusion groups). This study provides evidence for the potential clinical utility of administering local hypothermia to ischemic muscle tissue during the early reperfusion period.

The use of split-thickness dermal grafts to resurface full thickness skin defects.

Coverage of large burns may be difficult when skin graft donor sites are limited. This study explored the use of the split-thickness dermal graft (STDG), as an alternative to the standard split-thickness skin graft (STSG). STSGs and STDGs were compared experimentally by their ability to resurface full thickness skin defects in a pig model. Both types of grafts were harvested from the backs of six pigs and placed on full thickness wounds. From the same donor site a 0.012in. thick STSG and another two 0.012in. thick STDGs were harvested. Thus the deep surface of grafts measured 0.012, 0.024 and 0.036in. from the skin surface, respectively. All grafts were placed on 6cmx6cm full thickness wounds. The donor areas healed at 1 week. Epithelialization of the STDGs, was assessed by computerized planimetry, and was 100% at 4 weeks. Graft biopsies revealed that STSGs were significant thinner than STDGs at 1 week (P=0.0422, 0.0135), 2 weeks (P=0.0240) and 4 weeks (P=0.0516, 0.0425). We conclude that STDGs my provide definitive coverage of full thickness skin deficits in a pig wound model.