De novo generation of permanent neovascularized soft tissue appendages by platelet-derived growth factor.

Treatment of wounds with pharmacologic doses of the BB homodimeric form of recombinant PDGF (rPDGF-BB) induces the recruitment, activation, and proliferation of mesenchymal cells, resulting in the deposition of provisional, and subsequently collagen-containing extracellular matrix. In preliminary experiments with an in vitro growth chamber model in the rat consisting of a silicone shell containing a dissected femoral vascular bundle, we found that rPDGF-BB incorporated into a rapidly dissolving collagen type I film induces the generation of a marked, but transient amount of de novo tissue around the femoral vascular bundle. In the present studies, the new tissue generated around the femoral vascular bundle was wrapped with a full thickness syngeneic skin graft to determine if functional support of the graft would lead to sustained maintenance of the underlying generated tissue and create an epithelialized soft tissue appendage. The tissue generated after a single application of rPDGF-BB was skin grafted on the 10th day, exteriorized 20 d later, and observed for an additional month. This led to the formation of soft tissue appendages which demonstrated marked neovascularization, fibroblast migration and proliferation, and increased glycosaminoglycan, fibronectin, and collagen fibril deposition, now leading to preservation of the newly generated tissue. In contrast, minimal new tissue was generated in control-treated vascular bundles or bundles treated with inactive PDGF-BB, and grafting with skin failed to sustain the underlying tissue. Thus, rPDGF-BB coupled with skin grafting induced the formation of functional large soft tissue appendages which are potentially useful clinically to fill tissue defects or to serve as a cell delivery system for transfected genes.

Localization of topically applied TFPI binding sites in an intimectomized microvessel.

Tissue factor pathway inhibitor, TFPI, has been shown to be highly effective as a topically applied antithrombotic in an arterial model of vascular thrombosis. To elucidate the mechanism and site of TFPI action, recombinant TFPI was conjugated to 30 nm diameter gold particles and used to localize the sites of TFPI binding in a traumatized microvessel by transmission electron microscopy. The model, the central artery of the rabbit ear, was transected, denuded of endothelial lining (intimectomized), and re-anastomosed. Prior to the restoration of blood flow, TFPI-gold or unconjugated gold particles in solution were applied by irrigation to the intimectomized vessel lumen. After 10 min of blood flow, the artery was harvested for electron microscopy. TFPI-gold binding was localized to the fine strands of fibrin that lined the lumen of the intimectomized section of the artery. Little or no binding was found on platelets, exposed smooth muscle, cell membrane fragments, or uninjured vessel segments. The TFPI-gold binding could be competed with native TFPI. TFPI-gold was inhibitory, although less potent than native TFPI, in a prothrombin time assay. Unconjugated gold exhibited very little binding in the vascular model. Hence, the TFPI-gold conjugate behaved like native TFPI. Our observations have identified the fibrin complex as an in vivo binding site for TFPI and suggest that this is an in vivo site of action for TFPI as a topical antithrombotic agent.

Tissue generation with growth factors.

BACKGROUND: An in vivo experimental model was introduced to determine whether the mitogenic effect of recombinant platelet-derived growth factor (rPDGF) could be used to generate potentially useful tissue. METHODS: In Lewis rats, the extended femoral arteriovenous bundle was placed within silicone chambers containing collagen disks. The disks could deliver their content of rPDGF-BB (125 to 131 micrograms/disk) either as a rapid pulse or as a slow release. The time course of tissue generation was determined by harvesting the specimens at various postoperative days. The effect of continuous versus pulsed delivery was determined at 30 days. Analysis of the generated tissue was performed by use of histomorphometry. RESULTS: Pulsed delivery of rPDGF-BB induced the formation of a substantial amount of tissue that peaked at 10 to 15 days (145.9 +/- 13.8 vs 35.0 +/- 6.8 mm3, p < 0.0001); however, the generated tissue completely subsided by day 30. Sustained delivery of rPDGF-BB caused continuous growth of the tissue and was more effective than pulsed delivery. CONCLUSIONS: In an experimental model that approximates an in vivo tissue culture system, rPDGF-BB can induce a tenfold increase in tissue within the chamber. However, that tissue is labile and its survival necessitates continuous rPDGF-BB delivery. To become useful for reconstructive purposes, means to stabilize this new tissue growth are needed.

Endoscopic carpal tunnel release. The voice of polite dissent.

It is ironic for us that our own studies, showing that neurolysis of the median nerve does not confer benefit in the primary OCTR, have been cited frequently as a reason for not needing to visualize the median nerve and that this indirectly has supported ECTR. Our recognition that even OCTR can cause significant complications that may not be able to be corrected, however, makes us very respectful of carpal tunnel release, a procedure that has been called the simplest of all operations. We, therefore, reject any modification that in any way presents more risk to even a single patient.

Prevention of ischemia-reperfusion injury with a synthetic metalloprotein superoxide dismutase mimic, SC52608.

Superoxide dismutase (SOD) scavenges free superoxide radicals generated during reperfusion of ischemic tissue and decreases cellular injury. A synthetic manganese-based metalloprotein superoxide dismutase mimic, SC52608 (Monsanto Co.), was tested in the isolated rabbit rectus femoris muscle flap to determine its effects on ischemia-reperfusion injury. The results of our experiments analyzing 38 isolated rectus femoris muscles in 19 New Zealand White rabbits show that administration of SC52608 at the onset of 4 hours of warm ischemia and before reperfusion significantly increases the survival of the muscle from 20.0 +/- 4.9 percent (control, HEPES) to 81.5 +/- 4.6 percent (SC52608) (p < 0.001). It preserved functional contraction in 8 of 10 muscles; only 1 of 12 control muscles (control, HEPES) had contractions (p = 0.0015). SC52608 decreased the neutrophil density from 4.63 +/- 0.6 x 10(4) cells/mm2 in the control (HEPES) muscle to 2.71 +/- 0.6 x 10(4) cell/mm2 in muscles perfused with SC52608 (p = 0.03). The level of malonyldialdehyde decreased from 6.12 +/- 0.26 nmol/gm (control, HEPES) to 4.64 +/- 0.41 nmol/gm (SC52608) (p = 0.0028). Postoperative weights of the muscles showed no statistical difference (p = 0.14) between the controls (16.0 +/- 0.9 gm) and the SC52608 (18.1 +/- 0.7 gm). Our investigation shows that direct intraarterial infusion of a synthetic superoxide dismutase mimic at the onset of ischemia and prior to reperfusion can reduce reperfusion injury in skeletal muscle.

Repair of calvarial defects with flap tissue: role of bone morphogenetic proteins and competent responding tissues.

Bone morphogenetic proteins 2 through 8 have the ability to induce the in vivo transformation of extraskeletal mesenchymal tissue into bone. The aims of this investigation were to determine the optimal responding tissue and the specificity of the inductive effect of bone morphogenetic protein 3. The optimal responding tissue was found to be skeletal muscle. The specificity of this response to bone morphogenetic protein 3 was compared with that of recombinant human basic fibroblast growth factor, recombinant platelet-derived growth factor, and recombinant insulin-like growth factor. Bone morphogenetic protein 3 was the only factor that induced de novo bone formation. This ability to transform muscle into bone was tested in 7 x 7 mm irradiated skull defects in the rat. After 1500 rads of exposure, these defects showed no significant signs of healing by 8 months. When these defects were treated with the microvascular transfer of a nonirradiated muscle flap, they had 8 percent healing at 4 months and 37 percent healing by 8 months. Defects treated with 30 micrograms bone morphogenetic protein 3 (without the muscle flap) achieved 50 percent healing by 4 months and 64 percent healing by 8 months. When the defects were treated with both the muscle flap and bone morphogenetic protein 3, there was 96 percent healing by 4 months and 100 percent healing by 8 months (p < 0.015, compared with bone morphogenetic protein 3 alone at both time points). At 8 months, the transplanted muscle was entirely transformed into bone and healed the skull defect with newly generated bone indistinguishable from the surrounding calvarial tissue. These findings suggest a potential clinical utility of bone morphogenetic protein 3-induced bone formation in skeletal reconstructions. Furthermore, they also show that there is a collaborative requirement for both the osteoinductive factor bone morphogenetic protein 3 and the presence of competent responsive cells in the well-perfused muscle.

Prevention of microvascular thrombosis by topical application of recombinant tissue factor pathway inhibitor.

Tissue factor pathway inhibitor is a naturally occurring protein inhibitor of factor X and the tissue factor-factor VII complex of the extrinsic pathway of coagulation. The potential of tissue factor pathway inhibitor as a topical antithrombotic agent was evaluated in a rabbit model of thrombosis that combined intimal injury, anastomosis, and a twisted pedicle. In 207 rabbit ears, a near-complete amputation was performed, preserving the central ear artery and vein. The central ear artery was transected, the intima was removed mechanically over a 1-cm length, the artery was anastomosed, and the ear was twisted 360 degrees, wrapping the intact vein around the artery. Before recirculation, the lumen was irrigated on a blinded, randomized basis with either hirudin (100 or 500 units/ml), heparin (50 or 100 units/ml), tissue factor pathway inhibitor (10, 40, 125, or 250 microgram/ml), heparin and tissue factor pathway inhibitor together, or vehicle (control). Upon arterial reflow, the ears were observed for 7 days. Patency rates after 7 days were as follows: hirudin, 30 and 55 percent; heparin, 43 and 50 percent; tissue factor pathway inhibitor, 75 and 90 percent; heparin and tissue factor pathway inhibitor, 75 percent; and vehicle, 6 percent. The higher concentrations of tissue factor pathway inhibitor led to significantly higher patency rates than heparin, hirudin, or control solutions. Electron microscopic evaluation of specimens irrigated with gold- labeled tissue factor pathway inhibitor revealed the inhibitor bound to the injured intimal surface for at least 3 days postoperatively. Coagulation studies showed no change in the clotting profile upon intravascular infusion with tissue factor pathway inhibitor even at the highest dose used topically. We conclude that tissue factor pathway inhibitor is a more effective topical antithrombotic agent than either heparin or hirudin.

Rat strain differences in flap tolerance to ischemia.

The rat epigastric island flap model is commonly used to explore ischemia-related phenomena. We sought to evaluate strain differences in tolerance to ischemia using two commonly used rat strains: Sprague-Dawley and Lewis. Epigastric flaps (3 x 6 cm) based on the superficial epigastric artery and vein were raised bilaterally in each rat (2 flaps/rat). Ischemia was induced for 10,12,14, or 16 hours by placing temporary occlusion clamps on each vessel of the vascular pedicle. Surviving flap areas were assessed planimetrically after 7 days. The average area of surviving flap tissue was greater in the Lewis rats for all ischemia times; this achieved significance for 12 hours and 14 hours of ischemia (P < 0.005). These findings indicate that comparisons among studies on rat flap ischemia must take into account the particular strain used. Furthermore, these findings suggest an inherent capacity of Lewis rat tissue to withstand ischemia better than tissue of the Sprague-Dawley rat strain.

Utility of the lateral arm flap in head and neck reconstruction.

Soft-tissue defects of the head and neck are often reconstructed with fasciocutaneous free flaps. The radial forearm flap is used most commonly, however the lateral arm flap may be the flap of choice in certain situations. Advantages include flap elevation with simultaneous tumor ablation, avoidance of intraoperative patient position changes, and primary closure of the donor site. After extirpative procedures of the head and neck region, 4 patients were reconstructed with the lateral arm flap. Flap survival was 100%, a vein graft to supplement the short pedicle length was necessary in 1 patient, all donor sites were closed primarily, and secondary procedures to reduce flap bulk were necessary in 2 patients. The lateral arm flap is an excellent alternative to the radial forearm flap and should be included in the armamentarium of the reconstructive head and neck surgeon.

Multiple-component tissue reconstruction of a complex dorsal foot wound through a single gracilis muscle donor incision.

The authors describe the ability of the gracilis muscle to provide multiple tissue components- skin, muscle, nerve, and tendonin the reconstruction of a complex dorsal foot wound resulting from a sarcoma resection. The deficits of skin, deep peroneal nerve, anterior tibialis tendon, and dorsal extensor retinaculum were all reconstructed with the gracilis component flap through one medial thigh incision. This case illustrates two important points: (1) the gracilis flap is tremendously versatile and can serve as the donor for multiple tissue components in complex reconstructions, and (2) donor site morbidity can and should be minimized even in complex reconstructions.

Experimental reproduction of free flap errors: a new model of thrombosis.

Surgical errors in the length and orientation of a flap pedicle can result in kinks, twists, or tension on the pedicle vessels. When these errors occur together with the microvascular repairs associated with free flap transfer, thrombosis and flap failure may be the outcome. A model was designed to simulate these events. The rabbit central ear artery and vein were dissected and all other tissue connections were severed in a near-complete amputation. The artery was transected, shortened by 2 mm, and repaired standardly. The ear was then rotated 360 degrees, twisting the vein around the repaired artery. The ear cartilage was shortened by 6 mm, then sutured, and the skin was closed. In a series of 20 ears, 95% necrosed, 18 from arterial thrombosis and 1 from venous thrombosis. Several other groups were used in which aspects of the basic model were modified to determine the factors influencing failure. When the arterial anastomosis was not performed and the vascular pedicle was rotated with cartilage shortening, all ears survived; when the cartilage was not resected in this paradigm, leaving tension on the pedicle, 50% of the ears necrosed from venous thrombosis. In counterpoint, when the artery was repaired without twisting the pedicle, all ears survived, whether or not the cartilage was shortened. When the artery was repaired without shortening it and with rotation of the ear, 50% of the ears necrosed when the cartilage was shortened, 40% from venous thrombosis and 10% from arterial thrombosis; all ears necrosed (of venous thrombosis) when the cartilage was not shortened.(ABSTRACT TRUNCATED AT 250 WORDS)

Topical tissue factor pathway inhibitor improves free-flap survival in a model simulating free-flap errors.

Free flap failure is frequently due to tension, twisting, kinking, or compression of the vascular pedicle after the anastomosis is completed. A rabbit model simulating these errors was used to evaluate the capacity of topically-applied tissue factor pathway inhibitor (TFPI) to prevent microvascular thrombosis. The rabbit ear was isolated on the central artery and vein. The artery was transected, shortened, repaired, and twisted 360 degrees around the vein. Immediately following the anastomosis. TFPI in concentrations of 1, 4, 10, or 40 micrograms/ml was irrigated across the lumen. Topically-applied control buffer and heparin (50 U/ml) were compared to TFPI. Treatment with control buffer resulted in a 20 percent survival rate. Topically-applied heparin improved the survival rate to 60 percent (p < 0.05). In contrast, TFPI in concentrations of 4, 10, and 40 micrograms/ml yielded survival rates of 89, 100, and 97 percent, respectively. This was significantly greater than the heparin-treated ears (p < 0.05). TFPI in a concentration of 40 micrograms/ml was effective in preventing arterial thrombosis when applied for as little as 30 sec; 4 micrograms/ml was effective in preventing thrombosis when applied for 10 min. These results support the use of TFPI as a topical irrigation solution to help prevent microvascular arterial thrombosis in free-flap surgery.