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.

Prevention of spinal cord injury after transient aortic clamping with tissue factor pathway inhibitor.

BACKGROUND: Lower limb paralysis that occurs in 11% of patients after treatment of thoracic and thoracoabdominal aortic aneurysms is unpredictable and at present not preventable. The proposed cause for the neurologic changes is believed to be spinal cord ischemia combined with ischemia/reperfusion injury. Recombinant tissue factor pathway inhibitor (rTFPI), a multivalent Kunitz-type inhibitor that binds to tissue factor-VIIa complex, was evaluated. METHODS: The effectiveness of rTFPI as an agent to limit spinal cord ischemia/reperfusion injury was studied in a rabbit spinal cord made ischemic for 20 minutes. rTFPI or phosphate-buffered saline solution (control) was given in randomized blinded fashion at the onset and conclusion of ischemia. Animals underwent neurologic evaluation at 24 hours in a blinded fashion with a modified Tarlov Scale to rate the lower limb paralysis (score of 4 = normal function, score of 0 = complete paralysis). RESULTS: Seventy-five percent of the TFPI-treated animals had Tarlov scores of 3 to 4, whereas only 29% of the animals treated with phosphate-buffered saline solution had such scores (p < 0.0014). Spinal cord histologic findings correlated with the neurologic findings. CONCLUSIONS: We believe that TFPI has unique inhibitory properties that make it an effective agent in limiting postoperative paraplegia associated with spinal ischemia.

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.

Thigh adductor flap: an experimental model for free flap transfer in the rat.

A new model of rat muscle free flap transfer is presented. The flap is based on a long pedicle originating from the femoral vessels and continuing down to the distal saphenous margin at the ankle. The distal portion of the semitendinosus muscle is harvested along with the saphenous artery and great saphenous vein. A larger muscle flap may be harvested by including the distal portions of the posterior and anterior gracilis muscles. The flap can be transferred to remote sites on the animal, with a vascular pedicle of up to 3 cm in length. The diameters of the vessels repaired are of standard microsurgical size. This model has distinct advantages of simplicity, speed of dissection, high success rate after microvascular transfer, and versatility in placement of the muscle mass (due to the long pedicle).

Tissue transformation into bone in vivo. A potential practical application.

The transformation of mesenchymal tissue, such as muscle, into cartilage and bone can be induced by the recently purified osteoinductive factor, osteogenin, and by its parent substratum, demineralized bone matrix. We investigated the possibility of transforming readily available muscle flaps into vascularized bone grafts of various shapes that could be used as skeletal replacement parts. In a rat experimental model, thigh adductor muscle island flaps were placed inside bivalved silicone rubber molds. Prior to closure of the mold, 18 flaps were injected with osteogenin and coated with demineralized bone matrix. Five flaps served as controls and were injected with the vehicle only, and not coated with demineralized bone matrix. The molds were implanted subcutaneously in the rats' flanks and reopened 10 days later. The control flaps consisted of intact muscle without any evidence of tissue transformation, whereas the flaps treated with osteogenin and demineralized bone matrix were entirely transformed into cancellous bone that matched the exact shape of the mold. Using tissue transformation, we were able to generate in vivo, autogenous, well-perfused bones in the shapes of femoral heads and mandibles.

Modification of the Van Nes rotationplasty: report of a case.

Rotationplasty is a reconstructive option that provides a highly functional limb after tumor resection about the knee in the skeletally immature patient. However, in the very young child with large intramedullary extension of the tumor, leg length may not be possible to maintain. Described is a modification of the Van Nes rotationplasty that preserves the proximal tibial physis, allowing maximization of future growth.

Assessment of acute microcirculatory changes by color Doppler sonography.

We studied the efficacy of color Doppler sonography in the transcutaneous assessment of acute changes in microvascular flow. Arterial and venous occlusion studies were performed in rabbits after simple isolation of the common femoral artery and vein and after raising an epigastric island flap. Vessel diameters were measured through an operating microscope and compared to color Doppler determinations of the diameters. Vessel patency and blood flow in the major vessels were monitored before and after occlusion. The correlation of vessel diameters measured under the operating microscope and with color Doppler sonography was .91 for the arteries and .39 for the veins. A strong linear correlation existed between Doppler identification of arterial occlusion, but not of venous occlusion. This study demonstrated that color Doppler sonography can be used transcutaneously to reliably assess patency and blood flow in the small vessels. Time-dependent, reproducible patterns in the spectral waveform analysis were indicative of the onset of arterial or venous occlusion.

Prevention of thrombosis by topical application of tissue factor pathway inhibitor in a rabbit model of vascular trauma.

The extrinsic pathway of coagulation is initiated when tissue factor complexes with factor VII. A naturally occurring protein inhibitor of this complex, tissue factor pathway inhibitor (TFPI), has recently been isolated and the cDNA coding for this protein cloned. We used a rabbit ear artery model of crush/avulsion injury and microvascular repair to investigate the efficacy of TFPI as a topically applied antithrombotic agent. Traumatized arteries treated through lumenal irrigation with normal saline vehicle (controls) achieved patency rates of 8% and 0% at 1 and 7 postoperative days, respectively. Heparin irrigation (10 U/ml) resulted in patencies of 40% at both evaluation times. In contrast, TFPI at a dose of 20 micrograms/ml (0.2 ml total volume; 10 minute exposure) yielded a 91% patency rate at 1 day and 73% at 7 days postoperatively (p < 0.0005 vs controls). Prothrombin time and activated partial thromboplastin time values were not altered after topical treatment with TFPI. Scanning electron microscopy revealed dramatically inhibited thrombogenesis upon the injured surfaces of TFPI-treated vessels. These results suggest that TFPI used as a topically applied antithrombotic agent is effective for the prevention of thrombosis in microvascular anastomoses.

Tissue factor pathway inhibitor protects the ischemic spinal cord.

Tissue factor pathway inhibitor (TFPI) is a novel agent that binds to tissue factor/VIIa complex and factor-Xa, thereby reducing the effect of tissue factor (TF) on inflammation and the extrinsic pathway of coagulation. We hypothesize that systemic treatment with TFPI may limit ischemia-reperfusion (IR) injury. Our experiment was designed to evaluate the effects of TFPI on IR in the spinal cord. Twenty-three adult New Zealand white rabbits had snare occlusion devices placed circumferentially around the aorta and tunneled to a subcutaneous position. Forty-eight hours later, in the fully awake state, the animals were treated with either TFPI (1 mg/kg bolus followed by a 1-hr infusion of 20 microgram/kg/min), or heparin (100 U/kg bolus) followed by a 1-hr infusion of 10 ml/kg/hr of PBS while controls received phosphate buffered saline (20 ml followed by a 1-hr infusion of 10 ml/kg/hr). The infrarenal aorta was occluded for 21 min in all groups via the snare device. Animals were observed for 3 days and neurologic recovery was graded by the Tarlov criteria. Results were evaluated as percent of animals with hindlimb recovery (Tarlov 3 and 4). At 24 hr postocclusion, 88% of the TFPI-treated animals had recovered neurologic function versus only 20% of heparin-treated animals and 10% of the phosphate buffered saline group (P=0.031 and 0.009, respectively). At 72 hr, 63% of the TFPI animals retained neurologic function versus 20% of heparin-treated animals and 10% of phosphate buffered saline-treated animals (P=0.032, TFPI versus phosphate buffered saline). The mechanism of action of TFPI is not completely understood, yet this drug may hold promise in the prevention of IR injury of the spinal cord.