Blood flow evaluation of vascularized bone transfers in a canine model

Blood flow in vascularized bone transfers was evaluated in a canine model. Cortical bone blood flow was determined in undisturbed control bone, vascularized heterotopic bone transfers, and nonvascularized cortical bone grafts by means of the radionuclide-labeled microsphere technique. Four animals were studied at 2 days, three at 1 week, eight at 2 weeks, four at 4 weeks, and three at 6 weeks postoperatively. In the 2-day animals, the vascularized bone transfers exhibited intermediate levels of blood flow (1.4 ± 0.4 ml/100 g of bone/min) compared with undisturbed control bone (2.7 ± 0.5 ml/100 g of bone/min) and conventional cortical bone grafts (0.0 ± 0.0 ml/100 g of bone/min). At 1 week postoperatively, the differences in blood flow values of the vascularized transfers (1.7 ± 0.7 ml/100 g of bone/min) and control bone (1.4 ± 1.0 ml/100 g of bone/min) and of the nonvascularized grafts (0.2 ± 0.1 ml/100 g of bone/min) and control bone (1.6 ± 1.1 ml/100 g of bone/min) were no longer significant. At 2 weeks postoperatively, blood flow had increased in both graft groups. At 4 weeks postoperatively, the bone blood flow values in the vascularized transfers (4.8 ± 1.4 ml/100 g of bone/min) were significantly greater than the bone blood flow in control specimens (1.6 ± 0.5 ml/100 g of bone/min) and conventional cortical bone grafts (2.5 ± 2.0 ml/100 g of bone/min). At 6 weeks postoperatively, the bone blood flow values in the vascularized transfers (4.0 ± 2.1 ml/100 g of bone/min) remained greater than the bone blood flow in control specimens (1.7 ± 0.5 ml/100 g of bone/min) and conventional cortical bone grafts (2.5 ± 2.7 ml/100 g of bone/min), but the differences were no longer significant.     

Endoneural and epineural blood flow evaluation with free vascularized and conventional nerve grafts in the canine

Blood flow distribution in conventional and vascularized canine nerve grafts was analyzed. Between 4 and 6 days after operation, blood flow at both the fascicular (endoneural) and the nerve-sheath (epineural) levels was significantly greater in the nonvascularized nerve grafts than in their vascularized counterparts (P less than 0.025 and P less than 0.001, respectively). The superior flow rates of the conventional grafts illustrate the efficiency of nerve revascularization, even to the fascicular level. This finding does not support a possible endoneural vascular advantage of vascularized nerve graft and suggests the need for further analysis of the role of vascularized nerve grafting.     

Quantitative ultrasonic blood flow measurement in Dacron grafts.

A quantitative directional Doppler system was used to determine blood flow in 8 mm. aortoaortic Dacron grafts in dogs. The Doppler was calibrated with frequency standards and the output was integrated for one minute intervals. With the probe held at an angle of 60 degrees to the flow axis, the average frequency of the Doppler shift was determined. Frequency was converted to velocity with the Doppler equation. Volume flow was calculated from the product of velocity and cross-sectional area of the graft. When compared with an electromagnetic flowmeter, the Doppler system was highly accurate with flow rates up to 900 c.c per minute. At flow rates above 900 c.c. per minute, there was a significant decrease in Doppler output which probably is due to the effects of turbulence and background noise on the Doppler signal.     

Hepatic blood flow measurement III. Total hepatic blood flow measured by ICG clearance and electromagnetic flowmeters in a canine septic shock model.

The validity of the ICG clearance method for the measurement of THBF in abnormal circulatory states remains questionable. In this study THBF measured by this method is compared with the electromagnetic flow technique in a canine spetic model. Good correlation is demonstrated between the two in normal control animals. However, in the septic animals the ICG underestimated the electromagnetic flow result by 20%. This is true in both the high and the low cardiac output septic shock pictures that emerge. In the septic animals, the total hepatic blood flow as measured by the ICG was almost equal to the portal vein flow alone measured by the electromagnetic flowmeters suggesting that this was the quantity it was measuring in this abnormal state. Pathophysiologic mechanisms that may explain the discrepancy are given.     

Adrenergic control mechanisms of blood flow in a vascularized canine tibial allograft

A vascularized canine tibial allograft was used to study the alpha-adrenoreceptor subtypes of the microcirculation of bone. Bone transplantation was performed on six dogs, and the bone blood flow was estimated with the use of serial injections of radiolabeled 15 μm microspheres. After microvascular anastomosis, the mean cortical blood flow in the allografted tibia was 3.6 ± 2.1 ml/100 g/min. Alpha₁-adrenoreceptor blockade, with prazosin, caused an increase in normalized blood flow in five allografts and a slight decrease in one allograft. The mean increase was 32 ± 48%, but this was not statistically significant (p ≤ 0.2). The addition of an alpha₂-adrenoreceptor blockade, with rauwolszin, resulted in a significant increase (132 ± 88%) in normalized blood flow in all allografts (p ≤ 0.02). This ex vivo experiment confirms that both alpha₁ and alpha₂ adrenergic mechanisms play a role in controlling bone blood flow in centrally denervated allografts.     

Experimental study of vascularized bone grafts in rat: Effect of mechanical loading on bone dynamics

We studied the etiology of postoperative hypertrophy of vascularized bone grafts in a murine experimental model. Syngeneic grafting of revascularized ulna to rat tibia was performed with (group 1) or without (group 2) mechanical loading. The effect of simple overloading on intact bone was studied by segmental resection of the radius (group 3). Bone dynamics were examined by histomorphological measurements. Significant hypertrophy was observed in the early postoperative period in both groups 1 and group 2. After the initial phase, bone growth continued and extensive remodeling was observed in group 1, while marked bone resorption was observed in group 2. Adaptive remodeling was also observed in group 3 after surgery, but was slower than that in groups 1 and 2. Early hypertrophy of vascularized grafts did not correspond to mechanical loading. These results suggest that mechanical loading is the principal factor responsible for remodeling in vascularized bone grafts for their adaptation to a new environment.     

Simulated nasoalveolar palatal defects: a canine model to study bone grafts

A simple, surgical technique to create a simulated nasoalveolar palatal defect in the canine is described. The procedure has been performed in 60 canines, and we have not experienced closure of the created clefts. The surgically created clefts had to fulfill five criteria: (1) bilateral maxillary alveolar clefts had to exist in each research animal; (2) each cleft had to have a 1-cm bony width; (3) a demonstrable oronasal communication had to be present; (4) each cleft had to be lined by healthy epithelialized mucosa; (5) there had to be functional teeth adjacent to each side of every cleft.     

Management of bone in digital replantation: free vascularized and composite bone grafts.

Microvascular surgical techniques have been applied to the problem of digital amputation, persistent nonunion and complicated extremity injuries with segmental bone and soft tissue loss. In replantations, adequate bone shortening and fixation with intramedullary pins or rods is mandatory. Proper patient selection, preoperative management of the amputated parts, application of microsurgical disciplines, employment of an organized surgical sequence, and rigid postoperative monitoring has resulted in an 80% viability rate in 121 replantations. Recent advances in the management of skeletal injuries using microvascular techniques include free vascularized bone grafting and salvage of the preamputation limb using free composite grafts of skin, subcutaneous tissue and bone. These free composite grafts techniques are exacting and time consuming. Meticulous planning, preoperative rehearsal and careful coordination between the operating teams make the functional restoration of a severely injured limb possible.     

Role of conventional and vascularized bone grafts in scaphoid nonunion with avascular necrosis: A canine experimental study

The effectiveness of vascularized and conventional bone grafts in the treatment of carpal fracture nonunion with avascular necrosis was evaluated in 12 adult dogs. The proximal third of the radiocarpal bone was removed bilaterally and frozen in liquid nitrogen. Its replacement, leaving a 4-mm gap, simulated a scaphoid fracture nonunion with avascular necrosis. A dorsal radius inlay graft was placed across the gap. The graft was nonvascularized, or conventional on one side, and vascularized with a reverse-flow arteriovenous pedicle on the other. Following a healing period, quantitative assessment of bone blood flow, fracture healing, and bone remodeling was conducted. Seventy-three percent of the vascularized grafts and none of the conventional grafts healed. At 6 weeks, bone blood flow in the proximal pole was significantly higher on the side of the vascularized graft. Quantitative histomorphometry of the avascular proximal segment demonstrated significantly higher levels of fluorochrome-labeled osteoid- and osteoblast-covered trabecular surfaces on the vascularized graft side. These experimental data support the potential clinical application of pedicled reverse-flow vascularized grafts in the treatment of carpal fracture nonunions with avascular necrosis, including proximal pole scaphoid nonunions.     

Lysis of intraventricular blood clot with urokinase in a canine model: Part 1. Canine intraventricular blood cast model

To test the safety and feasibility of using direct instillation of urokinase to induce rapid lysis of intraventricular clots, an animal model of intraventricular blood cast is required. Injections of 11 ml of fresh, unclotted autologous blood into the ventricles of adult mongrel dogs did not produce a solid blood cast in the ventricular system, suggesting that the adult dogs have an unusual ability to clear uncoagulated whole blood from the ventricles and subarachnoid space. Injection of 9 ml of preclotted blood resulted in a subtotal cast of the ventricles, leaving only portions of the occipital horns free of solid clots. This volume of injected clots incurred no mortality and minimal morbidity, whereas injection of 10 to 12 ml resulted in a mortality of 42% and formidable morbidity. The technique of producing this intraventricular blood cast model, as well as that of implanting an indwelling ventricular catheter-reservoir system useful in chronic urokinase administration, is described.     

The relationship between inert gas wash-out and radioactive tracer microspheres in measurement of bone blood flow: Effect of decreased arterial supply and venous congestion on bone blood flow in an animal model

Several methods have been employed in the study of bone perfusion. We used a method of determining inert gas wash-out by mass spectrometry in the study of blood flow rates in pigs. The method was validated by comparison of the result obtained with inert gas wash-out to that with measurement by microspheres. Furthermore, the effect of decreased inlet flow and venous congestion on the bone perfusion data was tested. The undisturbed bone blood flow was not significantly different when measured with wash-out of inert gas (7 ± 0.7 ml/min/100 g) or with microspheres (9 ± 2.9 ml/min/100 g), and the methods were correlated. Perfusion was reduced significantly, to 20% of the original value, after arterial occlusion. The changes in wash-out curves and accumulation of radioactive tracer provided substantial evidence for impaired intraosseous circulation following venous obstruction also. In conclusion, the study showed that this method of determining inert gas wash-out is feasible for studies of local perfusion rates in bone. The flow rates obtained by wash-out correlated well with the results of microsphere studies. In this animal model, both methods detected a fivefold reduction in flow rate after clamping of the arterial inflow. Obstruction of the venous outflow also impaired blood flow and lowered the cellular supply.     

Impact of radiation therapy on healing and stability of vascularized bone grafts in a dog model

The purpose of the study was to observe the impact of radiation therapy on healing and biomechanical properties of vascularized bone grafts, and thus to establish an appropriate large animal model. Ten male beagles were divided into two experimental groups: radiation (R) and control (C). The left 5th to 7th ribs of the animals of group R were irradiated 3 and 2 weeks preoperatively, using a dose of 8 Gy each time. Each animal of both groups underwent the following operative procedures. The 5th and 7th ribs were removed, and the 5th rib was replaced by a vascularized pedicle transfer of the 4th rib. The 7th rib was reconstructed using a pedicle transplant of the 8th rib. The 5th and 7th ribs were used as nonvascularized bone grafts to replace the donor sites of the 4th and 8th ribs, respectively. Group R received two further irradiation cycles 2 and 3 weeks postoperatively. The vascularized rib grafts of group R demonstrated a higher number of delayed unions in plain x-rays and avascular bone segments in microangiography than the control group. The presence of vital osteocytes in histology was not significantly different between groups. Biomechanical tests focusing on the durability of vascularized ribs against bending and torsion forces demonstrated a reduction of average maximum bending moments by 56.6% after radiation compared to controls (P < 0.05). Twisting moments were reduced by 47.6% (P < 0.05). The data demonstrate a significant worsening in bone healing and stability after pre- and postoperative radiation therapy to the wound bed and bone grafts. Thus, a large animal model is established for further determination of different strategies of radiotherapy in combination with vascularized bone transfers.     

Experimental study of oversized grafts in a canine living-donor lobar lung transplantation model

BACKGROUND: For infants and small children, organ transplantation is limited by the size discrepancy between donor and recipient. To address this problem, the use of over-sized grafts from living-relative donors could potentially expand the donor pool. The aim of this experimental study was to evaluate the effect of oversized grafts on early pulmonary function and to identify an indicator for acceptable size discrepancy. METHODS: Fourteen bilateral lobar lung allotransplant operations were performed without cardiopulmonary bypass in weight mismatched pairs of dogs. Animals were divided into 2 groups: Group I (n = 7), donor/recipient lung volume ratio < 2.85; Group II (n = 7), donor/recipient lung volume ratio >2.85. Pulmonary function of the recipient was measured before chest closure, after chest closure, and after the ventilator was removed. RESULTS: Pulmonary vascular resistance and airway pressure significantly increased in Group II after chest closure (1493 +/- 195 dynes sec cm(-5) and 14.4 +/- 0.9 mm Hg vs 2784 +/- 140 dynes sec cm(-5) and 23.4 +/- 1.2 mm Hg, p < 0.001). After the ventilator was removed, all recipients in Group I showed PaO2 > 239 mm Hg and PaCO2 < 76 mm Hg, whereas, all recipients in Group II showed PaO2 < 116 mm Hg and PaCO2 > 169 mm Hg. The donor/recipient chest circumference ratio was less than 1.3 in all but 1 dog in Group I. CONCLUSIONS: Acceptable, oversized grafts provide adequate pulmonary function, although excessively oversized grafts cause significant impairment in pulmonary function after chest closure. Chest circumference provides useful size-match criteria when oversized grafts are used in this canine experimental model.     

Experimental study on vascularized island pedicle bone graft: bony fusion between the graft and the recipient floor.

To evaluate the process of bony fusion between the recipient floor and the bone graft, where a muscle sleeve has been interposed, an experimental model for a vascularized island pedicle bone graft was prepared using rats. The proximal two thirds of the tibia pedicled with the popliteal artery and vein was collected to be used as a vascularized bone. This was onlay grafted to the femur of the same limb in such a manner that the muscle sleeve was interposed between the bone graft and the recipient floor. The animals subjected to this procedure were designated as the vascularized island pedicle bone graft group (hereafter called group V, n = 32). In another group, vascular pedicles were interrupted by ligation and the animals were subjected to nonvascularized bone graft (hereafter called group N, n = 32). In the third group, an appropriate amount of bone chips was transplanted throughout the length of the grafted floor; then the animals were subjected to additional vascularized island pedicle bone graft (hereafter called group B, n = 13). In groups V and B, the vascularized bone graft formed new bone at the site where the bone graft faced the recipient floor 6 weeks after surgery. Hypertrophy of the bone graft was noted. Bony fusion at the section where a muscle sleeve had been interposed was recognized only in group B. Chondrocytes were found around the free bone graft, suggesting chondrocyte participation in osteogenesis. In group N, the bone graft had been absorbed and no bony fusion was recognized between the bone graft and recipient floor in any of the samples. These findings imply that it is important to minimize the size of the muscle sleeve attached to the vascularized island pedicle bone graft and add free bone chips around the grafted bone to assure bony fusion between a vascularized bone graft and the recipient floor in clinical practice.     

Subclavian steal syndrome. Part 2: Intraoperative vertebral artery blood flow measurement.

Intraoperative vertebral artery blood flow was measured in two patients with symptomatic subclavian steal syndrome, before and after proximal end-to-side vertebral to common carotid artery transposition. This confirmed retrograde flow in the vertebral artery before transposition, and antegrade flow after transposition. The measured flow rates were compared to values in other series involving different operative procedures for correction of symptomatic subclavian steal. The greatest mean antegrade flow rates in the vertebral artery were restored by proximal end-to-side vertebral to common carotid artery transposition.     

Direct measurement of arteriovenous anastomotic blood flow in the septic canine hindlimb.

Absolute blood flow through hindlimb capillaries and arteriovenous anastomoses (AVA) was determined by the radioactive microsphere technique of relative shunt measurement combined with direct (electromagnetic) measurement of femoral artery blood flow. Studies were performed before and 72 hours after infection was created in six dogs by implanting fecal-contaminated wicks in one hindlimb. Three control dogs had sterile wicks implanted. Septic dogs demonstrated decreased arterial pressure and increased core temperature with no change in mean cardiac index. Infected legs had increased total femoral blood flow, decreased peripheral resistance, decreased arteriovenous (A-V) O2 difference, but unchanged oxygen consumption, Paw temperature increased in both infected and normal contralateral limbs. Muscle blood flow (MBF) (Xe133 clearance) increased only in infected legs. A-V shunting increased five-fold and AVA flow increased seven-fold in infected hindlimbs, significantly more than that in contralateral or control limbs. Increased A-V shunting correlated highly (r=0.95) with increased total femoral blood flow in infected legs, but increased MBF was not correlated (r=0.05). Despite increased MBF, increased AVA flow occurs in septic canine hindlimbs and contributes to the low-resistance, high-flow, but unchanged oxygen consumption described in this model.     

Revascularization of nerve grafts: a qualitative and quantitative study of the soft-tissue bed contributions to blood flow in canine nerve grafts

Blood flow to canine saphenous nerve grafts either in contact with a healthy soft-tissue bed or isolated from the soft-tissue bed by entubulation was quantitated at 3, 7, 14, and 28 days after surgery with radionuclide-labeled microspheres and evaluated histomorphometrically. At 3 days there was no statistical difference between the 2 techniques in regard to nerve graft blood flow, and neither group had blood flow in the middle portion of the graft. In contrast, analysis at 7, 14, and 28 days found blood flow in the middle segments of the nonisolated conventional nerve grafts to exceed that of entubulated nerve grafts with evidence in these segments of both lateral and longitudinal revascularization. The results of this investigation support an important role for the soft-tissue bed in nerve graft revascularization and suggest that neovascularization from the soft-tissue bed is the primary mechanism for restoration of blood flow in such grafts.     

Impaction bone grafting with freeze-dried irradiated bone. Part II. Changes in stiffness and compactness of morselized grafts

In the technique of impaction bone grafting, implant stability depends on the mechanical properties of the impacted morselized grafts. Although the procedure is usually performed with fresh-frozen femoral heads, there is still some concern about their supply and safety. Bone processing is a potential solution, but the mechanical properties of this material during and after impaction need to be determined. We used 6 osteoarthrotic femoral heads to prepare two paired batches of morselized bone. One batch was morselized and frozen. The other batch was chemically treated, morselized, freeze-dried and then gamma-irradiated. We impacted 18 samples from each batch in a contained cylinder. Freeze-dried bone grafts were tested after 30 minutes of rehydration. The changes in the compactness and stiffness of the material were monitored during the impaction. The compaction of the freeze-dried bone was faster than that of their fresh-frozen control. The maximal stiffness reached by both materials was the same (55 MPa), but the freeze-dried grafts required three to four times fewer impactions to achieve that stiffness. After 3, 10 and 50 impactions the freeze-dried bone was stiffer than the fresh-frozen bone. As it is easier to impact, the freeze-dried bone may be mechanically more efficient than the fresh-frozen bone in surgical conditions. Moreover, the processed bone meets the highest safety standards, as regards the risk of disease transmission.     

Bilateral vascularized femoral bone transplant: a new model of vascularized bone marrow transplantation in rats, part I

We present a new model of vascularized bone marrow transplantation-bilateral vascularized femoral bone (BVFB) isograft transplant based on abdominal aorta and inferior vena cava. A total of 7 BVFB isograft transplants were performed between Lewis (RT1) rats. In the donor, both femoral bones were harvested based on the abdominal aorta and inferior vena cava. In the recipient, the harvested isograft transplants were transferred into the inguinal region (in 3 animals) and into the abdominal cavity (in 4 animals). The mean operation time was 3 hours and 35 minutes. The mean warm ischemic time was 35 minutes. The vascular pedicles of the transplants that were transferred into the inguinal region were thrombosed at day 7 posttransplantation. The vascular pedicles of transplants into the abdominal cavity were patent and the bones were viable during the follow-up period of 63 days posttransplant. We have confirmed the feasibility of BVFB transplantation based on abdominal aorta and inferior vena cava.