Shigeru Mizumoto, Yuji Inada, and Andrew J. Weiland
PRE-FORMED VASCULARIZED BONE GRAFTS USING
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POLYETHELYNE CHAMBERS ABSTRACT Previous work has shown that corticocancellous bone chips placed in a titanium chamber with an arteriovenous vascular pedicle will result in a pre-formed vascularized bone graft. The present study was designed to determine whether these grafts can be transferred as an island or free vascularized bone graft, and to examine the material properties of these grafts. Thirty-two male, New Zealand white rabbits were divided into four groups based on the time of sacrifice following the initial chamber implantation. Injected molded cylindrical polyethelyne chambers, measuring 22 mm long and with an 8-mm inner diameter, were used. Corticocancellous bone chips were placed in the chambers and each chamber was implanted in the mid-thigh, with saphenous vessels running through the chamber. The chambers were implanted into the right and left thigh of each animal. To test the hypothesis of the possibility of transferring this graft as an island or free vascularized graft, ligation of the distal vascular pedicle on one side was achieved at re-exploration at 3,6,9, and 12 weeks. The contralateral side served as a control in which the vascular pedicle was not ligated. In the controls, bony bridging between the corticocancellous bone chips was observed after 7 weeks. A solid bone graft was present within the chamber by 10 weeks. However, histomorphometric evaluation indicated significant bone resorption. By 13 weeks, resorption progressed to the point where only small islands of bone remained. Ligation of the distal vessels resulted in thrombosis of the vessels within the chamber. Necrosis of newly-formed bone was observed in the area adjacent to the vascular thrombosis. By 9 weeks, viable bone was observed within the chamber, despite thrombosis of the vascular pedicle. These findings were most probably due to the development of collateral circulation. This study supports the concept of creating a "molded vascularized bone graft" with a single vascular pedicle. The advent of free vascularized bone graft and its refinements over the past two decades has dramatically expanded the horizons of reconstructive surgery. Vascularized bone grafts, by virtue of their intrinsic blood supply, can obtain more rapid union and tolerate infection and mechanical loading better than conventional bone grafts, in a recipient site devoid of adequate blood supply.1-4 However, potential donor sites are limited by the anatomic configuration of the graft, morbidity of the donor site, or the difficult techniques needed for harvesting the graft.56 Freedom from these restrictions would enhance the reconstructive capability of free vascularized bone grafts. Nettelblad and colleagues in 1984 described a
method for creating "molded vascularized osteogenesis."7 Their study has shown that corticocancellous bone chips placed in a titanium chamber with an arteriovenous vascular pedicle will result in a suitable size and shape of vascularized bone graft. The possibility of transplantation of this type of graft as a microsurgical free vascularized bone graft is at the present time unknown. The present study was designed to determine if pre-formed vascularized bone grafts can be transferred as an island or free vascularized bone graft, and to determine the material properties of these grafts. Autogenous corticocancellous bone chips were placed in an injection-molded polyethelyne chamber, with a vas-
Department of Orthopaedic Surgery, The Hospital for Special Surgery, Cornell University Medical College, New York Materials in this article were reported at the 37th Annual Meeting of the Orthopaedic Research Society, Anaheim, CA, March 4, 1991 Reprint requests-. Dr. Weiland, Hospital for Special Surgery, 535 E. 70th St., New York, NY 10021 Accepted for publication February 25, 1992 Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 4
MATERIALS AND METHODS Thirty-two seven-month-old, male New Zealand white rabbits, weighing 3.8 to 4.2 kg, were divided into four groups based on the time of vascular pedicle ligation. An intramuscular injection of penicillin-G 70,000 IU was given for antibiotic prophylaxis, just prior to surgery and 6 hr following chamber implantation. Under general anesthesia, both iliac crests were approached through a transverse dorsal skin incision. Corticocancellous bone chips (I to 2 mm) were obtained using a rongeur, and were placed into injectionmolded, cylindrical polyethelyne chambers, which were custom made for this experiment (Fig. 1A). While preparing the vascular pedicle, the bone graft in the chamber was preserved in moistened gauze sponges to prevent desiccation. The right and left saphenous vascular pedicles, consisting of an artery and a vein, were exposed from mid-thigh to below the knee joint, and dissected free from the surrounding soft tissue. One-half of the chamber which was filled with bone chips was positioned beneath the vascular bundle. The other half was positioned on top of the first, permitting
the vessels to pass through the enclosed chamber without bending or compression. The chamber was closed with 4-0 monofilament nylon sutures, and anchored in the intramuscular space of the thigh. Another chamber was prepared on the opposite side as a control. Skin and subcutaneous tissue were closed with 4-0 monofilament nylon sutures (Fig. IB). The animals were placed in their cages and fed standard laboratory food. Following surgery, multiple fluorochrome bone labels were administered every three weeks. D.C.A.F. (2,4-bis amino-methyl fluorescein, 20 mg/kg) at 3 weeks, alizarin complexone (30 mg/kg) at 6 weeks, oxytetracycline (30 mg/kg) at 9 weeks, and xylenol orange (90 mg/kg) at 12 weeks, were injected subcutaneously The last labeling was given to document activity and viability of osteoblasts and osteocytes, immediately following ligation of the distal vascular bundle. The ligation on one side was achieved by a reexploration at 3, 6, 9, and 12 weeks. Prior to ligation, patency of the proximal vascular bundle was judged using a Doppler flowmeter. The contralateral side served as a control in which the distal vascular pedicle was not ligated. Following sacrifice 1 week after ligation of the distal vascular pedicle, a 30 percent barium sulfate solution was infused through an 18-G transabdominal intraaortic catheter under a controlled pressure (140 mmHg) and temperature (40°C). Chambers were dissected free from the surrounding tissue and the contents were carefully removed. A radiograph was taken
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cular bundle coursing through it in a heterotopic site. Ligation of the distal vascular pedicle was performed at different time intervals, and viability and bone volume of the graft were evaluated by histologic, histomorphometric, and angiographic studies.
JULY 1992
f B
326
Figure 1. A, Polyethelyne chamber implant, I.D. = 9 mm, length = 22 mm. B, Photograph showing the chamber filled with corticocancellous bone chips, with the saphenous vessels coursing through the chamber.
PRE-FORMED VASCULARIZED BONE GRAFTS/MIZUMOTO, INADA, WEILAND
ured in each undecalcified section stained with von Kossa, using a semi-automated image analyzer with a video camera. Each datum was analyzed statistically for significance, using multiple Student's t-tests to compare individual groups.
RESULTS Three rabbits died, and two were sacrificed due to local infection during the study. Additional animals were added to complete the four experimental groups. Primary occlusion of the vascular bundle was confirmed by histologic examination and Doppler examination in five chambers of the control group, and in three chambers of the ligated group. These specimens were discarded from the data. At sacrifice, the chambers were surrounded by fibrous tissue which did not adhere to the outer wall of the chamber. At 7 and 10 weeks, the contents of the chamber were solid and had the consistency of a piece of bone (Fig. 3A). However, partial collapse of the molded shape was noted in some of the chambers. At 13 weeks, the molded cylindrical shape of the graft was destroyed and collapsed in a significant portion of the specimen and had a rubbery consistency. (Fig. 3B). There was no difference between the ligated groups and control groups in the clinical findings, as described above.
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for microangiography, using Cabinet X-ray system Model 50/20 (TEMSI Clinton, MD) at (25 kV, 5 mA, 5 sec). The contents were divided into two transverse sections fixed in 70 percent ethanol, and embedded in methylmetracrylate. Six |xm undecalcified sections were obtained from the mid-portion of each specimen. After deplastication, one section was stained with hematoxylin and eosin for osteocyte counting and routine histologic study. The other section was stained with von Kossa for histomorphometry. The remaining unstained section was used for fluorescent microscopy. Therefore, there was a total of six histologic specimens for each chamber. MEASUREMENTS. TO evaluate the patency of the vessels ligated at the end of the chamber, the vascular pedicle within the chamber was divided into four equal zones on the microangiogram. The patency was evaluated in each zone, and a scoring system was devised (Fig. 2). The reason for using a scoring system was that the bone fragments interfered with the ability to visualize whether the distal aspect of the vessels was filled with barium. The number of osteocytes and lacunae were counted in 15 square fields (1 mm2 each), chosen in each section stained with hematoxylin and eosin, using a glass grid under a microscope. The percentage of osteocytes in the total number of lacunae was calculated. Total cross-sectional area and bone area were meas-
EVALUATION OF VASCULAR PATENCY ON MICROANGIOGRAM Scoring System score COI
I
4.0n
zone
0 proximal side
3.0-
2
8 1.0distal side ligation
o.o-
10
13 weeks
#, *: p < 0.05 Patent vessel Thrombosis Figure 2. Patency of the ligated vessels on the proximal and distal sides of the chamber.
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JULY 1992
Figure 3. A, The molded shape of the contents in the chamber is solid at 7 weeks in the control group. B, Photograph showing the collapse of the molded shape at 13 weeks in the control group.
B
328
HISTOLOGIC STUDY. In the controls, a large number of small vessels were observed in fibrous tissue surrounding the bone fragments at 4 weeks. Scant bone formation was present on the surface of acellular bone fragments adjacent to the implanted vessels. At 7 weeks, osteocytes and new bone formation were more widespread, and bony union between corticocancellous bone chips was observed, similar to the trabecular structure in normal cancellous bone. Newly-formed small arteries with a smooth muscle layer were often noted (Fig. 4A). However, wide acellular areas were noted in the center of the fragments. In addition, numerous osteoclasts were seen on the surface of bone (Fig. 4B). At 10 weeks, significant bone resorption resulted in a thinner and less dense trabecular structure of the bone. Lamellar bone formation was demonstrated under fluorescent microscopy as linear uptake on the newly-formed bone surface. Most of the interosseous space was occupied with bone marrow tissue. In several specimens, collapse of the graft was noted. (Fig. 5). At 13 weeks, resorption progressed to the point where only small bone islands surrounded with fatty tissue remained. Collapse of the molded shape was noted in most of the specimens at the time interval. Ligation of the vascular pedicle caused thrombosis of the vessels within the chamber at 4 and 7 weeks. In this period, necrosis of the newly-formed bone was observed, with disappearance of osteoblasts and osteocytes. (Fig. 6) This was observed most frequently in the distal part of the chamber. In the necrotic area, fluorescent microscopy showed uptake of
fluorochrome which was administered immediately after the ligation of the distal vascular pedicle. Viability of the bone may have been secondary to plasma diffusion. At 10 and 13 weeks, thrombosis was located predominantly at the distal end of the chamber. Small vessels were filled with barium, and viability of the bone was well maintained. PATENCY O F THE LIGATED V E S S E L S ( S E E FIG. 2).
The
patency score was increased after 10 weeks. Statistically significant differences were noted between weeks 4 and 10 and weeks 7 and 10 (p < 0.05). COUNTING OF OSTEOCYTES AND LACUNAE (FIG. 7).
Analysis of data on the proximal side of the chambers in the control group reveals that there was a significant decrease in the number of osteocytes after 10 weeks. By 13 weeks, there was a statistically significant decrease in osteocytes, when compared to weeks 4, 7 and 10 in the control group, but not in the ligated group. There was a statistically significant increase in the percent of osteocytes at 13 weeks, compared to 4 weeks. This can be explained by the relatively increased resorption of total bone that was noted histologically. There was no difference noted between the control and ligated group proximally at any time interval. Analysis of data on the distal side of the chambers in the control group revealed a statistically significant decrease in osteocytes between weeks 7 and 13. In the ligated group, there was a significant increase in the number of osteocytes between 4 and 13 weeks. A significant difference between the control and ligated groups
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...-
Downloaded by: University of Iowa Libraries. Copyrighted material.
PRE-FORMED VASCULARIZED BONE GRAFTS/MIZUMOTO, INADA, WEILAND
Figure 4. A, Photomicrograph of control specimen at 7 weeks, demonstrating increased new bone formation and bony union between the bone fragments. Acellular areas are present in the center of fragments. Numerous dilated vessels are observed in the surrounding soft tissue. (H&E, x 80). B, Arrows indicate numerous osteoclasts at high magnification, (von Kossa, x 320).
Figure 5. Photomicrograph of control at 13 weeks; arrows indicate collapse of the molded shape due to bone resorption. (von Kossa, x 20).
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JULY 1992
was noted only at weeks 4 and 7. As was noted on the proximal side of the graft, the percentage of osteocytes in the lacunae increased significantly from weeks 4 to 13 due to bone resorption. CROSS-SECTIONAL BONE AREA (FIG. 8). There was
no statistically significant difference in cross-sectional bone area on either the proximal or distal sides at any time interval between the control and ligated groups. A significant continuous decrease of bone was noted on both the proximal and distal sides from week 4
Distal Side
Proximal Side 100
100
E E
80-
80-
CD Q.
60-
60-
CD
40-
40-
E
20-
20-
EH3
Lacunae Control Group
Number of Osteocytes Control Group EZ3
Number of Osteocytes Ligated Group
=3 4 weeks
4 weeks 7 weeks 10 weeks 13 weeks 1
2
3
4
1
7 weeks 10 weeks 13 weeks 2
3
4
Note: Number of osteocytes and lacunae and standard deviation are shown. In comparing with the control group and the ligated group at the same time period, statistical significance was noted in the distal part of Group 1 and Group 2 (x: ligated G-1 distal p < 0.05; y: ligated G-2 distal vs. control G-2 distal p < 0.05). In ligated G-1, significant difference (z: p < 0.01) was noted between proximal and distal side. Other results are shown as follows:
control group proximal p value
distal
ligated group p value proximal
a: 1 vs. 4 < 0.05 d: 2 vs. 4
PRE-FORMED VASCULARIZED BONE GRAFTS USING
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POLYETHELYNE CHAMBERS ABSTRACT Previous work has shown that corticocancellous bone chips placed in a titanium chamber with an arteriovenous vascular pedicle will result in a pre-formed vascularized bone graft. The present study was designed to determine whether these grafts can be transferred as an island or free vascularized bone graft, and to examine the material properties of these grafts. Thirty-two male, New Zealand white rabbits were divided into four groups based on the time of sacrifice following the initial chamber implantation. Injected molded cylindrical polyethelyne chambers, measuring 22 mm long and with an 8-mm inner diameter, were used. Corticocancellous bone chips were placed in the chambers and each chamber was implanted in the mid-thigh, with saphenous vessels running through the chamber. The chambers were implanted into the right and left thigh of each animal. To test the hypothesis of the possibility of transferring this graft as an island or free vascularized graft, ligation of the distal vascular pedicle on one side was achieved at re-exploration at 3,6,9, and 12 weeks. The contralateral side served as a control in which the vascular pedicle was not ligated. In the controls, bony bridging between the corticocancellous bone chips was observed after 7 weeks. A solid bone graft was present within the chamber by 10 weeks. However, histomorphometric evaluation indicated significant bone resorption. By 13 weeks, resorption progressed to the point where only small islands of bone remained. Ligation of the distal vessels resulted in thrombosis of the vessels within the chamber. Necrosis of newly-formed bone was observed in the area adjacent to the vascular thrombosis. By 9 weeks, viable bone was observed within the chamber, despite thrombosis of the vascular pedicle. These findings were most probably due to the development of collateral circulation. This study supports the concept of creating a "molded vascularized bone graft" with a single vascular pedicle. The advent of free vascularized bone graft and its refinements over the past two decades has dramatically expanded the horizons of reconstructive surgery. Vascularized bone grafts, by virtue of their intrinsic blood supply, can obtain more rapid union and tolerate infection and mechanical loading better than conventional bone grafts, in a recipient site devoid of adequate blood supply.1-4 However, potential donor sites are limited by the anatomic configuration of the graft, morbidity of the donor site, or the difficult techniques needed for harvesting the graft.56 Freedom from these restrictions would enhance the reconstructive capability of free vascularized bone grafts. Nettelblad and colleagues in 1984 described a
method for creating "molded vascularized osteogenesis."7 Their study has shown that corticocancellous bone chips placed in a titanium chamber with an arteriovenous vascular pedicle will result in a suitable size and shape of vascularized bone graft. The possibility of transplantation of this type of graft as a microsurgical free vascularized bone graft is at the present time unknown. The present study was designed to determine if pre-formed vascularized bone grafts can be transferred as an island or free vascularized bone graft, and to determine the material properties of these grafts. Autogenous corticocancellous bone chips were placed in an injection-molded polyethelyne chamber, with a vas-
Department of Orthopaedic Surgery, The Hospital for Special Surgery, Cornell University Medical College, New York Materials in this article were reported at the 37th Annual Meeting of the Orthopaedic Research Society, Anaheim, CA, March 4, 1991 Reprint requests-. Dr. Weiland, Hospital for Special Surgery, 535 E. 70th St., New York, NY 10021 Accepted for publication February 25, 1992 Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
325
JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 4
MATERIALS AND METHODS Thirty-two seven-month-old, male New Zealand white rabbits, weighing 3.8 to 4.2 kg, were divided into four groups based on the time of vascular pedicle ligation. An intramuscular injection of penicillin-G 70,000 IU was given for antibiotic prophylaxis, just prior to surgery and 6 hr following chamber implantation. Under general anesthesia, both iliac crests were approached through a transverse dorsal skin incision. Corticocancellous bone chips (I to 2 mm) were obtained using a rongeur, and were placed into injectionmolded, cylindrical polyethelyne chambers, which were custom made for this experiment (Fig. 1A). While preparing the vascular pedicle, the bone graft in the chamber was preserved in moistened gauze sponges to prevent desiccation. The right and left saphenous vascular pedicles, consisting of an artery and a vein, were exposed from mid-thigh to below the knee joint, and dissected free from the surrounding soft tissue. One-half of the chamber which was filled with bone chips was positioned beneath the vascular bundle. The other half was positioned on top of the first, permitting
the vessels to pass through the enclosed chamber without bending or compression. The chamber was closed with 4-0 monofilament nylon sutures, and anchored in the intramuscular space of the thigh. Another chamber was prepared on the opposite side as a control. Skin and subcutaneous tissue were closed with 4-0 monofilament nylon sutures (Fig. IB). The animals were placed in their cages and fed standard laboratory food. Following surgery, multiple fluorochrome bone labels were administered every three weeks. D.C.A.F. (2,4-bis amino-methyl fluorescein, 20 mg/kg) at 3 weeks, alizarin complexone (30 mg/kg) at 6 weeks, oxytetracycline (30 mg/kg) at 9 weeks, and xylenol orange (90 mg/kg) at 12 weeks, were injected subcutaneously The last labeling was given to document activity and viability of osteoblasts and osteocytes, immediately following ligation of the distal vascular bundle. The ligation on one side was achieved by a reexploration at 3, 6, 9, and 12 weeks. Prior to ligation, patency of the proximal vascular bundle was judged using a Doppler flowmeter. The contralateral side served as a control in which the distal vascular pedicle was not ligated. Following sacrifice 1 week after ligation of the distal vascular pedicle, a 30 percent barium sulfate solution was infused through an 18-G transabdominal intraaortic catheter under a controlled pressure (140 mmHg) and temperature (40°C). Chambers were dissected free from the surrounding tissue and the contents were carefully removed. A radiograph was taken
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cular bundle coursing through it in a heterotopic site. Ligation of the distal vascular pedicle was performed at different time intervals, and viability and bone volume of the graft were evaluated by histologic, histomorphometric, and angiographic studies.
JULY 1992
f B
326
Figure 1. A, Polyethelyne chamber implant, I.D. = 9 mm, length = 22 mm. B, Photograph showing the chamber filled with corticocancellous bone chips, with the saphenous vessels coursing through the chamber.
PRE-FORMED VASCULARIZED BONE GRAFTS/MIZUMOTO, INADA, WEILAND
ured in each undecalcified section stained with von Kossa, using a semi-automated image analyzer with a video camera. Each datum was analyzed statistically for significance, using multiple Student's t-tests to compare individual groups.
RESULTS Three rabbits died, and two were sacrificed due to local infection during the study. Additional animals were added to complete the four experimental groups. Primary occlusion of the vascular bundle was confirmed by histologic examination and Doppler examination in five chambers of the control group, and in three chambers of the ligated group. These specimens were discarded from the data. At sacrifice, the chambers were surrounded by fibrous tissue which did not adhere to the outer wall of the chamber. At 7 and 10 weeks, the contents of the chamber were solid and had the consistency of a piece of bone (Fig. 3A). However, partial collapse of the molded shape was noted in some of the chambers. At 13 weeks, the molded cylindrical shape of the graft was destroyed and collapsed in a significant portion of the specimen and had a rubbery consistency. (Fig. 3B). There was no difference between the ligated groups and control groups in the clinical findings, as described above.
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for microangiography, using Cabinet X-ray system Model 50/20 (TEMSI Clinton, MD) at (25 kV, 5 mA, 5 sec). The contents were divided into two transverse sections fixed in 70 percent ethanol, and embedded in methylmetracrylate. Six |xm undecalcified sections were obtained from the mid-portion of each specimen. After deplastication, one section was stained with hematoxylin and eosin for osteocyte counting and routine histologic study. The other section was stained with von Kossa for histomorphometry. The remaining unstained section was used for fluorescent microscopy. Therefore, there was a total of six histologic specimens for each chamber. MEASUREMENTS. TO evaluate the patency of the vessels ligated at the end of the chamber, the vascular pedicle within the chamber was divided into four equal zones on the microangiogram. The patency was evaluated in each zone, and a scoring system was devised (Fig. 2). The reason for using a scoring system was that the bone fragments interfered with the ability to visualize whether the distal aspect of the vessels was filled with barium. The number of osteocytes and lacunae were counted in 15 square fields (1 mm2 each), chosen in each section stained with hematoxylin and eosin, using a glass grid under a microscope. The percentage of osteocytes in the total number of lacunae was calculated. Total cross-sectional area and bone area were meas-
EVALUATION OF VASCULAR PATENCY ON MICROANGIOGRAM Scoring System score COI
I
4.0n
zone
0 proximal side
3.0-
2
8 1.0distal side ligation
o.o-
10
13 weeks
#, *: p < 0.05 Patent vessel Thrombosis Figure 2. Patency of the ligated vessels on the proximal and distal sides of the chamber.
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 4
JULY 1992
Figure 3. A, The molded shape of the contents in the chamber is solid at 7 weeks in the control group. B, Photograph showing the collapse of the molded shape at 13 weeks in the control group.
B
328
HISTOLOGIC STUDY. In the controls, a large number of small vessels were observed in fibrous tissue surrounding the bone fragments at 4 weeks. Scant bone formation was present on the surface of acellular bone fragments adjacent to the implanted vessels. At 7 weeks, osteocytes and new bone formation were more widespread, and bony union between corticocancellous bone chips was observed, similar to the trabecular structure in normal cancellous bone. Newly-formed small arteries with a smooth muscle layer were often noted (Fig. 4A). However, wide acellular areas were noted in the center of the fragments. In addition, numerous osteoclasts were seen on the surface of bone (Fig. 4B). At 10 weeks, significant bone resorption resulted in a thinner and less dense trabecular structure of the bone. Lamellar bone formation was demonstrated under fluorescent microscopy as linear uptake on the newly-formed bone surface. Most of the interosseous space was occupied with bone marrow tissue. In several specimens, collapse of the graft was noted. (Fig. 5). At 13 weeks, resorption progressed to the point where only small bone islands surrounded with fatty tissue remained. Collapse of the molded shape was noted in most of the specimens at the time interval. Ligation of the vascular pedicle caused thrombosis of the vessels within the chamber at 4 and 7 weeks. In this period, necrosis of the newly-formed bone was observed, with disappearance of osteoblasts and osteocytes. (Fig. 6) This was observed most frequently in the distal part of the chamber. In the necrotic area, fluorescent microscopy showed uptake of
fluorochrome which was administered immediately after the ligation of the distal vascular pedicle. Viability of the bone may have been secondary to plasma diffusion. At 10 and 13 weeks, thrombosis was located predominantly at the distal end of the chamber. Small vessels were filled with barium, and viability of the bone was well maintained. PATENCY O F THE LIGATED V E S S E L S ( S E E FIG. 2).
The
patency score was increased after 10 weeks. Statistically significant differences were noted between weeks 4 and 10 and weeks 7 and 10 (p < 0.05). COUNTING OF OSTEOCYTES AND LACUNAE (FIG. 7).
Analysis of data on the proximal side of the chambers in the control group reveals that there was a significant decrease in the number of osteocytes after 10 weeks. By 13 weeks, there was a statistically significant decrease in osteocytes, when compared to weeks 4, 7 and 10 in the control group, but not in the ligated group. There was a statistically significant increase in the percent of osteocytes at 13 weeks, compared to 4 weeks. This can be explained by the relatively increased resorption of total bone that was noted histologically. There was no difference noted between the control and ligated group proximally at any time interval. Analysis of data on the distal side of the chambers in the control group revealed a statistically significant decrease in osteocytes between weeks 7 and 13. In the ligated group, there was a significant increase in the number of osteocytes between 4 and 13 weeks. A significant difference between the control and ligated groups
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...-
Downloaded by: University of Iowa Libraries. Copyrighted material.
PRE-FORMED VASCULARIZED BONE GRAFTS/MIZUMOTO, INADA, WEILAND
Figure 4. A, Photomicrograph of control specimen at 7 weeks, demonstrating increased new bone formation and bony union between the bone fragments. Acellular areas are present in the center of fragments. Numerous dilated vessels are observed in the surrounding soft tissue. (H&E, x 80). B, Arrows indicate numerous osteoclasts at high magnification, (von Kossa, x 320).
Figure 5. Photomicrograph of control at 13 weeks; arrows indicate collapse of the molded shape due to bone resorption. (von Kossa, x 20).
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JOURNAL OF RECONSTRUCTIVE MICROSURGERY/VOLUME 8, NUMBER 4
JULY 1992
was noted only at weeks 4 and 7. As was noted on the proximal side of the graft, the percentage of osteocytes in the lacunae increased significantly from weeks 4 to 13 due to bone resorption. CROSS-SECTIONAL BONE AREA (FIG. 8). There was
no statistically significant difference in cross-sectional bone area on either the proximal or distal sides at any time interval between the control and ligated groups. A significant continuous decrease of bone was noted on both the proximal and distal sides from week 4
Distal Side
Proximal Side 100
100
E E
80-
80-
CD Q.
60-
60-
CD
40-
40-
E
20-
20-
EH3
Lacunae Control Group
Number of Osteocytes Control Group EZ3
Number of Osteocytes Ligated Group
=3 4 weeks
4 weeks 7 weeks 10 weeks 13 weeks 1
2
3
4
1
7 weeks 10 weeks 13 weeks 2
3
4
Note: Number of osteocytes and lacunae and standard deviation are shown. In comparing with the control group and the ligated group at the same time period, statistical significance was noted in the distal part of Group 1 and Group 2 (x: ligated G-1 distal p < 0.05; y: ligated G-2 distal vs. control G-2 distal p < 0.05). In ligated G-1, significant difference (z: p < 0.01) was noted between proximal and distal side. Other results are shown as follows:
control group proximal p value
distal
ligated group p value proximal
a: 1 vs. 4 < 0.05 d: 2 vs. 4
