RESEARCH
The Effects of Desferroxamine on Bone and Bone Graft Healing in Critical-Size Bone Defects Serbulent Guzey, MD,* Andac Aykan, MD,† Serdar Ozturk, MD,† Hakan Avsever, DMD,‡ Yildirim Karslioglu, MD,§ and Atilla Ertan, DMDk Background: Autogenous bone grafts are still the criterion standard treatment option in critical-size bone defect reconstructions, and many therapies can affect its incorporation. In this study, it was aimed to research the effects of desferroxamine (DFO) application on bone and bone graft healing due to the effects of osteoblast and osteoclast regulation and stimulation of angiogenesis. Methods: Rat zygomatic arch critical-size bone defect model (5 mm) was used as the experimental model. Thirty-two Sprague-Dawley rats (64 zygomatic arches) were divided into 4 groups (16 zygomatic arches in each). In groups 1 and 2, defects were reconstructed with the bone grafts harvested from the other side, and the right arc was named as group 1, and the left was group 2. At group 1, 200 μM/300 μL dosage of DFO was injected at the zygomatic arch region starting at the seventh day preoperatively and lasting until the 45th day postoperatively. Group 2 animals were defined as the control group of group 1, and 0.9% NaCl injection was applied. In groups 3 and 4, there was no repair after the formation of defects, and the right arc region was treated with DFO, and left was treated with 0.9% NaCl for postoperative 45 days, respectively. Radiological (computed tomography), histological (hematoxylin-eosin), and biomechanical (3-point bending test) tests were used for the evaluation. Results: In radiological evaluation, there was a statistically significant decrease (P < 0.05) in bone defect size in group 3 animals at the 4th, 8th, and 12th weeks, and bone graft volume showed a statistical difference at all weeks (P < 0.05). In histological evaluation, it was observed that there was an increase in osteoblast number and vascularity rates (P < 0.05) in the DFO-treated groups at all weeks. Biomechanical evaluation of the subjects showed increase in bone strength in group 1 animals at 12 weeks. Conclusions: In this study, it was shown that DFO treatment increased bone graft incorporation and healing in critical-size bone defects. In this aspect, we suggest that DFO can be used to increase graft incorporation in risky areas and reduce the defect size in patients who are not suitable for vascularized bone graft transfer. Key Words: bone graft, bone healing, desferroxamine (Ann Plast Surg 2015;00: 00–00)
C
ritical-size bone defect is known as the smallest defect that cannot heal spontaneously and needs bone graft material for total recovery.1,2 Although many graft materials for the reconstruction of these defects have been used, autogenous bone grafts are still the criterion standard treatment option.3 Autogenous bone grafts can be used in either free or vascularized form because of certain limitations such as defect size, vascularization, and infection.4 The usage of vascularized forms becomes more frequent when the defect is large or the recipient bed has an insufficient vascular network. Although Received August 20, 2015, and accepted for publication, after revision November 7, 2015. From the *Department of Plastic, Reconstructive and Aesthetic Surgery, Kasımpasa Military Hospital, Istanbul; and Departments of †Plastic, Reconstructive and Aesthetic Surgery and ‡Oral Diagnosis and Radiology, Gulhane Military Medical Academy; §Department of Pathology, Gulhane Military Medical Academy; and kDepartment of Dentistry, Hacettepe University Medical Faculty, Ankara, Turkey. Conflicts of interest and sources of funding: none declared. Reprints: Serbulent Guzey, MD, Department of Plastic, Reconstructive and Aesthetic Surgery, Kasimpasa Military Hospital, Beyoglu, Istanbul, Turkey. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/0000–0000 DOI: 10.1097/SAP.0000000000000679
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
development of microsurgical techniques has made this procedure common, comorbidities, complication rates, long hospitalization times, and difficulties faced by inexperienced surgeons can limit its application.4 Therefore, such techniques, which can reduce the defect size or augment graft incorporation, become important in these cases. Bone graft incorporation success has many variables. In previous studies, it was shown that the bone graft structure, fixation, mechanical stress, protection of periosteum, and the recipient site were the main factors, which can all directly affect the healing process.4 Recipient bed is an important component of good healing as well.4 It can affect the process in different ways such as vascularization and infection. With vascularization, graft incorporation increases when the recipient bed has a rich vascular network or vascularization is stimulated.5–7 Desferroxamine (DFO), which is a siderophore produced by Streptomyces pilosus, has been used as an iron chelator for many years. In addition, DFO leads to prevention of the degradation of hypoxiainducible factor 1 α, which is a potent stimulator of vascular endothelial growth factor (VEGF) synthesis in the nucleus.8 Increased VEGF levels not only stimulate neoangiogenesis, but also regulate osteogenesis through the regulation of osteblastic and osteoclastic activation.9,10 In this study, the aim was to research the effects of DFO application on bone and bone graft healing due to the effects of osteoblast and osteoclast regulation, stimulation of angiogenesis, and reduction of oxygen radicals.
MATERIALS AND METHODS Thirty-two adult male Sprague-Dawley rats weighing between 350 and 400 g were included in the study and paired in cages on a 12-hour light-dark schedule. All experiments were performed in accordance with the approved protocol of the Scientific Research Board and Ethics Board of Animal Experiments. To reduce the number of animals used, the right zygomatic arch regions of the rats were planned as study groups, and the left sides as control groups.
Experimental Model and Surgical Procedure In the study, rat zygomatic arc critical-size bone defect model (5 mm), as described by Kim et al,11 was used as an experimental model. All surgical procedures were performed under general anesthesia and sterile conditions. Following a lateral incision on the zygomatic arch region, the arch was exposed and isolated. Five millimeters of bone segment was marked, with the zygomaticotemporal suture as the midpoint. Osteotomies were performed, and bone grafts were harvested (Figs. 1, 2). Grafts were used for the reconstruction process on 16 of 32 rats (later named groups 1 and 2). Fixation was performed with 7-0 of polypropylene (Dogsan; Trabzon, Turkey), which was passed through the microholes of the grafts (Fig. 2). These animals’ grafts were harvested from the left side, used for reconstruction of the right arch, and then named as Group 1. In this group, a 200 μM/300 μL dosage of DFO (Desferal 0.5 g flacon; Novartis, Switzerland) was applied to the right arch region beginning at 7 days preoperatively and lasting until 45 days, every other day. From those same animals, grafts were harvested from the right arch and used for the repair of the left side. The same dosage www.annalsplasticsurgery.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
1
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Guzey et al
FIGURE 1. Surgical procedure of the study. A, Zygomatic arch was exposed with lateral incision. B and C, Microholes were drilled. D, Osteotomies were performed. E, Five-millimeter defect was created.
of 0.9% NaCl was injected instead of DFO for the same period, and this group was designated as group 2 (Fig. 3). In groups 3 and 4 (n = 16 rats), no repair was performed. The right arch regions of these animals were named group 3, and a 200 μM/300 μL dosage of DFO was applied for 45 days. The left sides of these animals were called group 4, and a 0.9%NaCl was injected for the same amount of time (Fig. 3).
Assessment Methods Histological (hematoxylin-eosin), radiological (computed tomography), and biomechanical (the 3-point bending test) tests were used for evaluation (Fig. 4).
Clinical Observations Bone graft stability and bleeding differences were assessed macroscopically for the animals in groups 1 and 2, whereas tissue sampling was completed for the histological examination. The defect size was measured for animals in groups 3 and 4 at the same time.
Histological Examination Eight rats (at the first and sixth weeks) and then the remaining 16 rats (at the 12th week) were killed. Zygomatic arch regions were harvested and stained with hematoxylin-eosin. The graft regions and defect zones with adjacent bone tissue were examined under 40 magnification with a light microscope (Nikon Eclipse i80, Japan) for all groups.1–4 A pathologist, who was blinded to the study, counted the osteoblast, osteoclast, and vascular structures at 5 different zones, and the mean values of each were calculated.
Radiological Evaluation At the second and fourth weeks 24 rats and at the eighth and twelfth weeks 16 rats were examined with computed tomography (Toshiba Aquilion 320, Japan) with a slice thickness of 0.5 mm. (Eight rats were killed following the first and sixth weeks.) Radiological images were analyzed with a software (Mimics 16.0 for X64 and 3-Matic version 8.0; Materialise, Belgium), and bone graft volumes were calculated for the bone-grafted groups (Fig. 5). In groups 3 and 4, the defect sizes of the radiological images
FIGURE 2. Five-millimeter-length bone grafts were harvested bilaterally and fixed to the opposite side. 2
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© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Desferroxamine in Bone Graft Healing
FIGURE 3. Schematic view of the study groups. Grafts were harvested from the left side, used for reconstruction of the right arch, and then named as group 1, and DFO was injected to this region. From those same animals, grafts were harvested from the right arch and used for the repair of the left side, and same dosage of serum was injected instead of DFO (left photograph). In groups 3 (right arc) and 4 (left arc), no repair was performed, and DFO and serum were injected, respectively (right photograph).
were measured with specialized software (Hipax patient cd viewer; Steinhart, Germany).
addition, stress-strain graphics were analyzed, and Young modulus values were calculated.
Biomechanical Evaluation
Statistical Analysis
At the end of the 12th week, the remaining 8 rats of groups 1 and 2 were analyzed with the 3-point bending test (Lloyd LR 50 K; Lloyd Instruments, UK). The crosshead speed was 1 mm/min, and the load-bearing capacities were recorded for each sample. In
All statistical analyses were conducted using SPSS for Windows version 15.0 (SPSS, Inc, Chicago, Ill). The Kolmogorov-Smirnov test was used to determine the distribution of data. The Wilcoxon signed ranks test was used for analyzing bone graft volumes, bone defect
FIGURE 4. Assessment schedule of the study. © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
3
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Guzey et al
FIGURE 5. Volumetric analysis of a rat with software (Mimics 16.0 for X64 and 3-Matic version 8.0; Materialise). A, Bone graft boundaries were marked. B, Grafts were isolated, and the volumes were calculated.
FIGURE 6. Desferroxamine–treated regions had a more bleeding capacity than control sides. A, Right arch region. B, Left arch region of the same rat at the end of the first week.
FIGURE 7. At the end of the 12th week, all of the bone grafts were stable; graft volumes were greater (A vs B), and defect sizes were smaller in the DFO-treated groups (C vs D). 4
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Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Desferroxamine in Bone Graft Healing
FIGURE 8. Radiological images of a groups 1 and 2 rat at the 2nd, 4th, 8th, and 12th weeks.
sizes, and biomechanical data. The 2-tailed independent-samples t test was used for analyzing the following values: osteoblast number, osteoclast number, and vascularity rates. Results were accepted as statistically significant at (P < 0.05).
and P < 0.012. The evaluation of groups 3 and 4 at these weeks showed a statistical difference as well. P < 0.012 for each week (Tables 1 and 2, Fig. 9).
Histological Evaluation RESULTS Clinical Observations At the end of the first week, we observed that the soft tissue over the right zygomatic arch region of the rats had a greater bleeding capacity than the left side (Fig. 6). In terms of defect size and graft volume, there was no difference between the control and study groups. There was, however, a partial bone graft resorption in the animals of groups 1 and 2 in that first week. At the sixth week, there was no difference in bleeding volume, but bone graft volumes were much higher on the right side (group 1). The defect size was smaller in group 3 animals. At the end of the 12th week, all of the bone grafts were stable; graft volumes were greater and defect sizes were smaller in the DFO-treated groups (Fig. 7).
Biomechanical Evaluation
Radiological Evaluation The evaluation of radiological images at the second week showed a reduction in bone graft volumes. Although the proportion of reduction was low, and there were significant differences for group 1 (P < 0.034), no statistical differences were found in the evaluation of the defect sizes between groups 3 and 4 (P < 0.62). At the fourth week, the reduction of the graft volumes still existed, and the statistical analysis showed significant difference (P < 0.02) between the bone-grafted groups. Differently from the second week, statistically significant differences were found in the defect groups at this week (P < 0.02). At the 8th and 12th weeks, we observed an increase in bone graft volumes (Fig. 8). The median values of bone graft volumes were 16.41 and 18.20 for group 1 and 13.955 and 15.14 for group 2, respectively. The statistical analyses showed a difference between these groups, TABLE 1. Bone Graft Volume Changes of Groups 1 and 2 Animals Group
1 2
2nd Week (mm3)
Histological evaluation confirmed that the DFO-treated groups generated more bone formation and vascularity during all weeks of the study. Quantitative histological counting of osteoblast and vascular structures demonstrated a proliferation in these groups (Figs. 10, 11). At the 1st, 6th, and 12th weeks, statistical analyses of the osteoblast numbers and vascularity were significant for the DFOtreated groups. There was no statistical difference between the osteoclast numbers of the control and study groups at all weeks (Tables 3–5). When we compared the changes at the 1st, 6th, and 12th weeks for each group, we observed a decrease in the osteoclast number from the first week to the sixth. This difference was statistically significant in groups 1, 2, and 4 (Fig. 12). In addition, there was a statistically significant decrease in the osteoblast number between the 6th and 12th weeks in each group (Fig.13).
4th Week (mm3)
8th Week (mm3)
12th Week (mm3)
Median 15.74 14.415 16.41 18.20 Max-Min 16.74–13.64 15.65–12.54 17.75–13.98 19.98–16.64 Median 14.705 13.295 13.955 15.14 Max-Min 16.21–12.74 14.67–11.32 14.72–12.13 15.97–13.33 P
The Effects of Desferroxamine on Bone and Bone Graft Healing in Critical-Size Bone Defects Serbulent Guzey, MD,* Andac Aykan, MD,† Serdar Ozturk, MD,† Hakan Avsever, DMD,‡ Yildirim Karslioglu, MD,§ and Atilla Ertan, DMDk Background: Autogenous bone grafts are still the criterion standard treatment option in critical-size bone defect reconstructions, and many therapies can affect its incorporation. In this study, it was aimed to research the effects of desferroxamine (DFO) application on bone and bone graft healing due to the effects of osteoblast and osteoclast regulation and stimulation of angiogenesis. Methods: Rat zygomatic arch critical-size bone defect model (5 mm) was used as the experimental model. Thirty-two Sprague-Dawley rats (64 zygomatic arches) were divided into 4 groups (16 zygomatic arches in each). In groups 1 and 2, defects were reconstructed with the bone grafts harvested from the other side, and the right arc was named as group 1, and the left was group 2. At group 1, 200 μM/300 μL dosage of DFO was injected at the zygomatic arch region starting at the seventh day preoperatively and lasting until the 45th day postoperatively. Group 2 animals were defined as the control group of group 1, and 0.9% NaCl injection was applied. In groups 3 and 4, there was no repair after the formation of defects, and the right arc region was treated with DFO, and left was treated with 0.9% NaCl for postoperative 45 days, respectively. Radiological (computed tomography), histological (hematoxylin-eosin), and biomechanical (3-point bending test) tests were used for the evaluation. Results: In radiological evaluation, there was a statistically significant decrease (P < 0.05) in bone defect size in group 3 animals at the 4th, 8th, and 12th weeks, and bone graft volume showed a statistical difference at all weeks (P < 0.05). In histological evaluation, it was observed that there was an increase in osteoblast number and vascularity rates (P < 0.05) in the DFO-treated groups at all weeks. Biomechanical evaluation of the subjects showed increase in bone strength in group 1 animals at 12 weeks. Conclusions: In this study, it was shown that DFO treatment increased bone graft incorporation and healing in critical-size bone defects. In this aspect, we suggest that DFO can be used to increase graft incorporation in risky areas and reduce the defect size in patients who are not suitable for vascularized bone graft transfer. Key Words: bone graft, bone healing, desferroxamine (Ann Plast Surg 2015;00: 00–00)
C
ritical-size bone defect is known as the smallest defect that cannot heal spontaneously and needs bone graft material for total recovery.1,2 Although many graft materials for the reconstruction of these defects have been used, autogenous bone grafts are still the criterion standard treatment option.3 Autogenous bone grafts can be used in either free or vascularized form because of certain limitations such as defect size, vascularization, and infection.4 The usage of vascularized forms becomes more frequent when the defect is large or the recipient bed has an insufficient vascular network. Although Received August 20, 2015, and accepted for publication, after revision November 7, 2015. From the *Department of Plastic, Reconstructive and Aesthetic Surgery, Kasımpasa Military Hospital, Istanbul; and Departments of †Plastic, Reconstructive and Aesthetic Surgery and ‡Oral Diagnosis and Radiology, Gulhane Military Medical Academy; §Department of Pathology, Gulhane Military Medical Academy; and kDepartment of Dentistry, Hacettepe University Medical Faculty, Ankara, Turkey. Conflicts of interest and sources of funding: none declared. Reprints: Serbulent Guzey, MD, Department of Plastic, Reconstructive and Aesthetic Surgery, Kasimpasa Military Hospital, Beyoglu, Istanbul, Turkey. E-mail: [email protected]. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/0000–0000 DOI: 10.1097/SAP.0000000000000679
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
development of microsurgical techniques has made this procedure common, comorbidities, complication rates, long hospitalization times, and difficulties faced by inexperienced surgeons can limit its application.4 Therefore, such techniques, which can reduce the defect size or augment graft incorporation, become important in these cases. Bone graft incorporation success has many variables. In previous studies, it was shown that the bone graft structure, fixation, mechanical stress, protection of periosteum, and the recipient site were the main factors, which can all directly affect the healing process.4 Recipient bed is an important component of good healing as well.4 It can affect the process in different ways such as vascularization and infection. With vascularization, graft incorporation increases when the recipient bed has a rich vascular network or vascularization is stimulated.5–7 Desferroxamine (DFO), which is a siderophore produced by Streptomyces pilosus, has been used as an iron chelator for many years. In addition, DFO leads to prevention of the degradation of hypoxiainducible factor 1 α, which is a potent stimulator of vascular endothelial growth factor (VEGF) synthesis in the nucleus.8 Increased VEGF levels not only stimulate neoangiogenesis, but also regulate osteogenesis through the regulation of osteblastic and osteoclastic activation.9,10 In this study, the aim was to research the effects of DFO application on bone and bone graft healing due to the effects of osteoblast and osteoclast regulation, stimulation of angiogenesis, and reduction of oxygen radicals.
MATERIALS AND METHODS Thirty-two adult male Sprague-Dawley rats weighing between 350 and 400 g were included in the study and paired in cages on a 12-hour light-dark schedule. All experiments were performed in accordance with the approved protocol of the Scientific Research Board and Ethics Board of Animal Experiments. To reduce the number of animals used, the right zygomatic arch regions of the rats were planned as study groups, and the left sides as control groups.
Experimental Model and Surgical Procedure In the study, rat zygomatic arc critical-size bone defect model (5 mm), as described by Kim et al,11 was used as an experimental model. All surgical procedures were performed under general anesthesia and sterile conditions. Following a lateral incision on the zygomatic arch region, the arch was exposed and isolated. Five millimeters of bone segment was marked, with the zygomaticotemporal suture as the midpoint. Osteotomies were performed, and bone grafts were harvested (Figs. 1, 2). Grafts were used for the reconstruction process on 16 of 32 rats (later named groups 1 and 2). Fixation was performed with 7-0 of polypropylene (Dogsan; Trabzon, Turkey), which was passed through the microholes of the grafts (Fig. 2). These animals’ grafts were harvested from the left side, used for reconstruction of the right arch, and then named as Group 1. In this group, a 200 μM/300 μL dosage of DFO (Desferal 0.5 g flacon; Novartis, Switzerland) was applied to the right arch region beginning at 7 days preoperatively and lasting until 45 days, every other day. From those same animals, grafts were harvested from the right arch and used for the repair of the left side. The same dosage www.annalsplasticsurgery.com
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
1
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Guzey et al
FIGURE 1. Surgical procedure of the study. A, Zygomatic arch was exposed with lateral incision. B and C, Microholes were drilled. D, Osteotomies were performed. E, Five-millimeter defect was created.
of 0.9% NaCl was injected instead of DFO for the same period, and this group was designated as group 2 (Fig. 3). In groups 3 and 4 (n = 16 rats), no repair was performed. The right arch regions of these animals were named group 3, and a 200 μM/300 μL dosage of DFO was applied for 45 days. The left sides of these animals were called group 4, and a 0.9%NaCl was injected for the same amount of time (Fig. 3).
Assessment Methods Histological (hematoxylin-eosin), radiological (computed tomography), and biomechanical (the 3-point bending test) tests were used for evaluation (Fig. 4).
Clinical Observations Bone graft stability and bleeding differences were assessed macroscopically for the animals in groups 1 and 2, whereas tissue sampling was completed for the histological examination. The defect size was measured for animals in groups 3 and 4 at the same time.
Histological Examination Eight rats (at the first and sixth weeks) and then the remaining 16 rats (at the 12th week) were killed. Zygomatic arch regions were harvested and stained with hematoxylin-eosin. The graft regions and defect zones with adjacent bone tissue were examined under 40 magnification with a light microscope (Nikon Eclipse i80, Japan) for all groups.1–4 A pathologist, who was blinded to the study, counted the osteoblast, osteoclast, and vascular structures at 5 different zones, and the mean values of each were calculated.
Radiological Evaluation At the second and fourth weeks 24 rats and at the eighth and twelfth weeks 16 rats were examined with computed tomography (Toshiba Aquilion 320, Japan) with a slice thickness of 0.5 mm. (Eight rats were killed following the first and sixth weeks.) Radiological images were analyzed with a software (Mimics 16.0 for X64 and 3-Matic version 8.0; Materialise, Belgium), and bone graft volumes were calculated for the bone-grafted groups (Fig. 5). In groups 3 and 4, the defect sizes of the radiological images
FIGURE 2. Five-millimeter-length bone grafts were harvested bilaterally and fixed to the opposite side. 2
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© 2015 Wolters Kluwer Health, Inc. All rights reserved.
Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Desferroxamine in Bone Graft Healing
FIGURE 3. Schematic view of the study groups. Grafts were harvested from the left side, used for reconstruction of the right arch, and then named as group 1, and DFO was injected to this region. From those same animals, grafts were harvested from the right arch and used for the repair of the left side, and same dosage of serum was injected instead of DFO (left photograph). In groups 3 (right arc) and 4 (left arc), no repair was performed, and DFO and serum were injected, respectively (right photograph).
were measured with specialized software (Hipax patient cd viewer; Steinhart, Germany).
addition, stress-strain graphics were analyzed, and Young modulus values were calculated.
Biomechanical Evaluation
Statistical Analysis
At the end of the 12th week, the remaining 8 rats of groups 1 and 2 were analyzed with the 3-point bending test (Lloyd LR 50 K; Lloyd Instruments, UK). The crosshead speed was 1 mm/min, and the load-bearing capacities were recorded for each sample. In
All statistical analyses were conducted using SPSS for Windows version 15.0 (SPSS, Inc, Chicago, Ill). The Kolmogorov-Smirnov test was used to determine the distribution of data. The Wilcoxon signed ranks test was used for analyzing bone graft volumes, bone defect
FIGURE 4. Assessment schedule of the study. © 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
3
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Guzey et al
FIGURE 5. Volumetric analysis of a rat with software (Mimics 16.0 for X64 and 3-Matic version 8.0; Materialise). A, Bone graft boundaries were marked. B, Grafts were isolated, and the volumes were calculated.
FIGURE 6. Desferroxamine–treated regions had a more bleeding capacity than control sides. A, Right arch region. B, Left arch region of the same rat at the end of the first week.
FIGURE 7. At the end of the 12th week, all of the bone grafts were stable; graft volumes were greater (A vs B), and defect sizes were smaller in the DFO-treated groups (C vs D). 4
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Copyright © 2015 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.
Annals of Plastic Surgery • Volume 00, Number 00, Month 2015
Desferroxamine in Bone Graft Healing
FIGURE 8. Radiological images of a groups 1 and 2 rat at the 2nd, 4th, 8th, and 12th weeks.
sizes, and biomechanical data. The 2-tailed independent-samples t test was used for analyzing the following values: osteoblast number, osteoclast number, and vascularity rates. Results were accepted as statistically significant at (P < 0.05).
and P < 0.012. The evaluation of groups 3 and 4 at these weeks showed a statistical difference as well. P < 0.012 for each week (Tables 1 and 2, Fig. 9).
Histological Evaluation RESULTS Clinical Observations At the end of the first week, we observed that the soft tissue over the right zygomatic arch region of the rats had a greater bleeding capacity than the left side (Fig. 6). In terms of defect size and graft volume, there was no difference between the control and study groups. There was, however, a partial bone graft resorption in the animals of groups 1 and 2 in that first week. At the sixth week, there was no difference in bleeding volume, but bone graft volumes were much higher on the right side (group 1). The defect size was smaller in group 3 animals. At the end of the 12th week, all of the bone grafts were stable; graft volumes were greater and defect sizes were smaller in the DFO-treated groups (Fig. 7).
Biomechanical Evaluation
Radiological Evaluation The evaluation of radiological images at the second week showed a reduction in bone graft volumes. Although the proportion of reduction was low, and there were significant differences for group 1 (P < 0.034), no statistical differences were found in the evaluation of the defect sizes between groups 3 and 4 (P < 0.62). At the fourth week, the reduction of the graft volumes still existed, and the statistical analysis showed significant difference (P < 0.02) between the bone-grafted groups. Differently from the second week, statistically significant differences were found in the defect groups at this week (P < 0.02). At the 8th and 12th weeks, we observed an increase in bone graft volumes (Fig. 8). The median values of bone graft volumes were 16.41 and 18.20 for group 1 and 13.955 and 15.14 for group 2, respectively. The statistical analyses showed a difference between these groups, TABLE 1. Bone Graft Volume Changes of Groups 1 and 2 Animals Group
1 2
2nd Week (mm3)
Histological evaluation confirmed that the DFO-treated groups generated more bone formation and vascularity during all weeks of the study. Quantitative histological counting of osteoblast and vascular structures demonstrated a proliferation in these groups (Figs. 10, 11). At the 1st, 6th, and 12th weeks, statistical analyses of the osteoblast numbers and vascularity were significant for the DFOtreated groups. There was no statistical difference between the osteoclast numbers of the control and study groups at all weeks (Tables 3–5). When we compared the changes at the 1st, 6th, and 12th weeks for each group, we observed a decrease in the osteoclast number from the first week to the sixth. This difference was statistically significant in groups 1, 2, and 4 (Fig. 12). In addition, there was a statistically significant decrease in the osteoblast number between the 6th and 12th weeks in each group (Fig.13).
4th Week (mm3)
8th Week (mm3)
12th Week (mm3)
Median 15.74 14.415 16.41 18.20 Max-Min 16.74–13.64 15.65–12.54 17.75–13.98 19.98–16.64 Median 14.705 13.295 13.955 15.14 Max-Min 16.21–12.74 14.67–11.32 14.72–12.13 15.97–13.33 P
