The Influence of Cortical Perforation on Guided Bone Regeneration Using Synthetic Bone Substitutes: A Study of Rabbit Cranial Defects Sang-Hwa Lee, DDS, PhD1/Pil Lim, DDS, MSD2/Hyun-Joong Yoon, DDS, PhD3 Purpose: The purpose of this study was to investigate the influence of cortical perforation on angiogenesis and osteogenesis following guided bone regeneration using synthetic bone substitutes in rabbit cranial defects. Materials and Methods: The right and left sides of the calvaria were exposed in 11 rabbits. In each rabbit, two custom-made titanium domes were placed on either side of the midline. In experimental sites, the cortical surface inside the boundary of one of the two circular slits was then mechanically perforated five times with a round bur; in control sites, this was left intact. All sites received beta-tricalcium phosphate. The animals were sacrificed at 2, 4, and 8 weeks. Biopsy samples were examined histomorphometrically by light microscopy, and the expression of vascular endothelial growth factor (VEGF) and osteocalcin (OC) was determined immunohistochemically. Results: The percent area of newly formed bone was significantly higher in the experimental group than in the control group 2 weeks after surgery. Marrow cells reached the normal rabbit calvarial bone more rapidly in experimental sites than in control sites. Immunostaining intensity and the percentage of positively stained cells for VEGF were greater in the experimental group than in the control group at 2 weeks after surgery. At 4 weeks, immunostaining intensity and the percentage of positively stained cells for OC were greater in the experimental group than in the control group. However, there were no significant differences between the experimental and control groups in immunohistochemical findings for VEGF and OC. Conclusions: The results of this study suggest that cortical perforation of the receptor bone may improve angiogenesis in bone grafts and increase the amount of newly formed bone in grafted areas, especially in the early bony healing phase. Further studies in larger samples are needed to confirm these results. Int J Oral Maxillofac Implants 2014;29:464–471. doi: 10.11607/jomi.3221 Key words: beta-tricalcium phosphate, cortical perforation, osteocalcin, vascular endothelial growth factor
O
ne of the key requirements for achieving osseointegration is the presence of adequate osseous volume. In patients with inadequate osseous width or height, bone augmentation using guided bone regeneration (GBR) may be applied. Some studies have revealed intimate spatial and temporal correlations between newly formed blood vessels and de novo
1Associate
Professor, Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, Seoul, Republic of Korea. 2Private Practice, Seoul, Republic of Korea. 3Professor, Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, Seoul, Republic of Korea. Correspondence to: Dr Hyun-Joong Yoon: Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, #62 Yeouido-dong, Yeongdeungpo-gu, Seoul, 150-713, Republic of Korea. Fax: +82-2-769-1689. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
bone formation. These observations have emphasized the significance of angiogenesis in GBR.1,2 Intramarrow penetration or bone decortication is often performed as part of GBR. The biologic rationale for intramarrow penetration or decortication of bone is to allow for easy access of progenitor cells to GBR-treated sites and to facilitate prompt angiogenesis. It may also enhance the physical connection between bone grafts and recipient sites. However, decortication prior to GBR is controversial because there have been no human clinical trials regarding its effectiveness and because animal studies have provided opposing points of view. 3 Carvalho et al4 reported that autogenous bone grafts integrated with the receptor bed mainly when perforated or decorticated; the poorest results were seen in cortical bone. However, Adeyemo et al5 and Barbosa et al6 showed that perforated recipient cortical beds offered no advantage over nonperforated beds in terms of healing and integration of bone grafts. Although some studies have reported that intramarrow penetration accelerates bone neogenesis and results in increased bone
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Lee et al
fill and density,7–9 others have demonstrated that bone formation is not significantly different between procedures done with and without marrow penetration.10,11 This study was undertaken to investigate the influence of cortical perforation on the angiogenesis and osteogenesis of GBR using synthetic bone substitutes in rabbit cranial defects by assessing the histology and immunohistochemistry of the grafts.
Materials and Methods Animals
Eleven adult male New Zealand white rabbits, weighing between 2.8 and 3.5 kg each, were used in this study and were kept in individual metal cages at room temperature. All rabbits were checked beforehand by a single veterinarian to ensure their health. Each rabbit was allowed an acclimatization period of 2 weeks prior to each surgery. The study was approved by the Committee on the Use and Care of Animals and the Institutional Review Board of the Catholic University of Korea.
Fig 1 Two circular 1-mm-deep slits were prepared, one on each side of the midline. The cortical surface inside the boundary of one of the two circular slits was mechanically perforated five times (experimental group), while the bone surface within the perimeter of the other slit was left intact (control group).
Surgical Procedures
General anesthesia was induced with intramuscular injections of tiletamine/zolazepam (15 mg/kg; Zoletil, Virbac Korea) combined with xylazine hydrochloride (5 mg/kg; Rompun, Bayer Korea). Before the surgical procedure, each animal received a single subcutaneous dose of penicillin G (0.3 mL; Gentamycin, Kukje Pharm) and ketoprofen (1 mg/kg; Bukwang Pharm). In addition, 0.5 mL of 2% lidocaine (Xylestesin-A, 3M ESPE) was injected locally at the periphery of the surgical site. Immediately before surgery, the scalp was carefully shaved and disinfected with a povidone-iodine topical antiseptic. The skin and subcutaneous tissue were incised in the median region of the rabbit calvarial bone, extending from the frontal to the occipital bone. The periosteum was carefully incised and dissected bilaterally to expose the cortical bone in the region. Thereafter, two standardized circular 1-mm-deep slits one on either side of the midline were prepared in the bone under constant irrigation with 0.9% saline solution using a trephine bur with an inner diameter of 7.0 mm mounted on a low-speed handpiece. In experimental sites, the bone surface inside the boundary of the circular slit was then mechanically perforated five times with a 0.8-mm-diameter round bur; in the control group, the bone surface within the perimeter of the slit was left intact (Fig 1). Each of the experimental and control sites received 0.15 mL of synthetic bone substitute (Cerasorb, Curasan) (Fig 2). Experimental and control sites were designated on a random basis. Subsequently, one custom-made titanium (Ti > 99.5%) dome with
Fig 2 Each of the experimental and control groups received 0.15 mL of synthetic bone substitute (beta tricalcium phosphate).
Fig 3 Two custom-made titanium domes were tightly fitted to the prepared slits, allowing for a complete peripheral seal between the dome margins and the osseous surface.
an inner diameter of 7.0 mm and an inner height of 3.5 mm was tightly fitted into each prepared slit to create a complete peripheral seal between the dome margin and the bone surface. Each dome was equipped with a 0.5-mm-wide horizontal peripheral flange to ensure stability (Fig 3). Both the periosteum and the skin were The International Journal of Oral & Maxillofacial Implants 465
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Lee et al
repositioned, and primary closure was achieved with resorbable suture material (Vicryl, Ethicon). For 3 days postoperatively, the animals received subcutaneously administered penicillin G at a dose of 0.3 mL and ketoprofen at a dose of 1 mg/kg of body weight. The animals were sacrificed by intravenous administration of potassium chloride (KCl-40 injectable, Daihan Pharm) at 2 weeks (n = 4), 4 weeks (n = 3), and 8 weeks (n = 4).
Histologic Preparation
The skull bones were retrieved en bloc with the titanium domes in situ and immediately fixed in 10% neutral buffered formalin. After being embedded in paraffin, the blocks were sectioned transversely along a plane perpendicular to the bone surface to yield sections about 4 µm thick. The most central sections of each specimen were selected for each dome, stained with hematoxylin and eosin (H&E), and examined by light microscopy.
Immunostaining Procedure
Microtome sections were evaluated by immunohistochemical staining to detect vascular endothelial growth factor (VEGF) and osteocalcin (OC) using a Polink-2 Plus mouse kit (D58-15, Golden Bridge International). Microtome sections were deparaffinized using serial xylene and ethanol baths. After rehydration, slides were heated in a microwave oven with 0.01 mol/L citrate buffer for 3 minutes and then treated with 3% hydrogen peroxide in methanol for 15 minutes at room temperature. Slides were washed three times with Tris Buffer Saline Tween (TBST) (Triology buffer, Cellmarque) at pH 7.4 for 3 minutes and were then incubated for 1 hour with primary antibodies (VEGF: ab28775, abcam; OC: NB 600-1394, Nobus) at room temperature. Slides were again washed three times with TBST for 3 minutes and then incubated with an enhancer reagent (HK 518-50K, BioGenex) for 10 minutes at room temperature. After that, slides were incubated with Polymer HRP (HK 519-50K, BioGenex) for 10 minutes. After a final washing with TBST, slides were treated with diaminobenzidine chromogen (DAKO) to produce dark brown staining for immunoreaction. Sections were counterstained with Mayer hematoxylin for 1 minute, dehydrated in ethanol, cleared in xylene, and mounted in Canada balsam. All slides were evaluated by an independent observer using a light microscope (Olympus BX 51, Olympus). The observer was blinded to the groups and relative ages of the specimens.
Histomorphometric Analysis
The histomorphometric data for the central section obtained from each specimen were recorded using a computerized image analysis system. The newly formed
bone within the experimental and control domes was subjected to the following histomorphometric measurements. First, the longest vertical height of the newly generated tissue (consisting of mineralized bone and marrow space from the parent bone relative to the inner height of the titanium dome) was examined; the inner height of the titanium dome was designated as 5, so the newly generated tissue was expressed as a proportion of the height of newly formed bone (0 to 5). Second, the percent area of the newly formed bone relative to the area bounded by the titanium dome and parent bone was calculated. The parent bone area was designated as 50 and expressed as a percentage of the total area of the newly formed bone (0 to 50). Third, the amount of marrow cell formation in the grafted area relative to the amount in the normal rabbit calvarial bone was evaluated on a three-point scale (1 to 3). The amount of marrow cell formation for each specimen was expressed as less than (1), similar to (2), or more than (3) the normal rabbit calvarial bone. Fourth, the expression of VEGF and OC was evaluated as the proportion of positively stained cells (PSCs) and the immunostaining intensity with a four-point scale (0 to 3). After the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone was designated as 3, the immunostaining intensity for each specimen was evaluated by comparing it with the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone. Data were presented as n (%) or means ± standard deviations (medians, minimums, and maximums) according to their data types. Categorical variables were compared with the Fisher exact probability test because of the small expected values. In light of the small sample size, analyses at the bivariate level were completed using the Wilcoxon rank sum test (a nonparametric statistical test) to identify significant differences between the two groups. A two-sided P < .05 was considered statistically significant. All statistical analyses were performed using SAS software (version 9.2, SAS Institute).
Results Histomorphometric Findings
Histologic images and results are shown in Figs 4 to 7. Percentage of the height of newly formed bone. The height of the newly formed bone (HB) was greater in the experimental group (4.25) than in the control group (2.5) 2 weeks after surgery. The HB was consistently greater in the experimental group (5.0/4.75) than in the control group (4.0/4.0) at 4 and 8 weeks after surgery. However, no significant difference was noted between the groups (Table 1, Figs 4 and 7a).
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Lee et al
Figs 4a and 4b Histologic findings in the (a) control and (b) experimental groups 2 weeks after surgery showing newly formed tissues (yellow arrows) beyond the external cortical calvarial bone (black arrow) (H&E; original magnification ×12.5).
a
b
a
b
a
b
Figs 5a and 5b Histologic findings in the (a) control and (b) experimental groups 8 weeks after surgery showing a larger amount of newly formed bone than at 2 weeks after surgery (H&E; original magnification ×12.5).
Figs 6a and 6b Eight weeks after surgery, the amount of marrow cells appeared to be greater in the (a) control group than in the (b) experimental group (H&E; original magnification ×200).
Percent area of newly formed bone. The percent area of newly formed bone (AB) was consistently higher in the experimental group (42.5/56.7/47.5) than in the control group (12.5/40.0/40.0) at 2, 4, and 8 weeks after surgery. AB was significantly higher in the experimental group than in the control group at 2 weeks after surgery (P < .05). The differences in AB between the experimental and control groups decreased over time (Table 1, Figs 5 and 7b). Amount of marrow cell formation. No marrow cell formation (MCF) was found in the grafted bone area of the experimental and control groups 2 weeks after surgery. The amount of MCF was greater in the experimental group (2.0) than in the control group (1.34) at 4 weeks after surgery. However, by 8 weeks after surgery, the amount of MCF was smaller in the experimental group (2.25) than in the control group (2.5), but the difference was not statistically significant (Table 1, Figs 6 and 7c).
VEGF Expression A linear increase in immunostaining intensity was seen in both the experimental and control groups, whereas no statistically significant difference was noted between the control and experimental groups (Table 2, Fig 8a). No linear increase in the percentage of PSCs was seen in either the experimental or control groups. Two weeks after surgery, the greatest differences in the percentage of PSCs were 25.0 in the experimental group and 12.5 in the control group; however, no statistically significant difference was noted (Table 3, Figs 8b and 9).
OC Expression
Immunostaining intensity for OC was greater in the experimental group than in the control group 4 weeks after surgery. However, there were no significant differences in immunostaining intensity between the experimental and control groups (Table 2, Figs 10 and 11a). The International Journal of Oral & Maxillofacial Implants 467
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Lee et al
Control Test
Height of newly formed bone
7 6 5
3 2
2 wk
4 wk
8 wk
Control Test
Percentage of newly formed bone
70 60 50 40 30 20
0
2 wk
4 wk
Marrow cell formation
Marrow cell formation
Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
2.50 ± 1.00 (2; 2–4) 4.00 ± 1.00 (4; 3–5) 4.00 ± 0.82 (4; 3–5)
4.25 ± 0.96 (4.5; 3–5) 5.00 ± 0.00 (5; 5–5) 4.75 ± 0.50 (5; 4–5)
.0723
Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
12.50 ± 5.00 (10; 10–20) 40.00 ± 10.00 (40; 30–50) 40.00 ± 0.00 (40; 40–40)
42.50 ± 9.57 (45; 30–50) 56.67 ± 5.77 (60; 50–60) 47.50 ± 5.00 (50; 40–50)
2 (66.67) 0 (0.00) 1 (33.33)
1 (33.33) 1 (33.33) 1 (33.33)
.9999
1 (25.00) 3 (75.00) 0 (0.00)
1 (25.00) 2 (50.00) 1 (25.00)
.9999
.1967 .2059
MCF Week 4 (n = 6) Less Similar More Week 8 (n = 8) Less Similar More
.0256 .1157 .0603
8 wk
6
6
c
P
Data presented as mean ± standard deviation (median; range) or n (%). P values indicate differences between control and experimental group according to Fisher exact test and Wilcoxon rank sum test.
10
b
Experimental
AB
1 0
Control HB
4
a
5 4 3 2 1 0
Table 1 Histomorphometric Findings of Newly Formed Bone
Control Test 4 wk
Less Similar More
5 4 3 2 1 0
Control Test 8 wk
Figs 7a to 7c Results of histomorphometric analysis of newly formed bone.
The percentage of PSCs was consistently higher in the experimental group (55.0/53.3) than in the control group (50.0/30.0) at 2 and 4 weeks after surgery, but the difference between groups was not statistically significant (Table 3, Fig 11b).
Discussion Angiogenesis is a multiple-step process with its origin in preexisting vessels. Factors released after wounding
are responsible for the temporary activation of blood vessel formation.2 The importance of blood vessels in bone formation was noted as early as 1763, when it was found that bones rely on arteries to supply mineral elements. Angiogenesis has recently been found to be important for osteogenesis and bone repair.12–16 In GBR, any temporary removal of the overlying periosteum will tear some small vessels extending from the periosteum into the bone and will cause some vessel damage. This damage is sufficient to start the biologic cascade that results in new bone generation. Thus, many studies have indicated that successful bone regeneration occurs without further bone damage.2,17–19 Some reports have shown that cortical bone perforation enhances bone formation in GBR by allowing for the migration of angiogenic and osteogenic cells into the secluded space. Other studies have found that bone regeneration begins in the uninjured cortical layer. There have been few reports on the basic mechanisms underlying the effects of cortical bone perforation, although some experiments have been designed to evaluate the effects of cortical bone perforation on GBR.20 Rompen et al7 reported that predictable bone formation can be achieved beneath completely occlusive barriers over an uninjured cortical layer. They also demonstrated that stimulation of blood supply and bone-
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Table 2 Analysis of VEGF and OC Immunostaining Intensity Intensity rating VEGF Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8) OC Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 6)
Control
Table 3 Analysis of VEGF and OC Immunostaining (% PSCs)
Experimental
1 2 3 1 2 3 1 2 3
3 (75.00) 1 (25.00) – 0 (0.00) 2 (66.67) 1 (33.33) 0 (0.00) 1 (25.00) 3 (75.00)
1 (25.00) 3 (75.00) – 1 (33.33) 0 (0.00) 2 (66.67) 1 (25.00) 0 (0.00) 3 (75.00)
1 2 3 1 2 3 1 2 3
– 4 (100.00) – 2 (66.67) 1 (33.33) – 1 (33.33) 2 (66.67) –
– 4 (100.00) – 1 (33.33) 2 (66.67) – 1 (33.33) 2 (66.67) –
P .4857
.4000
.9999
.9999
Control
Experimental
P
VEGF Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
12.50 ± 5.00 (10; 10–20) 26.67 ± 5.77 (30; 20–30) 25.00 ± 12.91 (25; 10–40)
25.00 ± 10.00 (30; 10–30) 20.00 ± 10.00 (20; 10–30) 27.50 ± 12.58 (30; 10–40)
.1138
OC Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 6)
50.00 ± 24.49 (45; 30–80) 30.00 ± 17.32 (40; 40–80) 56.67 ± 20.82 (50; 40–80)
55.00 ± 26.46 (50; 30–90) 53.33 ± 23.09 (40; 40–80) 46.67 ± 25.17 (50; 20–70)
.7660
.4795 .8809
.3017 .8248
Data presented as mean ± standard deviation (median; range). P values indicate the differences between control and experimental groups by Wilcoxon rank sum test.
.9999
a
3 2 1 0
Control Test 2 wk
4 3 2 1 0
Control Test 4 wk
1 2 3
5
Percentage of VEFG-positive cells
4
5
Intensity of VEGF-positive cells
5
Intensity of VEGF-positive cells
Intensity of VEGF-positive cells
Data presented as n (%). P value; difference between control and experimental group by Fisher’s exact test.
4 3 2 1 0
Control Test 8 wk
b
Control Test
60 50 40 30 20 10 0
2 wk
4 wk
8 wk
Figs 8a and 8b Analysis of VEGF immunostaining.
forming cells by cortical perforation and/or through the addition of blood clot enhanced de novo bone formation in an experimental model. Majzoub et al8 documented that intramarrow penetration enhanced initial osteogenesis and resulted in increased bone density, suggesting that it can be beneficial to bone regenerative procedures. Min et al9 pointed out that bone augmentation was significantly greater in procedures with marrow penetration than in those without and that the number of osteoblastlike cells under the titanium caps was significantly larger at marrow penetration sites. However, Slotte and Lundgren11 demonstrated, in an experimental study, that cortical perforations of contiguous donor bone or the degree of immediate
blood filling of an extracalvarial space did not enhance augmented tissue volume beyond the skeletal envelope. They also stated that, although there were higher mean values for the density of augmented bone in the test sites, their large variations failed to show significant intergroup differences.11 Many studies have been designed to evaluate the histologic and histomorphometric effects of cortical bone perforation on GBR without bone grafting in rabbit calvaria. In the present study, the caps on the control and experimental sites of each rabbit were filled with granulated beta-tricalcium phosphate (β-TCP). Although blood supply is critical for successful bone augmentation, few studies have been conducted on The International Journal of Oral & Maxillofacial Implants 469
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Lee et al
Figs 9a and 9b VEGF expression was lower in the (a) control group than in the (b) experimental group 2 weeks after surgery (yellow arrows = stromal cells; black arrows = endothelial cells) (H&E; original magnification ×200).
a
b Figs 10a and 10b OC expression was lower in the (a) control group than in the (b) experimental group at 4 weeks after surgery (H&E; original magnification ×200).
a
4 3 2 1 0
Control Test 2 wk
5 4 3 2 1 0
Control Test 4 wk
1 2 3
5
Control Test Percentage of OC-positive cells
5
Intensity of OC-positive cells
b
Intensity of OC-positive cells
Intensity of OC-positive cells
a
4 3 2 1 0
Control Test 8 wk
b
100 80 60 40 20 0
2 wk
4 wk
8 wk
Figs 11a and 11b Analysis of OC immunostaining.
angiogenesis in augmented bone. Yamada et al16 evaluated angiogenesis in newly augmented bone and reported that newly generated blood vessels entered the space beyond existing calvarial bone and that angiogenesis occurred to a similar extent with intergranular β-TCP. In the present study, the AB and the percentage of HB were consistently greater in the experimental group than in the control group at all time points examined. The AB was significantly higher in the experimental group than in the control group 2 weeks after surgery (P < .05). This shows that bone augmentation may be greater, especially in the early bony healing period, following procedures with marrow penetration than in those without. Although the difference between the control group and the experimental group was not statistically significant, the amount of marrow cells approached that of normal rabbit calvarial bone
more rapidly in the experimental group than in the control group. Previous studies have indicated that VEGF and OC play important roles in the healing process through modulation of angiogenesis and osteogenesis following injury to the long bones in animals.21–24 However, few reports have been designed to evaluate the immunohistochemical effects of cortical bone perforation on GBR with bone grafting. In the present study, immunostaining intensity and the percentage of PSCs for VEGF were greater in the experimental group than in the control group 2 weeks after surgery, although no statistically significant difference was present. Immunostaining intensity and the percentage of PSCs for OC were greater in the experimental group than in the control group 4 weeks after surgery. However, there were no significant differences between the experimental and control groups.
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Conclusions Taken together, the results of this study suggest that cortical perforation of the receptor bone in addition to synthetic bone substitutes may improve angiogenesis and increase the amount of newly formed bone, especially at early bony healing stages. Further studies with a larger sample size are needed to confirm these results.
ACKNOWLEDGMENTs The authors reported no conflicts of interest related to this study.
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8. Majzoub Z, Berengo M, Giardino R, Aldini N, Cordioli G. Role of inramarrow penetration in osseous repair: A pilot study in the rabbit calvaria. J Periodontol 1999;70:1501–1510. 9. Min S, Sato S, Murai M, et al. Effects of marrow penetration on bone augmentation within a titanium cap in rabbit calvarium. J Periodontol 2007;78:1978–1984. 10. Lundgren AK, Lundgren D, Hämmerle CH, Nyman S, Sennerby L. Influence of decortication of the donor bone on guided bone augmentation. An experimental study in the rabbit skull bone. Clin Oral Implants Res 2000;11:99–106. 11. Slotte C, Lundgren D. Impact of cortical perforation of contiguous donor bone in a guided bone augmentation procedure: An experimental study in the rabbit skull. Clin Implant Dent Relat Res 2002;4:1–10. 12. Mousa SA. Angiogenesis promoters and inhibitors: Potential therapeutic implications. Mol Med Today 1996;2:140–142. 13. Polverini P. Cellular adhesion molecules. Newly identified mediators of angiogenesis. Am J Pathol 1996;4:1023–1029. 14. Carano RA, Filcaroff EH. Angiogenesis and bone repair. Drug Discov Today 2003;8:980–989. 15. Schliephake H. Bone growth factors in maxillofacial reconstruction. Int J Oral Maxillofac Surg 2002;31:469–484. 16. Yamada Y, Tamura T, Hariu K, Asano Y, Sato S, Ito K. Angiogenesis in newly augmented bone observed in rabbit calvarium using a titanium cap. Clin Oral Implants Res 2008;19:1003–1009. 17. Kostopoulos L, Karring T, Uraguchi R. Formation of jawbone tuberosities by guided tissue regeneration. Clin Oral Implants Res 1994;5:245–253. 18. Kostopoulos L, Karring T. Augmentation of the rat mandible using guided tissue regeneration. Clin Oral Implants Res 1994;5:75–82. 19. Lundgren D, Lundgren AK, Sennerby L, Nyman S. Augmentation of intramembranous bone beyond the skeletal using an occlusive titanium barrier. An experimental study in the rabbit. Clin Oral Implants Res 1995;6:67–72. 20. Nishimura I, Shimizu Y, Ooya K. Effects of cortical bone perforation on experimental guided bone regeneration. Clin Oral Implants Res 2004;15:293–230. 21. Wang CJ, Huang HY, Chen HS, et al. The effect of shockwave therapy on acute fracture of the tibia. A study in a dog model. Clin Orthop Relat Res 2001;387:112–118. 22. Wang CJ, Chen HS, Chen CE, et al. Treatment of non-unions of long bone fractures with shock waves. Clin Orthop Relat Res 2001;387:95–101. 23. Uchida S, Sakai A, Kudo H, et al. Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone 2003;32:491–501. 24. Wang CJ, Huang KE, Sun YC, et al. VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res 2011 Nov;171(1):114–119. Epub 2010 Feb 21.
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O
ne of the key requirements for achieving osseointegration is the presence of adequate osseous volume. In patients with inadequate osseous width or height, bone augmentation using guided bone regeneration (GBR) may be applied. Some studies have revealed intimate spatial and temporal correlations between newly formed blood vessels and de novo
1Associate
Professor, Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, Seoul, Republic of Korea. 2Private Practice, Seoul, Republic of Korea. 3Professor, Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, Seoul, Republic of Korea. Correspondence to: Dr Hyun-Joong Yoon: Department of Oral and Maxillofacial Surgery, Yeouido St. Mary’s Hospital, Catholic University of Korea, #62 Yeouido-dong, Yeongdeungpo-gu, Seoul, 150-713, Republic of Korea. Fax: +82-2-769-1689. Email: [email protected] ©2014 by Quintessence Publishing Co Inc.
bone formation. These observations have emphasized the significance of angiogenesis in GBR.1,2 Intramarrow penetration or bone decortication is often performed as part of GBR. The biologic rationale for intramarrow penetration or decortication of bone is to allow for easy access of progenitor cells to GBR-treated sites and to facilitate prompt angiogenesis. It may also enhance the physical connection between bone grafts and recipient sites. However, decortication prior to GBR is controversial because there have been no human clinical trials regarding its effectiveness and because animal studies have provided opposing points of view. 3 Carvalho et al4 reported that autogenous bone grafts integrated with the receptor bed mainly when perforated or decorticated; the poorest results were seen in cortical bone. However, Adeyemo et al5 and Barbosa et al6 showed that perforated recipient cortical beds offered no advantage over nonperforated beds in terms of healing and integration of bone grafts. Although some studies have reported that intramarrow penetration accelerates bone neogenesis and results in increased bone
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Lee et al
fill and density,7–9 others have demonstrated that bone formation is not significantly different between procedures done with and without marrow penetration.10,11 This study was undertaken to investigate the influence of cortical perforation on the angiogenesis and osteogenesis of GBR using synthetic bone substitutes in rabbit cranial defects by assessing the histology and immunohistochemistry of the grafts.
Materials and Methods Animals
Eleven adult male New Zealand white rabbits, weighing between 2.8 and 3.5 kg each, were used in this study and were kept in individual metal cages at room temperature. All rabbits were checked beforehand by a single veterinarian to ensure their health. Each rabbit was allowed an acclimatization period of 2 weeks prior to each surgery. The study was approved by the Committee on the Use and Care of Animals and the Institutional Review Board of the Catholic University of Korea.
Fig 1 Two circular 1-mm-deep slits were prepared, one on each side of the midline. The cortical surface inside the boundary of one of the two circular slits was mechanically perforated five times (experimental group), while the bone surface within the perimeter of the other slit was left intact (control group).
Surgical Procedures
General anesthesia was induced with intramuscular injections of tiletamine/zolazepam (15 mg/kg; Zoletil, Virbac Korea) combined with xylazine hydrochloride (5 mg/kg; Rompun, Bayer Korea). Before the surgical procedure, each animal received a single subcutaneous dose of penicillin G (0.3 mL; Gentamycin, Kukje Pharm) and ketoprofen (1 mg/kg; Bukwang Pharm). In addition, 0.5 mL of 2% lidocaine (Xylestesin-A, 3M ESPE) was injected locally at the periphery of the surgical site. Immediately before surgery, the scalp was carefully shaved and disinfected with a povidone-iodine topical antiseptic. The skin and subcutaneous tissue were incised in the median region of the rabbit calvarial bone, extending from the frontal to the occipital bone. The periosteum was carefully incised and dissected bilaterally to expose the cortical bone in the region. Thereafter, two standardized circular 1-mm-deep slits one on either side of the midline were prepared in the bone under constant irrigation with 0.9% saline solution using a trephine bur with an inner diameter of 7.0 mm mounted on a low-speed handpiece. In experimental sites, the bone surface inside the boundary of the circular slit was then mechanically perforated five times with a 0.8-mm-diameter round bur; in the control group, the bone surface within the perimeter of the slit was left intact (Fig 1). Each of the experimental and control sites received 0.15 mL of synthetic bone substitute (Cerasorb, Curasan) (Fig 2). Experimental and control sites were designated on a random basis. Subsequently, one custom-made titanium (Ti > 99.5%) dome with
Fig 2 Each of the experimental and control groups received 0.15 mL of synthetic bone substitute (beta tricalcium phosphate).
Fig 3 Two custom-made titanium domes were tightly fitted to the prepared slits, allowing for a complete peripheral seal between the dome margins and the osseous surface.
an inner diameter of 7.0 mm and an inner height of 3.5 mm was tightly fitted into each prepared slit to create a complete peripheral seal between the dome margin and the bone surface. Each dome was equipped with a 0.5-mm-wide horizontal peripheral flange to ensure stability (Fig 3). Both the periosteum and the skin were The International Journal of Oral & Maxillofacial Implants 465
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Lee et al
repositioned, and primary closure was achieved with resorbable suture material (Vicryl, Ethicon). For 3 days postoperatively, the animals received subcutaneously administered penicillin G at a dose of 0.3 mL and ketoprofen at a dose of 1 mg/kg of body weight. The animals were sacrificed by intravenous administration of potassium chloride (KCl-40 injectable, Daihan Pharm) at 2 weeks (n = 4), 4 weeks (n = 3), and 8 weeks (n = 4).
Histologic Preparation
The skull bones were retrieved en bloc with the titanium domes in situ and immediately fixed in 10% neutral buffered formalin. After being embedded in paraffin, the blocks were sectioned transversely along a plane perpendicular to the bone surface to yield sections about 4 µm thick. The most central sections of each specimen were selected for each dome, stained with hematoxylin and eosin (H&E), and examined by light microscopy.
Immunostaining Procedure
Microtome sections were evaluated by immunohistochemical staining to detect vascular endothelial growth factor (VEGF) and osteocalcin (OC) using a Polink-2 Plus mouse kit (D58-15, Golden Bridge International). Microtome sections were deparaffinized using serial xylene and ethanol baths. After rehydration, slides were heated in a microwave oven with 0.01 mol/L citrate buffer for 3 minutes and then treated with 3% hydrogen peroxide in methanol for 15 minutes at room temperature. Slides were washed three times with Tris Buffer Saline Tween (TBST) (Triology buffer, Cellmarque) at pH 7.4 for 3 minutes and were then incubated for 1 hour with primary antibodies (VEGF: ab28775, abcam; OC: NB 600-1394, Nobus) at room temperature. Slides were again washed three times with TBST for 3 minutes and then incubated with an enhancer reagent (HK 518-50K, BioGenex) for 10 minutes at room temperature. After that, slides were incubated with Polymer HRP (HK 519-50K, BioGenex) for 10 minutes. After a final washing with TBST, slides were treated with diaminobenzidine chromogen (DAKO) to produce dark brown staining for immunoreaction. Sections were counterstained with Mayer hematoxylin for 1 minute, dehydrated in ethanol, cleared in xylene, and mounted in Canada balsam. All slides were evaluated by an independent observer using a light microscope (Olympus BX 51, Olympus). The observer was blinded to the groups and relative ages of the specimens.
Histomorphometric Analysis
The histomorphometric data for the central section obtained from each specimen were recorded using a computerized image analysis system. The newly formed
bone within the experimental and control domes was subjected to the following histomorphometric measurements. First, the longest vertical height of the newly generated tissue (consisting of mineralized bone and marrow space from the parent bone relative to the inner height of the titanium dome) was examined; the inner height of the titanium dome was designated as 5, so the newly generated tissue was expressed as a proportion of the height of newly formed bone (0 to 5). Second, the percent area of the newly formed bone relative to the area bounded by the titanium dome and parent bone was calculated. The parent bone area was designated as 50 and expressed as a percentage of the total area of the newly formed bone (0 to 50). Third, the amount of marrow cell formation in the grafted area relative to the amount in the normal rabbit calvarial bone was evaluated on a three-point scale (1 to 3). The amount of marrow cell formation for each specimen was expressed as less than (1), similar to (2), or more than (3) the normal rabbit calvarial bone. Fourth, the expression of VEGF and OC was evaluated as the proportion of positively stained cells (PSCs) and the immunostaining intensity with a four-point scale (0 to 3). After the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone was designated as 3, the immunostaining intensity for each specimen was evaluated by comparing it with the immunostaining intensity of the marrow cells of the normal rabbit calvarial bone. Data were presented as n (%) or means ± standard deviations (medians, minimums, and maximums) according to their data types. Categorical variables were compared with the Fisher exact probability test because of the small expected values. In light of the small sample size, analyses at the bivariate level were completed using the Wilcoxon rank sum test (a nonparametric statistical test) to identify significant differences between the two groups. A two-sided P < .05 was considered statistically significant. All statistical analyses were performed using SAS software (version 9.2, SAS Institute).
Results Histomorphometric Findings
Histologic images and results are shown in Figs 4 to 7. Percentage of the height of newly formed bone. The height of the newly formed bone (HB) was greater in the experimental group (4.25) than in the control group (2.5) 2 weeks after surgery. The HB was consistently greater in the experimental group (5.0/4.75) than in the control group (4.0/4.0) at 4 and 8 weeks after surgery. However, no significant difference was noted between the groups (Table 1, Figs 4 and 7a).
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Lee et al
Figs 4a and 4b Histologic findings in the (a) control and (b) experimental groups 2 weeks after surgery showing newly formed tissues (yellow arrows) beyond the external cortical calvarial bone (black arrow) (H&E; original magnification ×12.5).
a
b
a
b
a
b
Figs 5a and 5b Histologic findings in the (a) control and (b) experimental groups 8 weeks after surgery showing a larger amount of newly formed bone than at 2 weeks after surgery (H&E; original magnification ×12.5).
Figs 6a and 6b Eight weeks after surgery, the amount of marrow cells appeared to be greater in the (a) control group than in the (b) experimental group (H&E; original magnification ×200).
Percent area of newly formed bone. The percent area of newly formed bone (AB) was consistently higher in the experimental group (42.5/56.7/47.5) than in the control group (12.5/40.0/40.0) at 2, 4, and 8 weeks after surgery. AB was significantly higher in the experimental group than in the control group at 2 weeks after surgery (P < .05). The differences in AB between the experimental and control groups decreased over time (Table 1, Figs 5 and 7b). Amount of marrow cell formation. No marrow cell formation (MCF) was found in the grafted bone area of the experimental and control groups 2 weeks after surgery. The amount of MCF was greater in the experimental group (2.0) than in the control group (1.34) at 4 weeks after surgery. However, by 8 weeks after surgery, the amount of MCF was smaller in the experimental group (2.25) than in the control group (2.5), but the difference was not statistically significant (Table 1, Figs 6 and 7c).
VEGF Expression A linear increase in immunostaining intensity was seen in both the experimental and control groups, whereas no statistically significant difference was noted between the control and experimental groups (Table 2, Fig 8a). No linear increase in the percentage of PSCs was seen in either the experimental or control groups. Two weeks after surgery, the greatest differences in the percentage of PSCs were 25.0 in the experimental group and 12.5 in the control group; however, no statistically significant difference was noted (Table 3, Figs 8b and 9).
OC Expression
Immunostaining intensity for OC was greater in the experimental group than in the control group 4 weeks after surgery. However, there were no significant differences in immunostaining intensity between the experimental and control groups (Table 2, Figs 10 and 11a). The International Journal of Oral & Maxillofacial Implants 467
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Lee et al
Control Test
Height of newly formed bone
7 6 5
3 2
2 wk
4 wk
8 wk
Control Test
Percentage of newly formed bone
70 60 50 40 30 20
0
2 wk
4 wk
Marrow cell formation
Marrow cell formation
Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
2.50 ± 1.00 (2; 2–4) 4.00 ± 1.00 (4; 3–5) 4.00 ± 0.82 (4; 3–5)
4.25 ± 0.96 (4.5; 3–5) 5.00 ± 0.00 (5; 5–5) 4.75 ± 0.50 (5; 4–5)
.0723
Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
12.50 ± 5.00 (10; 10–20) 40.00 ± 10.00 (40; 30–50) 40.00 ± 0.00 (40; 40–40)
42.50 ± 9.57 (45; 30–50) 56.67 ± 5.77 (60; 50–60) 47.50 ± 5.00 (50; 40–50)
2 (66.67) 0 (0.00) 1 (33.33)
1 (33.33) 1 (33.33) 1 (33.33)
.9999
1 (25.00) 3 (75.00) 0 (0.00)
1 (25.00) 2 (50.00) 1 (25.00)
.9999
.1967 .2059
MCF Week 4 (n = 6) Less Similar More Week 8 (n = 8) Less Similar More
.0256 .1157 .0603
8 wk
6
6
c
P
Data presented as mean ± standard deviation (median; range) or n (%). P values indicate differences between control and experimental group according to Fisher exact test and Wilcoxon rank sum test.
10
b
Experimental
AB
1 0
Control HB
4
a
5 4 3 2 1 0
Table 1 Histomorphometric Findings of Newly Formed Bone
Control Test 4 wk
Less Similar More
5 4 3 2 1 0
Control Test 8 wk
Figs 7a to 7c Results of histomorphometric analysis of newly formed bone.
The percentage of PSCs was consistently higher in the experimental group (55.0/53.3) than in the control group (50.0/30.0) at 2 and 4 weeks after surgery, but the difference between groups was not statistically significant (Table 3, Fig 11b).
Discussion Angiogenesis is a multiple-step process with its origin in preexisting vessels. Factors released after wounding
are responsible for the temporary activation of blood vessel formation.2 The importance of blood vessels in bone formation was noted as early as 1763, when it was found that bones rely on arteries to supply mineral elements. Angiogenesis has recently been found to be important for osteogenesis and bone repair.12–16 In GBR, any temporary removal of the overlying periosteum will tear some small vessels extending from the periosteum into the bone and will cause some vessel damage. This damage is sufficient to start the biologic cascade that results in new bone generation. Thus, many studies have indicated that successful bone regeneration occurs without further bone damage.2,17–19 Some reports have shown that cortical bone perforation enhances bone formation in GBR by allowing for the migration of angiogenic and osteogenic cells into the secluded space. Other studies have found that bone regeneration begins in the uninjured cortical layer. There have been few reports on the basic mechanisms underlying the effects of cortical bone perforation, although some experiments have been designed to evaluate the effects of cortical bone perforation on GBR.20 Rompen et al7 reported that predictable bone formation can be achieved beneath completely occlusive barriers over an uninjured cortical layer. They also demonstrated that stimulation of blood supply and bone-
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Lee et al
Table 2 Analysis of VEGF and OC Immunostaining Intensity Intensity rating VEGF Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8) OC Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 6)
Control
Table 3 Analysis of VEGF and OC Immunostaining (% PSCs)
Experimental
1 2 3 1 2 3 1 2 3
3 (75.00) 1 (25.00) – 0 (0.00) 2 (66.67) 1 (33.33) 0 (0.00) 1 (25.00) 3 (75.00)
1 (25.00) 3 (75.00) – 1 (33.33) 0 (0.00) 2 (66.67) 1 (25.00) 0 (0.00) 3 (75.00)
1 2 3 1 2 3 1 2 3
– 4 (100.00) – 2 (66.67) 1 (33.33) – 1 (33.33) 2 (66.67) –
– 4 (100.00) – 1 (33.33) 2 (66.67) – 1 (33.33) 2 (66.67) –
P .4857
.4000
.9999
.9999
Control
Experimental
P
VEGF Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 8)
12.50 ± 5.00 (10; 10–20) 26.67 ± 5.77 (30; 20–30) 25.00 ± 12.91 (25; 10–40)
25.00 ± 10.00 (30; 10–30) 20.00 ± 10.00 (20; 10–30) 27.50 ± 12.58 (30; 10–40)
.1138
OC Week 2 (n = 8) Week 4 (n = 6) Week 8 (n = 6)
50.00 ± 24.49 (45; 30–80) 30.00 ± 17.32 (40; 40–80) 56.67 ± 20.82 (50; 40–80)
55.00 ± 26.46 (50; 30–90) 53.33 ± 23.09 (40; 40–80) 46.67 ± 25.17 (50; 20–70)
.7660
.4795 .8809
.3017 .8248
Data presented as mean ± standard deviation (median; range). P values indicate the differences between control and experimental groups by Wilcoxon rank sum test.
.9999
a
3 2 1 0
Control Test 2 wk
4 3 2 1 0
Control Test 4 wk
1 2 3
5
Percentage of VEFG-positive cells
4
5
Intensity of VEGF-positive cells
5
Intensity of VEGF-positive cells
Intensity of VEGF-positive cells
Data presented as n (%). P value; difference between control and experimental group by Fisher’s exact test.
4 3 2 1 0
Control Test 8 wk
b
Control Test
60 50 40 30 20 10 0
2 wk
4 wk
8 wk
Figs 8a and 8b Analysis of VEGF immunostaining.
forming cells by cortical perforation and/or through the addition of blood clot enhanced de novo bone formation in an experimental model. Majzoub et al8 documented that intramarrow penetration enhanced initial osteogenesis and resulted in increased bone density, suggesting that it can be beneficial to bone regenerative procedures. Min et al9 pointed out that bone augmentation was significantly greater in procedures with marrow penetration than in those without and that the number of osteoblastlike cells under the titanium caps was significantly larger at marrow penetration sites. However, Slotte and Lundgren11 demonstrated, in an experimental study, that cortical perforations of contiguous donor bone or the degree of immediate
blood filling of an extracalvarial space did not enhance augmented tissue volume beyond the skeletal envelope. They also stated that, although there were higher mean values for the density of augmented bone in the test sites, their large variations failed to show significant intergroup differences.11 Many studies have been designed to evaluate the histologic and histomorphometric effects of cortical bone perforation on GBR without bone grafting in rabbit calvaria. In the present study, the caps on the control and experimental sites of each rabbit were filled with granulated beta-tricalcium phosphate (β-TCP). Although blood supply is critical for successful bone augmentation, few studies have been conducted on The International Journal of Oral & Maxillofacial Implants 469
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Lee et al
Figs 9a and 9b VEGF expression was lower in the (a) control group than in the (b) experimental group 2 weeks after surgery (yellow arrows = stromal cells; black arrows = endothelial cells) (H&E; original magnification ×200).
a
b Figs 10a and 10b OC expression was lower in the (a) control group than in the (b) experimental group at 4 weeks after surgery (H&E; original magnification ×200).
a
4 3 2 1 0
Control Test 2 wk
5 4 3 2 1 0
Control Test 4 wk
1 2 3
5
Control Test Percentage of OC-positive cells
5
Intensity of OC-positive cells
b
Intensity of OC-positive cells
Intensity of OC-positive cells
a
4 3 2 1 0
Control Test 8 wk
b
100 80 60 40 20 0
2 wk
4 wk
8 wk
Figs 11a and 11b Analysis of OC immunostaining.
angiogenesis in augmented bone. Yamada et al16 evaluated angiogenesis in newly augmented bone and reported that newly generated blood vessels entered the space beyond existing calvarial bone and that angiogenesis occurred to a similar extent with intergranular β-TCP. In the present study, the AB and the percentage of HB were consistently greater in the experimental group than in the control group at all time points examined. The AB was significantly higher in the experimental group than in the control group 2 weeks after surgery (P < .05). This shows that bone augmentation may be greater, especially in the early bony healing period, following procedures with marrow penetration than in those without. Although the difference between the control group and the experimental group was not statistically significant, the amount of marrow cells approached that of normal rabbit calvarial bone
more rapidly in the experimental group than in the control group. Previous studies have indicated that VEGF and OC play important roles in the healing process through modulation of angiogenesis and osteogenesis following injury to the long bones in animals.21–24 However, few reports have been designed to evaluate the immunohistochemical effects of cortical bone perforation on GBR with bone grafting. In the present study, immunostaining intensity and the percentage of PSCs for VEGF were greater in the experimental group than in the control group 2 weeks after surgery, although no statistically significant difference was present. Immunostaining intensity and the percentage of PSCs for OC were greater in the experimental group than in the control group 4 weeks after surgery. However, there were no significant differences between the experimental and control groups.
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Conclusions Taken together, the results of this study suggest that cortical perforation of the receptor bone in addition to synthetic bone substitutes may improve angiogenesis and increase the amount of newly formed bone, especially at early bony healing stages. Further studies with a larger sample size are needed to confirm these results.
ACKNOWLEDGMENTs The authors reported no conflicts of interest related to this study.
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8. Majzoub Z, Berengo M, Giardino R, Aldini N, Cordioli G. Role of inramarrow penetration in osseous repair: A pilot study in the rabbit calvaria. J Periodontol 1999;70:1501–1510. 9. Min S, Sato S, Murai M, et al. Effects of marrow penetration on bone augmentation within a titanium cap in rabbit calvarium. J Periodontol 2007;78:1978–1984. 10. Lundgren AK, Lundgren D, Hämmerle CH, Nyman S, Sennerby L. Influence of decortication of the donor bone on guided bone augmentation. An experimental study in the rabbit skull bone. Clin Oral Implants Res 2000;11:99–106. 11. Slotte C, Lundgren D. Impact of cortical perforation of contiguous donor bone in a guided bone augmentation procedure: An experimental study in the rabbit skull. Clin Implant Dent Relat Res 2002;4:1–10. 12. Mousa SA. Angiogenesis promoters and inhibitors: Potential therapeutic implications. Mol Med Today 1996;2:140–142. 13. Polverini P. Cellular adhesion molecules. Newly identified mediators of angiogenesis. Am J Pathol 1996;4:1023–1029. 14. Carano RA, Filcaroff EH. Angiogenesis and bone repair. Drug Discov Today 2003;8:980–989. 15. Schliephake H. Bone growth factors in maxillofacial reconstruction. Int J Oral Maxillofac Surg 2002;31:469–484. 16. Yamada Y, Tamura T, Hariu K, Asano Y, Sato S, Ito K. Angiogenesis in newly augmented bone observed in rabbit calvarium using a titanium cap. Clin Oral Implants Res 2008;19:1003–1009. 17. Kostopoulos L, Karring T, Uraguchi R. Formation of jawbone tuberosities by guided tissue regeneration. Clin Oral Implants Res 1994;5:245–253. 18. Kostopoulos L, Karring T. Augmentation of the rat mandible using guided tissue regeneration. Clin Oral Implants Res 1994;5:75–82. 19. Lundgren D, Lundgren AK, Sennerby L, Nyman S. Augmentation of intramembranous bone beyond the skeletal using an occlusive titanium barrier. An experimental study in the rabbit. Clin Oral Implants Res 1995;6:67–72. 20. Nishimura I, Shimizu Y, Ooya K. Effects of cortical bone perforation on experimental guided bone regeneration. Clin Oral Implants Res 2004;15:293–230. 21. Wang CJ, Huang HY, Chen HS, et al. The effect of shockwave therapy on acute fracture of the tibia. A study in a dog model. Clin Orthop Relat Res 2001;387:112–118. 22. Wang CJ, Chen HS, Chen CE, et al. Treatment of non-unions of long bone fractures with shock waves. Clin Orthop Relat Res 2001;387:95–101. 23. Uchida S, Sakai A, Kudo H, et al. Vascular endothelial growth factor is expressed along with its receptors during the healing process of bone and bone marrow after drill-hole injury in rats. Bone 2003;32:491–501. 24. Wang CJ, Huang KE, Sun YC, et al. VEGF modulates angiogenesis and osteogenesis in shockwave-promoted fracture healing in rabbits. J Surg Res 2011 Nov;171(1):114–119. Epub 2010 Feb 21.
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