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Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis
Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
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Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis Marcos Heidy Guskuma*, Eduardo Hochuli-Vieira, Flávia Priscila Pereira, Idelmo Rangel-Garcia Jr., Roberta Okamoto, Tetuo Okamoto, Osvaldo Magro Filho Dental School of Araçatuba, University of State of São Paulo “Júlio de Mesquita Filho” e UNESP, Rua José Bonifácio, 1193, CEP 16015-050 Araçatuba, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 April 2012 Accepted 28 May 2013
Aims: The purpose of this study was to evaluate the expression of proteins that participate in the osteoinduction stage (VEGF, BMP2 and CBFA1) of the process of bone regeneration of defects created in rat calvariae and filled with autogenous bone block grafts. Materials and methods: 10 adult male rats (Rattus norvegicus albinus, Wistar) were used, who received two bone defects measuring 5 mm each in the calvariae. The bone defects constituted two experimental groups (n ¼ 10): Control Group (CONT) (defects filled with a coagulum); Graft Group (GR) (defects filled with autogenous bone removed from the contralateral defect). The animals were submitted to euthanasia at 7 and 30 days post-operatively. Results: Quantitative analysis demonstrated significantly greater bone formation in Group GR, but the presence of the studied proteins was significantly greater in the CONT Group in both time intervals of observation. Conclusion: It was not possible in this study in cortical bone block groups to detect the osteoinductive proteins in a significant amount during the repair process. Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bone transplant Immunohistochemistry Bone Morphogenetic Protein 2 Core Binding Factor Alpha 1 Subunit Vascular Endothelial Growth Factor A
1. Introduction Autogenous bone is a well known and frequently used material, used as the gold standard for bone reconstruction procedures (Prósper et al., 2011; Kolk et al., 2012). In spite of not being ideal, autogenous bone has one characteristic that differentiates it from the majority of other biomaterials: osteoinductive properties (Pape et al., 2010). The concepts of osteoinduction were introduced by Urist in 1965. Later, Urist and Strates (1971) concluded that the factor responsible for osteogenesis was a protein denominated bone morphogenetic protein (BMP), involved in cell chemotaxis, mitosis and differentiation. In 1988, Wozney et al. made it possible to clone BMP, which began to be applied clinically in orthopaedics. Since then, various studies (Gutwald et al., 2010; Arpornmaeklong et al., 2012) have confirmed the osteoinductive capacity of the protein.
In a literature review we found no reference to how the proteins that participate in the induction process are expressed in autogenous bone grafts. This gave rise to the question: does autogenous bone always present the active osteoinductive property during the regeneration process of grafts? The aim of this study was to evaluate the osteoinductive property of autogenous bone by means of analysing the expression of the following proteins: VEGF (Vascular Endothelial Growth Factor), important for stimulation of angiogenesis (Street et al., 2002), differentiation (Eckardt et al., 2005) and migration of osteoblasts, and mineralization (Street et al., 2002; Leach et al., 2006); BMP2 (Bone Morphogenetic Protein-2), which promotes the differentiation of mesenchymal cells into osteoblasts, increasing bone formation and repair (Heckman et al., 1999); and CBFA1 (Core binding factor a1), which regulates the differentiation and gene expression of osteoblasts (Ducy et al., 1999). 2. Material and methods
* Corresponding author. Rua Ibiporã, 548, Jd. Santo Antonio, CEP 86060-510 Londrina, PR, Brazil. Tel.: þ55 (43) 30255272, 91013448. E-mail address: [email protected] (M.H. Guskuma).
This study was conducted in accordance with the ethical principles of animal experimentation adopted by the Brazilian College
1010-5182/$ e see front matter Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2013.05.022
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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M.H. Guskuma et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
of Animal Experimentation (“Colégio Brasileiro de Experimentação Animal e COBEA), and was submitted to and approved by the Ethics Committee on Animal Experimentation of the School of Dentistry of Araçatuba e UNESP. For the study 10 adult male rats (Rattus norvegicus albinus, Wistar) were used, aged from 3 to 4 months, with body weight of 200e300 g. For the surgical procedure, the rats were sedated with the combination of Ketamine (0.7 ml/kg) and Xylazine (0.3 ml/kg), administered intramuscularly. A linear incision was performed in the antero-posterior direction in the median region of the calvariae with detachment of the soft tissue. Two bone total thickness defects 5 mm in diameter were created in the parietal region bilaterally in each animal, using a trephine bur (Fig. 1). The bone defects constituted two experimental groups: Control Group (CONT) (n ¼ 10) e In this group, the bone defects were filled with their own coagulum. Graft Group (GR) (n ¼ 10) e In this group, the bone defects were filled with autogenous bone block removed from the contralateral defect in the same surgical procedure. After filling the defects, the tissues were replaced and sutured in planes, obtaining complete coverage of the defects. The animals were submitted to euthanasia in the time intervals of 7 and 30 postoperative days. The calvariae were removed and fixed in a 10% formaldehyde solution for 48 h, washed in running water for 24 h, decalcified in 20% EDTA for 5 weeks, dehydrated in a sequence of alcohols and diaphanized. The calvariae were cut in half in the longitudinal direction, separating the bone defects. The defects were cut in half in the latero-lateral direction and the parts obtained were separately embedded in paraffin. The parts were cut into slices 6 mm thick and mounted in slides. 2.1. Histometric analysis The slides destined for quantitative analysis were stained with haematoxylin and eosin (HE). To perform the analysis an optical microscope with a magnification objective of 2.5 was used, coupled to an image capturing camera (Leica DFC 300FX, Leica Microsystems, Heerbrugg, Switzerland) and connected to a Pentium III microcomputer with digitized image analyser software (Leica Camera Software Box, Leica Imaging Manager e IM50 Demo Software). With the aid of the ImageLab 2000, version 2.4 program, the extension of the area of bone formation in the defects was calculated. For this purpose, the following criteria were used: (Fig. 2). - Considering that the Total Area (TA) of the defect in the analysed region must correspond to the area occupied by bone from the calvariae in the same region before the defect was made, TA was determined in the following manner (Fig. 2): The height of the defect was established, with the parameters being the thickness of the calvarial bone at the edges of the defect, and the edges of the defect were connected by two lines (superficial and depth of the defect) separated by the height of the defect, following its anatomical curvature and delimiting the TA. The “calculation spreadsheet” tool of the program was used, and the TA value was obtained in pixels. The values obtained from each slide were added and divided in order to obtain a mean, which was established as a standard for all the defects, considering that the calvarial thickness was similar in all animals included in the sample.
Fig. 1. Scheme of defects created. Surgical defects located bilaterally in the parietal bones, with the respective treatments received.
- The regions corresponding to the newly formed bone areas (NBA) were delimited and the areas calculated separately in pixels. To obtain the value in percentage, we considered TA to be 100% and using a rule of three, we calculated the percentage of NBA for each group. Ten (10) percentage values were obtained for each group and the mean of these determined the final NBA in each time interval analysed. The values were submitted to the Analysis of Variance and Tukey post hoc tests to verify significance of differences between the groups. - In Group GR, the bone corresponding to the graft was considered the NBA, because at 30 days its incorporation was observed.
2.2. Immunohistochemical analysis The slides were separated and destined for immunohistochemical analysis. Endogenous peroxidase activity was inhibited with hydrogen peroxide, antigen recovery obtained with citrate buffer at 60 C for 20 min, and nonspecific reactions blocked with defatted milk and bovine serum albumin during antibody incubations.
Fig. 2. Delimitation of TA (traced line), respecting the criteria established, by means of the Imagelab 2000Ò Program. Within the TA neoformed bone is observed (arrows). HE (original 2.5).
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
M.H. Guskuma et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
Primary antibodies against CBFA1, BMP2 and VEGF (Santa Cruz Biotechnology), a biotinylated secondary antibody (Pierce Biotechnology), Streptavidin Biotin amplifier (Dako), and diaminobenzidine (Dako) as chromogen, which gives a brown colour to the markings, were used. On conclusion of reactions, the slides were counterstained with Harris Hematoxylin. Negative controls were performed (by omission of primary antibodies) to avoid the analysis of false positives. VEGF protein was only analysed at 7 days, because we believed that its action would occur mainly in the initial periods of repair. To perform the analysis an optical microscope with a magnification objective of 160 Leica Aristoplan Microsystems (Leitz, Benshein, Germany) was used, coupled to an image capturing camera (Leica DFC 300FX, Leica Microsystems, Heerbrugg, Switzerland) and connected to a Pentium III microcomputer with digitized image analyser software (Leica Camera Software Box, Leica Imaging Manager -IM50 Demo Software). Three images were obtained of each slide for analysis: The two edges and centre of the defect. For the effect of comparing the intensity of immunomarkings between the experimental groups, a score was created, in which the immunomarkings were classified according to a 6degree scale that ranged from absent to intense (Table 1). Two evaluators were calibrated to perform immunohistochemical analysis.
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Fig. 3. Histometric analysis of Group CONT at 7 and 30 days. It is observed the bone formation characteristics mainly at the edges of defects in both periods. A ¼ Group CONT 7 days/B ¼ Group CONT 30 days. HE (original 2.5).
osteoblasts. In Group GR (Fig. 7B), there was effectively an absence of positive markings for BMP2, especially in the centre of the defect. 3.2.1.3. CBFA1. The number of cells with positive immunomarkings for CBFA1 at 7 days, in Group CONT (Fig. 8A) was only moderate, but scattered throughout the entire extension of the defect, representing the pre-osteoblasts present. In Group GR (Fig. 8B) complete absence of CBFA1 expression was observed.
3. Results 3.1. Quantitative analysis (HE) (Fig. 5) 3.1.1. 7 days In this time interval of observation, Group GR (46.44%) (Fig. 4A) obtained the highest NBA index when compared with Group CONT (12.48%) (Fig. 3A) (Fig. 5). 3.1.2. 30 days At 30 days, there was no significant alteration in NBA in Group CONT (18.12 %) (Fig. 3B) whereas an appreciable increase in NBA was observed in Group GR (58.86%) (Fig. 4B) (Fig. 5).
3.2.2. 30 days 3.2.2.1. BMP2. At 30 days, Group GR (Fig. 9B) had no immunomarking for BMP2, in contrast with Group CONT (Fig. 9A), which had an intense expression of the protein, representing the areas of bone matrix formation. 3.2.2.2. CBFA1. Group CONT (Fig. 10A) was shown to have intense markings throughout the entire extension of the defect, in which there was bone matrix formation. In Group GR (Fig. 10B) CBFA1 expression was very discrete, with greater presence at the edges of the defect and graft surfaces. 4. Discussion
3.2. Qualitative analysis 3.2.1. 7 days 3.2.1.1. VEGF. Group CONT gave a higher number of positive immunomarkings for VEGF, marking the endothelial cells present in the conjunctive tissue and medullary spaces, both at the edges and in the centre of the defect (Fig. 6A). In contrast Group GR (Fig. 6B) had fewer immunomarkings, notably in the centre of the defect.
The osteoinductive capacity of autogenous bone has been poorly investigated and it is considered as being active in all situations of autogenous graft repair. Studies (Inoda et al., 2004) showing how
3.2.1.2. BMP-2. In this time interval of observation, Group CONT (Fig. 7A) was shown to have very discrete markings for this protein, equally scattered throughout the defect, observed mainly in Table 1 Intensity of immunomarkings for studied proteins. Protein
Group
7 Days
30 Days
VEGF
CONT GR CONT GR CONT GR
þþþþþþ þþ þþþ þ þþþþ þ
þþþþþþ þ þþþþþþ þþ
BMP2 CBFA1
Score: Absent ¼ þ/Very discrete ¼ þþ/Discrete ¼ þþþ/Moderate ¼ þþþþ/ Intense ¼ þþþþþ/Very intense ¼ þþþþþþ.
Fig. 4. Histometric analysis of Group GR at 7 and 30 days. Note the incorporation of the block and complete closure of the defects in both periods. A ¼ Group GR 7 days/ B ¼ Group GR 30 days. HE (original 2.5).
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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M.H. Guskuma et al. / Journal of Cranio-Maxillo-Facial Surgery xxx (2013) 1e7
Fig. 5. NBA in the Experimental Groups. The letters on the bars are comparative between the groups. The presence of a different letter denotes statistical significance between the results obtained in the groups compared. (P < 0.05) Analysis of Variance and Tukey’s post hoc Tests.
and when osteoinductive factors act during the repair of cortical, medullar, particulate or block autogenous grafts are still lacking. This study shows the incorporation of cortical autogenous block grafts into the calvariae of rats. On the one hand, the histometric analysis reaffirms the high biocompatibility of autogenous bone and its efficiency in closing defects (Fig. 4) and on the other, confirms the critical size (Hollinger and Kleinschmidt, 1990) of the studied defects (Fig. 3). Generally speaking, for all the studied proteins, the immunohistochemical analysis (Table 1) showed little or no expression of inductor proteins in Group GR and intense marking in Group CONT, which appears to be a result that contradicts the histometric analysis (Figs. 3 and 4). Autogenous bone, known for its osteoinductive potential, did not demonstrate the expression of proteins (Table 1) recognized as important in the initial process of bone formation, such as VEGF (Street et al., 2002; Dai and Rabie, 2007), BMP2 (Heckman et al., 1999), and CBFA1 (Ducy et al., 1999; Lien et al., 2007). Nevertheless, the defects in which it was placed were completely closed at 30 days (Fig. 4B), denoting a normal repair process, which was confirmed by the histological analysis. The autogenous bone blocks studied acted as an osteoconductive and highly compatible material. The structural and cellular conditions of the receptor bed appear to be essential for the integration of graft into bone (Ohtsubo et al., 2003). It is suggested that by some mechanism the animal’s body is able to recognize the presence of
bone and inhibit the signals for the release of osteoinductive factors. The presence of growth factors in autogenous bone grafts is known (Schmidmaier et al., 2006; Takemoto et al., 2010), and so is their expression during the repair process in osteogenic distraction (Campisi et al., 2003; Kroczek et al., 2010) and in bone fractures (Barnes et al., 1999). The principles that govern the repair process in fractures, grafts and osteogenic distraction are the same (Albrektsson, 1980), however, it would be reasonable to think that the grafted bone stimulates the regeneration of the defect. One of the factors that possibly contributed to the discrete expression of proteins in Group GR (Table 1) was the cortical nature of the graft. It has been recognized that cortical and medullary bone differ in some aspects other than macroscopic (Enneking and Morris, 1972). The presence of a large amount of vascularization, cells and growth factors in the medullary bone graft (Schmidmaier et al., 2006; Takemoto et al., 2010) favours more rapid repair, with high metabolism (Day et al., 2000; Khan et al., 2005) initially characterized by an osteogenic stage followed by a resorptive stage (Enneking and Morris, 1972; Kenzora et al., 1978). The cortical graft presents a much more resistant and dense structure, which limits the amount of vascularization, cells and growth factors (Day et al., 2000) inside the block. In these cases, repair initially goes through a stage of resorption followed by the osteogenic stage. Kenzora et al. (1978) suggested that the initial stimulus for repair in cortical and medullary grafts is the same, but as the density of cortical bone makes revascularization difficult and prevents cell proliferation, bone resorption prevails. The cortical graft areas that were previously revascularized initially go through the osteogenic stage. In this study, we could consider the hypothesis that in Group GR the stage of greater BMP2 and CBFA1 expression would be between the two periods observed, whereas at 7 days (Figs. 7A and 8A) the bone could still be passing through the resorption process, and at 30 days (Figs. 7B and 8B) one observes the previously formed bone (Fig. 4B). Studies with less space between observation time intervals are being conducted by our group and may help to explain these questions. Group CONT was characterized by intense marking of the studied proteins (Table 1) and their origin is an important factor to be considered. Ma et al. (2011) related that the periosteum has an osteoinductive property and may help in the bone repair process. The dura mater is also recognized as a source of osteogenic cells and osteoinductive factors (Gosain et al., 2003). Therefore, we concluded that the main source of the studied proteins in both groups was apparently the periosteum and dura mater, since the defect was of the complete thickness. In Group GR no bone neoformation was observed in the top part of the block, possibly due to
Fig. 6. VEGF protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The arrows indicate the vessels in neoformation, immersed in areas of intense marking (in brown) at one of the edges of the defect. B) Area of the interface between the graft and one of the edges of the defect, showing discrete VEGF expression.
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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Fig. 7. BMP2 protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The discrete markings in brown reflect the low number of osteoblasts and osteoid matrix in this period, at one of the edges of the defect. B) Complete absence of immunomarkings at the interface of the graft with one of the edges of the defect.
contact with the periosteum (Fig. 4A and B) which did not leave space for coagulum formation, as occurred with Choi et al. (2004) and Choi et al. (2005). At the base of the block and edges of the defects the layer of coagulum formed served as matrix for the migration of osteoinductive factors (Fig. 4), which is in agreement with the observations of Andrade et al. (2010). Similar to these findings, Alam et al. (2007), demonstrated that in rabbit mandibles, BMP2 is mainly expressed in the tissue formed in the gaps and in bone marrow at the limits of bone formation areas of grafts. In the Group CONT the characteristic of new bone formation, particularly at the edges of defects (Fig. 3) confirms that osteoinductive stimuli come from the bone in those regions. Nevertheless, we believe that this characteristic of new bone formation occurs due to the collapse of the periosteum in the deep and central part of the defect due to the lack of an osteoconductive structure, causing spaces for the formation of a coagulum matrix and migration of osteoinductive proteins only on the edges of defects, as has been demonstrated in other studies (Guskuma et al., 2010). VEGF expression at the edges of defects and absence of markings inside the bone block agree with the results of Ohtsubo et al. (2003) who, in a methodology similar to ours, demonstrated the initial formation of capillaries at 7 days, homogenization of the size of vessels at 14 days and decrease in the formation of new vessels at 21 days of autogenous graft repair. In this study the option was taken only to conduct the analysis of VEGF expression at 7 days,
however, an analysis over longer periods might have demonstrated a later revascularization of the cortical bone. Albrektsson (1980) determined the rate of revascularization of grafts in rabbits and concluded that the process of incorporation e creeping substitution e (Burchardt, 1987) of the cortical graft occurs more slowly. In medullary bones this rate was 0.2e0.4 mm/day and in cortical bones 0.15e0.30 mm/day. Considering these data, we believe that VEGF expression may be more intense in medullary grafts, which were not evaluated in this study. The presence of BMP2 protein was detected in Group GR, although in a discrete manner, only at 30 days (Fig. 9B) inside the channels of Havers and Volkmann in newly formed bone, with characteristics of an advanced ossification process. We believe that in this period the stage of BMP2 action had ceased and that is why the protein appeared discretely. According to Noël et al. (2004), 6 days of expression are necessary for bone formation to occur in rats. The immunomarking pattern for CBFA1 (Fig. 8; Fig. 10) was very similar to that of BMP2 (Table 1), possibly due to the fact that they act in conjunction in the repair process. In 2004, Isefuku et al. verified that, in spite of the important function of CBFA1 during foetal development, this protein may be of much less importance in bone repair processes, such as in fractures and osteogenic distraction. In this study Group CONT (Table 1) demonstrated a significant presence of all the studied proteins during the repair process.
Fig. 8. CBFA1 protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The arrows indicate areas of moderate CBFA1 expression. The darker brown regions are not considered markings. B) Complete absence of immunomarkings at the edge of the defect.
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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Fig. 9. BMP2 protein expression at 30 days in the 2 experimental Groups. (Original 160). A) The light arrows show the limit of the edge of the defect. The dark arrows show the regions of darker brown that are considered nonspecific markings. The large area lower down shows intense marking revealing the presence of osteoblasts and osteoid matrix. B) The region in the center of the defect in this group shows the complete absence of immunomarkings.
Fig. 10. CBFA1 protein expression at 30 days in the 2 experimental Groups. (Original 160). A) Intense markings throughout the entire analysed area. Only the region pointed out by the arrow is not considered as marking. B) Very discrete markings in the center of the defect.
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Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis Marcos Heidy Guskuma*, Eduardo Hochuli-Vieira, Flávia Priscila Pereira, Idelmo Rangel-Garcia Jr., Roberta Okamoto, Tetuo Okamoto, Osvaldo Magro Filho Dental School of Araçatuba, University of State of São Paulo “Júlio de Mesquita Filho” e UNESP, Rua José Bonifácio, 1193, CEP 16015-050 Araçatuba, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 April 2012 Accepted 28 May 2013
Aims: The purpose of this study was to evaluate the expression of proteins that participate in the osteoinduction stage (VEGF, BMP2 and CBFA1) of the process of bone regeneration of defects created in rat calvariae and filled with autogenous bone block grafts. Materials and methods: 10 adult male rats (Rattus norvegicus albinus, Wistar) were used, who received two bone defects measuring 5 mm each in the calvariae. The bone defects constituted two experimental groups (n ¼ 10): Control Group (CONT) (defects filled with a coagulum); Graft Group (GR) (defects filled with autogenous bone removed from the contralateral defect). The animals were submitted to euthanasia at 7 and 30 days post-operatively. Results: Quantitative analysis demonstrated significantly greater bone formation in Group GR, but the presence of the studied proteins was significantly greater in the CONT Group in both time intervals of observation. Conclusion: It was not possible in this study in cortical bone block groups to detect the osteoinductive proteins in a significant amount during the repair process. Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bone transplant Immunohistochemistry Bone Morphogenetic Protein 2 Core Binding Factor Alpha 1 Subunit Vascular Endothelial Growth Factor A
1. Introduction Autogenous bone is a well known and frequently used material, used as the gold standard for bone reconstruction procedures (Prósper et al., 2011; Kolk et al., 2012). In spite of not being ideal, autogenous bone has one characteristic that differentiates it from the majority of other biomaterials: osteoinductive properties (Pape et al., 2010). The concepts of osteoinduction were introduced by Urist in 1965. Later, Urist and Strates (1971) concluded that the factor responsible for osteogenesis was a protein denominated bone morphogenetic protein (BMP), involved in cell chemotaxis, mitosis and differentiation. In 1988, Wozney et al. made it possible to clone BMP, which began to be applied clinically in orthopaedics. Since then, various studies (Gutwald et al., 2010; Arpornmaeklong et al., 2012) have confirmed the osteoinductive capacity of the protein.
In a literature review we found no reference to how the proteins that participate in the induction process are expressed in autogenous bone grafts. This gave rise to the question: does autogenous bone always present the active osteoinductive property during the regeneration process of grafts? The aim of this study was to evaluate the osteoinductive property of autogenous bone by means of analysing the expression of the following proteins: VEGF (Vascular Endothelial Growth Factor), important for stimulation of angiogenesis (Street et al., 2002), differentiation (Eckardt et al., 2005) and migration of osteoblasts, and mineralization (Street et al., 2002; Leach et al., 2006); BMP2 (Bone Morphogenetic Protein-2), which promotes the differentiation of mesenchymal cells into osteoblasts, increasing bone formation and repair (Heckman et al., 1999); and CBFA1 (Core binding factor a1), which regulates the differentiation and gene expression of osteoblasts (Ducy et al., 1999). 2. Material and methods
* Corresponding author. Rua Ibiporã, 548, Jd. Santo Antonio, CEP 86060-510 Londrina, PR, Brazil. Tel.: þ55 (43) 30255272, 91013448. E-mail address: [email protected] (M.H. Guskuma).
This study was conducted in accordance with the ethical principles of animal experimentation adopted by the Brazilian College
1010-5182/$ e see front matter Ó 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2013.05.022
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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of Animal Experimentation (“Colégio Brasileiro de Experimentação Animal e COBEA), and was submitted to and approved by the Ethics Committee on Animal Experimentation of the School of Dentistry of Araçatuba e UNESP. For the study 10 adult male rats (Rattus norvegicus albinus, Wistar) were used, aged from 3 to 4 months, with body weight of 200e300 g. For the surgical procedure, the rats were sedated with the combination of Ketamine (0.7 ml/kg) and Xylazine (0.3 ml/kg), administered intramuscularly. A linear incision was performed in the antero-posterior direction in the median region of the calvariae with detachment of the soft tissue. Two bone total thickness defects 5 mm in diameter were created in the parietal region bilaterally in each animal, using a trephine bur (Fig. 1). The bone defects constituted two experimental groups: Control Group (CONT) (n ¼ 10) e In this group, the bone defects were filled with their own coagulum. Graft Group (GR) (n ¼ 10) e In this group, the bone defects were filled with autogenous bone block removed from the contralateral defect in the same surgical procedure. After filling the defects, the tissues were replaced and sutured in planes, obtaining complete coverage of the defects. The animals were submitted to euthanasia in the time intervals of 7 and 30 postoperative days. The calvariae were removed and fixed in a 10% formaldehyde solution for 48 h, washed in running water for 24 h, decalcified in 20% EDTA for 5 weeks, dehydrated in a sequence of alcohols and diaphanized. The calvariae were cut in half in the longitudinal direction, separating the bone defects. The defects were cut in half in the latero-lateral direction and the parts obtained were separately embedded in paraffin. The parts were cut into slices 6 mm thick and mounted in slides. 2.1. Histometric analysis The slides destined for quantitative analysis were stained with haematoxylin and eosin (HE). To perform the analysis an optical microscope with a magnification objective of 2.5 was used, coupled to an image capturing camera (Leica DFC 300FX, Leica Microsystems, Heerbrugg, Switzerland) and connected to a Pentium III microcomputer with digitized image analyser software (Leica Camera Software Box, Leica Imaging Manager e IM50 Demo Software). With the aid of the ImageLab 2000, version 2.4 program, the extension of the area of bone formation in the defects was calculated. For this purpose, the following criteria were used: (Fig. 2). - Considering that the Total Area (TA) of the defect in the analysed region must correspond to the area occupied by bone from the calvariae in the same region before the defect was made, TA was determined in the following manner (Fig. 2): The height of the defect was established, with the parameters being the thickness of the calvarial bone at the edges of the defect, and the edges of the defect were connected by two lines (superficial and depth of the defect) separated by the height of the defect, following its anatomical curvature and delimiting the TA. The “calculation spreadsheet” tool of the program was used, and the TA value was obtained in pixels. The values obtained from each slide were added and divided in order to obtain a mean, which was established as a standard for all the defects, considering that the calvarial thickness was similar in all animals included in the sample.
Fig. 1. Scheme of defects created. Surgical defects located bilaterally in the parietal bones, with the respective treatments received.
- The regions corresponding to the newly formed bone areas (NBA) were delimited and the areas calculated separately in pixels. To obtain the value in percentage, we considered TA to be 100% and using a rule of three, we calculated the percentage of NBA for each group. Ten (10) percentage values were obtained for each group and the mean of these determined the final NBA in each time interval analysed. The values were submitted to the Analysis of Variance and Tukey post hoc tests to verify significance of differences between the groups. - In Group GR, the bone corresponding to the graft was considered the NBA, because at 30 days its incorporation was observed.
2.2. Immunohistochemical analysis The slides were separated and destined for immunohistochemical analysis. Endogenous peroxidase activity was inhibited with hydrogen peroxide, antigen recovery obtained with citrate buffer at 60 C for 20 min, and nonspecific reactions blocked with defatted milk and bovine serum albumin during antibody incubations.
Fig. 2. Delimitation of TA (traced line), respecting the criteria established, by means of the Imagelab 2000Ò Program. Within the TA neoformed bone is observed (arrows). HE (original 2.5).
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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Primary antibodies against CBFA1, BMP2 and VEGF (Santa Cruz Biotechnology), a biotinylated secondary antibody (Pierce Biotechnology), Streptavidin Biotin amplifier (Dako), and diaminobenzidine (Dako) as chromogen, which gives a brown colour to the markings, were used. On conclusion of reactions, the slides were counterstained with Harris Hematoxylin. Negative controls were performed (by omission of primary antibodies) to avoid the analysis of false positives. VEGF protein was only analysed at 7 days, because we believed that its action would occur mainly in the initial periods of repair. To perform the analysis an optical microscope with a magnification objective of 160 Leica Aristoplan Microsystems (Leitz, Benshein, Germany) was used, coupled to an image capturing camera (Leica DFC 300FX, Leica Microsystems, Heerbrugg, Switzerland) and connected to a Pentium III microcomputer with digitized image analyser software (Leica Camera Software Box, Leica Imaging Manager -IM50 Demo Software). Three images were obtained of each slide for analysis: The two edges and centre of the defect. For the effect of comparing the intensity of immunomarkings between the experimental groups, a score was created, in which the immunomarkings were classified according to a 6degree scale that ranged from absent to intense (Table 1). Two evaluators were calibrated to perform immunohistochemical analysis.
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Fig. 3. Histometric analysis of Group CONT at 7 and 30 days. It is observed the bone formation characteristics mainly at the edges of defects in both periods. A ¼ Group CONT 7 days/B ¼ Group CONT 30 days. HE (original 2.5).
osteoblasts. In Group GR (Fig. 7B), there was effectively an absence of positive markings for BMP2, especially in the centre of the defect. 3.2.1.3. CBFA1. The number of cells with positive immunomarkings for CBFA1 at 7 days, in Group CONT (Fig. 8A) was only moderate, but scattered throughout the entire extension of the defect, representing the pre-osteoblasts present. In Group GR (Fig. 8B) complete absence of CBFA1 expression was observed.
3. Results 3.1. Quantitative analysis (HE) (Fig. 5) 3.1.1. 7 days In this time interval of observation, Group GR (46.44%) (Fig. 4A) obtained the highest NBA index when compared with Group CONT (12.48%) (Fig. 3A) (Fig. 5). 3.1.2. 30 days At 30 days, there was no significant alteration in NBA in Group CONT (18.12 %) (Fig. 3B) whereas an appreciable increase in NBA was observed in Group GR (58.86%) (Fig. 4B) (Fig. 5).
3.2.2. 30 days 3.2.2.1. BMP2. At 30 days, Group GR (Fig. 9B) had no immunomarking for BMP2, in contrast with Group CONT (Fig. 9A), which had an intense expression of the protein, representing the areas of bone matrix formation. 3.2.2.2. CBFA1. Group CONT (Fig. 10A) was shown to have intense markings throughout the entire extension of the defect, in which there was bone matrix formation. In Group GR (Fig. 10B) CBFA1 expression was very discrete, with greater presence at the edges of the defect and graft surfaces. 4. Discussion
3.2. Qualitative analysis 3.2.1. 7 days 3.2.1.1. VEGF. Group CONT gave a higher number of positive immunomarkings for VEGF, marking the endothelial cells present in the conjunctive tissue and medullary spaces, both at the edges and in the centre of the defect (Fig. 6A). In contrast Group GR (Fig. 6B) had fewer immunomarkings, notably in the centre of the defect.
The osteoinductive capacity of autogenous bone has been poorly investigated and it is considered as being active in all situations of autogenous graft repair. Studies (Inoda et al., 2004) showing how
3.2.1.2. BMP-2. In this time interval of observation, Group CONT (Fig. 7A) was shown to have very discrete markings for this protein, equally scattered throughout the defect, observed mainly in Table 1 Intensity of immunomarkings for studied proteins. Protein
Group
7 Days
30 Days
VEGF
CONT GR CONT GR CONT GR
þþþþþþ þþ þþþ þ þþþþ þ
þþþþþþ þ þþþþþþ þþ
BMP2 CBFA1
Score: Absent ¼ þ/Very discrete ¼ þþ/Discrete ¼ þþþ/Moderate ¼ þþþþ/ Intense ¼ þþþþþ/Very intense ¼ þþþþþþ.
Fig. 4. Histometric analysis of Group GR at 7 and 30 days. Note the incorporation of the block and complete closure of the defects in both periods. A ¼ Group GR 7 days/ B ¼ Group GR 30 days. HE (original 2.5).
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Fig. 5. NBA in the Experimental Groups. The letters on the bars are comparative between the groups. The presence of a different letter denotes statistical significance between the results obtained in the groups compared. (P < 0.05) Analysis of Variance and Tukey’s post hoc Tests.
and when osteoinductive factors act during the repair of cortical, medullar, particulate or block autogenous grafts are still lacking. This study shows the incorporation of cortical autogenous block grafts into the calvariae of rats. On the one hand, the histometric analysis reaffirms the high biocompatibility of autogenous bone and its efficiency in closing defects (Fig. 4) and on the other, confirms the critical size (Hollinger and Kleinschmidt, 1990) of the studied defects (Fig. 3). Generally speaking, for all the studied proteins, the immunohistochemical analysis (Table 1) showed little or no expression of inductor proteins in Group GR and intense marking in Group CONT, which appears to be a result that contradicts the histometric analysis (Figs. 3 and 4). Autogenous bone, known for its osteoinductive potential, did not demonstrate the expression of proteins (Table 1) recognized as important in the initial process of bone formation, such as VEGF (Street et al., 2002; Dai and Rabie, 2007), BMP2 (Heckman et al., 1999), and CBFA1 (Ducy et al., 1999; Lien et al., 2007). Nevertheless, the defects in which it was placed were completely closed at 30 days (Fig. 4B), denoting a normal repair process, which was confirmed by the histological analysis. The autogenous bone blocks studied acted as an osteoconductive and highly compatible material. The structural and cellular conditions of the receptor bed appear to be essential for the integration of graft into bone (Ohtsubo et al., 2003). It is suggested that by some mechanism the animal’s body is able to recognize the presence of
bone and inhibit the signals for the release of osteoinductive factors. The presence of growth factors in autogenous bone grafts is known (Schmidmaier et al., 2006; Takemoto et al., 2010), and so is their expression during the repair process in osteogenic distraction (Campisi et al., 2003; Kroczek et al., 2010) and in bone fractures (Barnes et al., 1999). The principles that govern the repair process in fractures, grafts and osteogenic distraction are the same (Albrektsson, 1980), however, it would be reasonable to think that the grafted bone stimulates the regeneration of the defect. One of the factors that possibly contributed to the discrete expression of proteins in Group GR (Table 1) was the cortical nature of the graft. It has been recognized that cortical and medullary bone differ in some aspects other than macroscopic (Enneking and Morris, 1972). The presence of a large amount of vascularization, cells and growth factors in the medullary bone graft (Schmidmaier et al., 2006; Takemoto et al., 2010) favours more rapid repair, with high metabolism (Day et al., 2000; Khan et al., 2005) initially characterized by an osteogenic stage followed by a resorptive stage (Enneking and Morris, 1972; Kenzora et al., 1978). The cortical graft presents a much more resistant and dense structure, which limits the amount of vascularization, cells and growth factors (Day et al., 2000) inside the block. In these cases, repair initially goes through a stage of resorption followed by the osteogenic stage. Kenzora et al. (1978) suggested that the initial stimulus for repair in cortical and medullary grafts is the same, but as the density of cortical bone makes revascularization difficult and prevents cell proliferation, bone resorption prevails. The cortical graft areas that were previously revascularized initially go through the osteogenic stage. In this study, we could consider the hypothesis that in Group GR the stage of greater BMP2 and CBFA1 expression would be between the two periods observed, whereas at 7 days (Figs. 7A and 8A) the bone could still be passing through the resorption process, and at 30 days (Figs. 7B and 8B) one observes the previously formed bone (Fig. 4B). Studies with less space between observation time intervals are being conducted by our group and may help to explain these questions. Group CONT was characterized by intense marking of the studied proteins (Table 1) and their origin is an important factor to be considered. Ma et al. (2011) related that the periosteum has an osteoinductive property and may help in the bone repair process. The dura mater is also recognized as a source of osteogenic cells and osteoinductive factors (Gosain et al., 2003). Therefore, we concluded that the main source of the studied proteins in both groups was apparently the periosteum and dura mater, since the defect was of the complete thickness. In Group GR no bone neoformation was observed in the top part of the block, possibly due to
Fig. 6. VEGF protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The arrows indicate the vessels in neoformation, immersed in areas of intense marking (in brown) at one of the edges of the defect. B) Area of the interface between the graft and one of the edges of the defect, showing discrete VEGF expression.
Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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Fig. 7. BMP2 protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The discrete markings in brown reflect the low number of osteoblasts and osteoid matrix in this period, at one of the edges of the defect. B) Complete absence of immunomarkings at the interface of the graft with one of the edges of the defect.
contact with the periosteum (Fig. 4A and B) which did not leave space for coagulum formation, as occurred with Choi et al. (2004) and Choi et al. (2005). At the base of the block and edges of the defects the layer of coagulum formed served as matrix for the migration of osteoinductive factors (Fig. 4), which is in agreement with the observations of Andrade et al. (2010). Similar to these findings, Alam et al. (2007), demonstrated that in rabbit mandibles, BMP2 is mainly expressed in the tissue formed in the gaps and in bone marrow at the limits of bone formation areas of grafts. In the Group CONT the characteristic of new bone formation, particularly at the edges of defects (Fig. 3) confirms that osteoinductive stimuli come from the bone in those regions. Nevertheless, we believe that this characteristic of new bone formation occurs due to the collapse of the periosteum in the deep and central part of the defect due to the lack of an osteoconductive structure, causing spaces for the formation of a coagulum matrix and migration of osteoinductive proteins only on the edges of defects, as has been demonstrated in other studies (Guskuma et al., 2010). VEGF expression at the edges of defects and absence of markings inside the bone block agree with the results of Ohtsubo et al. (2003) who, in a methodology similar to ours, demonstrated the initial formation of capillaries at 7 days, homogenization of the size of vessels at 14 days and decrease in the formation of new vessels at 21 days of autogenous graft repair. In this study the option was taken only to conduct the analysis of VEGF expression at 7 days,
however, an analysis over longer periods might have demonstrated a later revascularization of the cortical bone. Albrektsson (1980) determined the rate of revascularization of grafts in rabbits and concluded that the process of incorporation e creeping substitution e (Burchardt, 1987) of the cortical graft occurs more slowly. In medullary bones this rate was 0.2e0.4 mm/day and in cortical bones 0.15e0.30 mm/day. Considering these data, we believe that VEGF expression may be more intense in medullary grafts, which were not evaluated in this study. The presence of BMP2 protein was detected in Group GR, although in a discrete manner, only at 30 days (Fig. 9B) inside the channels of Havers and Volkmann in newly formed bone, with characteristics of an advanced ossification process. We believe that in this period the stage of BMP2 action had ceased and that is why the protein appeared discretely. According to Noël et al. (2004), 6 days of expression are necessary for bone formation to occur in rats. The immunomarking pattern for CBFA1 (Fig. 8; Fig. 10) was very similar to that of BMP2 (Table 1), possibly due to the fact that they act in conjunction in the repair process. In 2004, Isefuku et al. verified that, in spite of the important function of CBFA1 during foetal development, this protein may be of much less importance in bone repair processes, such as in fractures and osteogenic distraction. In this study Group CONT (Table 1) demonstrated a significant presence of all the studied proteins during the repair process.
Fig. 8. CBFA1 protein expression at 7 days in the 2 experimental Groups. (Original 160). A) The arrows indicate areas of moderate CBFA1 expression. The darker brown regions are not considered markings. B) Complete absence of immunomarkings at the edge of the defect.
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Fig. 9. BMP2 protein expression at 30 days in the 2 experimental Groups. (Original 160). A) The light arrows show the limit of the edge of the defect. The dark arrows show the regions of darker brown that are considered nonspecific markings. The large area lower down shows intense marking revealing the presence of osteoblasts and osteoid matrix. B) The region in the center of the defect in this group shows the complete absence of immunomarkings.
Fig. 10. CBFA1 protein expression at 30 days in the 2 experimental Groups. (Original 160). A) Intense markings throughout the entire analysed area. Only the region pointed out by the arrow is not considered as marking. B) Very discrete markings in the center of the defect.
5. Conclusions The methodology used allowed one to conclude that: Cortical block bone graft did not express the studied osteoinductive proteins significantly during the repair process and did not show an active osteoinductive property. The presence of the proteins studied in the autogenous bone graft did not appear to be critical for the regeneration of these type of defects. Biocompatibility and osteoconductivity were important properties for the graft in the regeneration of the defects. References Alam S, Ueki K, Marukawa K, Ohara T, Hase T, Takazakura D, et al: Expression of bone morphogenetic protein 2 and fibroblast growth factor 2 during bone regeneration using different implant materials as an onlay bone graft in rabbit mandibles. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 103: 16e26, 2007 Albrektsson T: Repair of bone grafts. Scand J Plast Reconstr Surg 14: 1e12, 1980 Andrade MGS, Moreira DC, Dantas DB, et al: Pattern of osteogenesis during onlay bone graft healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 110: 713e 719, 2010 Arpornmaeklong P, Pripatnanont P, Kittidumkerng W, Mitarnun W: Effects of autogenous growth factors on heterotopic bone formation of osteogenic cells in small animal model. J Craniomaxillofac Surg 40(4): 332e340, 2012 Barnes GL, Kostenuik PJ, Gerstenfeld LC: Growth factor regulation of fracture repair. J Bone Miner Res 14: 1805, 1999
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Please cite this article in press as: Guskuma MH, et al., Evaluation of the presence of VEGF, BMP2 and CBFA1 proteins in autogenous bone graft: Histometric and immunohistochemical analysis, Journal of Cranio-Maxillo-Facial Surgery (2013), http://dx.doi.org/10.1016/j.jcms.2013.05.022
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