Archives of Oral Biology (2004) 49, 1025—1033 www.intl.elsevierhealth.com/journals/arob Basic fibroblast growth factor up-regulates the expression of vascular endothelial growth factor during healing of allogeneic bone graft A. Bakr M. Rabie*, Mei Lu Hard Tissue Laboratory, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong SAR, China Accepted 28 May 2004 KEYWORDS VEGF; bFGF; DBMIM; Allogeneic bone graft; Revascularization Summary Recently we reported that basic fibroblast growth factor (bFGF) improved the healing of allogeneic bone grafts. However, the mechanism of action of the bFGF was not known. Therefore, the present study was designed to identify the expression pattern of vascular endothelial growth factor (VEGF) in the presence of bFGF reconstituted in demineralized intramembranous bone matrix (DBMIM) during the healing of allogeneic bone grafts. Eighteen critical size (15 mm  10 mm) defects were created on rabbit mandibles bilaterally. Three groups of six defects each were grafted with allogeneic bone alone, allogeneic bone and DBMIM, and allogeneic bone and bFGF reconstituted in DBMIM. Three weeks later, the defects were retrieved for immunohistochemistry and in situ hybridization for VEGF. The percentage of positive staining area was quantified by using image analyzer. The increase (517%) in the expression of VEGF mRNA was accompanied by an increase (492%) of immunoreactive VEGF protein in allogeneic bone graft augmented by bFGF reconstituted in DBMIM. A close correlation existed between levels of VEGF production and the amount of newly formed bone. The results show that bFGF reconstituted in DBMIM markedly upregulated the expression of VEGF in the grafted area. Basic FGF augments the healing of allogeneic bone grafts by enhancing vascularization through the up-regulation of VEGF. # 2004 Elsevier Ltd. All rights reserved. Abbreviations: bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor; DBMIM, demineralized intramembranous bone matrix. * Corresponding author. Tel.: +852 2859 0260; fax: +852 2559 3803. E-mail address: rabie@hkusua.hku.hk (A. Bakr M. Rabie). Introduction Recent work in our laboratory explored a new graft material that could possibly reduce the need to harvest autogenous bone from patients.1 In this 0003–9969/$ — see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.05.012 1026 research, composite graft material, consisted of allogeneic intramembranous (IM) bone similar to that obtained from bone bank and mixed with demineralized bone matrix prepared from IM bone (DBMIM) and enriched with human recombinant basic fibroblast growth factor (hrbFGF), produced 550% more new bone than allogeneic bone alone in the rabbit mandibular defect model.1 This combination of graft material showed faster integration and amalgamation with host bone. We speculated that the success of this graft material over allogeneic bone grafts could be due to its ability to promote revascularization.2 Revascularization by angiogenesis is considered a key step in the success of bone graft healing.3 The degree of revascularization is related to the stimuli present in the surrounding tissues that allow preexisting vessels to start budding into the freely applied grafts.3 Basic FGF is one of the endogenous factors present in the bone matrix,4 and it is apparently important in the initial vascularization of healing bone grafts.5 Furthermore, bFGF stimulated the cells involved in angiogenesis in vascularized bone grafts.6 In vitro experiments showed that bFGF caused a 6-fold increase in vascular endothelial growth factor mRNA expression in fetal rat calvarial cells.7 Vascular endothelial growth factor (VEGF), a heparin-binding growth factor, is a fundamental regulator of normal and abnormal angiogenesis.8 Cloning of VEGF cDNAs from several sources demonstrated that four different molecular species of VEGF are generated by alternative splicing of Mrna.9 Among them, VEGF165 is the most predominant molecular species produced by a variety of normal and transformed cells. The deletion of the VEGF165 and VEGF189 isoforms completely disturbed bone vascularization, and concomitantly resulted in a decreased trabecular bone volume and an abnormal growth plate morphology in perinatal mice.10 Moreover, secretion of VEGF in human fetal epiphyseal chondrocytes cultures was modulated in a 552% increase by bFGF after 48 h of treatment.11 Hence, there was a need to examine the up-regulation of the VEGF expression when hrbFGF is used to augment the healing of allogeneic bone grafts. Therefore, the purpose of the current study was to elucidate the mechanism by which bFGF enhances the osteoinductivity of allogeneic intramembranous bone grafts through (1) the identification of the expression pattern of VEGF during bone induction using the allogeneic bone grafts mixed with bFGF reconstituted in DBMIM; (2) to quantitatively correlate the level of expression of VEGF to the amount of new bone produced. A.B.M. Rabie, M. Lu Materials and methods Animals and materials Twenty-eight adult New Zealand White rabbits, 5— 12 months old and weighed 3.5—4 kg, were used in this study. The project was approved by the Committee for the Use of Living Animals in Teaching and Research at the University of Hong Kong (CULATR No. 368-99). Nine adult (5—12 months old) rabbits were used for preparation of freshfrozen allogeneic IM bone graft from bilateral mandibles, as previously described.1 Ten rabbits were used for preparation of DBMIM powder using the same procedures as previous one.1 The remaining nine rabbits were divided into three groups. Two defects per rabbit were created in the bilateral mandibles making a total of 18 defects. These three groups of six defects each were respectively grafted with fresh-frozen allogeneic IM bone alone, fresh-frozen allogeneic IM bone and DBMIM powder only, and fresh-frozen allogeneic IM bone and bFGF (Sigma) reconstituted in DBMIM powder. Surgical procedures Bone grafting surgical procedures are the same as previously described.1 In brief, after being anesthetized by intramuscular buprenorphine hydrochloride (5 mg/kg body weight) supplemented with diazepam (5 mg/kg body weight), nine rabbits underwent a central incision in hemicervical regions. A critical size 15 mm  10 mm ostectomy, which did not interrupt the mandibular continuity, was created bilaterally in mandibular body. Two bone grafts per rabbit were implanted in the mandibular defects bilaterally. Holes were drilled to allow for fixation of the bone grafts with stainless steel wires. In the group receiving allogeneic bone grafts, the fresh-frozen allogeneic IM bone was placed in the defect and fixed in place as described above. In the group receiving composite grafts of allogeneic IM bone + DBMIM, 500 mg DBMIM powder were placed in each defect with allogeneic IM bone. In the group receiving composite grafts of allogeneic IM bone + bFGF + DBMIM, 1250 ng hrbFGF reconstituted with 500 mg DBMIM powder were placed in each defect with allogeneic IM bone. All wounds were closed with 3-0 nylon sutures. No attempts were made to suture periosteum together. At 3 weeks post-surgically, animals were sacrificed with an intravenous overdose of pentobarbital sodium, 60 mg/kg body weight and the defects were harvested. Basic fibroblast growth factor Histological preparation The defect areas, including the surrounding tissues, were harvested for histological preparation. Tissues were fixed in 4% paraformaldehyde, decalcified in neutral ethylenediamine tetraacetic acid (EDTA, pH 7, Sigma), double-embedded in celloidin-paraffin, cut into 5 mm thick sections, and stained with hematoxylin and eosin. Immunohistochemistry Sections were dewaxed, rehydrated, immersed in methanol containing 0.3% H2O2, treated for nonspecific blocking, and incubated with a specific goat polyclonal antibody (A-20, Santa Cruz Biotechnology) at 4 mg/mL capable of recognizing the three variants (121aa, 165aa, and 189aa) of VEGF. Nonspecific binding was blocked again by 10% normal donkey serum before the second biotinylated donkey anti-goat antiserum (1:200, Santa Cruz Biotechnology) was applied, followed by an incubation in the presence of avidin biotin complex (DAKO). Negative controls were obtained in parallel avoiding the first antibody. Sections were visualized in diaminobezidine (Sigma) and counterstained with hematoxylin. In situ hybridization The mouse VEGF 165 cDNA (550 bp) was kindly provided by Dr. R.E. Gilbert (University of Melbourne, Australia). The plasmid was linearized using the appropriate restriction enzymes. The singlestranded antisense and sense probes were synthesized in the presence of Digoxigenin-11-UTP using SP6 and T7 RNA polymerase (Boehringer Mannheim), respectively. In situ hybridization was performed on wax sections.12 Briefly, 5 mm thick sections were dewaxed, cleared in xylene, and hydrated. The sections were then treated with 20 mg/mL of proteinase K (Roche) for 10 min, post-fixed in 4% paraformaldehyde, and incubated in pre-hybridization buffer at 42 8C for 3 h to prevent excessive background staining. Afterward, 1 mg/mL of digoxigenin-labeled antisense riboprobes were diluted in hybridization buffer (4 SSC, 10% dextran sulfate, 1 Denhardt’s solution, 2 mM EDTA, 0.1% SDS, 50% deionized formamide, 500 mg/mL herring sperm DNA, in DEPCtreated water) and added to the treated specimens. The sense probe was used as the control for nonspecific binding. The hybridization temperature was 50 8C, and the incubation time was 18 h. Post-hybridization wash was carried out under high-stringency conditions to remove unbound riboprobes. Locali- 1027 zation of hybridized transcripts in the specimen was visualized using alkaline phosphatase-conjugated antidigoxingenin Fab fragments (Roche) and NBT/ BCIP (Roche) as the chromagen. When the color was developed under dark the sections were immersed in stop buffer containing EDTA to terminate the reaction and then mounted by glycerol (Sigma). Quantitative analysis Quantitative analysis of neovascularization, represented by percentage of positive area produced by immunohistochemistry and in situ hybridization for VEGF, was carried out on serial sections of defects. The outlines of the defect were still detectable histologically. The 15 mm  10 mm defect was divided into 5 regions spaced 1500 mm apart. From among 20 sections in each region, 4 sections were randomly taken, giving a total of 20 sections from each defect. Half of these 20 sections were stained with immunohistochemistry, and half with in situ hybridization. Percentage of positive area via immunohistochemical staining and in situ hybridization within the surgically created defect was quantified with a computer-assisted image analyzer by one observer (M.L.). A three-channel system redgreen-blue color video camera (JVC) was attached to a LEICA DMLB microscopy, and each section was evaluated at a magnification of 40 by a computerassisted image analyzing system (Leica Q5501W) with Leica Q-win Pro.software (Version2.2). The production of VEGF (protein and mRNA) in defects was quantified by measuring percentage of positive area of signals (brown and blue, respectively). Statistical methods Data were analyzed with statistical software (Graphpad Instat, V.2.04a, 1994, San Diego). The one-way analysis of variance (ANOVA) method was used to compare sections drawn from the 5 regions in each defect. The arithmetic mean (the percentage of the area showing positive immunostaining), standard deviation (S.D.), and 95% confidence intervals were calculated for each experimental group. The means of each group were analysed with the Bonferroni multiple comparisons test. The critical level of statistical significance chosen was P < 0.05. The size of the method error in digitizing the area of positive was calculated by the formula ffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffistaining ( Sd 2 =2n) where d was the difference between the 2 registrations of a pair and n was the number of double registrations. Ten randomly drawn histological sections were digitized on two separate occasions at least 1 month apart by the same observer and also by an independent observer. Paired t-tests 1028 A.B.M. Rabie, M. Lu Figure 1 (a) Low magnification (length = 20 mm) of the allogenic IM + DBMIM + bFGF stained with anti-VEGF antibody (brown staining). (b) Higher magnification of the area marked with dark rectangle of (a). (c) Higher magnification of the area marked with dark rectangle of (b). Basic fibroblast growth factor were also performed to compare the intra- and inter-observer registrations. 1029 Results ited. Higher magnification showed that VEGF mRNA expression was mainly localized in the cytoplasm of proliferating osteogenic cells and osteoblasts (Fig. 3e). All control sections hybridized with the sense probe were negative (Fig. 3f). Clinical and physical examinations Quantitative analysis All animals remained in good health throughout the course of the experiment. There was no evidence of infection in any of animals. No weight change of the rabbits is remarkable during 3 weeks of experimental period. Fig. 4 showed the quantitative expression pattern of VEGF production (mRNA and protein) in three experimental groups for a total of 180 tissue sections digitized in Leica Q-win system. The percentage of positive immunohistochemical staining area in defects grafted with composite allogeneic IM bone-bFGF-DBMIM and in defects grafted with composite allogeneic IM bone-DBMIM was 492 and 216%, respectively more than that with allogeneic IM bone alone. A significant difference (P < 0.001) was found between those two groups. The percentage of positive area of VEGF mRNA signals in defects grafted with composite allogeneic IM bone-bFGF-DBMIM and in defects grafted with composite allogeneic IM bone-DBMIM was 517 and 208%, respectively more than that with allogeneic IM bone alone. A significant difference (P < 0.001) was found between those two groups. For the method error analysis, 10 sections were drawn at random and digitized, and the readings compared. The method error of the image analysis did not exceed 0.029 mm2, which was insignificant compared with the results. Hypothesis testing indicated no significant difference among the duplicate intra-observe (P = 0.134, t-test) and inter-observer registrations (P = 0.339, ttest) of the 10 randomly drawn sections. Immunolocalization of VEGF Histological examination showed integration of the new bone with the host bone throughout the defect in the allogenic bone, hrbFGF and DMBIM. DMBIM particles were seen in the middle of the defect interspersed between the newly formed bone (Fig. 1). Slightly VEGF positive staining (brown color) was localized around the new bone in allogeneic IM bone graft alone group, which is only a small amount near host bone (Fig. 2a). A stronger positive brown staining was evident among DBMIM particles where new bone was interposed in allogeneic IM bone graft + DBMIM group (Fig. 2b). Abundant and a much stronger brown staining was present around the new bone and in the region of mesenchyme that is destined to differentiate into osteoblasts in allogeneic IM bone graft + bFGF in DBMIM powder group (Fig. 2c). Higher magnification showed that VEGF expression was mainly localized in the cytoplasm of proliferating osteogenic cells and osteoblasts (Fig. 2d) and some osteoclasts (Fig. 2e). In bFGF-treated group invasion of numerous blood vessels among the newly formed bone was present in VEGF positive sections (Fig. 2c and d) and negative control (Fig. 2f). Expression of VEGF mRNA Slightly blue signal of VEGF mRNA was localized around the new bone in allogeneic IM bone graft alone group (Fig. 3a). Many blue signals of mRNA were shown among DBMIM particles where new bone was interposed on the side of bone graft in allogeneic IM bone graft + DBMIM group (Fig. 3b). Abundant and intensive blue signals of mRNA were present around the new bone (Fig. 3c) and especially in the region of capillary invasion in allogeneic IM bone graft + bFGF in DBMIM powder group (Fig. 3d). VEGF mRNA expression was associated with cells covering the surface of new bone in the region where osteoid and mineral are being depos- Discussion Results of the present study indicated that hrbFGF up-regulated the expression of VEGF in vivo (Fig. 4). VEGF is known to promote angiogenesis in threedimensional in vitro models, inducing confluent microvascular endothelial cells to invade collagen gel and form capillary-like structures.13 Furthermore, evidence for the important physiological role of VEGF in endochondral bone was found, as administration of a soluble VEGF receptor protein in juvenile mice suppressed blood vessel invasion into the hypertropic zone of long bone growth plate and concomitantly inhibited endochondral bone formation. The reintroduction of VEGF led to re-establishment of vascularization and resumption of bone growth.14 These results supported the results by Seghezzi et al., where bFGF was identified as an inducer of VEGF in endothelial cells during capillary formation.15 Furthermore, the present study 1030 A.B.M. Rabie, M. Lu Figure 2 Immunohistochemical analysis of VEGF in the healing of allogeneic bone grafts. (a) Slightly brown staining was localized around the new bone in allogeneic IM bone graft alone group. (b) A stronger brown staining was evident among DBMIM particles in allogeneic IM bone graft + DBMIM group. (c) Abundant and a much stronger brown staining was present around the new bone and in the region of mesenchyme that is destined to differentiate into osteoblasts in allogeneic IM bone graft + bFGF in DBMIM powder group. Higher magnification showed that VEGF expression was mainly localized in the cytoplasm of proliferating osteogenic cells (arrowhead) and osteoblasts (arrow) (d) and some in osteoclasts (arrow) (e). The invasion of numerous blood vessels among the newly formed bone was present in both VEGF positive sections (c and e) and negative control (f) which parallels the procedure avoiding the primary antibody. N: new bone; D: DBMIM; G: graft bone; BV: blood vessel. Note: regions expressing brown color are positive for VEGF expression. revealed that the addition of hrbFGF reconstituted in DBMIM caused increased expression of VEGF by the proliferating osteogenic cells (Fig. 2c; Fig. 3c) and by osteoblasts (Fig. 2d; Fig. 3d) present in the newly formed bone induced by the composite bone grafts. This point is of great importance to the integration and amalgamation of the allogeneic bone graft with the host bone through bridging by the newly formed bone.16 Since the osteogeneic cells (Fig. 2c; Fig. 3c) continue to express VEGF, which will in turn induce angiogenesis, thus bring new blood vessels to the proximity of the allogeneic bone graft (Fig. 2c; Basic fibroblast growth factor 1031 Figure 3 In situ hybridization analysis for VEGF mRNA expression in the healing of allogeneic bone grafts. (a) Slightly blue signal of mRNA was localized around the new bone in allogeneic IM bone graft alone group. (b) Numerous blue signals of mRNA were shown among DBMIM particles where new bone was interposed on the side of bone graft in allogeneic IM bone graft + DBMIM group. (c) Abundant and intensive signals of mRNA were present around the new bone and especially in the region of capillary invasion in allogeneic IM bone graft + bFGF in DBMIM powder group (d). (e) Higher magnification shown that VEGF mRNA expression was mainly localized in the cytoplasm of proliferating osteogenic cells (arrowhead) and osteoblasts (arrow). (f) Negative control section hybridized with the sense probe. N: new bone; D: DBMIM; G: graft bone; BV: blood vessel. Scale bar: 10 mm. Note: regions expressing blue signal are positive for gene expression. Fig. 3d and e). These new blood vessels are a rich source of nondifferentiated mesenchymal cells which could differentiate into bone-making cells. In vitro studies by Saadeh et al. showed that bFGF induced angiogenesis by osteoblastic cells. It was reported that physiologically relevant bFGF doses promoted VEGF mRNA and protein expression in clonal and nonimmortalized osteoblastic cells.7 A study by Wang et al. demonstrated that the expression of VEGF by bone cells stimulated the endothelial cells proximally located to express osteotrophic growth factors.17 These factors could be directly involved in the process of bone formation. The above data and others18,19 suggested a dual function of VEGF in bone formation: first, to recruit blood supply to enrich the population size of mesenchymal 1032 Figure 4 groups. A.B.M. Rabie, M. Lu Quantitative analysis of VEGF expression (protein and mRNA) in the healing of the three different treatment cells and second, to help with the differentiation of these cells into bone cells.19 Another factor that could influence the differentiation of these nondifferentiated mesenchymal cells is the family of BMPs and other cytokines present in the DBMIM20,21 present in the mixture of the composite graft used in the current study. This could, in part, explain the increased osteoinductivity of the allogeneic IM bone grafts when mixed with hrbFGF reconstituted in DBMIM.1 Another role that should be mentioned regarding bFGF is its ability to induce the expression of TGFß1, which, itself, modulates angiogenesis and stimulates VEGF production.7 Therefore, in the current graft material, it is possible that bFGF promotes angiogenesis through VEGF expression directly or through the activation of TGFß1 which in turn up-regulates VEGF expression. In the present study, there was a close correlation that existed between the level of expression of VEGF gene (mRNA or protein) and bone formation in the healing of allogeneic intramembranous bone grafts (Figure 5). This observation might provide an exmplanation on the direct correlation between increased vascularization and increased osteogenesis in vivo. Earlier, it was reported a close correlation between vascularization and bone formation during osteogenesis induced by DBM.2,21,22 Furthermore, a direct correlation existed between the level of expression of VEGF and bone formation during natural growth in the glenoid fossa.23 In the same study, the highest level of expression of VEGF preceded the highest level of bone formation in the glenoid fossa.23 Similar results were obtained in the mandibular condyle where increased levels of VEGF expression were accompanied by increased bone formation.24 In conclusion, the composite hrbFGF reconstituted in DBMIM enhances the healing of allogeneic bone grafts by possibly enhancing vascularization. The mode of action of hrbFGF is in part through the Figure 5 The allograft + DBMIM + bFGF produced the highest amount of VEGF mRNA, protein as well as new bone when compared to the allograft along and with DBMIM. Basic fibroblast growth factor up-regulation of VEGF expression which is a key regulator of angiogenesis. A close correlation might exist between VEGF and the amount of newly formed bone. 1033 11. 12. Acknowledgements 13. The authors thank Dr. Kenneth Lee and Dr. Mei-Kuen Tang from Dept. of Anatomy, Chinese University of Hong Kong for their guidance with the in situ hybridization. This study was sponsored by CRCG-HKU, HKSAR. No. 10203285-12767-08002-323-01. 14. 15. References 1. Lu M, Rabie AB. The effect of demineralized intramembranous bone matrix and basic fibroblast growth factor on the healing of allogeneic intramembranous bone grafts in the rabbit. Arch Oral Biol 2002;47:831—41. 2. Chow KM, Rabie AB. Vascular endothelial growth pattern of endochondral bone graft in the presence of demineralized intramembranous bone matrix—quantitative analysis. Cleft Palate Craniofac J 2000;37:385—94. 3. Habal MB. Different forms of bone grafts. In: Habal MB, Reddi AH, editors. Bone grafts and bone substitutes. Philadelphia: WB Saunders; 1992. p. 6—8. 4. Harris SE, Bonewald LF, Harris MA et al. Effects of transforming growth factor beta on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long-term cultures of fetal rat calvarial osteoblasts. J Bone Miner Res 1994;9:855—63. 5. Eppley BL, Doucet M, Connolly DT, Feder J. Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit. J Oral Maxillofac Surg 1988;46:391—8. 6. Weiss AP, Olmedo ML, Lin JC, Ballock RT. Growth factor modulation of the formation of a molded vascularized bone graft in vivo. J Hand Surg Am 1995;20:94—100. 7. 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Ultrastructural identification of cells involved in the healing of intramembranous bone grafts in both the presence and absence of demineralised intramembranous bone matrix. Aust Orthod J 2000;16: 88—97. Rabie AB, Wong RW, Hagg U. Bone induction using autogenous bone mixed with demineralised bone matrices. Aust Orthod J 1999;15:269—75. Rabie AB. Vascular endothelial growth pattern during demineralized bone matrix induced osteogenesis. Connect Tissue Res 1997;36:337—45. Rabie AB, Shum L, Chayanupatkul A. VEGF and bone formation in the glenoid fossa during forward mandibular positioning. Am J Orthod Dentofacial Orthop 2002;122: 202—9. Rabie ABM. Leung FYC. Chayanupatkul A, Hägg U. The correlation between neovascularization and bone formation in the condyle during forward mandibular positioning. Angle Orthodontist 2002;72:431—8.