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Osteopromotion for cranioplasty: An experimental study in rats using acidic fibroblast growth factor
ELSEVIER
OSTEOPROMOTION FOR CRANIOPLASTY: AN EXPERIMENTAL STUDY IN RATS USING ACIDIC FIBROBLAST GROWTH FACTOR Pedro Cuevas, M.D., Ph.D.,* Victor de Paz, M.D.,t Begofia Cuevas, B.Sc.,* Jesus Marin-Martinez, M.D.,t Manuel Picon-Molina, M.D.,y Antonio Fernandez-Pereira, M.D.,t and Guillermo Gimenez-Gallego, B.Sc., Ph.D.? *Servicios de Histologia y , fCirugfa Maxilo-facial, Hospital Ram& y Cajal, Madrid, Spain; and $Centro de Investigaciones BiolGgicas, CSIC, Madrid, Spain
Cuevas P, de Paz V, Cuevas B, Marin-Martinez .I, Picon-Molina M, Ferntidez-Pereira A, GimCnez-GalIego C. Osteopromotion for cranioplasty: an experimental study in rats using acidic fibroblast growth factor. Surg Neurol 1997;47:242-6. BACKGROUND
Many growth factors influence the bone healing cascade.
Furthermore, the occasional failure of bone repair may in part be due to perturbation in the activation of local growth factors. Local activation of fibroblast growth factors (FGFs) at the fracture site may serve to increase neovascularization, and induce early granulation formation that can affect bone healing. METHODS
We have performed a rat parietal (6 X 3 mm) critical size defect (CSD). Human recombinant acidic fibroblast growth factor (hraFGF’) imbided in agarosewas topically administered at the craniectomy site. Control animals received agarosealone in the same manner. Three weeks after surgery, osteopromotion was histologically evaluated. RESULTS
hraFGF-treated animals show a continuous bridge of regenerated bone extending from one edge of the defect to the other. None of the parietal defects that had been treated with agarose contained new bone in the central portion. CONCLUSION
Our results suggest an important
role of FGFs to promote
large cranioplasty repair and support the use of these proteins asan alternative choice for bone grafts and bone substitutes. 0 1997by Elsevier Science Inc. KEY
WORDS
Fibroblastgrowth Factor,bone regeneration, cranial defects, rats.
Address reprints requests to: Dr. Pedro Cuevas, Servicio Hospital Ram6n y Cajal, E28034-Madrid-Spain. Received January 17. 1996; accepted June 5, 1996. 0090-3019/97/$17.00 PI1 SOO90-3019(96)004387
de Histologfa,
T
he major indication for cranioplasty is to reestablish a rigid protection for the underlying brain. Cranioplasty may be performed by bone grafts or bone substitutes. The limited availability of autogenous bones for grafting after craniectomy, the difficulty in sculpturing them, the bone resorption that could destroy the aesthetic results, and the associated donor site morbidity have prompted searches for bone substitutes in recent years. However, the materials most frequently used for cranioplasty, such as metals and plastics, show disadvantages of toxicity, infection, and rejection [16,24]. Recently, bone growth factors have emerged as an alternative choice for treating bone defects [4]. Bone repair involves a complex series of cellular and extracellular matrix events [30], which is regulated by several growth factors secreted at the wound site [4]. The fibroblast growth factors (FGFs) are a family of nine heparin-binding mitogens for mesoderm-derived cells, which promote cell growth, induce differentiation, and act as tissue maintenance factors [29]. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor @FGF) are the best characterized of this group of proteins [ 141. FGFs are synthesized by osteoblasts and other mesenchymal cells and deposited in bone matrix [ 15,171. Furthermore, FGFs can stimulate the proliferation of osteogenic cells and chondrocytes [2,7,13,32], and enhance fracture healing in diabetic rats [ 191. In addition, FGFs are highly angiogenic proteins, causing the formation of capillary sprouts by migration, proliferation, and organization of endothelial cells [ 121. Thus, FGFs have several prop erties suggesting their ability to enhance osteopromotion. 655 Avenue
0 1997 by Elsevier Science Inc. of the Americas, New York, NY 10010
Osteopromotion
by aFGF
Several different formulations have been used as carriers for topical delivery of growth factors. They include collagen [ 251, multilamellar liposomes [ 51, hyaluronic acid [ 111, polyvinyl alcohol sponges [S], methylcellulose [3], nitrocellulose paper [ 281, sucralfate [9], and fibrin gel [31]. However, the ideal carrier for the local administration of a growth factor remains unknown. This report describes the delivery of hraFGF to a rat craniectomy using agarose gel as carrier.
MATERIALSANDMETHODS OPERATIVE PROCEDURE The smallest defect that will heal less than 10% bony growth is termed a critical size defect (CSD) [22]. In the adult rats, the calvarial CSD is 4 mm2 [ 181. In this study we created a defect so large that the environmental milieu is depleted of factors needed for regenerating bone [16]. Twenty adult Wistar rats (250300 g) of both sexes were used in accordance with the guidelines set by the European Community Council directives 86/6091 EEC. Animals were anesthetized by intraperitoneal injection of 3 ml/kg of the anesthetic mixture: ketamine hydrochloride 2.5 mg/ml, Valium 2 mg/ml, and atropine 0.1 mgfml. The hair over the calvaria was shaved and cleared with depilatory. Lidocaine (0.5 ml of 1%) was injected intradermally in the midline on top of the head. The rats were placed in a smallanimal stereotaxic head frame. Under a surgical operating microscope (Zeiss OPMl) a midline scalp incision from the occipital to the frontal region was made and full thickness flaps were unilaterally reflected exposing the calvaria. A 6 X 3 mm rectangular CSD was excavated over the left parietal bone using a drill with saline irrigation. Extreme care was taken not to damage the dura mater. AFGF TREATMENT Human recombinant acidic fibroblast growth factor (hraFGF) was obtained from Escherichia coli harboring proteinexpression plasmids that contain the gene coding the human aFGF protein in agreement with procedures previously described [35]. The 139 amino acid form of aFGF and agarose gel as the vehicle for hraFGF administration were used. hraFGF was imbided in agarose gel by mixing 100 ~1 of 2.6 pg hraFGF in PBS with 0.1% heparin. Once the mixture acquires gel consistency (-20 seconds), it was used to cover the craniectomy in 10 animals. In control animals (n = IO), the parietal bone defect was covered with 100 ~1 agarose gel in PBS containing 0.1% heparin. After scalp closure the rats were
Surg Neurol 1997;47:242-6
243
allowed to recover for 3 weeks, during which time food and water were provided ad libitum. MORPHOLOGIC ANALYSIS Three weeks after surgery, the animals were reanesthetized and retrogradely perfused through the abdominal aorta using a rinse solution of PBS containing 0.1% heparin and a fixative solution of 4% paraformaldehyde in O.lM phosphate buffer. The cranial defect areas including the surrounding tissues were removed and harvested for histologic preparation. The tissue blocks were postfixed in the same fixative and decalcified with ethylenediamine tetraacetic acid/HCl. Specimens were cut in a coronal plane in the center of the defect, embedded in paraffin, sectioned (10 pm thick), and stained with hematoxylin and eosin.
RESULTS After 3 weeks, none of the calvarial defects that had been treated with vehicle solution contained new bone in the central part. However, some appositional growth of new bone was observed on the periphery of defects adjacent to the lamellar calvarial bone. The parietal defect was occupied by supracalvarial soft tissue, located in direct contact with the dural tissue (Figure la). Animals that received 2.6 pg hraFGF showed a continuous bridge of regenerated bone extending from one edge of the defect to the other (Figure lb). This newly formed bone was the same thickness as the bone bordering the calvarial defect. The histologic aspect of regenerated bone is depicted in Figures lc and d. As these pictures show, regenerated calvarial defects are formed by mature (Figure lc) and inmature (Figure lc) lamellae of trabecular bone. The bone trabecullae are lined by cuboidal osteoblasts that form multilayers in some parts. Osteocytes are observed within the bone trabecullae. Few osteoclasts are present adjacent to excavated areas along some bone trabecullae. No signs of adverse tissue reactions were found in any of the specimens.
DISCUSSION Several local peptide growth factors are likely to play a significant role in the induction, growth, and differentiation of bone cells [4,34]. Of these, both acidic and basic FGF are powerful bone forming factors [ 1,2,10,12,18-20,32,33], possibly the most powerful currently known [23]. Using a parietal bone defect exceeding the CSD in rats, we show that local application of hraFGF to the cranial bone de-
244
Cuevas et al
Surg Neural 199714712424
Photomicrographs showing coronal sections through calvarial critical size defects at 3 weeks postcraniectomy. (a) Fibrous connective tissue throughout most of the defect is observed in control animals. Space between arrows indicates appositional growth of new bone on the periphery of the defect; @), (c), and (d) show new bone formation throughout the defect in animals treated with hraFGF; arrows in @) mark the edge of the original defect. Mature and immature lamellae of trabecular bone were depicted at high magnification in (b) and (d), respectively. In (d), the osteocytes (arrows) appear as small cells within the bone matrix. The osteoblasts (curued arrows) are larger on the
surface of the bone tissue (d). (a) and (b)
X
300; (c) and (d) X 750.
feet stimulates bone repair. The osteoinductive
ac-
tivity of hraFGF may be mediated by stimulation and proliferation of osteoblast precursors to form a
larger
pool of committed
progenitors
[19].
Al-
though hraFGF could stimulate bone repair directly, it is also possible that hraFGF stimulates vascular
Osteopromotion
by aFGF
invasion [ 121, which causes the influx of bone cells precursors. Additionally, the osteoinductive capacity of hraFGF may be related to its ability to induce TGFf3 gene expression in osteoblastic cells [26] that participates in bone formation [27]. Thus, hraFGF may participate in the cascade of events for bone repair by increasing the number of mesenchymal osteoblastic cells and capillaries at the craniectomy site, and enhancing the release of additional bone growth factors such as TGFP from mesenchymal cells. In studies on the effects of growth factors, questions remain about the dose of growth factor, the mode of topical delivery, and the number of applications. The hraFGF dose used in the present study was chosen according to dose-response studies of bFGF-stimulation of bone induction [ 11. The effect of hraFGF in cranial bone repair was evaluated applying hraFGF imbided in agarose, a method of slow release of this protein proved successful in an angiogenesis-induction model [8] and in axotomyinduced neuronal death [9]. The results of the present study are in agreement with the findings of Kawaguchi et al. and Mohler et al., showing that a single application of bFGF was sufficient to stimulate fracture repair in normal and streptozotocindiabetic rats [ 19,211. In conclusion, the findings presented here suggest a key role of FGF to achieve large surface bone repair in general, and in particular for osteopromotion for cranioplasty as an alternative choice for bone grafts and bone substitutes. This study was supported by the DGICYT and Fundacion Gregorio Maraiionand FundaciBn FutureBoehringer Ingelheim Esparia SA agreements. We are grateful to M. Guerricabeitia and C. Bourdier For technical and editorial assistance, respectively.
REFERENCES 1. Asperberg P, Thomgren KG, Lohmander LS. Dose dependent stimulation of bone induction by basic fibroblast growth factor in rats. Acta Orthop Stand 1991;62:481-4. Baron J, Klein KO, Yanovski JA, Novosad JA, Bather JD, Bolander ME, Cutler GB. induction of growth plate cartilage ossification by basic fibroblast growth factor. Endocrinology 1994;135:2790-3. Beck LS, Chen TL, Mikaianski P, Ausman AJ. Recombinant human transforming growth factor-p1 (rhTGF/31) enhances healing and strength of granulation skin wounds. Growth Factors 1990;3:267-75. Bolander ME. Regulation of fracture repair by growth factors. Proc Sot Exp Biol Med 1992;200:165-70. Brown GL, Curtsinger LJ, White M, Mitchell RO, Pietsch J, Nordquist R, von Fraunhoger A, Schultz GS. Acceleration of tensile strength of incisions treated with EGF and TFG-f3.Ann Surg 1988;208:788-94.
Surg Neurol 1997;47:242-6
245
6. Buckley A, Davidson JM, Kamerath CD, Wolt TR, Woodward SC.Sustained releaseof epidermal growth factor accelerates wound repair. Proc Nat1 Sci USA 1985;82:7340-4. 7. Cuevas P, Burgos J, Baird A. Basic fibroblast growth (FGF)promotes cartilage repair in vivo. Biochem Biophys Res Commun 1988;156:611-8. 8. Cuevas P, Gimenez-GallegoG, Carceller F, Cuevas B, CrespoA. Singletopical application of human recombinant basic fibroblast growth factor (rbFGF) promotes neovascularization in rat cerebral cortex. Surg Neurol 1993;39:380-4. 9. Cuevas P, Carceller F, Gimenez-GallegoG. Acidic fibroblast growth factor prevents post-axotomy neuronal death of the newborn rat facial nerve. Neurosci Lett 1995;197:183-6. 10. Dunstan CR, Boyce BF, Izbicka E, Adams R, Mundy GR. Acidic and basic fibroblast growth factor promotes bone growth in vivo comparable to that of TGB. J Bone Miner Res 1993;8:250. 11. Eppley BL, Connolly DT, Winkelmann T, Sadore AM, Heuvelman D, Feder J. Free bone graft reconstruction of irradiated facial tissue: experimental effects of basic fibroblast growth factor stimulation. Plast Reconstr Surg 1991;88:1-11. 12. Folkman J, Klagsburn M. Angiogenic factors. Science 1987;235:442-7. 13. Freukel SR, Grande DA, Collins M, Singh IJ. Fibroblast growth factor in chick osteogenesis. Biomaterials 1990;11:38-40. 14. Gimenez-GallegoG, Cuevas P. Fibroblast growth factors, proteins with a broad spectrum of biological activities. Neurol Res 1994;16:313-6. 15. Globus R, Plonet J, Gospodarowicz D. Cultured bovine bone cells synthesize fibroblast growth factor and store it in their extracellular matrix. Endocrinology 1989;124:1539-47. 16. Habal MB. Craniofacial surgery. In: Habal MB, Reddi AH, eds. Bone grafts and bone substitutes. Philadelphia: WB Saunders Company, 1992:316-65. 17. Hauschka PV, Maurakos AE, Jafrati MD, Doleman SE, Klagsbrun M. Growth factors in bone matrix: isolation of multiple types by affinity chromatography on heparin sepharose.J Biol Chem 1986;261:12665-74. 18. Hollinger JO, Kleinschmidt JC.The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1990;1:60-68. __ 19. Kawaguchi H, Kurokawa T, Hanada K, Kiyama Y, Tamura M, Ogata E, Matsumoto T. Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats. Endocrinology 1994;135:774-81. 20. Mayahara J, Ito T, Nagai H, Miyajima H, Tsukuda R, Taketami S, Mizoguchi J, Kato K. In vivo stimulation endosteal bone formation by basic fibroblast growth factor in rats. Growth Factors 1993;9:73-80. 21. Mohler DG, Lane JM, Fehnel DG. Effects of single dose basic fibroblast growth factor (WGF) in rat femoral defect model. J Bone Miner Res 1990;5:S147. 22. Mulliken JB, Glowacki JB. Induced osteogenesisfor repair and reconstruction in the craniofacial region. Plast Reconstr Surg 1980;65:553-9. 23. Mundy GR,Boyce B, HughesD, Wright K, Bonewald L, DallasS, Harris S, Ghosh-Choudhury N, Chen D, DunStan C, Izbicka E. Yoneda T. The effects of cytokines
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24. 25.
26. 27. 28.
Sure Neurol 199’j;47:242-6 and growth factors on osteoblastic cells. Bone 1995; 17(suppl.):71S-5s. Muschler GF, Lane JM. Orthopedic surgery. In: Habal MB, Reddi AH, eds. Bone grafts and bone substitutes. Philadelphia: WB SaundersCompany, 1992:375-407. Mutsoe TA, Pierce GF, Thomason A, Gramates P, Sporn MB, Deuel TF. Accelerated healing of incisional wounds in rats induced by transforming growth factor-p. Science 1987;237:1333-6. Noda M, Vogel R. Fibroblast growth factor enhances type /31 transforming growth factor gene expression in osteoblast-like cells. J Cell Biol 1989;109:2529-35. Noda M, Camilliere JJ. In vivo stimulation of bone formation by transforming growth factor-p. Endocrinology 1989;124:2991-4. Petmann B, Manthorpe M, Powell JA, Varon S.Biological activities of nerve growth factor bound to nitrocellulose paper by Western blotting. J Neurosci 1988;
8:3624-32. 29. Rifkin DB, Moscatelli D. Recent development in the 30. 31.
32.
33. 34.
35.
cell biology of basic fibroblast growth factor. J Cell Biol 1989:109-16. Simmons DJ. Fracture healing perspectives. Clin Orthop Rel Res 1985;200:100-13. Thomas KA, Rios-Candelore M, Gimenez-GallegoG, DiSalvo J, Bennett CL, Rodkey J, Fitzpatrick S. Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc Nat1 Acad Sci USA 1985;82:6409-13. Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR.Regulation of growth-plate chondrocytes by insulin-like growth factor 1 and basic fibroblast growth factor. J Bone Joint Surg 1993; 75:177-89. Wang JS, Aspenberg P. Basic fibroblast growth factor increasesallograft incorporation. Acta Orthop Stand 1994;65:27-31. Wornom IL, Buchman SR. Bone and cartilaginous tissue. In: Cohen IK, DiegelmannRF, Lindblad WJ. eds. Wound healing. Biochemical and clinical aspects. Philadelphia. WB. Saunders Company. 1992:356-83. Zazo M, Lozano RM, Ortega S,Varela J, Diaz-Orejas R, Ramirez JM, Gimenez-GallegoG. High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions. Gene 1992;113: 231-8.
COMMENTARY
The number and importance of growth factors has increased dramatically in the last months and years. We are most familiar with the now multiple nerve growth factors and their effect on the promotion of nerve recovery as well as the death and
Cuevas et al
removal of expendable nerve cells. This study presents interesting and important new information regarding the use of fibroblastic growth factors at the margins of fractures to affect bone healing and particularly for the regeneration of cranial bone in a well-controlled animal model. The authors suggest that the studied factors may be able to promote large cranioplastic repair, and they further suggest that these factors may be an alternative choice for bone grafts or bone substitutes. At the present time, a number of studies are being conducted in the use of bone morphogenic protein and other factors such as bovine fetal cartilage activation factor for acceleration of fusion development through the mechanism of chondrocyte replacement with subsequent calcification and conversion to bone. Intradiscal fibroblastic growth factors have been shown to accelerate the restoration of glycosaminoglycan synthesis in the intervertebral disc nucleus following the chemical damage resulting from chymopapain injection in an experimental animal model. It is important to note that in this study by Cuevas et al., they found that some growth of new bone was observed on the periphery of small (3 x 6 mm) cranial defects without any sign of adverse tissue reaction. However, the bone had not grown over the center of the defect at 3 weeks. One wonders how long it might take for a large defect to fully close. The method of topical delivery of the otherwise putative growth factor is important as it was embedded in agarose. There are a number of other hydrogels that are capable of slow release of small molecular species such as the growth factors and a few studies have utilized implantable AlzaTM micropumps for this purpose. Clearly, growth factors and their controlled r-elease will play an important role in tissue regeneration and repair in the future. This study presents exciting information regarding this and related factors and their profound effect on local tissue response. Charles D. Ray, M.D., F.A.C.S.
Neurosurgeon Minneapolis, Minnesota
OSTEOPROMOTION FOR CRANIOPLASTY: AN EXPERIMENTAL STUDY IN RATS USING ACIDIC FIBROBLAST GROWTH FACTOR Pedro Cuevas, M.D., Ph.D.,* Victor de Paz, M.D.,t Begofia Cuevas, B.Sc.,* Jesus Marin-Martinez, M.D.,t Manuel Picon-Molina, M.D.,y Antonio Fernandez-Pereira, M.D.,t and Guillermo Gimenez-Gallego, B.Sc., Ph.D.? *Servicios de Histologia y , fCirugfa Maxilo-facial, Hospital Ram& y Cajal, Madrid, Spain; and $Centro de Investigaciones BiolGgicas, CSIC, Madrid, Spain
Cuevas P, de Paz V, Cuevas B, Marin-Martinez .I, Picon-Molina M, Ferntidez-Pereira A, GimCnez-GalIego C. Osteopromotion for cranioplasty: an experimental study in rats using acidic fibroblast growth factor. Surg Neurol 1997;47:242-6. BACKGROUND
Many growth factors influence the bone healing cascade.
Furthermore, the occasional failure of bone repair may in part be due to perturbation in the activation of local growth factors. Local activation of fibroblast growth factors (FGFs) at the fracture site may serve to increase neovascularization, and induce early granulation formation that can affect bone healing. METHODS
We have performed a rat parietal (6 X 3 mm) critical size defect (CSD). Human recombinant acidic fibroblast growth factor (hraFGF’) imbided in agarosewas topically administered at the craniectomy site. Control animals received agarosealone in the same manner. Three weeks after surgery, osteopromotion was histologically evaluated. RESULTS
hraFGF-treated animals show a continuous bridge of regenerated bone extending from one edge of the defect to the other. None of the parietal defects that had been treated with agarose contained new bone in the central portion. CONCLUSION
Our results suggest an important
role of FGFs to promote
large cranioplasty repair and support the use of these proteins asan alternative choice for bone grafts and bone substitutes. 0 1997by Elsevier Science Inc. KEY
WORDS
Fibroblastgrowth Factor,bone regeneration, cranial defects, rats.
Address reprints requests to: Dr. Pedro Cuevas, Servicio Hospital Ram6n y Cajal, E28034-Madrid-Spain. Received January 17. 1996; accepted June 5, 1996. 0090-3019/97/$17.00 PI1 SOO90-3019(96)004387
de Histologfa,
T
he major indication for cranioplasty is to reestablish a rigid protection for the underlying brain. Cranioplasty may be performed by bone grafts or bone substitutes. The limited availability of autogenous bones for grafting after craniectomy, the difficulty in sculpturing them, the bone resorption that could destroy the aesthetic results, and the associated donor site morbidity have prompted searches for bone substitutes in recent years. However, the materials most frequently used for cranioplasty, such as metals and plastics, show disadvantages of toxicity, infection, and rejection [16,24]. Recently, bone growth factors have emerged as an alternative choice for treating bone defects [4]. Bone repair involves a complex series of cellular and extracellular matrix events [30], which is regulated by several growth factors secreted at the wound site [4]. The fibroblast growth factors (FGFs) are a family of nine heparin-binding mitogens for mesoderm-derived cells, which promote cell growth, induce differentiation, and act as tissue maintenance factors [29]. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor @FGF) are the best characterized of this group of proteins [ 141. FGFs are synthesized by osteoblasts and other mesenchymal cells and deposited in bone matrix [ 15,171. Furthermore, FGFs can stimulate the proliferation of osteogenic cells and chondrocytes [2,7,13,32], and enhance fracture healing in diabetic rats [ 191. In addition, FGFs are highly angiogenic proteins, causing the formation of capillary sprouts by migration, proliferation, and organization of endothelial cells [ 121. Thus, FGFs have several prop erties suggesting their ability to enhance osteopromotion. 655 Avenue
0 1997 by Elsevier Science Inc. of the Americas, New York, NY 10010
Osteopromotion
by aFGF
Several different formulations have been used as carriers for topical delivery of growth factors. They include collagen [ 251, multilamellar liposomes [ 51, hyaluronic acid [ 111, polyvinyl alcohol sponges [S], methylcellulose [3], nitrocellulose paper [ 281, sucralfate [9], and fibrin gel [31]. However, the ideal carrier for the local administration of a growth factor remains unknown. This report describes the delivery of hraFGF to a rat craniectomy using agarose gel as carrier.
MATERIALSANDMETHODS OPERATIVE PROCEDURE The smallest defect that will heal less than 10% bony growth is termed a critical size defect (CSD) [22]. In the adult rats, the calvarial CSD is 4 mm2 [ 181. In this study we created a defect so large that the environmental milieu is depleted of factors needed for regenerating bone [16]. Twenty adult Wistar rats (250300 g) of both sexes were used in accordance with the guidelines set by the European Community Council directives 86/6091 EEC. Animals were anesthetized by intraperitoneal injection of 3 ml/kg of the anesthetic mixture: ketamine hydrochloride 2.5 mg/ml, Valium 2 mg/ml, and atropine 0.1 mgfml. The hair over the calvaria was shaved and cleared with depilatory. Lidocaine (0.5 ml of 1%) was injected intradermally in the midline on top of the head. The rats were placed in a smallanimal stereotaxic head frame. Under a surgical operating microscope (Zeiss OPMl) a midline scalp incision from the occipital to the frontal region was made and full thickness flaps were unilaterally reflected exposing the calvaria. A 6 X 3 mm rectangular CSD was excavated over the left parietal bone using a drill with saline irrigation. Extreme care was taken not to damage the dura mater. AFGF TREATMENT Human recombinant acidic fibroblast growth factor (hraFGF) was obtained from Escherichia coli harboring proteinexpression plasmids that contain the gene coding the human aFGF protein in agreement with procedures previously described [35]. The 139 amino acid form of aFGF and agarose gel as the vehicle for hraFGF administration were used. hraFGF was imbided in agarose gel by mixing 100 ~1 of 2.6 pg hraFGF in PBS with 0.1% heparin. Once the mixture acquires gel consistency (-20 seconds), it was used to cover the craniectomy in 10 animals. In control animals (n = IO), the parietal bone defect was covered with 100 ~1 agarose gel in PBS containing 0.1% heparin. After scalp closure the rats were
Surg Neurol 1997;47:242-6
243
allowed to recover for 3 weeks, during which time food and water were provided ad libitum. MORPHOLOGIC ANALYSIS Three weeks after surgery, the animals were reanesthetized and retrogradely perfused through the abdominal aorta using a rinse solution of PBS containing 0.1% heparin and a fixative solution of 4% paraformaldehyde in O.lM phosphate buffer. The cranial defect areas including the surrounding tissues were removed and harvested for histologic preparation. The tissue blocks were postfixed in the same fixative and decalcified with ethylenediamine tetraacetic acid/HCl. Specimens were cut in a coronal plane in the center of the defect, embedded in paraffin, sectioned (10 pm thick), and stained with hematoxylin and eosin.
RESULTS After 3 weeks, none of the calvarial defects that had been treated with vehicle solution contained new bone in the central part. However, some appositional growth of new bone was observed on the periphery of defects adjacent to the lamellar calvarial bone. The parietal defect was occupied by supracalvarial soft tissue, located in direct contact with the dural tissue (Figure la). Animals that received 2.6 pg hraFGF showed a continuous bridge of regenerated bone extending from one edge of the defect to the other (Figure lb). This newly formed bone was the same thickness as the bone bordering the calvarial defect. The histologic aspect of regenerated bone is depicted in Figures lc and d. As these pictures show, regenerated calvarial defects are formed by mature (Figure lc) and inmature (Figure lc) lamellae of trabecular bone. The bone trabecullae are lined by cuboidal osteoblasts that form multilayers in some parts. Osteocytes are observed within the bone trabecullae. Few osteoclasts are present adjacent to excavated areas along some bone trabecullae. No signs of adverse tissue reactions were found in any of the specimens.
DISCUSSION Several local peptide growth factors are likely to play a significant role in the induction, growth, and differentiation of bone cells [4,34]. Of these, both acidic and basic FGF are powerful bone forming factors [ 1,2,10,12,18-20,32,33], possibly the most powerful currently known [23]. Using a parietal bone defect exceeding the CSD in rats, we show that local application of hraFGF to the cranial bone de-
244
Cuevas et al
Surg Neural 199714712424
Photomicrographs showing coronal sections through calvarial critical size defects at 3 weeks postcraniectomy. (a) Fibrous connective tissue throughout most of the defect is observed in control animals. Space between arrows indicates appositional growth of new bone on the periphery of the defect; @), (c), and (d) show new bone formation throughout the defect in animals treated with hraFGF; arrows in @) mark the edge of the original defect. Mature and immature lamellae of trabecular bone were depicted at high magnification in (b) and (d), respectively. In (d), the osteocytes (arrows) appear as small cells within the bone matrix. The osteoblasts (curued arrows) are larger on the
surface of the bone tissue (d). (a) and (b)
X
300; (c) and (d) X 750.
feet stimulates bone repair. The osteoinductive
ac-
tivity of hraFGF may be mediated by stimulation and proliferation of osteoblast precursors to form a
larger
pool of committed
progenitors
[19].
Al-
though hraFGF could stimulate bone repair directly, it is also possible that hraFGF stimulates vascular
Osteopromotion
by aFGF
invasion [ 121, which causes the influx of bone cells precursors. Additionally, the osteoinductive capacity of hraFGF may be related to its ability to induce TGFf3 gene expression in osteoblastic cells [26] that participates in bone formation [27]. Thus, hraFGF may participate in the cascade of events for bone repair by increasing the number of mesenchymal osteoblastic cells and capillaries at the craniectomy site, and enhancing the release of additional bone growth factors such as TGFP from mesenchymal cells. In studies on the effects of growth factors, questions remain about the dose of growth factor, the mode of topical delivery, and the number of applications. The hraFGF dose used in the present study was chosen according to dose-response studies of bFGF-stimulation of bone induction [ 11. The effect of hraFGF in cranial bone repair was evaluated applying hraFGF imbided in agarose, a method of slow release of this protein proved successful in an angiogenesis-induction model [8] and in axotomyinduced neuronal death [9]. The results of the present study are in agreement with the findings of Kawaguchi et al. and Mohler et al., showing that a single application of bFGF was sufficient to stimulate fracture repair in normal and streptozotocindiabetic rats [ 19,211. In conclusion, the findings presented here suggest a key role of FGF to achieve large surface bone repair in general, and in particular for osteopromotion for cranioplasty as an alternative choice for bone grafts and bone substitutes. This study was supported by the DGICYT and Fundacion Gregorio Maraiionand FundaciBn FutureBoehringer Ingelheim Esparia SA agreements. We are grateful to M. Guerricabeitia and C. Bourdier For technical and editorial assistance, respectively.
REFERENCES 1. Asperberg P, Thomgren KG, Lohmander LS. Dose dependent stimulation of bone induction by basic fibroblast growth factor in rats. Acta Orthop Stand 1991;62:481-4. Baron J, Klein KO, Yanovski JA, Novosad JA, Bather JD, Bolander ME, Cutler GB. induction of growth plate cartilage ossification by basic fibroblast growth factor. Endocrinology 1994;135:2790-3. Beck LS, Chen TL, Mikaianski P, Ausman AJ. Recombinant human transforming growth factor-p1 (rhTGF/31) enhances healing and strength of granulation skin wounds. Growth Factors 1990;3:267-75. Bolander ME. Regulation of fracture repair by growth factors. Proc Sot Exp Biol Med 1992;200:165-70. Brown GL, Curtsinger LJ, White M, Mitchell RO, Pietsch J, Nordquist R, von Fraunhoger A, Schultz GS. Acceleration of tensile strength of incisions treated with EGF and TFG-f3.Ann Surg 1988;208:788-94.
Surg Neurol 1997;47:242-6
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COMMENTARY
The number and importance of growth factors has increased dramatically in the last months and years. We are most familiar with the now multiple nerve growth factors and their effect on the promotion of nerve recovery as well as the death and
Cuevas et al
removal of expendable nerve cells. This study presents interesting and important new information regarding the use of fibroblastic growth factors at the margins of fractures to affect bone healing and particularly for the regeneration of cranial bone in a well-controlled animal model. The authors suggest that the studied factors may be able to promote large cranioplastic repair, and they further suggest that these factors may be an alternative choice for bone grafts or bone substitutes. At the present time, a number of studies are being conducted in the use of bone morphogenic protein and other factors such as bovine fetal cartilage activation factor for acceleration of fusion development through the mechanism of chondrocyte replacement with subsequent calcification and conversion to bone. Intradiscal fibroblastic growth factors have been shown to accelerate the restoration of glycosaminoglycan synthesis in the intervertebral disc nucleus following the chemical damage resulting from chymopapain injection in an experimental animal model. It is important to note that in this study by Cuevas et al., they found that some growth of new bone was observed on the periphery of small (3 x 6 mm) cranial defects without any sign of adverse tissue reaction. However, the bone had not grown over the center of the defect at 3 weeks. One wonders how long it might take for a large defect to fully close. The method of topical delivery of the otherwise putative growth factor is important as it was embedded in agarose. There are a number of other hydrogels that are capable of slow release of small molecular species such as the growth factors and a few studies have utilized implantable AlzaTM micropumps for this purpose. Clearly, growth factors and their controlled r-elease will play an important role in tissue regeneration and repair in the future. This study presents exciting information regarding this and related factors and their profound effect on local tissue response. Charles D. Ray, M.D., F.A.C.S.
Neurosurgeon Minneapolis, Minnesota