Acta Neurochir (Wien) (2004) 146: 279–283 DOI 10.1007/s00701-003-0190-3

Experimental Study The value of expanded polytetrafluoroethylene in preventing early re-ossification after craniosynostosis surgery: an experimental animal study in the rat T. Calisaneller1, M. Bavbek1 , B. Demirhan2 , H. Caner1, and N. Altino¨rs1 1 2

Department of Neurosurgery, Baskent University Faculty of Medicine, Ankara, Turkey Department of Pathology, Baskent University Faculty of Medicine, Ankara, Turkey

Published online January 8, 2004 # Springer-Verlag 2004

Summary Background. Early re-ossification at the suturectomy site after craniosynostosis surgery remains an important problem. Many surgical methods have been used to address this, including placement of various types of absorbable and non-absorbable material between the bone edges at the site. This experimental study investigated the value of expanded polytetrafluoroethylene (ePTFE) membrane as a barrier to calvarial reclosure after craniosynostosis surgery in rats. Method. Thirty-five 2-week-old Sprague-Dawley rats were divided into two groups. In Group A (n ¼ 17), ePTFE membranes were placed in the defect formed by a left coronal suturectomy. The Group B rats (n ¼ 18) underwent left coronal suturectomy only. Animals were sacrificed at 1, 2 and 4 months postoperatively. In each case, the skull was removed and the operative site was examined for fibrosis, new bone formation, bone bridging, neovascularization and inflammatory response. Findings. The two groups were similar with respect to neovascularization and new bone formation. By the end of the fourth postoperative month, 50% of the Group B specimens showed fibrosis and 50% showed bridging between the bone edges at the suturectomy site. In contrast, at the same stage in Group A, only 16.6% of the specimens exhibited a small amount of fibrosis, and none showed bone bridging between the edges. Interpretation. Expanded PTFE is one of the most inert materials used in surgery. The study showed that inserting ePTFE membrane as a barrier between the bone edges at the suturectomy site prevents early re-ossification after craniosynostosis surgery in rats. Keywords: Craniosynostosis; ePTFE; re-ossification; rat.

Introduction Craniosynostosis is a complex disease which causes both functional and cosmetic problems. The underlying etiology of this condition is poorly understood, but

several disorders are suspected causes, including genetic and metabolic diseases. Craniosynostosis is treated surgically, with early resection of synostotic sutures using open or endoscopic surgical techniques [15, 22, 26, 33, 39]. One of the most important limitations to the effectiveness of this surgery is the early reossification at the suturectomy site [5, 22, 29]. Many surgical methods have been used in attempts to prevent this, including placement of absorbable and nonabsorbable material between the edges [8, 24]; placement of a silicone barrier [6]; folding of the dura [41]; fixation of the suturectomy site using acrylic [34], steel, titanium or polyglactic plates [10, 18, 19]; and performing a large craniectomy [35]. However, all of these procedures have associated problems. For example, non-absorbable material can cause foreign-body reactions, increase the risk of infection, produce artifacts during radiodiagnostic testing, or even migrate [7, 9, 21, 42]. Folding the dura may damage underlying brain tissue, and large craniectomy may result in poor healing of the bone edges, which can lead to serious cosmetic deformities [35]. Currently, there is no satisfactory surgical treatment for early re-ossification of the suturectomy site after craniosynostosis surgery. The aim of this study was to determine the value of expanded polytetrafluoroethylene (ePTFE) membrane as barrier material for preventing this problem.

280

T. Calisaneller et al.

Findings at 1 month

Fig. 1. Surgical technique used to place the ePTFE membrane in the suturectomy defect: A 3 mm-wide left coronal suturectomy extending from the midline to the left temporalis muscle was performed. The dura was dissected from the overlying skull anteriorly, and a 5
10
0.3-mm piece of ePTFE membrane was placed between the dura and the overlying skull anteriorly, and between the skull and the scalp posteriorly

Methods and material Thirty-five 2-week-old Sprague-Dawley rats were used in the experiment. The animals were divided into two groups. Group A (n ¼ 17) underwent left coronal suturectomy and had ePTFE membrane placed between the bone edges at the surgery site, and Group B (n ¼ 18) underwent left coronal suturectomy only. Each rat was anesthetized with intramuscular (i.m.) injections of ketamine 60 mg=kg (Ketalar, Parke Davis, Eczacibasi, Istanbul) and xylazine 9 mg=kg (Rompun, Bayer, Istanbul). The body was immobilized by taping the extremities to the operating table, and the scalp was shaved and cleaned with betadine solution. A midline sagittal scalp incision was made, and the scalp was dissected from the pericranium. For left coronal suturectomy, the left coronal suture was exposed and a 3 mmwide strip was removed from the midline to the left temporalis muscle. In Group A, the dura anterior to the suturectomy site was carefully dissected from the overlying skull. Then a 5
10
0.3-mm strip of ePTFE membrane was placed such that half was between the dura and skull anteriorly, and half was between the skull and scalp posteriorly (Fig. 1). Nothing was inserted to hold the ePTFE membrane in position. In both groups, the skin was closed in one layer with 4=0 silk suture, and each animal received a single i.m. dose of cefazolin 25 mg=kg (Sefazol, Mustafa Nevzat, Istanbul). Upon recovery from anesthesia, each rat was returned to its cage. There was no mortality during follow-up. Animals from both groups were sacrificed at 1, 2 and 4 months postoperatively. In each case, the skull was removed and a specimen including the operative site was prepared for histological examination. All specimens were embedded in paraffin and stained with hematoxylin–eosin. A Zeiss Axioplan light microscope was used to assess for fibrosis, new bone formation, bone bridging, neovascularization and inflammatory response.

At the end of the first postoperative month, one of the five (20%) specimens from Group A showed minimal fibrosis, four showed new bone formation (80%), all five showed neovascularization (100%), and one showed inflammatory changes (20%). In Group B, four of the six specimens showed minimal fibrosis (66.6%), three showed new bone formation (50%), four showed neovascularization (66.6%), and two showed inflammatory changes (33.3%). None of the specimens from either group showed bone bridging between the edges of the suturectomy site. All three of the specimens which exhibited inflammatory changes contained hair follicles, and the hair was suspected to have caused the inflammation. Findings at 2 months At 2 months post-surgery, none of the six specimens from Group A showed fibrosis, all six showed new bone formation (100%), and four showed neovascularization (66.6%). In Group B, two of the six specimens showed fibrosis (33.3%), five showed new bone formation (83.3%), and five showed neovascularization (83.3%). None of the tissues from either group exhibited bone bridging or inflammatory changes. Findings at 4 months At 4 months postoperatively, one of the six specimens from Group A showed minimal fibrosis (16.6%), all six rats showed new bone formation (100%), and all six

Results At the end of the first postoperative month, five rats from Group A and six from Group B were sacrificed. At 2 months post-surgery, six rats from Group A and six from Group B were sacrificed. The remaining six rats from each group were sacrificed at 4 months post-surgery.

Fig. 2. Histological appearance of the ePTFE membrane between the bone edges at the suturectomy site at 4 months post-surgery: Note the neovascularization and new bone formation at both edges, but no bone bridging between the edges. There is no foreign-body reaction. (H&E
50)

The value of ePTFE in preventing early re-ossification after craniosynostosis surgery

showed neovascularization (100%). There was no bridging between the bone edges of the suturectomy site, nor any inflammatory changes in the operative field (Fig. 2). In Group B, three of the six specimens showed fibrosis (50%), all six showed new bone formation (100%), and all six showed neovascularization (100%). There were no inflammatory changes at the suturectomy site in Group B; however, three of the six specimens (50%) showed bridging between the bone edges of the site.

Discussion Craniosynostosis is a heterogeneous disease with severe cosmetic and functional consequences. The etiology is still not clear, but recent studies suggest that mutations of the genes for fibroblast growth factor receptors (FGFR-1, FGFR-2 and FGFR-3), MSX2 and TWIST may cause this condition [12, 14, 16, 25, 32, 36]. It has also been argued that transforming growth factor beta (TGF-beta) subtypes have a strong influence on suture patency in settings where TGF-beta 3 is necessary for suture patency and TGF-beta 2 induces sutural closure [30, 38]. Aberrant paracrine interactions between cranial sutures and the underlying dura can also induce early closure of cranial sutures [4]. Despite some encouraging experimental results with gene therapy [11, 23], the current treatment for all types of craniosynostosis involves early resection of the problem suture or sutures by open or endoscopic surgical techniques. The main impediment to success with this procedure is the early re-ossification at the suturectomy site. As mentioned above, the literature describes several surgical methods for addressing this issue, including placement of absorbable and non-absorbable material between the edges of the suturectomy site. However, inserting foreign material has potential problems, such as foreignbody reaction, increased infection risk, creation of artifacts during radiodiagnostic tests, migration of inserted material, and damage to underlying brain tissue. In this study, we placed ePTFE membrane in suturectomy defects in rats and assessed whether this material prevented or delayed re-ossification. Expanded PTFE is an inert material with a broad range of surgical uses. Cardiovascular surgeons use it for cardiovascular grafting, general surgeons use it to repair abdominal wall defects and tears in other structures, and neurosurgeons use it as a substitute for cranial and spinal dura [1, 3, 13, 31, 40, 43]. In an experimental study in rats, Mardas et al. showed that covering cerebral and galeal aspects of suturectomy defects with ePTFE membranes for guided

281

tissue regeneration, which allows a desired suturectomy defect healing by ensuring the determined suture precursor cells from the remaining part of the suture to migrate into the defective area, provided a suture-like tissue healing in the suturectomy field [20]. Expanded PTFE is composed of microfibers and is highly biocompatible [17]. It has no systemic toxic, carcinogenic or mutagenic effects, and does not produce radiodiagnostic artifacts. In addition, it is not allergenic, pyrogenic or hemolytic, and the tissue response in areas where ePTFE is employed is as favorable as that observed with other materials [2]. Our results indicate that placing ePTFE membrane in the suturectomy defect between the dura and the overlying skull has no effect on neovascularization or new bone formation. However, at 4 months post-surgery, we observed that ePTFE effectively prevented bone bridging between the edges of the suturectomy site. This may be due to direct effects of the material as a mechanical barrier between the bone edges, or as a blocker of paracrine interactions between the dura and the overlying skull. In order to find out how our treatment model affects future ossification in the suturectomy defects at a given age, one should use a spontaneous craniosynostosis model and perform both cephalometric measurements and histopathological examinations in the very late stages of the postoperative period. In the literature, only a few in vivo spontaneous craniosynostosis models are available for experimental studies [27]. However, it must be underlined that the animals in this study were naive Sprague-Dawley rats who were not predisposed to craniosynostosis. In naive rats the posterior-frontal sutures fuses between postnatal days 12 and 22 while all other sutures including the sagittal and coronal suture remain patent for the rest of the life [28]. Since it has been suggested in the literature that surgical treatment for craniosynostosis would be optimal when performed before postnatal sixth month, we thought choosing an operation time at the end of second postnatal week of rats might represent this period in man. Also, unimpeded dura-suture interaction is necessary for normal suture closure, and research has shown that inserting a silastic membrane to separate the dura from the overlying suture delays suture closure in Sprague-Dawley rats [37]. We observed no foreign-body reactions to ePTFE, but the surrounding tissue in one rat showed an inflammatory response. This was the only animal in the ePTFE group in which the surgery site was contaminated with hair, and we believe the hair caused the inflammatory response in this case.

282

Conclusions Although the etiology of craniosynostosis remains unclear, the results of this study suggest that ePTFE functions as a physical barrier between the bone edges at the suturectomy site, and between the dura and the overlying cranium. Further research is needed in order to contemplate insertion of ePTFE as an alternative to other surgical methods for preventing early re-ossification of the suturectomy site after non-syndromic single suture craniosynostosis surgery. References 1. Balen EM, Diez-Caballero A, Hernandez-Lizoain JL, Pardo F, Torramade JR et al (1998) Repair of ventral hernias with expanded polytetrafluoroethylene patch. Br J Surg 85: 1415–1418 2. Batniji RK, Hutchison JL, Dahiya R, Lam SL, Williams EF 3rd (2002) Tissue response to expanded polytetrafluoroethylene and silicone implants in a rabbit model. Arch Facial Plast Surg 4: 111–113 3. Bhatnagar G, Fremes SE, Christakis GT, Goldman BS (1998) Early results using an ePTFE membrane for pericardial closure following coronary bypass grafting. J Card Surg 13: 190–193 4. Bradley JP, Han VK, Roth DA, Levine JP, McCarthy JG, Longaker MT (1999) Increased IGF-I and IGF-II mRNA and IGF-I peptide in fusing rat cranial sutures suggest evidence for a paracrine role of insulin-like growth factors in suture fusion. Plast Reconstr Surg 104: 129–138 5. Drake DB, Persing JA, Berman DE, Ogle RC (1993) Calvarial deformity regeneration following subtotal craniectomy for craniosynostosis: a case report and theoretical implications. J Craniofac Surg 4: 85–89; discussion 90 6. Duff TA, Mixter RC (1991) Midline craniectomy for sagittal suture synostosis: comparative efficacy of two barriers to calvarial reclosure. Surg Neurol 35: 350–354 7. Duke BJ, Mouchantat RA, Ketch LL, Winston KR (1996) Transcranial migration of microfixation plates and screws. Case report. Pediatr Neurosurg 25: 31–34; discussion 35 8. Fatah MF, Ermis I, Poole MD, Shun-Shin GA (1992) Prevention of cranial reossification after surgical craniectomy. J Craniofac Surg 3: 170–172 9. Fiala TG, Novelline RA, Yaremchuk MJ (1993) Comparison of CT imaging artifacts from craniomaxillofacial internal fixation devices. Plast Reconstr Surg 92: 1227–1232 10. Gewalli F, da Silva Guimaraes-Ferreira JP, Maltese G, Ortengren U, Lauritzen C (2001) Expander elements in craniofacial surgery: an experimental study in rabbits. Scand J Plast Reconstr Surg Hand Surg 35: 149–156 11. Greenwald JA, Mehrara BJ, Spector JA, Warren SM, Fagenholz PJ et al (2001) In vivo modulation of FGF biological activity alters cranial suture fate. Am J Pathol 158: 441–452 12. Gripp KW, McDonald-McGinn DM, Gaudenz K, Whitaker LA, Bartlett SP et al (1998) Identification of a genetic cause for isolated unilateral coronal synostosis: a unique mutation in the fibroblast growth factor receptor 3. J Pediatr 132: 714–716 13. Ishikawa M, Yamazaki T, Yano H, Fujikawa T, Konagai N et al (1993) Six year experience with wrinkled ePTFE vascular prostheses for arteriosclerosis obliterans. Asaio J 39: M522–M525 14. Jabs EW, Muller U, Li X, Ma L, Luo W et al (1993) A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 75: 443–450

T. Calisaneller et al. 15. Jimenez DF, Barone CM, Cartwright CC, Baker L (2002) Early management of craniosynostosis using endoscopic-assisted strip craniectomies and cranial orthotic molding therapy. Pediatrics 110: 97–104 16. Johnson D, Horsley SW, Moloney DM, Oldridge M, Twigg SR et al (1998) A comprehensive screen for TWIST mutations in patients with craniosynostosis identifies a new microdeletion syndrome of chromosome band 7p21.1. Am J Hum Genet 63: 1282–1293 17. Kowligi RR, Taylor HH, Wollner SA (1993) Physical properties and testing methods for PTFE cardiovascular patches. J Biomater Appl 7: 353–361 18. Losken A, Williams JK, Burstein FD, Cohen SR, Hudgins R et al (2001) Outcome analysis for correction of single suture craniosynostosis using resorbable fixation. J Craniofac Surg 12: 451–455; discussion 456–457 19. Maggi G, Petrone G, Aliberti F, Pittore L (1993) The operative treatment of craniosynostosis using bone plate and screw fixation (titanium micro system). J Neurosurg Sci 37: 157–160 20. Mardas N, Kostopoulos L, Karring T (2002) Bone and suture regeneration in calvarial defects by e-PTFE-membranes and demineralized bone matrix and the impact on calvarial growth: an experimental study in the rat. J Craniofac Surg 13: 453–462; discussion 462–464 21. Matic D, Phillips JH (2002) A contraindication for the use of hydroxyapatite cement in the pediatric population. Plast Reconstr Surg 110: 1–5 22. McCarthy JG, Epstein F, Sadove M, Grayson B, Zide B (1984) Early surgery for craniofacial synostosis: an 8-year experience. Plast Reconstr Surg 73: 521–533 23. Mehrara BJ, Spector JA, Greenwald JA, Ueno H, Longaker MT (2002) Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion. Plast Reconstr Surg 110: 506–514 24. Merikanto JE, Alhopuro S, Ritsila VA (1987) Free fat transplant prevents osseous reunion of skull defects. A new approach in the treatment of craniosynostosis. Scand J Plast Reconstr Surg Hand Surg 21: 183–188 25. Meyers GA, Orlow SJ, Munro IR, Przylepa KA, Jabs EW (1995) Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans. Nat Genet 11: 462–464 26. Mohr G, Hoffman HJ, Munro IR, Hendrick EB, Humphreys RP (1978) Surgical management of unilateral and bilateral coronal craniosynostosis: 21 years of experience. Neurosurgery 2: 83–92 27. Mooney MP, Losken HW, Tschakaloff A, Siegel MI, Losken A et al (1993) Congenital bilateral coronal suture synostosis in a rabbit and craniofacial growth comparisons with experimental models. Cleft Palate Craniofac J 30: 121–128 28. Moss ML (1958) Fusion of the frontal suture in the rat. Am J Anat 102: 141–166 29. Norwood CW, Alexander E Jr, Davis CH Jr, Kelly DL Jr (1974) Recurrent and multiple suture closures after craniectomy for craniosynostosis. J Neurosurg 41: 715–719 30. Opperman LA, Adab K, Gakunga PT (2000) Transforming growth factor-beta 2 and TGF-beta 3 regulate fetal rat cranial suture morphogenesis by regulating rates of cell proliferation and apoptosis. Dev Dyn 219: 237–247 31. Park YK, Tator CH (1998) Prevention of arachnoiditis and postoperative tethering of the spinal cord with Gore-Tex surgical membrane: an experimental study with rats. Neurosurgery 42: 813–823; discussion 23–24 32. Passos-Bueno MR, Sertie AL, Richieri-Costa A, Alonso LG, Zatz M et al (1998) Description of a new mutation and characterization of FGFR1, FGFR2, and FGFR3 mutations among Brazilian patients with syndromic craniosynostoses. Am J Med Genet 78: 237–241

The value of ePTFE in preventing early re-ossification after craniosynostosis surgery 33. Persing J, Babler W, Winn HR, Jane J, Rodeheaver G (1981) Age as a critical factor in the success of surgical correction of craniosynostosis. J Neurosurg 54: 601–606 34. Persing JA, Cronin AJ, Delashaw JB, Edgerton MT, Henson SL, Jane JA (1987) Late surgical treatment of unilateral coronal synostosis using methyl methacrylate. J Neurosurg 66: 793–799 35. Prevot M, Renier D, Marchac D (1993) Lack of ossification after cranioplasty for craniosynostosis: a review of relevant factors in 592 consecutive patients. J Craniofac Surg 4: 247–254; discussion 55–56 36. Reardon W, Winter RM, Rutland P, Pulleyn LJ, Jones BM, Malcolm S (1994) Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet 8: 98–103 37. Roth DA, Bradley JP, Levine JP, McMullen HF, McCarthy JG, Longaker MT (1996) Studies in cranial suture biology: part II. Role of the dura in cranial suture fusion. Plast Reconstr Surg 97: 693–699 38. Roth DA, Gold LI, Han VK, McCarthy JG, Sung JJ et al (1997) Immunolocalization of transforming growth factor beta 1, beta 2, and beta 3 and insulin-like growth factor I in premature cranial suture fusion. Plast Reconstr Surg 99: 300–309; discussion 310–316 39. Stelnicki EJ (2002) Endoscopic treatment of craniosynostosis. Atlas Oral Maxillofac Surg Clin North Am 10: 57–72 40. van der Lei B, Bleichrodt RP, Simmermacher RK, van Schilfgaarde R (1989) Expanded polytetrafluoroethylene patch for the repair of large abdominal wall defects. Br J Surg 76: 803–805 41. van der Werf AJ (1977) [Craniosynostoses (17 years of experience with a new surgical technic)]. Neurochirurgie 23: 491–502 42. Winkler PA, Herzog C, Weiler C, Krishnan KG (2000) Foreignbody reaction to silastic burr-hole covers with seroma formation: case report and review of the literature. Pathol Res Pract 196: 61–66

283

43. Yamagata S, Goto K, Oda Y, Kikuchi H (1993) Clinical experience with expanded polytetrafluoroethylene sheet used as an artificial dura mater. Neurol Med Chir (Tokyo) 33: 582–585

Comment This is a well written paper dealing with the use of a relatively new membrane (polytetrafluoroethylene {ePTFE}) to act as a barrier to prevent re-ossification after surgery for craniosynostosis. While I feel the study has been well performed, I would like to make the following points: While the authors describe restenosis as a problem associated with simple suturectomy, they do not discuss the fact that this is now rarely performed (other than for sagittal synostosis in very young babies). Once one is into remodelling of the head shape, the use of a membrane to prevent re-ossification becomes of much less relevance. Furthermore, although the authors say that re-ossification is a problem, they do not quantify it. My own impression (clinical and from other literature) is that with modern craniofacial techniques, re-operation for re-ossification is relatively rare and would seem to be related to the age at which surgery is undertaken. The authors acknowledge the fact that this is obviously not a model for synostosis and therefore the reproducibility of the results in a clinical setting may be different. P. D. Chumas

Correspondence: Murad Bavbek, M.D., Baskent University Faculty of Medicine, Department of Neurosurgery, Fevzi C° akmak Caddesi 10. Sok No: 45, C-Blok Kat:1, Ankara, Turkey 06490. e-mail: mbavbek@ baskent-ank.edu.tr