EXPERIMENTAL Guided Tissue Regeneration Enhances Bone Formation in a Rat Model of Failed Osteogenesis Tony D. Fang, M.D. Randall P. Nacamuli, M.D. Han Joon M. Song, M.D. Kenton D. Fong, M.D. Yun-Ying Shi, B.S. Michael T. Longaker, M.D., M.B.A. Stanford, Calif.

Background: Guided tissue regeneration is a technique that uses barrier materials to enhance tissue regeneration. Although previously demonstrated to be an effective way of enhancing craniofacial osteogenesis in several animal models, the ability of guided tissue regeneration to augment bone formation in the context of distraction osteogenesis is unknown. In the current study, the authors applied the principle of guided tissue regeneration to their rat mandibular distraction osteogenesis model in an attempt to enhance bone regeneration. Methods: Twelve (n ⫽ 6 per group) adult Sprague-Dawley rats underwent routine gradual distraction (5 days’ latency, 4-mm distraction over 8 days, 4 to 6 weeks of consolidation) and acute distraction (immediate lengthening to 4 mm, 6 to 8 weeks of consolidation). An additional 10 animals underwent acute distraction followed by application of bioabsorbable Gore Resolut XT membranes (acute distraction plus guided tissue regeneration). Membranes were completely wrapped around the distraction gap. Animals were killed 6 and 8 weeks postoperatively and mandibles analyzed radiographically and histologically. Results: Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Gradual distraction mandibles achieved bony union by 6 weeks with 86 percent bone formation, which increased to 98 percent by 8 weeks. Acute distraction mandibles healed with a fibrous nonunion and only 37 percent bone formation by 8 weeks. In contrast, acute distraction plus guided tissue regeneration–treated mandibles formed significantly more bone than acute distraction mandibles by 6 weeks (57 percent) and achieved bony bridging by 8 weeks, with 88 percent new bone formation. Conclusion: The authors’ data demonstrate that guided tissue regeneration can significantly enhance bone formation in a fibrous nonunion model of mandibular distraction osteogenesis. (Plast. Reconstr. Surg. 117: 1177, 2006.)

D

istraction osteogenesis is a form of endogenous tissue engineering that uses controlled mechanical distraction to guide regeneration of bone. Gavril Ilizarov first described this technique and elicited the principles of distraction osteogenesis using a long bone distraction model in the 1950s.1–3 Since 1989, From the Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine. Received for publication September 15, 2004; revised November 30, 2004. The first two authors contributed equally to this work. Copyright ©2006 by the American Society of Plastic Surgeons DOI: 10.1097/01.prs.0000204581.59190.53

distraction osteogenesis has been widely applied to the craniofacial skeleton to treat both congenital and acquired bony deficiencies.4 –14 Despite its clinical success, distraction osteogenesis usually requires substantial treatment time and is intermittently complicated by device dislodgement, infection, and nonunion.15 Methods that accelerate bone formation and decrease the chance of nonunion would be a welcome advance for surgeons performing distraction osteogenesis on the craniofacial skeleton. Guided tissue regeneration, a technique first described by Nyman et al.16 and Karring and Warrer17 over 10 years ago, was initially used to restore lost alveolar bone before further periodontal treatment. Since that time, guided tissue

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Plastic and Reconstructive Surgery • April 1, 2006 regeneration has been widely used clinically to treat alveolar and periodontal bony defects and to augment bone formation in conjunction with dental implants.18 Conceptually, guided tissue regeneration enhances osteogenesis by creating a relatively isolated space and prevents the prolapse of surrounding soft tissues into the bony gap. Maintaining the regenerative space is thought to augment repopulation of the defect with local, regional, or systemically derived progenitor cells, thereby promoting tissue induction. A variety of scaffolding materials have been used as barrier materials for osseous guided tissue regeneration. These materials may be derived from natural substances, such as bovine or porcine type I collagen, or from synthetic polymers, such as expanded polytetrafluoroethylene, polylactic acid, and polyglycolic acid.19 Some barrier materials are nonabsorbable (expanded polytetrafluoroethylene), whereas others are biodegradable (polylactic acid and polylactic acid).19 In addition, some of the scaffolding materials used are osteoconductive, facilitating osteoblastic cell attachment, migration, proliferation, and differentiation.20 –22 One of these materials is Gore Resolut XT (W. L. Gore and Associates, Inc., Flagstaff, Ariz.), a membrane material made of a random fiber matrix of polylactic acid, polyglycolic acid, and trimethylene carbonate on either side of a film that is impermeable to cells.23 In our rat mandibular distraction osteogenesis model, acute distraction, or immediate lengthening at the time of operation followed by 6 weeks of consolidation, leads to fibrous nonunion, whereas gradual distraction (5 days of latency followed by 0.25-mm distraction twice a day for 8 days, with 4 weeks of consolidation) yields a bony union.24,25 Guided tissue regeneration has been used experimentally in animal models to enhance bone formation in several portions of the craniofacial skeleton, including the calvarium, zygoma, hard palate, and mandible.26 –32 For example, Stal et al. used a resorbable polymer of polylactic acid and polyglycolic acid to enhance bone formation in a lagomorph model of calvarial defect healing.33 However, it has not been studied in detail as an adjunct to enhance mandibular osteogenesis and prevent fibrous nonunion in the context of mandibular lengthening procedures. In the current study, we determined the ability of guided tissue regeneration using the Gore Resolut XT membrane to enhance bone formation during mandibular lengthening in our non-

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healing rat acute distraction model. The outcomes were compared with mandibles undergoing either acute distraction alone (without the membrane) or gradual distraction at 6 and 8 weeks postoperatively. Our data demonstrate that by 8 weeks acute distraction mandibles treated with guided tissue regeneration exhibited bony bridging and had filled 88 percent of the distraction gap with new bone, nearly achieving the levels of bone formation seen attained using gradual distraction (98 percent). In contrast, mandibles treated with acute distraction alone demonstrated only 37 percent bone formation and had no bony bridging 8 weeks after surgery. These findings support the application of guided tissue regeneration techniques to enhance osteogenesis of the craniofacial skeleton and, in particular, the mandible.

MATERIALS AND METHODS Animals All experiments were performed in accordance with Stanford University Animal Care and Use Committee guidelines. Adult male SpragueDawley rats weighing between 300 and 400 g were purchased from Simonsen Laboratories, Inc. (Gilroy, Calif.). Animals were housed in a light- and temperature-controlled environment and given food and water ad libitum. Gore Resolut XT Membrane and Suture The Gore Resolut XT membranes and sutures used in this study were generously provided by W. L. Gore and Associates. They were both bioabsorbable. The material for the membrane is composed of a cell-occlusive, absorbable membrane sandwiched between a porous random matrix composed of polylactic acid, polyglycolic acid, and trimethylene carbonate copolymer fibers. This configuration prevents surrounding soft tissue from prolapsing into the wound area. The suture is made of polyglycolic acid coated with polycaprolactone. Both membrane and suture remain essentially unchanged for 8 to 10 weeks before beginning to biodegrade. Thus, they were intact throughout the entire time course of our study. Surgery The surgical procedures for our rat distraction osteogenesis model have been previously described.24,25,34 Briefly, animals were anesthetized (20 mg/kg Ketaset (Bristol Laboratories, Syracuse, N.Y.), 4 mg/kg xylazine, and 0.5 mg/kg

Volume 117, Number 4 • Guided Tissue Regeneration acepromazine maleate), given a preoperative dose of antibiotics (10 mg/kg cefazolin), prepared with povidone-iodine, and the incisors clipped. An incision was made over the right hemimandible, the masseter muscle was divided, and the mandible was exposed. An osteotomy was performed between the second and third molars using a diamond disk saw under constant saline irrigation. Two Flexi-Post pins (Essential Dental Systems, Hackensack, N.J.) were placed 4 mm anterior and posterior to the osteotomy. For gradual distraction– and acute distraction–treated animals, the muscle and skin were then closed in layers and a custom-made distraction device was fixed to the pins. Acute distraction mandibles were then immediately distracted 4 mm. For the acute distraction plus guided tissue regeneration group, the distraction device was fitted onto the two Flexi-Post pins before the skin closure. An on-table distraction of 4 mm was performed at this point. The inferior border and lingual portion of the right mandible was then bluntly dissected free of soft tissue. A 15 ⫻ 20-mm Gore Resolut XT membrane (Fig. 1, above) was wrapped around the distraction gap circumferentially. The final configuration of the membrane covered the entire space of the distraction gap and extended to the anterior and posterior pins (Fig. 1, center and below). The membrane was approximated at the superior portion of the mandible with a 4-0 Gore Resolut suture. At this point, the soft tissue and skin were closed as described above. Postoperatively, animals received sterile food and drinking water containing sulfamethoxazole for infection prophylaxis. Distraction Protocols Animals were divided into three experimental groups in this study: acute distraction (n ⫽ 6), gradual distraction (n ⫽ 6), and acute distraction plus guided tissue regeneration (n ⫽ 10). Both gradual distraction and acute distraction treatments were carried out as previously described, with acute distraction plus guided tissue regeneration animals being treated identically to acute distraction animals postoperatively.25 Our gradual distraction treatment consisted of a 5-day latency period after initial osteotomy and fixation of the distraction device, 8 days of distraction (0.25 mm two times per day), and 4 or 6 weeks of consolidation, for a total procedure time of 6 to 8 weeks. For our nonhealing acute distraction protocol, the distraction device was placed after immediate, on-table lengthening to 4 mm (same as total displacement in the gradual distraction group) and left in place with no distraction performed dur-

Fig. 1. (Above) The size and architecture of the Gore Resolut XT membrane used in the study. (Center) Schematic of a mandible undergoing acute distraction. The center white area between the Flexi-Post pins represents the distraction gap. (Below) Schematic depiction of the acute distraction plus guided tissue regeneration model. Note that the distraction gap was completely wrapped with Gore Resolut XT membrane.

ing the entire 6- to 8-week treatment period. Thus, all animals in all groups were subjected to the same total amount of mandibular lengthening. All three groups of animals were killed and mandibles were harvested at 6 and 8 weeks (n ⫽ 3 for gradual distraction and acute distraction at each time point; n ⫽ 5 for acute distraction plus guided tissue regeneration at each time point) postoperatively. Radiographic Imaging and Histomorphometric Analysis After harvesting at 6 and 8 weeks postoperatively, the distraction devices were removed from

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Plastic and Reconstructive Surgery • April 1, 2006 the mandibles. All the specimens were fixed in 4% paraformaldehyde for 48 hours before imaging with a high resolution Faxitron cabinet x-ray system (model 43855A; Faxitron X-Ray Corporation, Wheeling, Ill.). All the images were acquired at 40 kVP for 15 seconds. After imaging, the specimens were demineralized with Formical 2000 (Decal Company, Congers, N.Y.) and embedded in paraffin, and 5-␮m were sections cut. Hematoxylin and eosin staining was performed following standard protocols.35 Digital photomicrographs were taken with a Leica Axioplan, and histomorphometrically analyzed using Scion image analysis software (Scion Corporation, Frederick, Md.). Briefly, the area of new trabecular bone within the distraction gap was calculated and compared with the total distraction gap area, with the ratio of these two values representing the percentage of new bone formed within the distraction gap. Ten slides per specimen were analyzed in this manner and the average new bone percentage calculated for each specimen. Two examiners reviewed all slides in a blinded and independent fashion. Oneway analysis of variance with Bonferroni post hoc analysis was used to compare average bone formation within the gap between groups at each time point, with a value of p ⱕ 0.01 considered to be statistically significant.

RESULTS The operative procedures were well tolerated by all animals. There were no intraoperative or postoperative deaths, and all 22 animals com-

pleted the study without evidence of device infection or dislodgement. Faxitron Images At 6 weeks postoperatively, specimens in the acute distraction group clearly showed a radiolucency between the anterior and posterior pins, consistent with the expected outcome of a fibrous nonunion (Fig. 2, above, left). Specimens from the gradual distraction group were radiopaque within the distraction gaps (Fig. 2, above, center), consistent with the bony union normally seen 6 weeks after operation. Images of specimens from the acute distraction plus guided tissue regeneration group were similar in appearance to the gradual distraction group, demonstrating increased bone formation versus acute distraction and suggesting that a bony union was attained (Fig. 2, above, right). At 8 weeks following the initial operation, images of acute distraction specimens still showed evidence of a nonunion (Fig. 2, below, left). In marked contrast, the radiographic images of acute distraction plus guided tissue regeneration and gradual distraction appeared almost identical to an apparent complete bony union (Fig. 2, below, center and right). Hematoxylin and Eosin Staining and Histomorphometric Analysis In our rat mandibular distraction osteogenesis model, animals undergoing gradual distraction achieve nearly complete bony union 6 weeks after

Fig. 2. Radiographic images of mandibles. (Above) Mandibles after 6 weeks of total treatment time. (Below) Mandibles after 8 weeks of total treatment time.

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Volume 117, Number 4 • Guided Tissue Regeneration surgery, whereas those animals undergoing acute distraction form a fibrous nonunion.24,25 The results of the current study were consistent with our previous reports. Six weeks after surgery, there was only minimal new bone formation in the acute distraction specimens, with the area of new bone formation limited to a zone immediately adjacent to the osteotomies (Fig. 3, above, left). Quantification of new bone formation demonstrated an increase in bone in the distraction gap of 28 ⫾ 4.8 percent (Fig. 4) The gradual distraction group showed the highest level of bone formation (Fig. 3, above, center), with bony union seen in all specimens and new bone nearly completely filling the distraction gaps (86 ⫾ 11 percent) (p ⱕ 0.001), gradual distraction versus acute distraction (Fig. 4). In contrast, acute distraction plus guided tissue regeneration specimens demonstrated significant bone formation when compared with acute distraction specimens (Fig. 3, above, right), with immature trabecular bone occupying more than half of the distraction gap (57 ⫾ 17 percent) (p ⬍ 0.001) (Fig. 4). Eight weeks after surgery, all acute distraction specimens continued to demonstrate a fibrous nonunion (Fig. 3, below, left), with only a moderate, statistically insignificant increase in the percentage of new bone formation when compared with acute distraction specimens collected after 6 weeks of consolidation (37 ⫾ 4.2 percent) (p ⬎ 0.05) (Fig. 4). Gradual distraction specimens con-

tinued to have the greatest amount of bone formation (98 ⫾ 2.0 percent) within the distraction gap (Figs. 3, below, center and 4). Specimens from the acute distraction plus guided tissue regeneration group demonstrated markedly increased bone formation (Fig. 3, below, center), which remained significantly greater than bone formation in the acute distraction group (88 ⫾ 11 percent) (p ⱕ 0.001), acute distraction plus guided tissue regeneration versus acute distraction (Fig. 4). This also represented a significant increase in the amount of bone seen at 6 weeks in the acute distraction plus guided tissue regeneration group (p ⱕ 0.001) (Fig. 4). The distribution and morphology of the new bone was nearly identical to that of the gradual distraction specimens, with newly formed trabecular bone in the center of the distraction gap and more mature woven bone at the periphery adjacent to the osteotomies. Interestingly, by the 8-week time point, there was no significant difference in the amount of bone seen in gradual distraction specimens versus acute distraction plus guided tissue regeneration specimens (p ⬎ 0.05) (Fig. 4).

DISCUSSION Despite the overall excellent clinical outcomes obtained when using distraction osteogenesis to treat disorders such as craniofacial microsomia, Treacher-Collins syndrome, and micrognathia, numerous complications may still arise at signifi-

Fig. 3. Hematoxylin and eosin staining of mandibles. (Above) Mandibles after 6 weeks of total treatment time. (Below) Mandibles after 8 weeks of total treatment time. f, fibrous tissue; t, new trabecular bone; bu, bony union; AD, acute distraction; GTR, guided tissue regeneration; dotted line, site of original osteotomy.

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Fig. 4. Histomorphometric analysis of new bone formation. (Above) Illustration of the methods for calculating the percentage of new bone within the distraction gap. a, new trabecular bone area within the distraction gap; b, center mesenchymal tissue; percentage new bone formation ⫽ (a/b) ⫻ 100. (Below) Plot of new bone formation within the distraction gap. *p ⬎ 0.05, **p ⱕ 0.001; all statistical comparisons are to the acute distraction plus guided tissue regeneration group. AD, acute distraction; GTR, guided tissue regeneration.

cant rates even for the most experienced of surgeons. In a recent comprehensive review of outcomes for over 3000 patients undergoing craniofacial distraction, the incidence of several complications was assessed, including hardware failure (4.5 percent), device dislodgement (3.0 percent), and infection (5.2 percent).36 Serious infection requiring premature removal of hardware occurred in 0.9 percent of patients. As these complications are logically associated to a greater degree with the overall treatment time, techniques to enhance osteogenesis and thereby either decrease the amount of time spent on dis-

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traction or consolidation could very well lead to a decrease in the rate of complications. In this study, we asked whether or not guided tissue regeneration would enhance osteogenesis in our acute distraction model of fibrous union. Our data demonstrate that guided tissue regeneration enhanced bone formation in a mandibular model of failed osteogenesis, converting a fibrous nonunion to a bony union. Importantly, these results demonstrate that guided tissue regeneration can be used to achieve bony union in the setting of acute distraction, with the resultant total mandibular lengthening being equal to that of gradual distraction in our model. We recognize, however, that this does not take into account potential limitations to the degree of acute lengthening achievable in the clinical setting, where the soft-tissue envelope surrounding the mandible may limit the total distraction acutely attempted and promote relapse. These results are very encouraging, as the ability of guided tissue regeneration to convert a nonhealing environment into a healing environment and convert a fibrous nonunion to bony union suggests that this technique may be a powerful method with which to augment osteogenesis during skeletal regeneration. Several potential mechanisms may be responsible for the increased healing seen in our study. In our previous studies, we demonstrated that the mechanical environment induced by our fibrous union model (acute distraction) was clearly different from the model leading to bony union (gradual distraction).37 This mechanical environment, which consists of initial high strain and relatively less fixation (because of the larger distraction gap) favors the generation of fibrous tissue or fibrocartilage tissue.38 Additional analyses have demonstrated that the transcriptional response induced in the regenerate by acute distraction and gradual distraction is also specific, with marked differences observed in the pattern of expression of genes associated with osteogenesis, angiogenesis, mechanotransduction, and hypoxia.39 In summary, the osteogenic environment created by acute distraction is inferior to that of the gradual distraction model as evidenced by the inability of acute distraction–treated mandibles to form significant bone or produce bony bridging. As such, one potential explanation for the enhanced osteogenesis seen with acute distraction plus guided tissue regeneration may be a favorable alteration in the mechanical environment. It is conceivable that by wrapping the distraction gap with a barrier membrane, additional stabilization was provided,

Volume 117, Number 4 • Guided Tissue Regeneration modifying the mechanical environment in favor of bone formation.38 Improved stability could also have helped to maintain the structural integrity of new osteoid and newly formed blood vessels, enhancing angiogenesis, which is normally disrupted during acute distraction.39 However, it is unlikely that the major factor leading to increased bone formation was increased stability of the osteotomy, as the distraction device provides rigid fixation, and we would predict that minimal (if any) stabilization would be provided by application of the relatively flexible barrier membrane. One of the most commonly held beliefs regarding the ability of guided tissue regeneration to enhance wound healing is that the barrier membrane prevents the prolapse of surrounding soft tissues into the wound, preserving the space for cells with regenerative capabilities.26 This ensures that the majority of the cells in the wound are capable of repairing the damaged tissue, enhancing tissue regeneration. In the context of an osseous defect, wrapping the ends of a fractured or osteotomized bone in a barrier membrane excludes muscle and connective tissue, allowing repopulation of the fracture site with locally or systemically derived cells capable of osteoblastic differentiation. The Gore Resolut XT membrane used in our current study contains a cell-impermeable film, which would theoretically further enhance retention of osteoprogenitor cells and exclude external, confounding progenitor cells such as muscle satellite cells.23 Another possible explanation for the enhanced bone formation and bony union seen in acute distraction plus guided tissue regeneration–treated mandibles lies in the osteoconductive properties of the Gore Resolut XT membrane. Osteoconductive materials are those that promote the attachment, proliferation, and migration of committed osteoprogenitor cells, therefore potentiating the deposition of new bone.40 In a study addressing the ability of various barrier materials to promote cellular attachment, Wang et al. found that Gore Resolut XT membranes enhanced osteoblast attachment when compared with traditional expanded polytetrafluoroethylene membranes.22 A separate study has investigated the ability of osteoblastic cells to migrate on various types of guided tissue regeneration membranes. Takata et al. showed that cell migration on various Gore Resolut membranes was greater than migration on natural scaffold materials such as collagen or other synthetic materials including expanded polytetrafluoroethylene and cellulose.21 Considered together, the results of these studies suggest that guided tissue regeneration treatment of

acute distraction mandibles resulted in bony union for several reasons, not solely attributable to the potential barrier effect, including the membrane’s ability to enhance cell migration, attachment, proliferation, and differentiation. The clinical implications of this study relate to accelerating osteogenesis with guided tissue regeneration during distraction procedures, with the goal being to decrease the overall treatment time. Benefits of decreased treatment time would theoretically include decreased rates of infection and device failure, and an increase in compliance and patient comfort. These goals could be accomplished in two ways, either by augmenting bone formation in the setting of distraction osteogenesis or by converting nonhealing, critical sized gaps to healing defects. In the former case (augmentation of distraction osteogenesis), guided tissue regeneration could be used as an adjuvant therapy to accelerate distraction-mediated osteogenesis. Thus, it is possible that increased rates of distraction and/or an increase in the length of daily distraction could be achieved, allowing similar distraction lengths to be realized in a shorter period of time. Furthermore, the ability of guided tissue regeneration to stimulate bone formation in a nonhealing model argues strongly that it would also be capable of accelerating bone formation during the consolidation phase, the longest single phase of distraction osteogenesis and therefore the phase most likely to benefit from techniques that enhance osteogenesis.36 For the latter potential clinical application, conversion of a nonhealing critical-sized bony defect to a healing defect could possibly reduce the need for distraction in selected clinical situations, providing immediate benefits both in patient comfort and in use of hardware. Minimizing removable hardware, or switching to an entirely absorbable stabilization system, should also diminish rates of infection and further increase patient comfort. Either way, the use of guided tissue regeneration membranes should not necessitate larger incisions than currently used, as membranes should be easily placed through either intraoral or extraoral incisions used to make the required osteotomies. Although guided tissue regeneration treatment of acute distraction mandibles did not surpass the degree of bone formation seen in gradual distraction–treated mandible, the levels of healing seen in acute distraction plus guided tissue regeneration specimens after 8 weeks approximated those seen in gradual distraction specimens after 6 weeks. However, before clinical implementation, additional studies, such as those comparing

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Plastic and Reconstructive Surgery • April 1, 2006 the mechanical properties of the guided tissue regeneration regenerate to endogenous bone and distraction osteogenesis-mediated regenerate, and comparing the regenerate induced by various types of membranes, would need to be performed. Finally, the benefits of any clinical translation must be weighed against the risks. Use of a guided tissue regeneration membrane adds another foreign body to the equation, theoretically increasing the risk of infection, especially in the intraoral environment. Despite this fact, no infection was seen in any of our study animals, either with or without a membrane. The biodegradable nature of the Gore Resolut XT membrane ensures that it will eventually be absorbed and not be a permanent potential nidus for infection. Similarly, various barrier membranes are used routinely by periodontists to augment osteogenesis in the oral cavity, with success rates of over 98 percent up to 5 years after treatment and minimal rates of infection.41,42 Thus, it is possible that no increase in infection rates would be seen clinically over and above those currently observed, and it is hoped that infection rates may even be diminished by shortening overall treatment time. Michael T. Longaker, M.D., M.B.A. Children’s Surgical Research Program Stanford University Medical Center 257 Campus Drive West Stanford, Calif. 94305-5148 [email protected]

ACKNOWLEDGMENT

This study was supported by RO1 DE-13028 and the Oak Foundation. REFERENCES 1. Ilizarov, G. A. The tension-stress effect on the genesis and growth of tissues: Part I. The influence of stability of fixation and soft-tissue preservation. Clin. Orthop. 238: 249, 1989. 2. Ilizarov, G. A. The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction. Clin. Orthop. 239: 263, 1989. 3. Ilizarov, G. A. Clinical application of the tension-stress effect for limb lengthening. Clin. Orthop. 250: 8, 1990. 4. McCarthy, J. G., Schreiber, J., Karp, N., Thorne, C. H., and Grayson, B. H. Lengthening the human mandible by gradual distraction. Plast. Reconstr. Surg. 89: 1, 1992. 5. Mahatumarat, C., Chokrungvaranont, P., and Rojvachiranonda, N. Mandibular distraction osteogenesis in unilateral craniofacial microsomia: Preliminary report. J. Med. Assoc. Thai. 84: 811, 2001. 6. Imola, M. J., Hamlar D. D. Thatcher, G., and Chowdhury, K. The versatility of distraction osteogenesis in craniofacial surgery. Arch. Facial Plast. Surg. 4: 8, 2002. 7. McCarthy, J. G., Stelnicki, E. J., Mehrara, B. J., and Longaker, M. T. Distraction osteogenesis of the craniofacial skeleton. Plast. Reconstr. Surg. 107: 1812, 2001.

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8. McCarthy, J. G., Stelnicki, E. J., and Grayson, B. H. Distraction osteogenesis of the mandible: A ten-year experience. Semin. Orthod. 5: 3, 1999. 9. McCarthy, J. G., Katzen, J. T., Hopper, R., and Grayson, B. H. The first decade of mandibular distraction: Lessons we have learned. Plast. Reconstr. Surg. 110: 1704, 2002. 10. Gosain, A. K. Distraction osteogenesis of the craniofacial skeleton. Plast. Reconstr. Surg. 107: 278, 2001. 11. Cohen, S. R., Burstein, F. D., and Williams, J. K. The role of distraction osteogenesis in the management of craniofacial disorders. Ann. Acad. Med. Singapore 28: 728, 1999. 12. Toth, B. A., Kim, J. W., Chin, M., and Cedars, M. Distraction osteogenesis and its application to the midface and bony orbit in craniosynostosis syndromes. J. Craniofac. Surg. 9: 100, 1998. 13. Matsumoto, K., Nakanishi, H., Koizumi, Y., et al. Segmental distraction of the midface in a patient with Crouzon syndrome. J. Craniofac. Surg. 13: 273, 2002. 14. Papageorge, M. B. Distraction osteogenesis for augmentation of the deficient alveolar ridge. J. Mass. Dent. Soc. 51: 24, 2002. 15. Mofid, M. M., Manson, P. N., Robertson, B. C., et al. Craniofacial distraction osteogenesis: A review of 3278 cases. Plast. Reconstr. Surg. 108: 1103, 2001. 16. Nyman, S., Gottlow, J., Lindhe, J., Karring, T., and Wennstrom, J. New attachment formation by guided tissue regeneration. J. Periodontal Res. 22: 252, 1987. 17. Karring, T., and Warrer, K. Development of the principle of guided tissue regeneration. Alpha Omegan 85: 19, 1992. 18. Lindhe, J., Karring, T., and Lang, N. P. Clinical Periodontology and Implant Dentistry, 4th Ed. Malden, Mass.: Blackwell, 2003. 19. Hutmacher, D., Hurzeler, M. B., and Schliephake, H. A review of material properties of biodegradable and bioresorbable polymers and devices for GTR and GBR applications. Int. J. Oral Maxillofac. Implants 11: 667, 1996. 20. Takata, T., Wang, H. L., and Miyauchi, M. Attachment, proliferation and differentiation of periodontal ligament cells on various guided tissue regeneration membranes. J. Periodontal Res. 36: 322, 2001. 21. Takata, T., Wang, H. L., and Miyauchi, M. Migration of osteoblastic cells on various guided bone regeneration membranes. Clin. Oral Implants Res. 12: 332, 2001. 22. Wang, H. L., Miyauchi, M., and Takata, T. Initial attachment of osteoblasts to various guided bone regeneration membranes: An in vitro study. J. Periodontal Res. 37: 340, 2002. 23. Instructions for Use: Gore Resolut Adapt Regenerative Membrane. Flagstaff, AZ: W.L. Gore & Associates, Inc. 2004. 24. Rowe, N. M., Mehrara, B. J., Dudziak, M. E., et al. Rat mandibular distraction osteogenesis: Part I. Histologic and radiographic analysis. Plast. Reconstr. Surg. 102: 2022, 1998. 25. Warren, S. M., Mehrara, B. J., Steinbrech, D. S., et al. Rat mandibular distraction osteogenesis: Part III. Gradual distraction versus acute lengthening. Plast. Reconstr. Surg. 107: 441, 2001. 26. Dahlin, C., Linde, A., Gottlow, J., and Nyman, S. Healing of bone defects by guided tissue regeneration. Plast. Reconstr. Surg. 81: 672, 1988. 27. Valdevit, A., Turegun, M., Kambic, H., Siemionow, M., and Zins, J. Cranial defect repair using e-PTFE: Part I. Evaluation of bone stiffness. J. Biomed. Mater. Res. 53: 62, 2000. 28. Matzen, M., Kostopoulos, L., and Karring, T. Healing of osseous submucous cleft palates with guided bone regeneration. Scand. J. Plast. Reconstr. Surg. Hand Surg. 30: 161, 1996.

Volume 117, Number 4 • Guided Tissue Regeneration 29. Kostopoulos, L., and Karring, T. Regeneration of the sagittal suture by GTR and its impact on growth of the cranial vault. J. Craniofac. Surg. 11: 553, 2000. 30. Mooney, M. P., Mundell, R. D., Stetzer, K., et al. The effects of guided tissue regeneration and fixation technique on osseous wound healing in rabbit zygomatic arch osteotomies. J. Craniofac. Surg. 7: 46, 1996. 31. Dahlin, C., Gottlow, J., Linde, A., and Nyman, S. Healing of maxillary and mandibular bone defects using a membrane technique: An experimental study in monkeys. Scand. J. Plast. Reconstr. Surg. Hand Surg. 24: 13, 1990. 32. Wiltfang, J., Merten, H. A., and Peters, J. H. Comparative study of guided bone regeneration using absorbable and permanent barrier membranes: A histologic report. Int. J. Oral Maxillofac. Implants 13: 416, 1998. 33. Stal, S., Tjelmeland, K., Hicks, J., et al. Compartmentalized bone regeneration of cranial defects with biodegradable barriers: An animal model. J. Craniofac. Surg. 12: 41, 2001. 34. Mehrara, B. J., Rowe, N. M., Steinbrech, D. S., et al. Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression. Plast. Reconstr. Surg. 103: 536, 1999. 35. Mcmanus, J., and Mowry, R. W. Staining Methods: Histologic and Histochemical. New York: Hoeber Medical Division, Harper and Row, 1963.

36. Mofid, M. M., Manson, P. N., Robertson, B. C., et al. Craniofacial distraction osteogenesis: A review of 3278 cases. Plast. Reconstr. Surg. 108: 1103, 2001. 37. Loboa, E. G., Fang, T. D., Warren, S. M., et al. Mechanobiology of mandibular distraction osteogenesis: Experimental analyses with a rat model. Bone 34: 336, 2004. 38. Carter, D. R., Beaupre, G. S., Giori, N. J., and Helms, J. A. Mechanobiology of skeletal regeneration. Clin. Orthop. 355 (Suppl.): S41, 1998. 39. Fang, T. D., Salim, A., Xia, W., et al. Angiogenesis is required for successful bone induction in distraction osteogenesis and is determined by the mechanical environment. Bone Miner. Res. 20: 1114, 2005. 40. Lanza, R. P., Langer, R. S., and Vacanti, J. Principles of Tissue Engineering, 2nd Ed. San Diego, Calif.: Academic Press, 2000. 41. Hammerle, C. H., Jung, R. E., and Feloutzis, A. A systematic review of the survival of implants in bone sites augmented with barrier membranes (guided bone regeneration) in partially edentulous patients. J. Clin. Periodontol. 29 (Suppl. 3): 226, 2002. 42. Buser, D., Ingimarsson, S., Dula, K., et al. Long-term stability of osseointegrated implants in augmented bone: A 5-year prospective study in partially edentulous patients. Int. J. Periodontics Restorative Dent. 22: 109, 2002.

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