Experimental Studies Microstructural and Strength Evaluation of Regenerate Tissue during the Consolidation Period after Vertical Mandibular Ramus Distraction David F. Gomez, DDS,*y§ Eduardo F. Sant’Anna, DDS, MS,*yk Robert M. Leven, PhD,y Sjdran A. Ostric, MD,* Alvaro A. Figueroa, DDS, MS,*y Thomas J. Royston, PhD,z Dale R. Sumner, PhD,y John W. Polley, MD* Chicago, Illinois

Mandibular ramus height restoration by distraction osteogenesis (DO) is a key procedure in mandibular hypoplasia reconstruction. The objective of this study was to evaluate short-term skeletal changes in the regenerated bone after vertical mandibular ramus DO using a buried distraction device. Eight subadult beagle dogs underwent bilateral vertical mandibular ramus DO. After a 7-day latency period, distraction was performed at a rate of 0.5 mm twice a day for 12 days. Four dogs were killed at 1 month and four dogs at 2 months after the end of distraction. One intact beagle was included as an unoperated control. After sacrifice, micro computed tomography (mCT) and mechanical testing of distracted sites were used to measure bone volume (BV), total volume (TV), and mechanical peak load strength, respectively. The mCT images showed wide variation in the response, with some animals demonstrating considerable bone formation and reconstitution of the canal for the inferior alveolar nerve. Quantitatively, BV was no more than 67% and BV/TV was less than 25% of the intact control, and strength was approximately 33% of the intact control value. The 1 and 2 month values were similar. These results suggest that internal distractors can successfully reconstitute bone but that the

*Rush Craniofacial Center, Department of Plastic and Reconstructive Surgery, yDepartment of Anatomy and Cell Biology, Rush University Medical Center, Chicago, Illinois; zDepartment of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, Illinois; §Department of Orthodontics, College of Dentistry, Instituto de Ciencias de la Salud, CES University, Medellin, Colombia; kDepartment of Orthodontics, School of Dentistry, Federal University of Rio de Janeiro, Brazil. Address correspondence to Alvaro A. Figueroa, DDS Rush Craniofaical Center, 1725 West Harrison Street, Suite 425, Chicago, IL, 60612; E-mail: [email protected].

regenerated tissue did not regain structural and mechanical characteristics of native bone within the 2 month study period.

Key Words: Canine, mandible, distraction osteogenesis, bone regeneration, micro computed tomography

H

emifacial microsomia (HFM) is the second most common congenital craniofacial anomaly after cleft lip and palate. Reconstruction of the hypoplastic mandible is one of the main treatment objectives in the skeletal rehabilitation of HFM patients. However, successful mandibular reconstruction remains a challenge.1,2 Distraction osteogenesis (DO) is a procedure often used to treat craniofacial skeletal deficiencies. DO is a treatment technique that results in the genesis of new bone through the application of gradual tensile stress.3 DO can be applied to the surgical correction of hypoplasias of the craniofacial skeleton to replace extensive bone tissue deficiencies without requiring the use of bone grafts.4–7 This technique also provides the benefit of allowing for some expansion of overlying soft tissues, which are frequently deficient in these patients. Surgical mandibular lengthening with DO is the preferred technique in cases with moderate to severe vertical and horizontal mandibular hypoplasia.5 Ortiz et al8 developed the maxillomandibular DO technique for the treatment of severe facial asymmetry caused by vertical maxillary and mandibular hypoplasia as seen commonly in HFM. Several reports demonstrated significant improvement in achieving facial symmetry and reduction in the need for extensive orthodontic treatment.8–10 805

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Vertical mandibular ramus DO (VMRDO) can be used by clinicians to generate new bone, thus enlarging the hypoplastic mandibular ramus, and, in combination with maxillary osteotomies, improve facial and occlusal symmetry. However VMRDO presents challenges not seen in DO of the body of the mandible, which has been more extensively investigated.11–13 VMRDO requires application of tension on soft tissues, in particular the muscles of mastication and application of pressure on the temporomandibular joint. Tension generated by the surrounding musculature may be detrimental to the long-term success of this procedure. This difficulty has been demonstrated clinically where long-term stability of mandibular lengthening was not accompanied by stable vertical ramus elongation.14 However, other clinical studies have demonstrated relative stability.15,16 In addition, clinicians using simultaneous maxillary and mandibular distraction allude to the clinical efficiency of the technique as well as its stability.8–10 Therefore, the surgical technique (mandible only vs. simultaneous maxilla and mandible), timing of the procedure (growing vs. nongrowing patients), and severity of the patients treated are all critical and will influence the development and quality of the new bone formed after mandibular distraction. Therefore, it is important to understand which clinical protocols are most effective in creating the highest quality bone (similar to native bone), which in turn should provide stable, long-term clinical outcomes. Experimental models of mandibular body DO have been developed in dog, goat, pig, rat, rabbit, and primates,14,17–25 but information on the quality of new bone generated from vertical lengthening of the mandibular ramus and its stability is still limited. The purpose of this study was to evaluate the shortterm bone changes associated with bilateral vertical mandibular ramus distraction in a canine model by micro computed tomography (mCT) analysis and a mechanical three-point bending test to evaluate peak load (PL) strength.

MATERIALS

AND

insure proper humane treatment. Eight dogs underwent DO, and one dog served as an intact control. Surgical Procedures The surgery was performed with modifications, after Karp et al.4 The surgical technique was as follows: after a 5-day period of acclimation to the research facility, the dogs received Ampicillin (250 mg/mL, 1 mL), intramuscularly, 1 hour before surgery. The average duration of the procedure was 2 hours. It was performed under general anesthesia with intravenous Pentobarbital (25 mg/kg), endotracheal intubation, and halothane inhalation. Strict sterile technique during the surgical procedure was used. The surgical site was shaved and cleaned with 10% isopropanol followed by 10% Betadine solution and quartered with sterile towels. A skin incision was made on the right and left mandibular angles. On each side, the masseter muscle was sectioned horizontally, and the mandible was exposed in the supraperiosteal plane. The osteotomy was placed along a line located from a point equidistant between the antegonial notch and the articular surface of the condyle and a point posterior to the molars at the anterior end, avoiding injury to the inferior alveolar nerve (Fig 1). Internal distraction devices (KLS Martin L.P. Zu¨rich Distractor, Cloverleaf Plate, 15 mm, Tuttlingen, Germany and Jacksonville, FL, Fig 2) were used to mark and perforate the pilot holes for the fixation screws (KLS Martin L.P. Centre-Drive Drill-Free screws, 1.5 mm wide, 5 and 7 mm long Tuttlingen,

METHODS

Animals

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ine male beagle dogs (6–9 months old; Hodgkins Kennels, Howell, MI) were used for the current study. Appropriate institutional animal care and use committees approved the protocol and guidelines for the current study. Preoperative and postoperative care of these animals was overseen by certified veterinarians and husbandry staff to

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Fig 1 Osteotomy and the distractor placement. The direction of the distractor is perpendicular to the osteotomy and as parallel as possible to the direction of the mandibular condyle.

EVALUATION OF REGENERATE TISSUE / Gomez et al

Sacrifice

Fig 2 Internal distraction device used for the current study. A KLS Martin L.P. Zu¨rich Distractor, Cloverleaf Plate, 15 mm was used with KLS Martin L.P. Centre-Drive Drill-Free fixation screws, 1.5 mm wide, 5 and 7 mm long.

Germany and Jacksonville, FL). The devices were located following a plane parallel to the canine mandibular ramus and avoiding the inferior alveolar nerve. The outlined osteotomy was performed using a conventional surgical reciprocating saw (Stryker surgical instruments, Stryker Corp., Kalamazoo, MI). After this, the bone-lengthening device was fixed to the mandible. The dissection remained extraoral to minimize contamination of the operative site. The wound was closed in layers. 3–0 Dexon sutures for the platysma, and 3–0 nylon sutures for the skin were used. Skin sutures were removed 7 days postoperatively. Postsurgically, the dogs received Ampicillin 250 mg/mL, 1mL intramuscularly, once a day for 5 days and as needed thereafter. They also received Buprenorphine 0.3 mg/mL, 1 mL intramuscularly, twice a day for 7 days postoperatively as needed. They were placed on a soft mechanical diet as desired beginning on the second postoperative day. During this 7-day latency period, the fracture was maintained in rigid internal fixation by the lengthening device.

The intact control animal had no surgery. Its sacrifice took place 1 month after the arrival at the animal facility. Four experimental animals were killed after 1 month of consolidation, and four experimental animals were killed after 2 months of consolidation. All animals were killed under anesthesia using intravenous KCl (5 mL; 0.3 mg/mL). The mandibular ramus, including the regenerated bone (Fig 3), was placed in a saline soaked gauze, doubled packed in two successive plastic bags, and frozen at 20°C for mCT scanning and mechanical testing. mCT-Scan Analysis The distracted area and adjacent bone were scanned by mCT (Scanco Model 40, Wayne, PA) after removal of the distractor but with screws re-inserted to serve as markers of the margin between adjacent bone and the distraction site. The slices were perpendicular to the direction of distraction with isotropic resolution of 36 mm. The scout view allowed the identification of the two screws that defined the region to be scanned. The entire region between the two screws was then scanned. For the distracted region, the total volume (TV) and the bone volume (BV) were measured. TV was defined by the soft tissue margin and would be equivalent to the volume enveloped by the periosteum in an intact bone and as the callus envelope in the distracted specimens. BV was defined as the mineralized tissue within the TV and was

Distraction Osteogenesis Protocol Bilateral vertical mandibular distraction was performed in all experimental groups at a rate of 1 mm/day (0.5 mm every 12 hours) for 12 consecutive days after the latency period. After the completion of distraction, the internal distraction devices were left in place, but not activated, during the remainder of the study.

Radiographic Evaluation Occlusal radiographs of the mandibular ramus region were taken in all groups before surgery, after surgery, and every 4 weeks during the healing process. Radiographs were used to keep records to compare mandibular vertical changes before surgery, after surgery, after distraction, and monthly during the length of the consolidation period (1 or 2 months).

Fig 3 A mandibular ramus, including the regenerated bone. The rami were placed in a saline soaked gauze, doubled packed in two successive plastic bags, and frozen at 20°C for micro computed tomography scanning and mechanical testing.

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calculated after Gaussian filtering and then applying a threshold of 225. Mechanical Evaluation The equipment used was an Instron 5500 servohydraulic Universal Testing Machine (Instron Corp, Norwood, MA). The hemimandibles were stored in phosphate-buffered saline solution at 4°C after the completion of the mCT scanning. At the testing site, each sample was positioned in a flat, simple supported configuration on the lateral aspect of each hemimandible. The distance between the two supporting points was 20 mm. The Instron machine was configured to perform a three-point bending test at a rate of 1 mm/s, with a maximum displacement of 7 mm from initial contact with the bone. The PL was recorded. Statistical Analysis The data were analyzed statistically with the aid of commercial software (SPSS v11.0, SPSS Inc., Chicago, IL). For each animal, the values for the two sides were found not to be significantly different (paired t-tests), and the average of the two sides was used. The 1 and

2 month groups were compared with Student’s t-test. One-sample t-tests were used to evaluate differences between the two experimental groups and the one intact specimen.

RESULTS Subjective Radiographic Assessment

I

n the control specimen and in the preoperative radiographs from the test animals, the mandible was intact, and its cortical and trabecular patterns were clearly identifiable. Note that the path for the inferior dental canal followed the curvature of the canine mandible (Fig 4A). A clear osteotomy cut was apparent after the surgical procedure. The space between the boundaries of bone segments was clearly defined. No sign of bone deposition was found (Fig 4B). In the immediate postDO stage, a gap was found between the two margins of the osteotomized bone (Fig 4C). Bone spicules had formed at the periphery of the gap and started to migrate toward the center of the distracted zone, especially in the region lateral to the inferior third molar. After 1 month of consolidation, the gap was largely filled with mineralized

Fig 4 Radiographs of distraction site. In vivo radiographs taken at the indicated time points in the same animal. (A) control, preoperative radiographs. (B) A clear osteotomy cut is seen after the surgical procedure (arrow). (C) Beginning of consolidation. (D) After 1 month of consolidation. (E) After 2 months of consolidation.

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EVALUATION OF REGENERATE TISSUE / Gomez et al

Fig 5 Micro computed tomography of regenerate in distracted mandibular ramus specimens demonstrating transverse sections and transverse and longitudinal reconstructions on samples with low to high bone volume (BV). Far right sample represents a control specimen.

tissue, although there were occasional radiolucent areas within the distraction site (Fig 4D). The bone created in the periphery of the distracted zone had begun to be incorporated into the native bone. After 2 months of consolidation stage, the gap was almost entirely filled with mineralized tissue, and this tissue appeared to be totally incorporated into the native bone (Fig 4E). mCT Scan Evaluation After both 1 and 2 months of consolidation, there was considerable variability in the amount of bone that had formed (Fig 5). Figure 5 shows mCT images of representative samples from the distraction zone after 1 month of consolidation, including samples with low, medium, and high BV measurements as well as the intact control. The top two rows show threedimensional reconstructions, whereas the bottom row shows actual slices, demonstrating the considerable volume of soft callus tissue around the newly formed bone.

BV, TV, and BV/TV BV was significantly higher in the control specimen than in the 1-month group (P = 0.028), but there was no difference between the control and 2-month group or between the 1- and 2-month groups (Fig 6A). TV was larger in the 1-month group than the control (P = 0.022), but was not significantly different in the 2-month group compared with the control (Fig 6B), even though the mean at 2 months tended to be higher than the mean at 1 month. TV did not differ significantly between the 1- and 2-month groups. BV/TV of the control specimen was significantly higher than the 1-month mean (P = 0.001) and 2-month mean (P , 0.001) (Fig 6C). BV/TV did not differ significantly between the 1- and 2-month groups.

Peak Load PL of the control specimen was significantly higher than the 1-month (P = 0.01) and 2-month (P , 0.001)

Fig 6 Graphs of micro computed tomography and peak load data. Value for the intact animal and the mean values for the treated animals with standard deviations are shown. *Significant difference from the intact control animal. There were no significant differences between the 1 month and 2 month values for any of the measured parameters (bone volume [BV], total volume [TV], BV/TV, and peak load).

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groups (Fig 6D). PL did not differ significantly between the 1- and 2-month groups. DISCUSSION

T

he purpose of this study was to evaluate the regenerated bone after bilateral vertical mandibular ramus distraction in a canine model using mCT and mechanical testing. We found that at 2 months, the regenerated bone had not re-established the morphology or mechanical competence observed in the intact control specimen. We did find increased TV, decreased BV, and decreased BV/TV for both the 1-month and 2-month samples compared with the intact control. It is likely that the increase in TV reflects the continued presence of fibrous callus. The lower BV and BV/TV ratio represents incomplete bone regeneration within the distraction site even after 2 months of consolidation. In agreement with the morphologic findings, the PL in the experimental groups was less than in the intact control, suggesting that the development of new bone remained incomplete, and the mechanical integrity of the newly formed tissue was significantly less than native bone. A longer-term study would be needed to determine when the intact bone values would be reached. Other techniques have been used in the past to assess characteristics of distracted bone, including histologic and fluorescent microscopy 4microradiograph, cephalometric radiographs,17 normal CT26 mechanical testing, and ultrasound assessment.27 Our findings are consistent with these previous studies in which new bone formed in the distraction site, although the new bone does not have the same characteristics as native bone. A limitation of the current study was the small sample size; however, other canine studies4,28 have also used similar or even smaller sample sizes to study the effects of distraction on bone regeneration. The reason behind this limitation relates to the expense in keeping the animals long term. The limited sample size is especially true for the intact control. Assuming the intact control values would be reasonably consistent, we used a ‘‘one sample t-test’’ in which the experimental values were compared with the control values. The inherent assumption is that there is no variation in the control. Although this is obviously not entirely true, the experimental values were quite different than the control values, and it is likely that they really were different. This will need to be confirmed in a study with a larger control sample size. The correct choice of animal model to resemble human clinical conditions is critical in the development of new medical techniques. We used dogs because 810

they have mandibles that allow for the use of the same distractors used for human patients. This preliminary study demonstrates that the canine model is useful to evaluate techniques that may be applied to humans. The use of internal devices had been proposed for the design of animal models.4 In this study, internal distractors were chosen over intraoral devices used by other authors.17,26 Internal distractors have the advantage of easier activation than intraoral or external distractors in the canine model. External distractors have the additional problem of being accessible to the dog’s paws and can be dislodged. Additional studies will provide a more complete understanding of the development of regenerate tissue to determine whether the regenerate will eventually be equal to the native bone and at what point the regenerate is clinically stable even though it may not have the strength of native bone and finally whether the mechanics of mandibular function are changed after DO, and whether the regenerate is stable or would it resorb with a significant part of the initial gain being lost over time. Future studies also need to include the addition of bone morphogenetic factors that may accelerate the consolidation process and improve the structural characteristics of the regenerate.

The authors thank the Department of Periodontics, College of Dentistry, University of Illinois at Chicago (James Drummond, PhD) for allowing the use of the Instron Testing Machine, and KLS Martin Company, which provided the distractors used in the current study. The authors also acknowledge the technical assistance of Eileen Broderick, Susan Infanger, and support from the Grainger Foundation.

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microsomia in adults: Avoiding occlusal disasters. Plast Reconstr Surg 1997;100:852–861 Molina F. Combined maxillary and mandibular distraction osteogenesis. Semin Orthod 1999;5:41–45 Figueroa AA, Polley JW. Mandibular distraction osteogenesis. Oper Tech Otoloaryngol Head Neck Surg 2002;13:17–28 Zhou HZ, Hu M, Liu HC, Yao J, Xie M, Xiao HX. Reconstruction of segmental mandibular defect of canine using titanium-nickel distractor. Zhonghua Kou Qiang Yi Xue Za Zhi 2003;38:333–335 Cope JB, Samchukov ML, Muirhead DE. Distraction osteogenesis and histogenesis in beagle dogs: The effect of gradual mandibular osteodistraction on bone and gingiva. J Periodontol 2002;73:271–282 Cho BC, Park JW, Baik BS, Kwon IC, Kim IS. The role of hyaluronic acid, chitosan, and calcium sulfate and their combined effect on early bony consolidation in distraction osteogenesis of a canine model. J Craniofac Surg 2002;13:783–793 Marquez IM, Fish LC, Stella JP. Two-year follow-up of distraction osteogenesis: Its effect on mandibular ramus height in hemifacial microsomia. Am J Orthod Dentofac Orthop 2000; 117:130–139 Hollier LH, Kim JH, Grayson B, McCarthy JG. Mandibular growth after distraction in patients under 48 months of age. Plast Reconstr Surg 1999;103:1361–1370 Kusnoto B, Figueroa AA, Polley JW. A longitudinal threedimensional evaluation of the growth pattern in hemifacial microsomia treated by mandibular distraction osteogenesis: A preliminary report. J Craniofac Surg 1999;10:480–486 Altuna G, Walker DA, Freeman E. Rapid orthopedic lengthening of the mandible in primates by sagittal split osteotomy and distraction osteogenesis: A pilot study. Int J Adult Orthodont Orthognath Surg 1995;10:59–64 Annino DJ Jr, Goguen LA, Karmody CS. Distraction osteogenesis for reconstruction of mandibular symphyseal defects. Arch Otolaryngol Head Neck Surg 1994;120:911–916

19. Bell WH, Gonzalez M, Samchukov ML, Guerrero CA. Intraoral widening and lengthening of the mandible in baboons by distraction osteogenesis. J Oral Maxillofac Surg 1999;57:548–562 20. Guerrissi J, Ferrentino G, Margulies D, Fiz D. Lengthening of the mandible by distraction osteogenesis: Experimental work in rabbits. J Craniofac Surg 1994;5:313–317 21. Kaban LB, Thurmuller P, Troulis MJ, Glowacki J, Wahl D, Linke B, Rahn B, Perrott DH. Correlation of biomechanical stiffness with plain radiographic and ultrasound data in an experimental mandibular distraction wound. Int J Oral Maxillofac Surg 2003;32:296–304 22. King GJ, Liu ZJ, Wang LL, Chiu IY, Whelan MF, Huang GJ. Effect of distraction rate and consolidation period on bone density following mandibular osteodistraction in rats. Arch Oral Biol 2003;48:299–308 23. Li J, Hu J, Wang D, Tang Z, Gao Z. Biomechanical properties of regenerated bone by mandibular distraction osteogenesis. Chin J Traumatol 2002;5:67–70 24. Liu ZJ, King GJ, Herring SW. Alterations of morphology and microdensity in the condyle after mandibular osteodistraction in the rat. J Oral Maxillofac Surg 2003;61:918–927 25. Makarov MR, Harper RP, Cope JB, Samchukov ML. Evaluation of inferior alveolar nerve function during distraction osteogenesis in the dog. J Oral Maxillofac Surg 1998;56:1417–1423 26. Smith SW, Sachdeva RC, Cope JB. Evaluation of the consolidation period during osteodistraction using computed tomography. Am J Orthod Dentofac Orthop 1999;116:254–263 27. el Bialy TH, Royston TJ, Magin RL, Evans CA, Zaki A, Frizzell LA. The effect of pulsed ultrasound on mandibular distraction. Ann Biomed Eng 2002;30:1251–1261 28. Liou EJ, Polley JW, Figueroa AA. Distraction osteogenesis: The effects of orthodontic tooth movement on distracted mandibular bone. J Craniofac Surg 1998;9:564–571

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Experimental Studies Microstructural and Strength ...

David F. Gomez, DDS,*y§ Eduardo F. Sant'Anna, DDS, MS,*yk Robert M. Leven, PhD,y. Sjdran A. .... and use committees approved the protocol and guidelines ...

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