Cleft palate is one of the most common birth

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1 Experimental Study Roles of Different Areas of Palatine Bone Denudation on Growth and Development of the Maxilla and Dental Arch: An Experimental Study Tian Meng, MM, MD, PhD,* Bing Shi, MD, PhD,* Xu Huang, DDS,* Qian Zheng, MD, PhD,* Yan Wang, PhD,* Min Wu, MD, PhD, 1 Yong Lu, DDS,* Sheng Li, DDS* Chengdu, People s Republic of China This study was designed to explore the relationship of the growth of the maxilla and dental arch with different sizes of the denudation area on the hard palate. We tested the hypothesis that different sizes of denudation areas on the hard palate following palatoplasty would significantly influence the growth of the maxilla and dental arch differently. Fifty-one three-week-old Sprague-Dawley male rats were randomly divided into a control group and 3 experimental groups. In the three experimental groups, bilateral palatal mucoperiosteum was excised to three different proportions, i.e. onequarter, one-half and three-quarters of the palate. Then, the animals were sequentially injected weekly with different fluorescent markers alternately. Three in each group were sacrificed randomly every two weeks with maxilla dissected following injections. The widths of the maxilla and dental arch were measured and the fluorescent labeling investigated at different phases. The results showed that the growth of the maxilla and dental arch here was inversely correlated with the area of denuded bone on palate. Therefore, our data may provide references for optimal treatment of cleft palate. Key Words: Bone denudation, maxillary growth, dental arch growth, triple fluorescent labeling From the *Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, People s Republic of China; and 1 Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota. Address correspondence and reprint requests to Dr. Bing Shi, Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, No. 14, Section 3, Ren Min Nan Road, Chengdu , People s Republic of China; menglj123@126.com Supported by the National Science Funds of China ( ). Cleft palate is one of the most common birth defects in humans. Surgery has by far been the primary way to treat cleft palate. It was indicated by clinical trials and animal experiments that bone denudation on the hard palate after surgery was the main course for the developmental deformity. 1,2 The position of bone denudation correlates highly with the deformity of the maxilla and dental arch. 3,4 However, there has been no report concerning the relationship between different sizes of the palate bone denudation and the degree of deformity. We hypothesized that the size of bone denudation correlates with the extent of the maxilla and dental arch. Testing this hypothesis may help surgeons design and improve the management of cleft palate. MATERIALS AND METHODS Animals and Surgical Procedures Fifty-one purebred 3-week-old Sprague-Dawley male rats (Experimental Animal Center, Sichuan University, China) were used for this study. Throughout the experiments, the regulations of laboratory animal care (National Research Council of China, 1996) were enforced. The weights of rats were about 50 g (50 T 5 g). They were randomly assigned into a control group and three experimental groups. The animals were anesthetized with muscular injections of sodium pentobarbital (7 mg/kg, Nembutal, Abbott, Laboratories, Chicago, IL). Different proportions of bilateral palatal mucoperiosteum were excised, i.e. one-quarter, one-half and three-quarters of the palate, the incision began at posterior margin of anterior palatine foramen and ended on the level of distal surface of the maxillary second molar (Fig 1). The mucoperiosteum flaps were removed completely. 391

2 THE JOURNAL OF CRANIOFACIAL SURGERY / VOLUME 18, NUMBER 2 March 2007 Fig 1 Diagram of the rat palate. Hatched areas are the mucoperiosteal excision areas. (A) Group I (control group); (B) Group II, animals with surgically created defects simulating a one-quarter bone denudation of the hard palate bilaterally; (C) Group III, animals with surgically created defects simulating a one-half bone denudation of the hard palate bilaterally; (D) Group IV, animals with surgically created defects simulating a three-quarter bone denudation of the hard palate bilaterally. Group I: control group (n = 12) Group II: animals with surgically created defects simulating a one-quarter bone denudation of the hard palate bilaterally (n = 12) Group III: animals with surgically created defects simulating a one-half bone denudation of the hard palate bilaterally (n = 12) Group IV: animals with surgically created defects simulating a three-quarter bone denudation of the hard palate bilaterally (n = 12) Control group: normal animals without fluorescent injection (n = 3) Fig 2 Photograph of the hard tissue cutting machine (LEICA AP1600). Fig 3 Schematic drawing of the width of the maxilla and dental arch measured. The centers of the lines connecting buccal and lingual enamelo-cemental junctions of maxillary first molar were defined as A and A points, respectively. On the lines perpendicular to the above connecting lines, points 30 mm higher than the connecting line were defined as B and B points. The lines between A and A points represented the width of the maxilla, the lines between B and B points represented the width of the dental arch. A-A value minus B-B value were defined as C-C, representing the degree of tooth obliquity. 392

3 PALATINE BONE DENUDATION ON GROWTH & DEVELOPMENT OF MAXILLA & DENTAL ARCH / Meng et al Fig 4 Fluorescent microscope used in the study (LEICA DMI6000B, Leica Microsystems AG, Heerbrugg, Switzerland). Fluorescent Labeling The animals were sequentially injected with three fluorescent markers at one-week intervals, namely calcein (20 mg/kg, Sigma, US) by hypodermic injection on the backs of rats, cefracycline (50 mg/kg, Sigma, US) by intramuscular injection, and xylenol orange (90 mg/kg, Sigma, US) by hypodermic injection on the backs of rats. Treatment of the Specimen Three rats in each group were sacrificed with muscular injection of sodium pentobarbital (7 mg/kg, Nembutal) on the second, forth, sixth, and eighth week after fluorescent injection. The maxilla were dissected and imbedded in resin. Sections of the coronal plane of maxilla were cut between the maxillary first molar mesiolingual cusps and perpendicular to the occlusal plane by a hard tissue cutting machine (LEICA AP1600) at 100 mm (Fig 2). The sections were fixed on microscope slides, observed by stereomicroscope, and graphed by digital camera. Quantification of Data The scale plate photos were measured at 26.5 amplification. The experiments by Kim were performed according to the method of Kim et al: 5 1) To delineate the contour line of maxillary between the first molar and hard palate; 2) To define A and A, B and B, and C and C points (Fig 3). Distances between A and A point, B and B point were measured three times by a sliding caliper, to obtain the mean value. Using ultraviolet light exposure at 380 nm, the Fig 5 Gross appearance of the hard palate eight weeks after operation (stereomicroscope image original magnification 1.5): Group I (unoperated control group) (A). The palate in Group II (B) did not form evident scar tissue. Soft tissue contraction of wound surface on palate in Group III (C) and Group IV (D) can be found at eight weeks after operation. It appeared that sarciniform scar tissue penetrated to the palate close to the maxillary first molar, but the dental arch in Group II (B) did not have obvious changes. 393

4 THE JOURNAL OF CRANIOFACIAL SURGERY / VOLUME 18, NUMBER 2 March 2007 section was observed by fluorescence microscope (LEICA DMI6000B) (Fig 4), and the difference of bone fluorescent labeling between control group and experimental groups was analyzed after operation. RESULTS Gross Appearance The tresis vulnus of bone denudation on palate was healed in Group II and Group III two weeks after operation, the mucoperiosteal flaps on the palate were not necrotic and perforated, but the tresis vulnus in Group IV was not yet healed. The palate in experimental groups did not form evident scar tissue. Soft tissue contraction of the wound surface on the palate can be found at four, six, and eight weeks postoperative. The dental arch in Group III and Group IV appeared to be horseshoe shaped, the antero-palate became narrower, and palate near the maxillary first molar was introverted. Group IV among the total was conspicuous. It appeared that sarciniform scar tissue penetrated to the palate close to the maxillary first molar, but the dental arch in Group II did not have obvious changes. (Fig 5AYD) Histological Findings The fluorescence labeling line in Group I can be often found by aligning from the side of the nose to the side of the mucoperiosteal flaps. The nearer to the fluorescence labeling the side of palate was, the narrower the distance between one marker line and the other. Strong fluorescence labeling was seen on the median suture of palatine, corresponding to the last time of mark (Fig 6A). The distance of bone lamella of the hard palate from right to left in Group IV became bigger two weeks postsurgery, and the bone lamella of the hard palate lost the normal anatomy and structure. Much fluorescence labeling in the surface of bone trabecula was dispersed and unclear (Fig 6B). The bone lamella of the hard palate in Group III remained normal shape and structure in general, but showed much irregular fluorescence labeling, with a strong fluorescence labeling band seen on the median palatine suture (Fig 6C). The labeling lines in Group II, corresponding to the time of mark, are similar to Group I (Fig 6D). The dispersed fluorescence labeling was found on the whole bone lamella of the hard palate in Group IV four weeks after operation. The fluorescence labeling lines, corresponding to the time of mark, arose in Fig 6 Triple fluorescent labeling of the hard palate two weeks after operation. Frontal section of the rat palate (fluorescence microscope image; original magnification 40; calcein: yellow; cefracycline: green; xylenol orange: orange. (A) Group I, labeling lines corresponding to the injection times are clearly observed with apparent layers of different colors; (B) Group IV, labeling lines corresponding to the injection times cannot be clearly distinguished; (C) Group III, strong fluorescent labeling can be found on the median suture of palatine; (D) Group II, labeling lines corresponding to the injection times are similar to Group I. Pictures are representatives of three different animals. 394

5 PALATINE BONE DENUDATION ON GROWTH & DEVELOPMENT OF MAXILLA & DENTAL ARCH / Meng et al Fig 7 Triple fluorescent labeling of the hard palate eight weeks postsurgery. Frontal section of the rat palate (fluorescence microscope image; original magnification 40; calcein: yellow; cefracycline: green; xylenol orange: orange.) (A) Group I, labeling lines corresponding to the injection times are clearly observed, the labeling lines can be found to align regularly from the side of nose to the side of mucoperiosteal flaps; (B) Group IV, the sporadic irregular fluorescent labeling was disappeared; (C) Group III, the sporadic irregular fluorescent labeling did not disappear; (D) Group II, labeling lines corresponding to the injection times can be distinguished. Pictures are representatives of three different animals. Group IV and Group III. The bilateral hard palate was found to be around the median palatine suture six weeks after operation. The bone lamella of the hard palate near the median palatine suture was thicker than other experimental groups and the control group. The sporadic irregular fluorescence labeling presented on the first and second week of mark in Group IV disappeared eight weeks after operation (Fig 7B). However, sporadic irregular fluorescence labeling did not disappear in Group III (Fig 7C). The regular fluorescence cement line can be found in Group II (Fig 7D), similar to the control group (Fig 7A). CHRONOLOGICAL CHANGES IN THE MAXILLARY WIDTH, DENTAL ARCH WIDTH Table 1 and Figure 8 showed the chronological changes of the maxillary width, dental arch width, and the comparison between maxillary Table 1. Comparison of the Width of Maxilla and Dental Arch on the Different Phases in the Four Groups. x ± s (mm) Group IV Group III Group II Group I 0w AA T BB T CC T ws AA T T T T BB T T T T CC T T T T ws AA T T T T BB T T T T CC T T T T ws AA T T T T BB T T T T CC T T T T ws AA T T T T BB T T T T CC T T T T

6 THE JOURNAL OF CRANIOFACIAL SURGERY / VOLUME 18, NUMBER 2 March 2007 width and dental arch width in the four groups which were measured in different phases. Software SPSS10.0 was used for statistical analysis of the data. Multi-factorial analysis of variance showed that three indices of the four different phases had significant differences (P G 0.05; P = 0.001); only the CC value in all comparative indices on the sixth and eighth week was not considered statistically different, the other indices had statistical significance. One-way ANOVA was used to analyze the same indices of the four groups from the same period; all the groups had homogeneity of variance and statistical significance. Least significant difference (LSO) was used to analyze the subset of the groups between one group and the other, respectively. The data showed that all the groups except the CC value between Group IV and Group III grows at the sixth and eighth week had statistical significance (P G 0.05). Fig 8 Chronological changes of maxillary width, dental arch width, and comparison between maxillary width and dental arch width in the four groups that were measured on different phases. (A) Changes of maxillary width (A-A ); (B) changes of dental arch width (B-B ); (C) changes of tooth inclination distance (C-C ); (D) comparison between maxillary width and dental arch width. DISCUSSION Seal s work 4 showed that rats were suitable for studying the growth and development of maxilla after an operation. Rats are easy to maintain and their growth cycle is short. The maxilla in male rats develops more quickly and the size is bigger than female rats. Factors that affect the development of maxilla are easy to observe. It is easy to analyze the changes as expected (relationship between the operation factors and development of maxilla). Change of weight in experimental animals after operation did not interfere with the development of cranial bones. 6 Searls described the growth curve of maxilla in rats from days 23Y25 after birth. Our animal experiments were performed according to Kim s methods. The study lasted eight weeks long. 4,7,8 To avoid cleft of the hard palate that may influence the results, bones of the hard palate were not operated, with the only method of involving mucosperiosteal flaps on the palate. The rotation center of teeth in the experiment can not be defined by Kim et al. 5 The middle points of enamel-cement junction between buccal and lingual surface of the maxillary first molar (A and A points) assumed as the rotation center of teeth. The distance between A and A points represented the width of maxilla. On the lines perpendicular to the above connecting lines, there are 30 mm points higher than the connecting line defined as B and B points, the lines between B and B points representing the width of dental arch. The value of width of palate measured was a 396

7 PALATINE BONE DENUDATION ON GROWTH & DEVELOPMENT OF MAXILLA & DENTAL ARCH / Meng et al maximal value, while the distance of teeth obliquity measured was a minimal value. Four groups had statistical significance by analyzing the width of the maxilla and dental arch. The results showed that different sizes of denuded hard palate may lead to different degrees of inhibition in maxilla and dental arch development, the larger the denuded hard palate was, the more severely limited development of the maxilla and dental arch. The width of the maxilla and dental arch grew and developed, while the rats growing in the control group had the distance of teeth obliquity also become bigger gradually. It showed that maxilla and alveolar bone growing upwardandectadleadtoincreasedwidthofthe hard palate and dental arch. Although the maxilla and dental arch developed with time in Group III and Group IV, the growth of the maxilla and dental arch was slower. The width of the maxilla and dental arch in Group I increased 0.6 and 0.3 mm, respectively, but no growth in Group IV. It also showed that the development and growth of the maxilla and dental arch was restrained. As seen in Figure 7, the width of the maxilla and dental arch was increased, but the distance of tooth obliquity was decreased, suggesting that width of maxilla increased more than dental arch. It was evident that introversion of teeth lead to the above outcomes. The increase in the maxillary width and dental arch in Group II is less than that in Group I. It showed that the denuded hard palate acquired by operation influenced the development of the maxilla and dental arch, but the distance of teeth obliquity increased with time (Fig 8). It showed that the teeth had not developed inward as in Group III and Group IV. We compared the width of the maxilla and dental arch in the 3 experimental groups and the control Group 8 weeks postsurgery, which changed 2.7 and 3.8%, respectively. However, Group III comparing with the control, the width of the maxilla and dental arch changed 6.7 and 10.7%, respectively. These results showed the size of denuded hard palate was beyond onequarter of the hard palate, and thereby, altered the development and growth of the maxilla and dental arch significantly. Multiple factors may influence the growth and development of the craniofacial region. 9 Historical development and hormones regulated development of the skeleton in the body. Distribution of blood vessels, nutrition of nerve, and mechanical forces dominant growth and development of the local skeleton. The pathological conditions that influence any of these factors will lead to the abnormal development of maxillofacial region. 9 Surgical procedures will alter the previous regulation factors, while the animal growing and developing thereby results in the abnormal development of maxillofacial region. In this study, 3-week-old S-D rats were used as study objects; the hard palates of the rats was surgically modified to make different sizes of denuded hard palates, which may change blood vessel, nerve nutrition and local mechanical force of palate. Exogenous regulation factors, such as pressure of soft tissue, distention of tumors, tensile force of muscles, continued compression of exogenous objects and contraction of scar tissues can influence the bone formation of the craniofacial regions. The granulation tissue related to denuded hard palates contracted and the scar tissue subsequently formed, attributing to developmental deformity of the maxilla and dental arch. 10 By contrast, decreased blood vessel and insufficient nutrition of the denuded hard palate may destroy the growth center of the maxilla, affecting the development of maxilla eventually. Many methods can be used to study the growth, rebuilding and calcification of bone tissue, fluorescent labeling indicates the beginning of calcification, and also as a method to confirm formation of new bone. Triple fluorescent labeling was widely used in the field of orthopedics research since Olerad initiated the method in In this experiment, we compared fluorescent labeling on different phases. There were significant differences in fluorescent labeling between Group I and Group IV, or Group I and Group III. It showed that the formation of new bone was inhibited. Fluorescence labeling of part of bone trabecula was dispersed, indicating necrosis of bone tissue. In addition, the circumscription was wide, and the normal structure of the hard palate disappeared, and the interspaced of median palatine suture widen significantly. We conclude that the size of denuded hard palate was correlated to the hampered formation of new bone. Thus, our study demonstrates that the larger size of denuded hard palate is, the more severely limited the growth of the maxilla and dental arch is. REFERENCES 1. Shetye PR. Facial growth of adults with unoperated clefts. Clinics in Plastic Surgery 2004;31:361Y Kremenak CR Jr, Searls JC. Experimental manipulation of midfacial growth: a synthesis of five years of research at the Iowa Maxillofacial Growth Laboratory. J Dent Res 1971; 50:1488Y Bardach J, Kelly KM. Does interference with mucoperiosteum and palatal bone affect craniofacial growth? An experimental study in beagles. Plast Reconstr Surg 1990;86:1093Y

8 THE JOURNAL OF CRANIOFACIAL SURGERY / VOLUME 18, NUMBER 2 March Searls J, Biggs D. Surgically induced maxillary growth inhibition in rats. Cleft Palate J 1974;11:1Y16 5. Kim T, Ishikawa H, Chu S, et al. Constriction of the maxillary dental arch by mucoperiosteal denudation of the palate. Cleft Palate Craniofac J 2002;39:425Y Alberius P. Cranial suture growth as correlated with weight gain in rabbits. Arch Oral Biol 1987;32:637Y Kim T, Chu S, Ishikawa H. Influence of scar tissue and periosteum on the palatal growth. Hokkaido J Dent Sci 1999; 2:21Y30 8. Shi B, Song QG, Huang X, et al. Maxillary growth following tissue engineered oral mucosal implantation on mucoperiosteal denuded palate process in rats. West China J Stomatology 2003;21: Brennan M, Antonyshyn O. The effects of temporalis muscle manipulation on skull growth: an experimental study. Plast Reconstr Surg 1996;97:13Y Ishikawa H, Iwasaki H, Tsukada H, et al. Dentoalveolar growth inhibition induced by bone denudation on palates: a study of two isolated cleft palates with asymmetric scar tissue distribution. Cleft Palate Craniofac J 1999;36:450Y Olerad S, Lorenzi G. Triple fluorochrome labeling in bone formation and bone resorption. J Bone Joint Surg 1970;52: A