J Oral Maxillofac 49:605611,
Surg
1991
Fetal Cleft Lip Repair in Rabbits: Postnatal Facial Growth After Repair THOMAS B. DODSON, DMD, MPH,* BRIAN SCHMIDT, BS,t MICHAEL T. LONGAKER, MD,* AND LEONARD B. KABAN, DMD, MD5 We have previously described a model for in utero cleft lip repair in rabbits. Cleft lip and alveolus (CL) were created in fetal rabbits at 24 days gestation (term, 31 days). In this study, postnatal maxillary growth was evaluated in three groups of animals: 1) unoperated controls, 2) unrepaired CL, and 2) repaired CL. The animals were killed at 4, 12, and 26 weeks after birth. Direct cephalometry was performed on dry skulls to evaluate premaxillary width, anterior maxillary length and width, and posterior maxillary width. The results of this study indicate that rabbits that undergo an in utero CL procedure, with or without repair, exhibit no significant decrease in maxillary length and width when compared with controls.
growth inhibition follows operative repair of CL and CL/P.‘-” It has been hypothesized that scar formation secondary to soft-tissue dissection and multiple procedures restricts maxillary growth. Fetal wound healing is characterized by tissue regeneration without inflammation and fibrosis.i3-” The ability of such wounds to heal without the restrictive forces of scar tissue makes fetal intervention for craniofacial anomalies potentially attractive. Alternatively, with a better understanding of fetal wound healing, it may be possible to manipulate postnatal wounds to make them more “fetallike.” We hypothesize that a CL repaired in utero will heal without inflammation and fibrosis. Secondary maxillary growth deficiency, therefore, should not develop. To study this hypothesis, we used a fetal rabbit model, developed in our laboratory,** to evaluate postnatal growth after in utero CL repair.
Patients who have had multiple surgical procedures for correction of cleft lip/palate (CL/P) and related defects often develop characteristic midface growth deformities. These secondary anomalies may include nasal asymmetry and septal deviation, as well as maxillary hypoplasia in the sagittal, transverse, and vertical dimensions. The patients frequently have a class III malocclusion, with relative mandibular prognathism. Data from experimental and human clinical studies suggest that maxillary
Received from the University of California San Francisco. * Adjunct Assistant Clinical Professor, Department of Oral and Maxillofacial Surgery. t Professor and Chairman. Denartment of Oral and Maxillofacial Surgery. $ Summer Research Fellow, Department of Oral and Maxillofacial Surgery. 8 Research Fellow, Department of Surgery and the Fetal Treatment Program. This work was supported by a Research Support Grant from the American Association of Oral and Maxillofacial Surgeons (LBK), a University of California San Francisco School of Dentistry Research Grant (LBK), NIH Grant R03 DE0 9325-01, and the Department of Oral and Maxillofacial Surgery Research Fund. Presented at the 72nd Annual Meeting of the American Association of Oral and Maxillofacial Surgeons, New Orleans, September 15, 1990. Address correspondence and reprint requests to Dr Kaban: Department of Oral and Maxillofacial Surgery, University of California San Francisco, 513 Pamassus Ave. San Francisco, CA 94143-0440. 0 1991 geons 0278-2391
American
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Materials and Methods The operative procedure to create and repair a fetal rabbit CL has been previously described in detail.‘* Time-dated pregnant New Zealand albino rabbits (White Hair Rabbitry, Stark City, MO), in mid-third trimester (24/31 days gestational period), were housed separately in the University of California San Francisco (UCSF) Animal Care Facility and fed food and water ad libitum. On the day of operation, the does were anesthetized with halothane/oxygen by mask. After exposing the bicor-
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nate uterus through a midline laparotomy, a fetus was exposed. Under x6 loupe magnification, a lmm paramedian section of the left upper lip and maxillary alveolus were excised to simulate a complete CL. The fetus was returned to the uterus, the hysterotomy closed, and a second fetus was exposed on the contralateral uterine horn. A similar surgical defect was created. This defect was closed in a linear fashion with two 10-O chromic gut sutures. Care was taken to accurately approximate the nasal sill and vermilion border. The fetus was returned to the uterus and the hysterotomy was closed. The laparotomy wound was then repaired in layers. Does delivered spontaneously per vagina. At birth, the litter was examined to identify the operated and control newborn rabbits. The remainder of the newborns were euthanized by lethal intracardiac injection to reduce competition for food and water. The rabbits were labeled for identification, weighed, and photographed. They were then divided into three sets, each consisting of eight specimens: unoperated controls (n = 4), unrepaired CL (U-CL, n = 2), and repaired CL (R-CL, n = 2). Four weeks after delivery, all rabbits were sedated and then weighed and photographed. One set of rabbits was killed (n = 8). Twelve weeks after delivery, the two remaining sets of rabbits were sedated, weighed, and photographed. A second set of rabbits was killed (n = 8). Twenty-six weeks after delivery, the last set of rabbits was sedated, weighed, and photographed. This set of rabbits was also killed (n = 7; there was 1 death). Rabbits completed facial growth by 26 weeks.23924 Following death, gross observations were made and then the rabbit heads were cleaned and the skulls bleached dry. Direct cephalometry was performed on the dry skulls to document premaxillary width, anterior maxillary length and width, and posterior maxillary width (Fig 1).5 To assess premaxillary width, the distances K to J and L to J were measured. The difference between K to J and L to J was calculated to document the difference in width between cleft and noncleft sides. To assess anterior maxillary length, the distance A to B was measured. To determine the difference in length between cleft and noncleft sides, A to B (cleft side) was subtracted from A to B (noncleft side). Finally, to assess anterior maxillary width, landmarks M to NS, N to NS, and the difference between M to NS and N to NS were measured. The distance between landmarks 0 and P was used to evaluate posterior maxillary width. Data were entered and edited using Lotus l-2-3 (Lotus Development Corporation, Cambridge,
FETAL CLEFT LIP REPAIR: FACIAL GROWTH
FIGURE 1. Ventral view of dry skull illustrating landmarks used to measure premaxillary width (K-J,L-J),anterior maxillary length (A-B on cleft and noncleft sides), anterior maxillary width (M-NS, N-NS) and posterior maxillary width (O-P).(Adapted from Bardach et al.‘)
MA). Data analysis was carried out using the statistical analysis software package SAS (SAS Institute, Inc, Cary, NC). Descriptive statistics were computed for each of the three rabbit sets. Results SURVWAL
Cleft lip/alveolus was created in 40 fetuses in 20 does. Nineteen operated fetuses were born alive (47.5% survival) and 11 (27.5%) survived to be killed at the preselected times (4, 12, and 26 weeks after birth). Experimental specimens consisted of: 1) unoperated controls (n = 12), 2) U-CL (n = 5; 1 death), and 3) R-CL (n = 6).
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Table 1.
Average Weight by Age and Specimen Typs (Reported as Average Weight f SEM)
Unrepaired Repaired Control P Value*
Weight at Birth (g)
Weight at 4 Weeks (g)
n=2 53.85 2 8.85 n=3 54.00 2 2.31 n=7 59.94 t 4.36 0.63
n=5 453.78 2 59.73 n=6 612.62 f 56.72 n = 12 603.78 2 47.90 0.16
Weight at 12 Weeks(g) n=4 2447.45 2 157.22 n=4 2670.80 2 107.17 n=7 2693.58 2 119.28 0.39
Weight at
26Weeks (g) n=2 4103.70 2 180.90 n=2 3851.75 2 787.25 n=3 3769.13 -t 369.27 0.89
*One-wayANOVA.
The average litter size was 6.0 +_ 2.4 newborns (range, 0 to 10). If an operated newborn rabbit survived the first week after birth, it remained healthy and thrived as evidenced by weight gain approximately equal to the controls (Table 1).
structures. This is demonstrated by the dorsal views of the dry skull preparations of typical 6month control, U-CL, and R-CL rabbits (Fig 4AC). Of note is the minimal blunting of the anterior aspect of the nasal bones in both repaired and unrepaired specimens.
CLINICAL APPEARANCE
Ventral Vienl Early postnatal (up to 12 days after birth) clinical appearance has been previously reported.22 At 4 weeks after birth, the unrepaired animals demonstrated a complete cleft of the lip and alveolus with a well-formed oronasal fistula (Fig 2). There was little difference between those and 12-week animals except for increase in size. At 26 weeks, the control rabbits (Fig 3A) had symmetrical nares, a midline “cleft” (actually, an epithelial lined depression in the skin and not a true cleft), symmetrical labial commissures, and upright central incisors. The U-CL rabbits (Fig 3B) had marked nasal asymmetry with narrow and depressed nares and nasal base. The labial commissures were markedly asymmetrical, with the cleft side being considerably shorter than the noncleft side. Finally, there was considerable deviation of the remaining central incisor toward the cleft side. In contrast, the R-CL animals (Fig 3C) had only a slightly asymmetrical nasal configuration. The lip was healed without scar and the thickness of the lip and skin appendages were similar to that on the noncleft side. The distance from the midline to the commissure on the affected side was decreased when compared with control animals. This was a result of the amount of tissue resected in creating the initial CL.
Dry skull cephalometric analysis (Figs 1 and 5) showed a deviation of the nasal septum toward the cleft side in both unrepaired and repaired specimens that was too small to measure. Premaxillary width. Premaxillary width (K-J, L-J) was consistently narrower in the unrepaired group than in the repaired or control group (Table 2). To assess asymmetry between the cleft and noncleft sides, the difference between KJ and LJ was computed. At 4 weeks, the differences in width between the right and left sides among the three study groups ranged from 0.08 mm to 0.3 mm. At 12
DRY SKULL CEPHALOMETRY
Dorsal View The fetal CL procedure appeared to have little effect on the symmetry of the nasal bones, despite the marked asymmetry of the soft-tissue nasal
FIGURE 2. Photograph of 4-week-old rabbit with unrepaired cleft lip spread apart to demonstrate the oronasal fistula and deviation of the remaining incisors.
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Frontal view photographs of 6-month-old rabbits. control. Note symmetry of the nares, midline “cleft,” symmetry of the labial commissures, and upright maxillary incisors. B, Unrepaired animal. Note marked asymmetry of the nares, unrepaired cleft (arrow), and decreased distance from midline to commissure on cleft side in comparison with noncleft side. C, Repaired animal. Note the symmetry of the nares, symmetry and fullness of repaired lip, and lack of scar. The distance from midline to commissure on cleft side remains decreased because of the amount of lip removed when creating the cleft. FIGURE 3.
A, Unoperated
FIGURE 4. Dorsal view of 6-month-old rabbit dry skulls. A, Unoperated control. Note the symmetry and shape of nasal bones. B, Unrepaired animal. C, Repaired animal. The unrepaired and repaired animals demonstrate little effect of this CL operation on the nasal bones. The bones are blunted at the tip in comparison to unoperated controls, but there is essentially no asymmetry.
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Table 2. Premaxillary Width (Mean Value in Millimeters f SEM)
Four weeks Unrepaired (n = 1) Repaired (n = 2) Control (n = 5) Twelve weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 4) Twenty-six weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 3)
KJ
LJ
KL-LJ
1.90 2.24 20.10 2.58 to.15
1.80 2.10 20.10 2.50 20.09
0.10 0.30 kO.00 0.08 kO.11
3.45 20.25 4.05 to.35 3.63 to.19
2.85 20.35 3.45 20.25 3.70 20.23
0.60 “0.10 0.60 k-o.60 -0.07 20.04
3.25 20.45 4.15 to.05 3.56 40.06
3.00 20.30 3.65 to.15 3.5 -r-o.05
0.25 TO.15 0.50 20.20 0.06 ‘0.12
Table 3. Anterior Maxillary Length (Mean Value in Millimeters + SEM)
Four weeks Unrepaired (n = 1) Repaired (n = 2) Control (n = 5) Twelve weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 4) Twenty-six weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 3)
ABR
ABL
13.40 13.45 21.05 14.04 to.37
13.40 13.40 k1.00 13.98 kO.28
0.05 *0.05 0.06 TO.17
23.35 21.15 23.65 -1-0.65 25.43 kO.37
23.70 21.30 23.35 kO.15 25.17 20.33
-0.35 20.15 0.30 rto.50 0.25 20.27
30.55 kO.35 29.70 ?I.10 29.50 LO.35
28.95 20.55 28.90 20.60 29.23 kO.09
1.60 *0.90 0.80 20.50 0.27 LO.26
ABR-ABL 0.00
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weeks, the right-left differences were U-CL = RCL = 0.6 mm (control = -0.07 mm). At 26 weeks, the right-left differences were U-CL = 0.25 mm, R-CL = 0.5 mm, control = 0.06 mm. Anterior maxillary length. Table 3 summarizes the data for mean maxillary length. The overall mean anterior maxillary length (A-B) was approximately equal in controls, U-CL, and R-CL rabbits at the three time points. However, there was individual variation. In several instances (Fig 5), the anterior maxillary length asymmetry was worse in the unrepaired animals. At 4 weeks of age, the difference between the right (noncleft) and the left (cleft side) was U-CL = 0 mm, R-CL = 0.05 mm, control = 0.06 mm. At 12 weeks, the right-left differences were U-CL = -0.35 mm, R-CL = 0.3 mm, control = 0.25 mm. At 26 weeks, the right-left differences were U-CL = 1.6 mm, R-CL = 0.8 mm, control = 0.27 mm. Anterior and posterior maxillary width. Table 4 summarizes mean maxillary width data. There were
FIGURE 5. Ventral view of 6-month-old rabbit dry skulls. This view corresponds to that which illustrates the cephalometric landmarks in Figure 1. A, Unoperated control. B, Unrepaired animal. Note the decreased distance A-B on the cleft side in comparison with the noncleft side in this specimen. This photograph also illustrates the deviation of the remaining incisors on the normal side and the slight deviation of the nasal septum toward the cleft. C, Repaired animal. Note the much better symmetry in this animal in comparison with the unrepaired.
no consistent differences between the widths at MNS and N-NS among the different groups over time. At 4 weeks, right-left differences were U-CL = 0.6 mm, R-CL = 0.4 mm, control = 0.02 mm. At 12 weeks, right-left differences were U-CL = R-CL = 0.25 mm, control = 0.15 mm. At 26 weeks, rightleft differences were U-CL = 0.1 mm, R-CL = 0.2 mm, control = 0.67 mm. There were no differences in posterior maxillary width in control, U-CL, and R-CL groups at all three time points. Discussion The goal of operative correction of craniofacial abnormalities is to “restore form and function by surgery but keep the conditions for normal skull growth intact.“‘3 However, maxillary growth in untreated CL/P patients is frequently closer to normal than in those who have had operative correction of the deformity.‘6 Early and/or multiple surgical pro-
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Table 4. Maxillary Width (Mean Value in Millimeters f SEM)
Four weeks Unrepaired (n = 1) Repaired (n = 2) Control (n = 5) Twelve weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 4) Twenty-six weeks Unrepaired (n = 2) Repaired (n = 2) Control (n = 3)
MNS
NNS
MN
MNS-NNS
OP
5.70 4.90 -to.30
5.10 4.50 kO.70
9.90 8.85 +0.95
5.36 a0.29
5.34 kO.25
9.60 kO.18
0.60 0.40 fO.40 0.02 50.15
10.20 9.95 kO.75 10.38 fO.17
7.00 +0.50 6.65 20.35
6.75 ~0.25 6.40 kO.20
11.85 20.05 11.80 20.40
7.08 kO.24
6.93 20.19
12.00 kO.16
0.25 kO.75 0.25 kO.55 0.15 +-0.10
13.50 20.20 13.80 20.50 14.25 to.12
7.00 r0.60 8.20 -co.20
6.90 TO.40 8.00 to.70
12.95 +0.55 13.35 kO.55
8.33 kO.35
7.67 *0.09
14.07 kO.30
0.10 ‘1.00 0.20 20.50 0.67 kO.44
14.50 kO.20 15.90 20.80 15.57 20.26
cedures to repair CL/P and associated defects are thought to produce secondary midface growth inhibition.8-12 It has been hypothesized that the dissection and tissue trauma associated with the operative procedures produce inflammation and scarring of the soft tissue, which in turn restrict midface growth. This hypothesis has been tested in postnatal rabbit and beagle dog models. ‘-’ Bardach created CL/P in these animals and measured changes in lip pressure and facial growth over time in unrepaired, repaired, and control specimens.1’3-7 Lip pressure was greater in the repaired versus unrepaired specimens and the repaired animals showed the greatest amount of growth inhibition. The amount of growth inhibition was also shown to be proportional to the magnitude of dissection and therefore the amount of scar formation. In addition, it was noted that repair of cleft lip produced a decrease in anterior maxillary length, but had little effect on maxillary width. Repair of CL/P produced growth inhibition in both anterior-posterior and transverse dimensions. Bardach hypothesized that CL repair produced fibrosis that resulted in increased lip pressure. The increase in lip pressure correlated well with anterior maxillary growth inhibition. Fetal wound healing, in contrast to that in postnatal animals, is characterized by tissue regeneration and an absence of inflammation and fibrosis.13-** In previous articles, we reported that CL repaired in utero healed without intIammation** and with regeneration of muscle.*’ We therefore hypothesized that there would be less inhibition of
FACIAL
GROWTH
midface growth. In addition, creating an intact oral muscular sphincter would provide a more normal “functional matrix” for the growing fetus. If this hypothesis proved to be correct, we expected that midface length and width would be similar in unoperated controls, unrepaired, and repaired specimens using the model described previously by our laboratory.** This preliminary study is the first to report results of long-term facial growth in rabbits after in utero repair of CL. Based on the clinical appearance and cephalometric measurements, there was little evidence of anterior maxillary growth inhibition in specimens with a repaired CL. This finding is in contrast to Bardach’s results in postnatal animals6 In the postnatal model, repaired specimens showed the greatest amount of midface growth inhibition when compared to controls and unrepaired animals. Some have argued that the rabbit may be an inappropriate model in which to study CL/P.28 However, in our experience, the rabbit has served as an excellent experimental animal. It is relatively inexpensive, with a short gestation and multiple fetuses per pregnancy. The does and fetuses tolerate surgery well. Based on studies by Verwoerd et al,25 the presence of facial clefts in rabbits produces abnormal facial development that is constant and specific. In addition, the deformity created in rabbits parallels that found in humans. The overall survival of fetal rabbits operated in utero in this study was 27.5%. In a previous publication,*’ we reported a survival rate of 76.7% in fetuses that had the CL operation. The discrepancy occurred because of the different nature of the investigations. In the previous publication, fetuses were delivered by cesarian section 24 to 96 hours after operation and then killed. The current study was a much more rigorous test of survivability. The fetus had to remain viable for the entire duration of the pregnancy and had to be delivered alive, compete for nutrition, and then thrive and grow for as much as 6 months after birth. We found, however, that if the newborn rabbit survived the first week, the probability of surviving to time of sacrifice was 75%. Preliminary studies in this laboratory have failed to demonstrate inflammation or scar in midthirdtrimester rabbit wounds. *’ This, coupled with the absence of growth inhibition, indicates that the third-trimester rabbit model is acceptable to study fetal cleft lip healing. It is possible that we might find earlier or more complete muscle regeneration and less deformity in a second-trimester model. The second-trimester model, however, would be technically prohibitive and the mortality rate of both fetuses and does would be too high in the rabbit.
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1. Bardach J, Eisbach KJ: The influence of primary unilateral cleft lip repair on facial growth. Part 1. Lip pressure. Cleft Palate J 14:88, 1977 2. Eisbach KJ, Bardach J: Effect of lip closure on facial growth in the surgically induced cleft rabbit. ORL J Otorhinolaryngol Relat Spec 86:786, 1978 3. Bardach J, Klausner EC, Eisbach KJ: The relationship between lip pressure and facial growth after cleft lip repair: An experimental study. Cleft Palate J 16: 137, 1979 4. Bardach J, Roberts DM, Yale R, et al: The influence of simultaneous cleft lip and palate repair on facial growth in rabbits. Cleft Palate J 17:309, 1980 5. Bardach J, Mooney M, Giedrojc-Juraha ZL: A comparative study of facial growth following cleft lip repair with or without soft-tissue undermining: An experimental study in rabbits. Plast Reconstr Surg 69:745, 1982 6. Bardach J, Mooney MP: The relationship between lip pressure following lip repair and craniofacial growth: An experimental study in beagles. Plast Reconstr Surg 73:544, 1984 7. Bardach J, Mooney M, Bardach E: The influence of two-flap palatoplasty on facial growth in beagles. Plast Reconstr Surg 69:927, 1982 8. Graber T: Cranial facial morphology in cleft palate and cleft lip deformities. Surg Gynecol Obstet 88:359, 1949 9. Graber T: The congenital cleft palate deformity. J Am Dent Assoc 48:375, 1954 10. Graber T: Changing philosophies in cleft palate management. J Pediatr 37400, 1950 11. Kremenak C, Huffman W, Olin W: Growth of maxillae in dogs after palatal surgery. Cleft Palate J 4:6, 1967 12. Kremenak C, Huffman WC, Olin WH: Maxillary growth inhibition by mucoperiosteal denudation of palatal shelf bone in non-cleft beagles. Cleft Palate J 7:6, 1970 13. Robinson BW, Goss AN: Intrauterine healing of fetal rat cheeks wounds. Cleft Palate J 18:251, 1981 14. Rowsell AR: The intra-uterine healing of foetal muscle wounds: Experimental study in the rat. Br J Plast Surg 37:635, 1984
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IS. Goss AN: The intrauterine healing of fetal rat oral mucosal, skin, and cartilage wounds. J Oral Path01 6:35, 1987 16. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury in the rabbit. J Pediatr Surg 22640, 1987 17. Adzick NS. Harrison MR. Glick PL. et al: Comoarison of fetal, newborn, and adult wound ‘healing by histologic, enzyme-histochemical, and hydroxyproline determinations. J Pediatr Surg 20:315, 1985 18. Krummel TM. Nelson JM. Dieeelmann RF. et al: Fetal response to injury and its modulation with transforming growth factor-beta. Surg For 38:622, 1987 19. Delozier J, Nanney LB, Hagan K, et al: Epidermal growth factor enhances fetal epithelialization. Surg For 38:623, 1987 20. DePalma RL, Krummel TM, Nelson JM, et al: Fetal wound matrix is composed of proteoglycan rather than collagen. Surg For 38:626, 1987 21. Krummel TM, Nelson JM, Diegelmann RF, et al: Wound healing in the fetal and neonatal rabbit. Surg For 37:595, 1986 22. Longaker MT, Eckardt A, Dodson TB, et al: Fetal cleft lip repair in rabbits: I. The model. J Oral Maxillofac Surg 48:714, 1990 23. Urbanus NAM: The significance of skull growth studies for the treatment of cleft palate patients. Acta Morphol Neerl Stand 23:369, 1985 24. Engdahl E: Bone regeneration in maxillary defects. Stand J Plast Reconstr Surg 8: 1, 1972 (suppl) 25. Verwoerd CDA, Verwoerd-Verhoef HL, Urbanus NAM: Skulls with facial clefts. Acta Otolaryngol 81:249, 1976 26. Ortiz-Monasterio F: Cephalometric measurements on adult patients with non-operated cleft palates. Plast Reconstr Surg 24:53, 1959 27. Stem MS, Dodson TB, Schmidt B, et al: Histology and role of hyaluronic acid in fetal cleft lip repair. Abstract presented at the 72nd Annual Meeting, American Association of Oral and Maxillofacial Surgery, New Orleans, 1990 28. Samat BG: Discussion in response to: Bardach J, Mooney M, Giedrojc-Juraha ZL. A comparative study of facial growth following cleft lip repair with or without soft-tissue undermining: An experimental study in rabbits. Plast Reconstr Surg 69:754, 1982