J Oral Maxillofac
Surg
48:714-719.1990
A Rabbit Model for Fetal Cleft Lip Repair MICHAEL T. LONGAKER, MD,* THOMAS B. DODSON, DMD, MPH,t LEONARD B. KABAN, DMD, MD+
AND
Recent clinical and experimental data indicate that fetal wound healing occurs without the accompanying inflammation and scar formation typical of postnatal wounds. The lack of scar tissue may have significant implications for craniomaxillofacial surgery. This report documents the development of a model for fetal cleft lip repair in rabbits. The survival rate for the first 47 fetuses was 76.6%.
Materials and Methods
Recent laboratory and clinical studies have demonstrated that fetal wound healing occurs without inflammation and scar formation.‘-‘4 In this respect, at least, the process is fundamentally different from that of postnatal wound healing. The ability of wounds to heal without the restrictive forces of scar tissue makes fetal intervention for craniofacial anomalies potentially useful. Cleft lip/palate is the most common congenital craniofacial anomaly. Although excellent esthetic and functional results are routinely achieved with postnatal repair, consecutive surgical procedures lead to scar formation and secondary midface growth retardation in a significant number of patients. We hypothesized that a cleft lip repaired in utero would heal without inflammation and scar formation, and therefore secondary midface growth retardation would not occur. In preparation for testing this hypothesis, we developed a model for fetal cleft lip repair.
Time-dated pregnant New Zealand white rabbits at 24 days gestation (term = 32 days) were sedated by a ketamine HCl-acepromazine maleate mixture (OS-mL intramuscular injection followed by 0.5 mL intravenously into an ear vein). General anesthesia (0.5% halothane and oxygen) was achieved by mask. Penicillin G (3 x lo5 U) was given as an intramuscular (IM) injection. The maternal abdomen was shaved and scrubbed with povidone iodine. A sterile midline laparotomy exposed the bicornuate uterus. Each pregnant doe carries at least four fetuses (Fig 1A). On one side of the bicomuate uterus, a pursestring was placed in the uterine wall and a hysterotomy was made using electrocautery (Fig 1B). Clamps were used to secure the amniotic membranes and the fetal head was exposed. Under surgical loupe magnification (6x) a l-mm section of fetal lip and anterior maxillary alveolus was excised halfway between the midline and labial commissure (Fig 1C). The excision created a full-thickness oronasal Iistula (Fig 2A). In one group, the lip was repaired with two 9-O nylon interrupted sutures through skin, muscle, and mucosa (Fig 2B). Meticulous technique was used to align the vermillion border and nasal sill during the closure. In another fetus, on the opposite side of the uterus, the lip was left unrepaired. The fetus was returned to the uterus, sterile saline was used to replace the amniotic fluid, and the pursestring was tied to close the hysterotomy. For controls, fetuses were exposed and returned to the uterus without having an operation. For harvesting, the does were sedated and anesthetized in a similar fashion as for the initial operation. The midline laparotomy was reopened and the uterus and operated fetuses were exposed. The
Received from the University of California, San Francisco. * Research Fellow, Department of Surgery. t Assistant Clinical Professor, Oral and Maxillofacial Surgery. $ Professor and Chairman, Department of Oral and Maxillofacial Surgery. Supported by The Department of OMFS Research Fund, and an A.P. Giannini Medical Research Fellowship (MTL). Presented at the 71st annual meeting of The American Association of Oral and Maxillofacial Surgeons, San Francisco, CA, September 22, 1989. Address correspondence and reprint requests to Dr Kaban: Department of Oral and Maxillofacial Surgery-S 738, UCSF Medical Center. Third and Pamassus Aves, San Francisco, CA 94143-0440. 0 1990 American geons
Association
of Oral and Maxillofacial
Sur-
0278-2391/90/4807-0009$3.00/O
714
LONGAKER,
DODSON, AND KABAN
FIGURE 1. Operative anatomy. A, Representative drawing demonstrating bicornuate uterus. There are usually multiple fetuses. Arrows indicate relative position of the two fetuses selected for in utero cleft lip model. B, Purse-string suture shown surrounding the hysterotomy. C, Head exposed showing full-thickness right-sided oronasal fistula created by excision of a l-mm paramedian section of fetal lip halfway between the midline and labial commissure.
fetuses were removed, observed, weighed, and photographed to compare size and facial morphology to control (unoperated) fetuses. After documenting the viability of the control and experimental fetuses, they were killed by pentobarbital overdose. The heads were then separated, fixed in 10% formalin, and embedded in paraffin. Serial horizontal sections, 7 km thick, from the nasal floor to below the upper lip, were cut and stained with hematoxylin and eosin. Histologic results will be reported in the second phase of this study.
FIGURE 2. Intraoperative 6~ magnification.
Results
Forty-seven fetuses in 25 pregnant does underwent fetal surgery. Thirty-six fetuses survived the operation and were alive at the time of harvest, for a survival rate of 76.6%. This included 15 of 20 fetuses in the repaired group (75% survival), 11 of 17 fetuses in the unrepaired group (64.7% survival), and 10 of 10 controls (100% survival). There was no statistically significant difference in survival rates
photograph of cleft lip model. A, Unrepaired cleft. B, Straight line repair using two 9-O nylon sutures under
716
between the repaired and unrepaired fetuses (P = .747). The first group of fetuses (Fig 3) was harvested at 4 days postoperatively (28 days gestation). There was a marked facial asymmetry in the unrepaired group. This consisted of retraction of the lip segments into the underlying full-thickness defect and elevation and flattening of the nose on the cleft side
FETAL CLEFT LIP, FETAL WOUND HEALING
(Fig 3B). The cleft lip edges were epithelialized, but the lip remained open. There was no epitheliahzation or scar formation across the gap. In the repaired fetuses, at 28 days’ gestation, there was epithelial continuity across the cleft lip and the facial asymmetry was less severe than in the unrepaired group (Fig 3C). The second group of fetuses (Fig 4) were killed at
FIGURE 3. Results, 28 days’ gestation (4 days postoperatively). A, Unoperated control fetus demonstrating midline cleft, symmetry of the nares and upper lip. B, Unrepaired fetus. Note marked facial asymmetry with retraction of the lip segments into the underlying full-thickness defect and flattening of the nose on the cleft side. C, Repaired fetus. There is considerably less asymmetry of the nose and lip. Resorbable sutures remain in place.
LONGAKER,
DODSON, AND KABAN
FIGURE 4. Results, newborn specimens (7 days postoperatively). A, Unoperated control. B. Unrepaired newborn. Note the lack of epithelial continuity and retraction of lip toward underlying full-thickness defect (arrow). C. Repaired newborn. The asymmetry of the nose and lip are considerably less than in the unrepaired newborn. Resorbable sutures remain in place.
birth (7 days postoperatively) after spontaneous vaginal delivery. In the unrepaired fetuses, the cleft remained open, without scar or epithelial bridging. The cleft side of the nose remained flat and elevated (Fig 4B). In repaired fetuses, there was epithelialization across the cleft which was closed, and the facial asymmetry was less than that at 4 days postoperatively (Fig 4C). The third group of fetuses (Fig 5) also delivered spontaneously per vagina. These fetuses were cared for by the mother as long as all normal newborns were removed from the litter except for one control. With multiple normal fetuses in the litter, the cleft animals were cannibalized or neglected. By 12 days after birth (19 days postoperatively), gross observation revealed that the unrepaired cleft animals had slight flattening and asymmetry of the nose. The cleft remained open. It was epithelialized at the edges but there was no continuity across the cleft (Fig 5B). In the repaired animals the nose was symmetrical and the lip was full (Fig SC). There was a linear depression in the area of the previous cleft, but this was covered with epithelium and there was no scar. The distance from the midline to the commissure was decreased on the cleft side as a result of the magnitude of resection and not formation of scar tissue.
Discussion The purpose of this study was to develop a model for fetal cleft lip repair. We selected the rabbit because there is available data on fetal wound healing in this species from our laboratory and others.4*5 In addition, the rabbit model is relatively inexpensive, offers multiple fetuses per pregnancy, and both the doe and fetus tolerate anesthesia and hysterotomy with a low mortality rate. Finally, there is a body of literature documenting the adverse effects of postnatal repair on facial growth in rabbits.i5~ig The midface deformity after cleft lip/palate repair appears to be proportional to the extent of dissection (and therefore scar formation) carried out at the time of operation. Experimental models for the study of cleft lip/palate have existed for many years.““’ These have been used primarily to document the embryonic events leading to development of the deformity. In addition, cleft lip/palate models have been used to document the secondary growth effects of postnatal cleft lip/palate repair. More recently, investigators have examined the feasibility of fetal cleft lip repair.22-24 It is well known that midface retrusion and deficiency in maxillary width are often the result of
718
FETAL CLEFT LIP, FETAL WOUND HEALING
FIGURE 5. Results, 12 days after birth (19 days postoperatively). A, Unoperated control. B, Unrepaired animal. There is slight flattening and asymmetry of nose. The cleft remains open, epithelialized at the edge, but without continuity across the cleft. C, Repaired animal. The nose is symmetrical. The lip is full on the cleft side. There is a linear depression in the area of the cleft, but this is covered with epithelium, and grossly there is no scar. The distance from midline to commissure is decreased on the cleft side, a reflection of the resected tissue.
surgically induced scar formation.25 Bardach et al have demonstrated that increased lip pressure (as a result of soft tissue dissection) following postnatal repair of cleft lip in animals is associated with progressive midface hypoplasia. The severity of midface distortion was proportional to the amount of soft tissue undermining which occurred at the time of lip repair. 15-‘*Hallock has demonstrated in utero repair of cleft lip is possible in mice and monkeys.22,23 A simple linear repair of cleft lip in utero might result in healing without scar formation. The resultant oral sphincter, with intact muscle, would provide a normal functional matrix for the growing fetus. Also, the esthetic result would be greatly improved. The major advantage, however, would be unimpaired facial growth without the adverse effect of the lip and/or palate scar. The genetics, epidemiology, and predicted recurrence risk for cleft lip/palate are known. Women who are at risk for subsequent pregnancies may undergo fetal ultrasound. Fortunately, ultrasound is a reliable technique to diagnose fetal cleft lip and palate as early as 15 to 20 weeks’ gestation.26-28 Thus, a mother who is carrying a fetus with a cleft lip may be referred to a fetal treatment program. In the future, an in utero repair could potentially be offered as a therapeutic option. It is important to note, however, that before human fetal repair is even consid-
ered, a reproducible animal model in higher vertebrates must be developed. This model would be used to establish the technical feasibility of the operation, the risks and benefits for the fetus, and the safety for the mother. The rabbit model is a first step in this process. When this study was begun there was a question about whether fetal cleft lip repair in rabbits was technically feasible, with an acceptable survival rate. The results indicate that the model is indeed feasible and the survival rate was better than that for other reported fetal models.29 It was not clear whether the deformed newborns would be able to feed, grow, and thrive. We have demonstrated that they will feed and thrive, Cannibalization has not occurred if all normal newborns except one control are removed from the litter. Finally, with increasing experience, the survival rate is improving and quality of both the surgically created defect and the repair have improved. We plan to use this model to analyze the histology of fetal wound healing at various time points following surgery, focusing on tissue regeneration, inflammation, and the extracellular matrix (specifically hyaluronic acid). Histologic results will be reported in the next phase of this study. It appears from our preliminary gross results that the third trimester rabbit model demonstrates healing without scar formation. This may explain why
LONGAKER,
719
DODSON. AND KABAN
animals with a repaired cleft in this study developed less midface distortion than those with postnatal repair reported by Bardach et al.‘* It remains to be demonstrated by histologic and biochemical techniques, however, whether this is, in fact, healing without scar formation and with tissue regeneration (muscle, mucosa, and skin). In the future, we plan to investigate the characteristics of rabbit fetal wound healing in the second and early third trimester to determine the ideal stage of gestation during which to perform fetal cleft lip repair. It should be noted that it is clinically possible to perform open fetal surgery in the second trimester in humans and other animals.30 Finally, we plan to use this model to document the longterm effect of prenatal cleft lip repair on maxillofacial growth and compare the results to known data on postnatal repair in the rabbit model.
11. 12.
13.
14.
15.
16.
17.
Acknowledgment The authors gratefully acknowledge the contribution of Michael R. Harrison, MD, for his advice on the experimental design and his assistance in developing the experimental model. We also acknowledae Andre Eckardt. DDS. MD. who. as a fellow in-the Depart&nt of OMFS, helped to’develop the experimental model.
References 1. Robinson BW, Goss AN: Intrauterine healing of fetal rat cheeks wounds. Cleft Palate J 18:251, 1981 2. Rowsell AR: The intra-uterine healing of foetal muscle wounds: Experimental study in the rat. Br J Plast Surg 37635, 1984 3. Goss AN: The intrauterine healing of fetal rat oral mucosal, skin, and cartilage wounds. J oral Path01 6:35, 177 4. Krummel TM. Nelson JM. Dieeelmann RF. et al: Fetal response to injury in the rabbitrJ Pediatr S&g 22640, 1987 5. Adzick NS, Harrison MR, Glick PL, et al: Comparison of fetal, newborn, and adult wound healing by histologic, enzyme-histochemical, and hydroxyproline determinations. J Pediatr Surg 20:315, 1985 6. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury and it modulation with transforming growth factor-beta. Surg Forum 38:622, 1987 7. Delozier J, Nanney LB, Hagan K, et al: Epidermal growth factor enhances fetal epithelialization. Surg Forum 38:623, 1987 8. DePalma RL, Krummel TM, Nelson JM, et al: Fetal wound matrix is composed of proteoglycan rather than collagen. Surg Forum 38:626, 1987 9. Krummel TM, Nelson JM, Diegelmann RF, et al: Wound healing in the fetal and neonatal rabbit. Surg Forum 37~595, 1986 10. Longaker MT, Harrison MR, Crombleholme TM, et al:
18.
19.
20. 21.
22. 23.
24. 25.
26. 27. 28. 29. 30.
Studies in fetal wound healing. I. A factor in fetal serum that stimulates deposition of hyaluronic acid. J Pediatr Surg 24789, 1989 Longaker MT, Harrison MR, Langer JC, et al: Studies in fetal wound healing. II. A fetal environment accelerates fibroblast migration in vitro. J Pediatr Surg 24:793, 1989 Loneaker MT. Whitbv DJ. Ferauson MWJ. et al: Studies in feyal wound healing. III. E&ly deposition of fibronectin distinguishes fetal from adult wound healing. J Pediatr Surg 24~799, 1989 Longaker MT, Chiu ES, Harrison MR, et al: Studies in fetal wound healing. IV. Hyaluronic acid stimulating activity distinguishes fetal from adult wound healing. Ann Surg 210:667, 1989 Longaker MT, Whitby DW, Adzick NS, et al: Studies in fetal wound healing. VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J Pediatr Surg 25:63, 1990 Bardach J, Troberts 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 Bardach J, Klousner EC, Eisbach KJ: The relationship between lip pressure and facial growth after cleft lip repair: An experimental study. Cleft Palate J 16:137, 1979 Bardach J, Roberts DM, Klausner EC: Influence of two-flap palatoplasty on facial growth in rabbits. Cleft Palate J 16:402, 1979 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 McClire HM, Wilk AL, Horigan EA, et al: Induction of craniofacial malformations in rhesus monkeys (Mucaca mulatta) with cyclophosphamide. Cleft Palate J 16:248, 1979 Gibson JE, Becker BA: The teratogenicity of cyclophosphamide in mice. Cancer Res 28:475, 1968 Sulik KK, Johnson MC, Ambrose LJH, et al: Phenytoin (Dilantinl-induced cleft liu and palate in A/J mice: A scanning and transmission electron microscopic study. Anat Ret 195:243, 1979 Hallock, GG: In utero cleft lip repair in A/J mice. Plast Reconstr Surg 75:785, 1985 Hallock GG, Rice DC, McClure HM: In utero lip repair in the rhesus monkey: An update. Plast Reconstr Surg 80:855, 1987 Christ JE: Fetal surgery: A new frontier for plastic surgery. Plast Reconstr Surg 77645, 1986 Ross RB: Treatment variables affecting facial growth in complete unilateral cleft lip and palate. I. Treatment variables affecting growth. Cleft Palate J 24:5, 1987 Christ JE. Meinineer MG: Ultrasound diaenosis of cleft lio and palate before birth. Plast Reconstr Surg 68:854, 1985 Christ JE, Meininger MG: Ultrasound study of the nose and upper lip before birth. Ann Plast Surg 11:308, 1983 Savoldelli G, Schmid W, Schnigel A: Prenatal diagnosis of cleft lip and palate by ultrasound. Prenatal Diagn 2:313, 1982 Thomasson BJ, Ravitch MM: Fetal surgery in the rabbit. Surgery 66: 1092, 1%9 Crombleholme TM, Harrison MR, Langer JC, et al: Early experience with open fetal surgery for congenital hydronephrosis. J Pediatr Surg 23: 1114. 1988
Surg
48:714-719.1990
A Rabbit Model for Fetal Cleft Lip Repair MICHAEL T. LONGAKER, MD,* THOMAS B. DODSON, DMD, MPH,t LEONARD B. KABAN, DMD, MD+
AND
Recent clinical and experimental data indicate that fetal wound healing occurs without the accompanying inflammation and scar formation typical of postnatal wounds. The lack of scar tissue may have significant implications for craniomaxillofacial surgery. This report documents the development of a model for fetal cleft lip repair in rabbits. The survival rate for the first 47 fetuses was 76.6%.
Materials and Methods
Recent laboratory and clinical studies have demonstrated that fetal wound healing occurs without inflammation and scar formation.‘-‘4 In this respect, at least, the process is fundamentally different from that of postnatal wound healing. The ability of wounds to heal without the restrictive forces of scar tissue makes fetal intervention for craniofacial anomalies potentially useful. Cleft lip/palate is the most common congenital craniofacial anomaly. Although excellent esthetic and functional results are routinely achieved with postnatal repair, consecutive surgical procedures lead to scar formation and secondary midface growth retardation in a significant number of patients. We hypothesized that a cleft lip repaired in utero would heal without inflammation and scar formation, and therefore secondary midface growth retardation would not occur. In preparation for testing this hypothesis, we developed a model for fetal cleft lip repair.
Time-dated pregnant New Zealand white rabbits at 24 days gestation (term = 32 days) were sedated by a ketamine HCl-acepromazine maleate mixture (OS-mL intramuscular injection followed by 0.5 mL intravenously into an ear vein). General anesthesia (0.5% halothane and oxygen) was achieved by mask. Penicillin G (3 x lo5 U) was given as an intramuscular (IM) injection. The maternal abdomen was shaved and scrubbed with povidone iodine. A sterile midline laparotomy exposed the bicornuate uterus. Each pregnant doe carries at least four fetuses (Fig 1A). On one side of the bicomuate uterus, a pursestring was placed in the uterine wall and a hysterotomy was made using electrocautery (Fig 1B). Clamps were used to secure the amniotic membranes and the fetal head was exposed. Under surgical loupe magnification (6x) a l-mm section of fetal lip and anterior maxillary alveolus was excised halfway between the midline and labial commissure (Fig 1C). The excision created a full-thickness oronasal Iistula (Fig 2A). In one group, the lip was repaired with two 9-O nylon interrupted sutures through skin, muscle, and mucosa (Fig 2B). Meticulous technique was used to align the vermillion border and nasal sill during the closure. In another fetus, on the opposite side of the uterus, the lip was left unrepaired. The fetus was returned to the uterus, sterile saline was used to replace the amniotic fluid, and the pursestring was tied to close the hysterotomy. For controls, fetuses were exposed and returned to the uterus without having an operation. For harvesting, the does were sedated and anesthetized in a similar fashion as for the initial operation. The midline laparotomy was reopened and the uterus and operated fetuses were exposed. The
Received from the University of California, San Francisco. * Research Fellow, Department of Surgery. t Assistant Clinical Professor, Oral and Maxillofacial Surgery. $ Professor and Chairman, Department of Oral and Maxillofacial Surgery. Supported by The Department of OMFS Research Fund, and an A.P. Giannini Medical Research Fellowship (MTL). Presented at the 71st annual meeting of The American Association of Oral and Maxillofacial Surgeons, San Francisco, CA, September 22, 1989. Address correspondence and reprint requests to Dr Kaban: Department of Oral and Maxillofacial Surgery-S 738, UCSF Medical Center. Third and Pamassus Aves, San Francisco, CA 94143-0440. 0 1990 American geons
Association
of Oral and Maxillofacial
Sur-
0278-2391/90/4807-0009$3.00/O
714
LONGAKER,
DODSON, AND KABAN
FIGURE 1. Operative anatomy. A, Representative drawing demonstrating bicornuate uterus. There are usually multiple fetuses. Arrows indicate relative position of the two fetuses selected for in utero cleft lip model. B, Purse-string suture shown surrounding the hysterotomy. C, Head exposed showing full-thickness right-sided oronasal fistula created by excision of a l-mm paramedian section of fetal lip halfway between the midline and labial commissure.
fetuses were removed, observed, weighed, and photographed to compare size and facial morphology to control (unoperated) fetuses. After documenting the viability of the control and experimental fetuses, they were killed by pentobarbital overdose. The heads were then separated, fixed in 10% formalin, and embedded in paraffin. Serial horizontal sections, 7 km thick, from the nasal floor to below the upper lip, were cut and stained with hematoxylin and eosin. Histologic results will be reported in the second phase of this study.
FIGURE 2. Intraoperative 6~ magnification.
Results
Forty-seven fetuses in 25 pregnant does underwent fetal surgery. Thirty-six fetuses survived the operation and were alive at the time of harvest, for a survival rate of 76.6%. This included 15 of 20 fetuses in the repaired group (75% survival), 11 of 17 fetuses in the unrepaired group (64.7% survival), and 10 of 10 controls (100% survival). There was no statistically significant difference in survival rates
photograph of cleft lip model. A, Unrepaired cleft. B, Straight line repair using two 9-O nylon sutures under
716
between the repaired and unrepaired fetuses (P = .747). The first group of fetuses (Fig 3) was harvested at 4 days postoperatively (28 days gestation). There was a marked facial asymmetry in the unrepaired group. This consisted of retraction of the lip segments into the underlying full-thickness defect and elevation and flattening of the nose on the cleft side
FETAL CLEFT LIP, FETAL WOUND HEALING
(Fig 3B). The cleft lip edges were epithelialized, but the lip remained open. There was no epitheliahzation or scar formation across the gap. In the repaired fetuses, at 28 days’ gestation, there was epithelial continuity across the cleft lip and the facial asymmetry was less severe than in the unrepaired group (Fig 3C). The second group of fetuses (Fig 4) were killed at
FIGURE 3. Results, 28 days’ gestation (4 days postoperatively). A, Unoperated control fetus demonstrating midline cleft, symmetry of the nares and upper lip. B, Unrepaired fetus. Note marked facial asymmetry with retraction of the lip segments into the underlying full-thickness defect and flattening of the nose on the cleft side. C, Repaired fetus. There is considerably less asymmetry of the nose and lip. Resorbable sutures remain in place.
LONGAKER,
DODSON, AND KABAN
FIGURE 4. Results, newborn specimens (7 days postoperatively). A, Unoperated control. B. Unrepaired newborn. Note the lack of epithelial continuity and retraction of lip toward underlying full-thickness defect (arrow). C. Repaired newborn. The asymmetry of the nose and lip are considerably less than in the unrepaired newborn. Resorbable sutures remain in place.
birth (7 days postoperatively) after spontaneous vaginal delivery. In the unrepaired fetuses, the cleft remained open, without scar or epithelial bridging. The cleft side of the nose remained flat and elevated (Fig 4B). In repaired fetuses, there was epithelialization across the cleft which was closed, and the facial asymmetry was less than that at 4 days postoperatively (Fig 4C). The third group of fetuses (Fig 5) also delivered spontaneously per vagina. These fetuses were cared for by the mother as long as all normal newborns were removed from the litter except for one control. With multiple normal fetuses in the litter, the cleft animals were cannibalized or neglected. By 12 days after birth (19 days postoperatively), gross observation revealed that the unrepaired cleft animals had slight flattening and asymmetry of the nose. The cleft remained open. It was epithelialized at the edges but there was no continuity across the cleft (Fig 5B). In the repaired animals the nose was symmetrical and the lip was full (Fig SC). There was a linear depression in the area of the previous cleft, but this was covered with epithelium and there was no scar. The distance from the midline to the commissure was decreased on the cleft side as a result of the magnitude of resection and not formation of scar tissue.
Discussion The purpose of this study was to develop a model for fetal cleft lip repair. We selected the rabbit because there is available data on fetal wound healing in this species from our laboratory and others.4*5 In addition, the rabbit model is relatively inexpensive, offers multiple fetuses per pregnancy, and both the doe and fetus tolerate anesthesia and hysterotomy with a low mortality rate. Finally, there is a body of literature documenting the adverse effects of postnatal repair on facial growth in rabbits.i5~ig The midface deformity after cleft lip/palate repair appears to be proportional to the extent of dissection (and therefore scar formation) carried out at the time of operation. Experimental models for the study of cleft lip/palate have existed for many years.““’ These have been used primarily to document the embryonic events leading to development of the deformity. In addition, cleft lip/palate models have been used to document the secondary growth effects of postnatal cleft lip/palate repair. More recently, investigators have examined the feasibility of fetal cleft lip repair.22-24 It is well known that midface retrusion and deficiency in maxillary width are often the result of
718
FETAL CLEFT LIP, FETAL WOUND HEALING
FIGURE 5. Results, 12 days after birth (19 days postoperatively). A, Unoperated control. B, Unrepaired animal. There is slight flattening and asymmetry of nose. The cleft remains open, epithelialized at the edge, but without continuity across the cleft. C, Repaired animal. The nose is symmetrical. The lip is full on the cleft side. There is a linear depression in the area of the cleft, but this is covered with epithelium, and grossly there is no scar. The distance from midline to commissure is decreased on the cleft side, a reflection of the resected tissue.
surgically induced scar formation.25 Bardach et al have demonstrated that increased lip pressure (as a result of soft tissue dissection) following postnatal repair of cleft lip in animals is associated with progressive midface hypoplasia. The severity of midface distortion was proportional to the amount of soft tissue undermining which occurred at the time of lip repair. 15-‘*Hallock has demonstrated in utero repair of cleft lip is possible in mice and monkeys.22,23 A simple linear repair of cleft lip in utero might result in healing without scar formation. The resultant oral sphincter, with intact muscle, would provide a normal functional matrix for the growing fetus. Also, the esthetic result would be greatly improved. The major advantage, however, would be unimpaired facial growth without the adverse effect of the lip and/or palate scar. The genetics, epidemiology, and predicted recurrence risk for cleft lip/palate are known. Women who are at risk for subsequent pregnancies may undergo fetal ultrasound. Fortunately, ultrasound is a reliable technique to diagnose fetal cleft lip and palate as early as 15 to 20 weeks’ gestation.26-28 Thus, a mother who is carrying a fetus with a cleft lip may be referred to a fetal treatment program. In the future, an in utero repair could potentially be offered as a therapeutic option. It is important to note, however, that before human fetal repair is even consid-
ered, a reproducible animal model in higher vertebrates must be developed. This model would be used to establish the technical feasibility of the operation, the risks and benefits for the fetus, and the safety for the mother. The rabbit model is a first step in this process. When this study was begun there was a question about whether fetal cleft lip repair in rabbits was technically feasible, with an acceptable survival rate. The results indicate that the model is indeed feasible and the survival rate was better than that for other reported fetal models.29 It was not clear whether the deformed newborns would be able to feed, grow, and thrive. We have demonstrated that they will feed and thrive, Cannibalization has not occurred if all normal newborns except one control are removed from the litter. Finally, with increasing experience, the survival rate is improving and quality of both the surgically created defect and the repair have improved. We plan to use this model to analyze the histology of fetal wound healing at various time points following surgery, focusing on tissue regeneration, inflammation, and the extracellular matrix (specifically hyaluronic acid). Histologic results will be reported in the next phase of this study. It appears from our preliminary gross results that the third trimester rabbit model demonstrates healing without scar formation. This may explain why
LONGAKER,
719
DODSON. AND KABAN
animals with a repaired cleft in this study developed less midface distortion than those with postnatal repair reported by Bardach et al.‘* It remains to be demonstrated by histologic and biochemical techniques, however, whether this is, in fact, healing without scar formation and with tissue regeneration (muscle, mucosa, and skin). In the future, we plan to investigate the characteristics of rabbit fetal wound healing in the second and early third trimester to determine the ideal stage of gestation during which to perform fetal cleft lip repair. It should be noted that it is clinically possible to perform open fetal surgery in the second trimester in humans and other animals.30 Finally, we plan to use this model to document the longterm effect of prenatal cleft lip repair on maxillofacial growth and compare the results to known data on postnatal repair in the rabbit model.
11. 12.
13.
14.
15.
16.
17.
Acknowledgment The authors gratefully acknowledge the contribution of Michael R. Harrison, MD, for his advice on the experimental design and his assistance in developing the experimental model. We also acknowledae Andre Eckardt. DDS. MD. who. as a fellow in-the Depart&nt of OMFS, helped to’develop the experimental model.
References 1. Robinson BW, Goss AN: Intrauterine healing of fetal rat cheeks wounds. Cleft Palate J 18:251, 1981 2. Rowsell AR: The intra-uterine healing of foetal muscle wounds: Experimental study in the rat. Br J Plast Surg 37635, 1984 3. Goss AN: The intrauterine healing of fetal rat oral mucosal, skin, and cartilage wounds. J oral Path01 6:35, 177 4. Krummel TM. Nelson JM. Dieeelmann RF. et al: Fetal response to injury in the rabbitrJ Pediatr S&g 22640, 1987 5. Adzick NS, Harrison MR, Glick PL, et al: Comparison of fetal, newborn, and adult wound healing by histologic, enzyme-histochemical, and hydroxyproline determinations. J Pediatr Surg 20:315, 1985 6. Krummel TM, Nelson JM, Diegelmann RF, et al: Fetal response to injury and it modulation with transforming growth factor-beta. Surg Forum 38:622, 1987 7. Delozier J, Nanney LB, Hagan K, et al: Epidermal growth factor enhances fetal epithelialization. Surg Forum 38:623, 1987 8. DePalma RL, Krummel TM, Nelson JM, et al: Fetal wound matrix is composed of proteoglycan rather than collagen. Surg Forum 38:626, 1987 9. Krummel TM, Nelson JM, Diegelmann RF, et al: Wound healing in the fetal and neonatal rabbit. Surg Forum 37~595, 1986 10. Longaker MT, Harrison MR, Crombleholme TM, et al:
18.
19.
20. 21.
22. 23.
24. 25.
26. 27. 28. 29. 30.
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