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Osseous healing kinetics after apicoectomy in monkeys: I. An isodensitometric interpretation of radiographic images
0099-2399/84/1006-0233/$02.00/0 JOURNALOF ENDODONTICS
Printed in U.S.A.
Copyright 9 1984 by the American Association of Endodontists
VOL. 10, NO. 6, JUNE1984
SCIENTIFIC ARTICLES Osseous Healing Kinetics after Apicoectomy in Monkeys: I. An Isodensitometric Interpretation of Radiographic Images Cinetica de la Cicatrizacion Osea Despues de la Apicectomia en Monos. I. Interpretacion Isodensimetrica de las Imagenes Radiograficas Steven M. Sieraski, DDS,and John F. Corcoran, DDS,MS
A study was conducted using mandibular second premolar and first molar teeth from four adult monkeys. Each tooth was subjected to conventional root canal treatment followed by a standardized apicoectomy of the distal root. The standardized surgical defects were allowed to heal for 2, 4, 8, or 16 wk following apicoectomy. The animals were sacrified and mandibular posterior block sections harvested. Each block section was radiographed in conjunction with an aluminum step-wedge standard. Isodensitometric print-outs of radiographic images were assessed for a chronological progression of osseous regeneration. Radiographic images of defects that had healed for 2, 4 or 8 wk had similar densities below a range estimated for undisturbed interradicular bone. The images of defects that had healed for 16 wk were within the radiographic density range for undisturbed interradicular bone.
habian cicatrizado despues de 2, 4, t~ 8 semanas tenian densidad similar por debajo del espectro estimado para el hueso interradicular sano. Las imagenes de los defectos que cicatrizaron despues de 16 semanas estuvieron dentro del espectro de densidad radiogrMica a la del hueso interradicular sano. All of the health-related fields have as their ultimate purpose the maintenance of a "healed" state through the prevention and treatment of disease. The efficacy of diagnosis and therapy is reflected in assessment of healing kinetics. Healing after endodontic periapical surgery is primarily monitored by interpretation of periodic recall radiographs. Rud et al. (1) categorized radiographic criteria for the assessment of healing after periapical surgery. They reviewed material from 120 cases that had been observed for a minimum of 1 yr. Each case was judged for change in the size and shape of a previous radiolucency and the character of reestablished bony and periodontal architecture. These were then classified into one of four groups: complete healing, incomplete healing (scar tissue), uncertain healing, and unsatisfactory healing (failures). Others (2-9) have used similar criteria in the radiographic assessment of success and failure following periapical surgery. The quantitation of radiographic imagery is possible using microdensitometry. Matsue et al. (10, 11) used microdensitometry to analyze radiographic healing patterns of autogenous bone implants. Other investigators (12, 13) have used microdensitometry to produce isodensitometric print-outs in the study of radiographic imagery. Microdensitometric methods have also been
Este estudio se realiz6 en segundos premolares y primeros molares inferiores de cuatro monos adultos. Cada diente fue sometido a un tratamiento de Conductos convencional seguido de una apicectornia estandardizada de la raiz distal. Los defectos quirurgicos standard se dejaron cicatrizar durante 2, 4, 8, 6 16 semanas luego de la apicectomia. Luego de sacrificados los animales se obtuvieron cortes de bloques posteriores de la mandibula. Cada secci6n fue radiografiada con un bloque standard de aluminio. La impresion isodensimetrica a partir de la imagen radiogrMica fue evaluada respecto a la progresion cronologica de la regeneracion osea. Las im:~genes radiogrMicas de los defectos que 233
234
Sieraski and Corcoran
used in the evaluation of radiographic techniques, response of radiographic films to variations in exposure or processing, and variation in resolution between film types and brands (14-17). The purpose of this study was to assess radiographic osseous healing kinetics after standardized apicoectomy using isodensitometric methods. This is the first of a series on osseous healing kinetics after apicoectomy, to include: II. A Quantitative Histological Appraisal and III. Correlation Between Histology and Radiography. MATERIALS AND METHODS
A study was conducted on four 8- to 12-kg adult monkeys (Macaca rhesus) with completed root end development. Mandibular lateral incisor, first premolar, second premolar, and first molar teeth were used. Each tooth was subjected to conventional root canal treatment, followed 6 to 7 days later by standardized apicoectomy procedures. In each posterior tooth the apicoectomy procedures were limited to the distal root. In this radiographic investigation only second premolar and first molar teeth were suitable for controlled projections. A total of 16 teeth in the four healing periods were involved. Osseous healing was studied after periods of 2, 4, 8, and 16 wk after apicoectomy. Radiographic images of osseous regeneration were quantified using microdensitometric tracings in conjunction with an aluminum step-wedge standard. These readings were converted into isodensitometric print-outs which were compared and assessed for a chronological progression of radiographic osseous regeneration. An operative schedule was devised which assigned two teeth from each monkey to a healing group so that the resultant groups each contained an equal distribution of tooth types. The schedule also separated operations on adjacent teeth by a minimum of 4 wk. The animals were sedated and then anesthetized by intramuscular injections of Xylazine (2 mg/kg, Rompun; Cutter Laboratories, Inc., Shawnee, KA) and ketamine hydrochloride (10 mg/kg, Vetalar; Parke-Davis, Morris Plains, NJ), respectively. Two percent lidocaine (Xylocaine; Astra Pharmaceutical Products, Inc., Worcester, MA) with 1:100,000 epinephrine was used as a local anesthetic to aid in hemorhage control and management. An effort to preserve asepsis was exercised during all procedures. Initially and periodically all teeth were scaled and polished to maintain oral hygiene. Standard sterile instruments, materials, and techniques were used in the surgical and nonsurgical procedures. Conventional root canal procedures were completed in a single treatment. Cotton roll isolation was used in that salivation was eliminated by the anticholinergic effects of the Xylazine. The teeth were preoperatively radiographed and opened with appropriate endodontic access cavities. All root canals of these teeth were
Journal of Endodonticl
endodontically prepared to a # 2 5 K-type file using sterile saline as an irrigant. Working lengths were esti. mated from "trial length" radiographs and established when negotiable as 1 to 1.5 mm short of each respec. tive radiographic apex. These were then each dried and filled with a fitted #25 gutta-percha point, a Grossman type sealer (Roth Drug Co., Chicago, IL), and lateral condensation of accessory #25 gutta-percha points. The access openings were filled with a reinforced zinc oxide and eugenol and the teeth were radiographed.. The apicoectomies were performed with a specially developed 3-mm trephine bur with a self-limiting depth of 7 mm (Fig. 1). A semilunar mucoperiosteal flap was opened and the appropriate root apex was radiographically located using a lead foil marker (Fig. 2). The trephine bur was then used perpendicular to the cortical bone surface in a low-speed handpiece. Intermittent sterile saline irrigation was used as a coolant as the bur performed each apicoectomy and reached its self-limiting depth in the production of a standardized periapical surgical defect (Fig. 3). Residual debris from the trephine core were removed from the surgical site (Fig. 4) and the area was flushed with sterile saline. The mucoperiosteal flap was repositioned and sutured into place with either (4-0) black silk or (5-0) Dacron sutures. The teeth were then radiographed, and the sutures were removed 5 to 7 days after these procedures. The body weight of each monkey was periodically recorded as a health monitor. In one case a monkey had dropped 2 kg over the first 12 wk. At that time, 2 ml of aqueous penicillin G was administered by intramuscular injection. This was followed 1 and 5 days later by 1 ml of aqueous penicillin G injections. The animals weight stabilized for the remainder of the experiments. No other animal presented with significant weight loss. At the end of the experiment, the animals were sedated and perfused through the left ventricle using 0.9% saline followed by fixation with glutaraldehydephosphate-buffered paraformaldehyde (18). The man-
FiG 1. Trepine bur schematic (3-mm diameter, 7-mm self-limiting depth).
lfol. 10, No. 6, June 1984
Radiographic Healing after Apicoectomy
235
dibles were sectioned distal to the third molar teeth and postfixed in glutaraldehyde-phosphate-buffered paraformaldehyde for 1 wk. Each mandible was then sectioned distal to the cuspid teeth, producing two posterior pieces and one anterior piece. Each posterior piece was radiographed lingual-side down with an aluminum step-wedge projected on the same film (Fig. 5). The aluminum step-wedge ranged in thickness from 0.5 to 8 mm. Occlusal dental X-ray film (Kodak Ultra-Speed DF 57) was used with a film to
FIG 4. Standardized volumetric surgical defect. A, Trephine core. B, Surgical site prior to closure.
FIG 2. Radiographic location of surgical sites. A, Lead foil marker clinically. B, Radiopaque marker on radiograph.
FiG 3. Surgical site prior to trephine core removal.
source distance of 12 inches (long cone). Each film was exposed for 8 impulses at 90 kVp and 10 mA with a General Electric 100 X-ray machine. The exposed films were developed at the same time in a Phillips automatic film processor, set at 20~ and 4.5 min. The radiographs were analyzed using a Joyce-Loebl automatic recording microdensitometer, model MK III B (Joyce-Loebl & Co. Ltd., Gateshead, IL). The films were cut to separate the specimen images from those of the step-wedge. Each specimen image was then cut approximately 3 to 4 mm anterior to the image of the second premolar defect on a perpendicular to a line bisecting both defects to be studied. This cut edge was aligned along with the corresponding step-wedge image for scanning by the microdensitometer. Densitometric measurements of each specimen image consisted of a series of parallel scans resulting in 25 density tracings (Fig. 6). These scans were spaced at 0.25-mm intervals and positioned so that the defect images centered the 6-mm range. A density tracing was also made of the step-wedge from a scan positioned to measure all of the step images. The tracings were analyzed using a transparent grid overlay and calculated aluminum equivalents from corresponding step-wedge tracings. A series of 12 vertical
236
Sieraski and Corcoran
deflection ranges were defined for each radiograph. The vertical deflection ranges were chosen to standardize their aluminum equivalent values and establish values which equalized the amount of pen deflection between ranges. Each vertical deflection range was assigned a code symbol as well as a visual density symbol which were equated to an aluminum equivlent thickness (Table 1). Each tracing was then recorded as a series of codes coresponding to vertical pen deflection readings at consecutive horizontal coordinates spaced at 0.2-inch intervals. An isodensitometric image was produced from each series of 25 tracing code strings. The code strings were entered into a home computer system (Apple I1+, Apple Computer, Inc., Cupertino, CA) using a word processing program (Gutenberg, Micromation Ltd., Toronto, Ontario, Canada), each code string representing one line of text. Each code symbol was then searched for and replaced by its corresponding visual density symbol. The resultant print-out was an isodensitometric image of the original radiograph in the area of the defects (Fig. 7). Isodensitometric images of periapical surgical defects were quantitated and averaged as aluminum equivalent values. Each surgical defect was outlined on the print-
Journal of Endodontics
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TABLE 1. Equivalency chart between symbol sets
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outs as an ellipse corresponding to the distortion of the 3-mm diameter circular cut. The positions of these ellipse outlines were then transposed onto similar printouts into which corresponding code symbols and intervening dashes had been substituted back for the visual density symbols (Fig. 8). The codes inside each ellipse were then converted into their aluminum equivalent values and calculated as an average aluminum equivalent value for each defect. An average aluminum equivalent value and standard deviation were similarly calculated for areas of undisturbed interradicular bone to use for comparative purposes. RESULTS
FIG 5. Radiographic technique. A, Positioning of specimen and stepwedge. B, Radiographic image.
An aluminum equivalent value was calculated for each surgical defect image and an average value determined for each healing period (Table 2). Radiographic images of defects that had healed for 2, 4, 8, and 16
Vol. 10, No. 6, June 1984
Radiographic Healing after Apicoectomy
237
TABLE 2. Radiographic densities* for individual surgical defect images T O Monkeyno. 1 Monkeyno. 2 Monkeyno. 3 Monkeyno. 4 , A O ,9,zo 1 29 3 0 , 9 20 29 30 19 20 29 30 19 2 o ~ 3 o
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POST-SURGICAL PERIOD (Weeks) FIG 9. Radiographic osseous healing kinetics after apicoectomy. Density ranges were illustrated for each postsurgical period and a curve from average values was used to represent osseous healing kinetics. The radiographic density range of undisturbed interradicular bone was represented by the patterned area.
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wk after surgery had average aluminum equivalent values of 2.6, 2.5, 2.7, and 3.6 mm, respectively. These values were used to graphically represent radiographic osseous healing kinetics after apicoectomy (Fig. 9). The average aluminum equivalent value for areas of undisturbed interradicular bone was 3.5 mm with a standard deviation of 0.39 mm. This average value _+1 SD was used as an aluminum equivalent range to represent healed bone density. "t"(~--'-t
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FJG 8. Defect localization and translation into code symbols. A, Isodensitometric image with estimated surgical site boundaries, B, Image translation into code symbols.
An animal model system for the radiologic study of osseous healing kinetics after apicoectomy is dependent on methods of standardizing the surgical wounds and radiographic techniques. Unfortunately, anatomical considerations (sinus cavities, mandibular canals) limit the number of available surgical sites per animal. Radiographic standardization further restricts the sample size in this study. The limited data obtained in this study cannot serve to provide any biological conclusions; rather, it should be used to establish the credibility of the methods used in the model system and provide an initial pool of base line data. The trephine bur designed in this study served to
238
Sieraski and Corcoran
perform an apicoectomy and produce a standardized volumetric surgical defect. Variation in the radiographic location of the burring sites would seem to be the weakness in standardization of these techniques. A semilunar flap was used to establish surgical access and enhance undisturbed healing when sutured. Coolant was used during surgical burring to minimize any thermal necrosis of bone. Radiographic techniques were standardized to validate interpretation of densitometric findings. Each specimen was radiographed with an effort to maintain uniform distances and angulation between the film, specimen, and X-ray source. The films were exposed and processed consecutively as a group using the same machines and identical settings. The same aluminum step-wedge was placed in each projection to produce a standard image for densitometric comparison. The Joyce-Loebl microdensitometer produced graphic recordings from direct readings off the radiographic images. The microdensitometric data were quantitated and processed using the graphic imagery provided by a home computer system. The use of isodensitometric images allowed identification of density readings localized to the surgical defect sites. The use of density ranges and interpretation of image position were sources of standard error for all specimens. Earlier studies (12, 13) have used typewriter symbols singularly or in combination to produce isodensitometric images. The advent of the dot matrix printer and computer software to create user-definable symbols provides an optimum mechanism for the production of superior isodensitometric imagery. The results indicate that the data can be divided into two groups: that from the 2-, 4-, and 8-wk healing periods and that from the 16-wk healing period. The images of defects that had healed for 2, 4, or 8 wk had similar radiographic densities. These were below that density range estimated for undisturbed interradicular bone. The images of defects that had healed for 16 wk were within radiographic density range for undisturbed interradicular bone. These results would seem to indicate that the majority of the first 8 wk of healing occurs as soft tissue organization and that healing between 8 and 16 wk occurs with a significant amount of mineralization. The radiographic images of surgical sites after 16 wk of healing would seem to indicate complete osseous regeneration. These results would seem compatible with the radiographic patterns of early bone resorption followed by increased bone densities in implant studies (10, 11 ). The radiographic densities at any single healing period occurred as a range varying from 0.5- to 0.8-mm aluminum equivalents. These variations were not statistically analyzed due to the limited sample size. Monkey 2 was treated with an antibiotic during the course of treatments, which may account for its tendency toward
Joumal of Endodontics
greater than average radiographic densities. Anatomical location may also affect radiographic healing as seen in pairings of teeth within the same monkey (monkeys 1 and 2). These suggest a tendency for periapical regions of premolar teeth to reach a greater radiographic density than those of molar teeth in the same healing period. This is most likely attributable to the superimposition of larger marrow spaces common in more posterior regions of the mandible. The methods presented in this investigation define an animal model system for the study of osseous healing kinetics after apicoectomy. Despite the efforts to standardize all procedures, it would seem that radiographic healing occurs in broad ranges. Correlation between quantitative radiographic and histological osseous regeneration would have great implications in the transference of histological results in animal studies to radiographic results in clinical studies. This initiates a series of works to establish a complete system for the study of implant materials to enhance periapical osseous regeneration. SUMMARY
An animal model system for the study of osseous healing kinetics after apicoectomy is presented. A home computer system was used to produce isodensitometric images of radiographic healing after apicoectomy. The results of this study serve to validate the methodology and provide an initial pool of base line data for future studies. Se presentO un sistema de modelo en animales para el estudio de la cinetica de la cicatrizacion 6sea despues de una apicectomia. Se utilizo un sistema de computaci6n casero para producir im&genes isodensim6tricas de cicatrizaci6n radiogr&fica despues de la apicectomia. Los resultados de este estudio sirvieron para convalidar la metodologia y proveer una base de datos para futuros estudios. We want to express our thanks and appreciation to the following individuals for their support and efforts of this investigation: Maynard Ruud, Hu-Friedy Corp.; Randal Kadykowski, draftsman; Henry Kazlauskas, director of animal care, University of Michigan, Dental Research Institute; Charles Cox, associate research scientist and instructor in dentistry, University of Michigan, Dental Research Institute; Thomas Dunn, PhD, professor of chemistry, University of Michigan; Robert Ause, research assistant in chemsitry, University of Michigan; and, Chris Jung, artist. Dr. Sieraski is clinical instructor and Dr. Corcoran is associate professor and chairman, Department of Endodontics, School of Dentistry, University of Michigan, Ann Arbor, MI.
References 1. Rud J, Andreasen JO, Moller-Jensen JF. Radiographic criteria for the assessment of healing after endodontic surgery. Int J Oral Surg 1972;1:195214. 2. Mattila K, Altonen M. A CUnicat and roentgenological study of apicoectomized teeth. Odontol Tidskr 1968;76:389-406. 3. Harty FJ, Parkins BJ, Wengraf AM. The success rate of apicoectomy. Br Dent J 1970;129:407-13.
V~. 10, No. 6, June 1984 4. Nord PG Retrograde rootfilling with Cavit: a clinical and roentgenological study. Sven Tandlak Tidskr 1970;63:261-73. 5. Nordenram A, Svardstrom G. Results of apicoectomy. Sven Tandlak Tclskr 1970;63:593-604. 6. Perrson G. Prognosis of reoperation after apicoectomy. A clinical radiolOgiCalinvestigation. Sven Taodlak Tidskr 1973;66:49-68. 7. Ericson S, Finne K. Perrson G. Results of apicoectomy of maxillary canines, premolars and molars with special reference to orantral communication a prognostic factor. Int J Oral Surg 1974;3:386-93. 8. Perrson G, Lennartson B, Lundstrom I. Results of retrograde root filling with special reference to amalgam and Cavit as root-filling materials. Sven Tandlak Tidskr 1974;67:123-34. 9. Hirsch J-M, Ahlstrom U, Henrikson P-A, Heyden G, Peterson L-E. Perlapical surgery. Int J Oral Surg 1979;8:173-85. 10. Matsue I, Collings CK, Zirnmerman ER, Vail WC. Microdensitometric analysis of human autogenous alveolar bone implants. J Periodont 1970;41:489-95. 11. Matsue I, Zimmerman ER, Collings CK, Best JT. Microdensitometric analysis of human autogenous bone implants. I1. Two dimensional density and pattern analysis of interproximal alveolar bone. J Periodont 1971 ;42:435-8.
Radiographic Healing after Apicoectomy
239
12. Isenberg G, Goldman HM, Spira J, Parsons FG, Street PN. Radiograph analysis by two-dimensional microdensitometry. J Am Dent Assoc 1968;77:1069-73. 13. Duinkerke ASH, Van de Pohl ACM, Doesburg WH, Lemmens WAJG. Densitometric analysis of experimentally produced periapical radiolucencies. Oral Surg 1977;43:782-97. 14. Duinkerke ASH, Van de Poel ACM, Doesburg WH, Lemmens WAJG. Compensation of differences in density of radiographs by densitometry. Oral Surg 1978;45:635-42. 15. Sivasriyanond C, Manson-Hing LR. Microdensitometric and visual evaluation of the resolution of dental films. Oral Surg 1978;45:811-22. 16. Price C. A densitometric evaluation of two radiographic duplicating films under diffenng conditions of exposure and processing. Oral Surg 1980;50:1904. 17. Beyer-Olsen EM, Eggen S. Evaluation of the reproducibility of two bitewing techniques by means of microdensitometric recording method. Oral Surg 1983;55:103-7. 18. Cox CF, Heys DR, Heys RJ. A gravity perfusion technique for lab animals. Lab Animal 1977;6:18-22.
Printed in U.S.A.
Copyright 9 1984 by the American Association of Endodontists
VOL. 10, NO. 6, JUNE1984
SCIENTIFIC ARTICLES Osseous Healing Kinetics after Apicoectomy in Monkeys: I. An Isodensitometric Interpretation of Radiographic Images Cinetica de la Cicatrizacion Osea Despues de la Apicectomia en Monos. I. Interpretacion Isodensimetrica de las Imagenes Radiograficas Steven M. Sieraski, DDS,and John F. Corcoran, DDS,MS
A study was conducted using mandibular second premolar and first molar teeth from four adult monkeys. Each tooth was subjected to conventional root canal treatment followed by a standardized apicoectomy of the distal root. The standardized surgical defects were allowed to heal for 2, 4, 8, or 16 wk following apicoectomy. The animals were sacrified and mandibular posterior block sections harvested. Each block section was radiographed in conjunction with an aluminum step-wedge standard. Isodensitometric print-outs of radiographic images were assessed for a chronological progression of osseous regeneration. Radiographic images of defects that had healed for 2, 4 or 8 wk had similar densities below a range estimated for undisturbed interradicular bone. The images of defects that had healed for 16 wk were within the radiographic density range for undisturbed interradicular bone.
habian cicatrizado despues de 2, 4, t~ 8 semanas tenian densidad similar por debajo del espectro estimado para el hueso interradicular sano. Las imagenes de los defectos que cicatrizaron despues de 16 semanas estuvieron dentro del espectro de densidad radiogrMica a la del hueso interradicular sano. All of the health-related fields have as their ultimate purpose the maintenance of a "healed" state through the prevention and treatment of disease. The efficacy of diagnosis and therapy is reflected in assessment of healing kinetics. Healing after endodontic periapical surgery is primarily monitored by interpretation of periodic recall radiographs. Rud et al. (1) categorized radiographic criteria for the assessment of healing after periapical surgery. They reviewed material from 120 cases that had been observed for a minimum of 1 yr. Each case was judged for change in the size and shape of a previous radiolucency and the character of reestablished bony and periodontal architecture. These were then classified into one of four groups: complete healing, incomplete healing (scar tissue), uncertain healing, and unsatisfactory healing (failures). Others (2-9) have used similar criteria in the radiographic assessment of success and failure following periapical surgery. The quantitation of radiographic imagery is possible using microdensitometry. Matsue et al. (10, 11) used microdensitometry to analyze radiographic healing patterns of autogenous bone implants. Other investigators (12, 13) have used microdensitometry to produce isodensitometric print-outs in the study of radiographic imagery. Microdensitometric methods have also been
Este estudio se realiz6 en segundos premolares y primeros molares inferiores de cuatro monos adultos. Cada diente fue sometido a un tratamiento de Conductos convencional seguido de una apicectornia estandardizada de la raiz distal. Los defectos quirurgicos standard se dejaron cicatrizar durante 2, 4, 8, 6 16 semanas luego de la apicectomia. Luego de sacrificados los animales se obtuvieron cortes de bloques posteriores de la mandibula. Cada secci6n fue radiografiada con un bloque standard de aluminio. La impresion isodensimetrica a partir de la imagen radiogrMica fue evaluada respecto a la progresion cronologica de la regeneracion osea. Las im:~genes radiogrMicas de los defectos que 233
234
Sieraski and Corcoran
used in the evaluation of radiographic techniques, response of radiographic films to variations in exposure or processing, and variation in resolution between film types and brands (14-17). The purpose of this study was to assess radiographic osseous healing kinetics after standardized apicoectomy using isodensitometric methods. This is the first of a series on osseous healing kinetics after apicoectomy, to include: II. A Quantitative Histological Appraisal and III. Correlation Between Histology and Radiography. MATERIALS AND METHODS
A study was conducted on four 8- to 12-kg adult monkeys (Macaca rhesus) with completed root end development. Mandibular lateral incisor, first premolar, second premolar, and first molar teeth were used. Each tooth was subjected to conventional root canal treatment, followed 6 to 7 days later by standardized apicoectomy procedures. In each posterior tooth the apicoectomy procedures were limited to the distal root. In this radiographic investigation only second premolar and first molar teeth were suitable for controlled projections. A total of 16 teeth in the four healing periods were involved. Osseous healing was studied after periods of 2, 4, 8, and 16 wk after apicoectomy. Radiographic images of osseous regeneration were quantified using microdensitometric tracings in conjunction with an aluminum step-wedge standard. These readings were converted into isodensitometric print-outs which were compared and assessed for a chronological progression of radiographic osseous regeneration. An operative schedule was devised which assigned two teeth from each monkey to a healing group so that the resultant groups each contained an equal distribution of tooth types. The schedule also separated operations on adjacent teeth by a minimum of 4 wk. The animals were sedated and then anesthetized by intramuscular injections of Xylazine (2 mg/kg, Rompun; Cutter Laboratories, Inc., Shawnee, KA) and ketamine hydrochloride (10 mg/kg, Vetalar; Parke-Davis, Morris Plains, NJ), respectively. Two percent lidocaine (Xylocaine; Astra Pharmaceutical Products, Inc., Worcester, MA) with 1:100,000 epinephrine was used as a local anesthetic to aid in hemorhage control and management. An effort to preserve asepsis was exercised during all procedures. Initially and periodically all teeth were scaled and polished to maintain oral hygiene. Standard sterile instruments, materials, and techniques were used in the surgical and nonsurgical procedures. Conventional root canal procedures were completed in a single treatment. Cotton roll isolation was used in that salivation was eliminated by the anticholinergic effects of the Xylazine. The teeth were preoperatively radiographed and opened with appropriate endodontic access cavities. All root canals of these teeth were
Journal of Endodonticl
endodontically prepared to a # 2 5 K-type file using sterile saline as an irrigant. Working lengths were esti. mated from "trial length" radiographs and established when negotiable as 1 to 1.5 mm short of each respec. tive radiographic apex. These were then each dried and filled with a fitted #25 gutta-percha point, a Grossman type sealer (Roth Drug Co., Chicago, IL), and lateral condensation of accessory #25 gutta-percha points. The access openings were filled with a reinforced zinc oxide and eugenol and the teeth were radiographed.. The apicoectomies were performed with a specially developed 3-mm trephine bur with a self-limiting depth of 7 mm (Fig. 1). A semilunar mucoperiosteal flap was opened and the appropriate root apex was radiographically located using a lead foil marker (Fig. 2). The trephine bur was then used perpendicular to the cortical bone surface in a low-speed handpiece. Intermittent sterile saline irrigation was used as a coolant as the bur performed each apicoectomy and reached its self-limiting depth in the production of a standardized periapical surgical defect (Fig. 3). Residual debris from the trephine core were removed from the surgical site (Fig. 4) and the area was flushed with sterile saline. The mucoperiosteal flap was repositioned and sutured into place with either (4-0) black silk or (5-0) Dacron sutures. The teeth were then radiographed, and the sutures were removed 5 to 7 days after these procedures. The body weight of each monkey was periodically recorded as a health monitor. In one case a monkey had dropped 2 kg over the first 12 wk. At that time, 2 ml of aqueous penicillin G was administered by intramuscular injection. This was followed 1 and 5 days later by 1 ml of aqueous penicillin G injections. The animals weight stabilized for the remainder of the experiments. No other animal presented with significant weight loss. At the end of the experiment, the animals were sedated and perfused through the left ventricle using 0.9% saline followed by fixation with glutaraldehydephosphate-buffered paraformaldehyde (18). The man-
FiG 1. Trepine bur schematic (3-mm diameter, 7-mm self-limiting depth).
lfol. 10, No. 6, June 1984
Radiographic Healing after Apicoectomy
235
dibles were sectioned distal to the third molar teeth and postfixed in glutaraldehyde-phosphate-buffered paraformaldehyde for 1 wk. Each mandible was then sectioned distal to the cuspid teeth, producing two posterior pieces and one anterior piece. Each posterior piece was radiographed lingual-side down with an aluminum step-wedge projected on the same film (Fig. 5). The aluminum step-wedge ranged in thickness from 0.5 to 8 mm. Occlusal dental X-ray film (Kodak Ultra-Speed DF 57) was used with a film to
FIG 4. Standardized volumetric surgical defect. A, Trephine core. B, Surgical site prior to closure.
FIG 2. Radiographic location of surgical sites. A, Lead foil marker clinically. B, Radiopaque marker on radiograph.
FiG 3. Surgical site prior to trephine core removal.
source distance of 12 inches (long cone). Each film was exposed for 8 impulses at 90 kVp and 10 mA with a General Electric 100 X-ray machine. The exposed films were developed at the same time in a Phillips automatic film processor, set at 20~ and 4.5 min. The radiographs were analyzed using a Joyce-Loebl automatic recording microdensitometer, model MK III B (Joyce-Loebl & Co. Ltd., Gateshead, IL). The films were cut to separate the specimen images from those of the step-wedge. Each specimen image was then cut approximately 3 to 4 mm anterior to the image of the second premolar defect on a perpendicular to a line bisecting both defects to be studied. This cut edge was aligned along with the corresponding step-wedge image for scanning by the microdensitometer. Densitometric measurements of each specimen image consisted of a series of parallel scans resulting in 25 density tracings (Fig. 6). These scans were spaced at 0.25-mm intervals and positioned so that the defect images centered the 6-mm range. A density tracing was also made of the step-wedge from a scan positioned to measure all of the step images. The tracings were analyzed using a transparent grid overlay and calculated aluminum equivalents from corresponding step-wedge tracings. A series of 12 vertical
236
Sieraski and Corcoran
deflection ranges were defined for each radiograph. The vertical deflection ranges were chosen to standardize their aluminum equivalent values and establish values which equalized the amount of pen deflection between ranges. Each vertical deflection range was assigned a code symbol as well as a visual density symbol which were equated to an aluminum equivlent thickness (Table 1). Each tracing was then recorded as a series of codes coresponding to vertical pen deflection readings at consecutive horizontal coordinates spaced at 0.2-inch intervals. An isodensitometric image was produced from each series of 25 tracing code strings. The code strings were entered into a home computer system (Apple I1+, Apple Computer, Inc., Cupertino, CA) using a word processing program (Gutenberg, Micromation Ltd., Toronto, Ontario, Canada), each code string representing one line of text. Each code symbol was then searched for and replaced by its corresponding visual density symbol. The resultant print-out was an isodensitometric image of the original radiograph in the area of the defects (Fig. 7). Isodensitometric images of periapical surgical defects were quantitated and averaged as aluminum equivalent values. Each surgical defect was outlined on the print-
Journal of Endodontics
A
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TABLE 1. Equivalency chart between symbol sets
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outs as an ellipse corresponding to the distortion of the 3-mm diameter circular cut. The positions of these ellipse outlines were then transposed onto similar printouts into which corresponding code symbols and intervening dashes had been substituted back for the visual density symbols (Fig. 8). The codes inside each ellipse were then converted into their aluminum equivalent values and calculated as an average aluminum equivalent value for each defect. An average aluminum equivalent value and standard deviation were similarly calculated for areas of undisturbed interradicular bone to use for comparative purposes. RESULTS
FIG 5. Radiographic technique. A, Positioning of specimen and stepwedge. B, Radiographic image.
An aluminum equivalent value was calculated for each surgical defect image and an average value determined for each healing period (Table 2). Radiographic images of defects that had healed for 2, 4, 8, and 16
Vol. 10, No. 6, June 1984
Radiographic Healing after Apicoectomy
237
TABLE 2. Radiographic densities* for individual surgical defect images T O Monkeyno. 1 Monkeyno. 2 Monkeyno. 3 Monkeyno. 4 , A O ,9,zo 1 29 3 0 , 9 20 29 30 19 20 29 30 19 2 o ~ 3 o
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POST-SURGICAL PERIOD (Weeks) FIG 9. Radiographic osseous healing kinetics after apicoectomy. Density ranges were illustrated for each postsurgical period and a curve from average values was used to represent osseous healing kinetics. The radiographic density range of undisturbed interradicular bone was represented by the patterned area.
;'(ONLY NO.4
wk after surgery had average aluminum equivalent values of 2.6, 2.5, 2.7, and 3.6 mm, respectively. These values were used to graphically represent radiographic osseous healing kinetics after apicoectomy (Fig. 9). The average aluminum equivalent value for areas of undisturbed interradicular bone was 3.5 mm with a standard deviation of 0.39 mm. This average value _+1 SD was used as an aluminum equivalent range to represent healed bone density. "t"(~--'-t
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FJG 8. Defect localization and translation into code symbols. A, Isodensitometric image with estimated surgical site boundaries, B, Image translation into code symbols.
An animal model system for the radiologic study of osseous healing kinetics after apicoectomy is dependent on methods of standardizing the surgical wounds and radiographic techniques. Unfortunately, anatomical considerations (sinus cavities, mandibular canals) limit the number of available surgical sites per animal. Radiographic standardization further restricts the sample size in this study. The limited data obtained in this study cannot serve to provide any biological conclusions; rather, it should be used to establish the credibility of the methods used in the model system and provide an initial pool of base line data. The trephine bur designed in this study served to
238
Sieraski and Corcoran
perform an apicoectomy and produce a standardized volumetric surgical defect. Variation in the radiographic location of the burring sites would seem to be the weakness in standardization of these techniques. A semilunar flap was used to establish surgical access and enhance undisturbed healing when sutured. Coolant was used during surgical burring to minimize any thermal necrosis of bone. Radiographic techniques were standardized to validate interpretation of densitometric findings. Each specimen was radiographed with an effort to maintain uniform distances and angulation between the film, specimen, and X-ray source. The films were exposed and processed consecutively as a group using the same machines and identical settings. The same aluminum step-wedge was placed in each projection to produce a standard image for densitometric comparison. The Joyce-Loebl microdensitometer produced graphic recordings from direct readings off the radiographic images. The microdensitometric data were quantitated and processed using the graphic imagery provided by a home computer system. The use of isodensitometric images allowed identification of density readings localized to the surgical defect sites. The use of density ranges and interpretation of image position were sources of standard error for all specimens. Earlier studies (12, 13) have used typewriter symbols singularly or in combination to produce isodensitometric images. The advent of the dot matrix printer and computer software to create user-definable symbols provides an optimum mechanism for the production of superior isodensitometric imagery. The results indicate that the data can be divided into two groups: that from the 2-, 4-, and 8-wk healing periods and that from the 16-wk healing period. The images of defects that had healed for 2, 4, or 8 wk had similar radiographic densities. These were below that density range estimated for undisturbed interradicular bone. The images of defects that had healed for 16 wk were within radiographic density range for undisturbed interradicular bone. These results would seem to indicate that the majority of the first 8 wk of healing occurs as soft tissue organization and that healing between 8 and 16 wk occurs with a significant amount of mineralization. The radiographic images of surgical sites after 16 wk of healing would seem to indicate complete osseous regeneration. These results would seem compatible with the radiographic patterns of early bone resorption followed by increased bone densities in implant studies (10, 11 ). The radiographic densities at any single healing period occurred as a range varying from 0.5- to 0.8-mm aluminum equivalents. These variations were not statistically analyzed due to the limited sample size. Monkey 2 was treated with an antibiotic during the course of treatments, which may account for its tendency toward
Joumal of Endodontics
greater than average radiographic densities. Anatomical location may also affect radiographic healing as seen in pairings of teeth within the same monkey (monkeys 1 and 2). These suggest a tendency for periapical regions of premolar teeth to reach a greater radiographic density than those of molar teeth in the same healing period. This is most likely attributable to the superimposition of larger marrow spaces common in more posterior regions of the mandible. The methods presented in this investigation define an animal model system for the study of osseous healing kinetics after apicoectomy. Despite the efforts to standardize all procedures, it would seem that radiographic healing occurs in broad ranges. Correlation between quantitative radiographic and histological osseous regeneration would have great implications in the transference of histological results in animal studies to radiographic results in clinical studies. This initiates a series of works to establish a complete system for the study of implant materials to enhance periapical osseous regeneration. SUMMARY
An animal model system for the study of osseous healing kinetics after apicoectomy is presented. A home computer system was used to produce isodensitometric images of radiographic healing after apicoectomy. The results of this study serve to validate the methodology and provide an initial pool of base line data for future studies. Se presentO un sistema de modelo en animales para el estudio de la cinetica de la cicatrizacion 6sea despues de una apicectomia. Se utilizo un sistema de computaci6n casero para producir im&genes isodensim6tricas de cicatrizaci6n radiogr&fica despues de la apicectomia. Los resultados de este estudio sirvieron para convalidar la metodologia y proveer una base de datos para futuros estudios. We want to express our thanks and appreciation to the following individuals for their support and efforts of this investigation: Maynard Ruud, Hu-Friedy Corp.; Randal Kadykowski, draftsman; Henry Kazlauskas, director of animal care, University of Michigan, Dental Research Institute; Charles Cox, associate research scientist and instructor in dentistry, University of Michigan, Dental Research Institute; Thomas Dunn, PhD, professor of chemistry, University of Michigan; Robert Ause, research assistant in chemsitry, University of Michigan; and, Chris Jung, artist. Dr. Sieraski is clinical instructor and Dr. Corcoran is associate professor and chairman, Department of Endodontics, School of Dentistry, University of Michigan, Ann Arbor, MI.
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V~. 10, No. 6, June 1984 4. Nord PG Retrograde rootfilling with Cavit: a clinical and roentgenological study. Sven Tandlak Tidskr 1970;63:261-73. 5. Nordenram A, Svardstrom G. Results of apicoectomy. Sven Tandlak Tclskr 1970;63:593-604. 6. Perrson G. Prognosis of reoperation after apicoectomy. A clinical radiolOgiCalinvestigation. Sven Taodlak Tidskr 1973;66:49-68. 7. Ericson S, Finne K. Perrson G. Results of apicoectomy of maxillary canines, premolars and molars with special reference to orantral communication a prognostic factor. Int J Oral Surg 1974;3:386-93. 8. Perrson G, Lennartson B, Lundstrom I. Results of retrograde root filling with special reference to amalgam and Cavit as root-filling materials. Sven Tandlak Tidskr 1974;67:123-34. 9. Hirsch J-M, Ahlstrom U, Henrikson P-A, Heyden G, Peterson L-E. Perlapical surgery. Int J Oral Surg 1979;8:173-85. 10. Matsue I, Collings CK, Zirnmerman ER, Vail WC. Microdensitometric analysis of human autogenous alveolar bone implants. J Periodont 1970;41:489-95. 11. Matsue I, Zimmerman ER, Collings CK, Best JT. Microdensitometric analysis of human autogenous bone implants. I1. Two dimensional density and pattern analysis of interproximal alveolar bone. J Periodont 1971 ;42:435-8.
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12. Isenberg G, Goldman HM, Spira J, Parsons FG, Street PN. Radiograph analysis by two-dimensional microdensitometry. J Am Dent Assoc 1968;77:1069-73. 13. Duinkerke ASH, Van de Pohl ACM, Doesburg WH, Lemmens WAJG. Densitometric analysis of experimentally produced periapical radiolucencies. Oral Surg 1977;43:782-97. 14. Duinkerke ASH, Van de Poel ACM, Doesburg WH, Lemmens WAJG. Compensation of differences in density of radiographs by densitometry. Oral Surg 1978;45:635-42. 15. Sivasriyanond C, Manson-Hing LR. Microdensitometric and visual evaluation of the resolution of dental films. Oral Surg 1978;45:811-22. 16. Price C. A densitometric evaluation of two radiographic duplicating films under diffenng conditions of exposure and processing. Oral Surg 1980;50:1904. 17. Beyer-Olsen EM, Eggen S. Evaluation of the reproducibility of two bitewing techniques by means of microdensitometric recording method. Oral Surg 1983;55:103-7. 18. Cox CF, Heys DR, Heys RJ. A gravity perfusion technique for lab animals. Lab Animal 1977;6:18-22.