Jorrmul of Orrhop,pnrdicRrsrnrch 15221-226 The Journal of Bone and Joint Surgery, Inc 0 1997 Orthopedic Research Society

Rat Model of Distraction Osteogenesis *t$J. Aronson, $X. C. Shen, *R. A. Skinner, *W. R. Hogue, t$T. M. Badger, and t$C. K. Lumpkin, Jr. Departments of "Orthopaedics and ?Pediatrics, University of Arkansas for Medical Sciences and Laboratory f o r

Limb Regeneration Research, and $Arkansas Children's Hospital Research Institute, Little Rock, Arkansas, U.S.A.

Summary: Prior studies of distraction osteogenesis in dog and rabbit models have shown predominantly intramembranous bone formation. Other models of fracture healing normally display mixtures of both endochondral and intramembranous bone formation. We have established a rat model of tibial lengthening that reliably reproduces the pattern of zonal osteogenesis previously observed in dog and rabbit models. A distraction rate of 0.25 mm twice a day with a 0-day latency period produced intramembranous bone with zones of progressive mineralization from collagen. With this protocol, rats bridged the distraction gap with a 25% increase in the tibial bone length. After 20 days of distraction and 50 days of consolidation, the three-point bending stiffness. as a percentage of the contralateral control. reached a level equivalent to that measured in the canine model for a 15% lengthening (28-day distraction and 84-day consolidation). Radiodensitometric analysis of the regenerate bones measured 97% of the unaffected contralateral tibial densities, and mineral analyses demonstrated that calcium and phosphorus levels in the regenerate bone reached 78% of contralatera1 tibial levels by day 70. We concluded that a rat model of distraction osteogenesis will be useful for a wide range of studies involving rapid intramembranous bone formation.

Distraction osteogenesis is a unique clinical method for regenerating local bone deficiencies in length, width, or alignment or in bones with intercalary gaps, nonunions, or osteomyelitis. Clinically and in experimental models, the new bone sections appear to be radiographically equivalent in cross section and in quality to the adjacent sites in the host bone. This laboratory has previously used both a dog and rabbit model for distraction osteogenesis, and much of this work has recently been summarized (2). Although the dog model has been the gold standard for distraction osteogenesis research, certain disadvantages, most prominently cost and availability of molecular probes, prompted us to develop a rat model for tibial distraction osteogenesis. The inherent advantages of the rat model include comprehensive understanding of rat endocrine systems. nutritional biochemistry (requirements similar to those of humans), and bone metabolism (7). Rats are less expensive and easier to handle than dogs, and a growing number of molecular probes are available for them. In addition, our labora-

tory has had experience with rats using cannulated delivery systems for both intragastric and intravenous supplements (4). We selected the tibia for lengthening for several reasons: the original dog model utilized the tibia, thereby providing a basis for comparison; the tibia is one of the longest bones in the rat hind limb and the most accessible surgically; and with the ring fixator attached to the tibia, the rat can bear weight in a nearly normal posture, permitting regular daily activities.

METHODS Animals A total of 54 virus-free, adult (450-500 g) male Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN, U.S.A.) for use in the rate, latency, and time course studies. They were housed in individual cages in temperature (22°C) and humidity (50%) controlled rooms having a 12-hour light/dark cycle with lights on at 0600 hours.

Experimental Groups The effects on osteogenesis of varying the rate of distraction (the total daily distraction distance) were investigated.To compare distraction rates (0.5 mm/day or 2.0 mm/day), two groups of 10 rats each underwent tibial lengthening of 10 mm, creating a 23-25% elongation. The effects of varying the latency period (the time from osteotomy to initial distraction) were studied. To compare latency periods, a group of 10 (0-day) rats and a group of 14 (7-day) rats underwent tibial lengthening at 0.5 mmlday for 20 days, followed by a 50-day consolidation period. Radiographs were made weekly for 10 rats from each group, from first distraction to day

Received May 13,1996; accepted November 8, 1996. Address correspondence and reprint requests to J. Aronson at Arkansas Children's Hospital. Department of Orthopaedics, Slot 839. 800 Marshall Street. Little Rock. AR 72202, U.S.A. Email: [email protected] Presented in part at the 38th Annual Meeting of the Orthopaedic Research Society, Washington. D.C.. U.S.A.,February 17. 1992.

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R A T M O D E L OF DISTRACTION OSTEOGENESIS 70. The tibiae were also analyzed by gravimetric, biomechanical, and chemical methods. For sequential qualitative histology, rat tibiae (n = 10) that had been lengthened after a 7-day latency period were collected 7,14, 21, 28,35, and 42 days after osteotomy and were compared with tibiae that had been lengthened after a 0-day latency period and were collected 7,14,21, and 28 days after osteotomy.

Operative Techniques All rats were handled by animal care personnel 5-7 days prior to the surgeries. A stainless-steel ring fixator (scaled down from the dog model) was placed on the same tibia of each rat (Fig. 1). The rat was anesthetized with an intraperitoneal injection of Nembutal (sodium pentobarbital), 50 mg/kg. The rat limb was shaved and wiped with 70% ethanol, and a sterile drape was placed around the operating field. The mid-fibula was manually broken by direct lateral compression. Four spinal needles (22 gauge = 0.7112 mm diameter, 3.5 inch [88.9 mm] length) were power drilled (model 275; Dremel, Racine, WI, U.S.A.) transosseously to attach the external fixator to the tibia. Two needles were drilled into the proximal tibia just below the knee joint (at a standardized angle of 55" relative to each other by using the ring as a template) in the transverse plane and fixed under tension to slotted bolts on both sides of the proximal ring. Two needles were similarly placed above the ankle and attached to the distal ring. The tibia was thus secured by these four transosscous needles, two above and two below the mid-diaphyseal osteotomy site. The fixator (fabricated in our machine shop) was designed to manually distract the proximal needles from the distal needles in a controlled fashion by two threaded telescoping support rods connecting the two rings. After stabilization of the fixator, a low-energy osteotomy was created by percutaneously predrilling from the lateral to the medial side with a 1.0 mm Kirschner wire (Simplex Medical, Beaverton, OR, U.S.A.), midway between the proximal and distal needles. After the ring support rods were loosened. counter-opposed digital pressure was applied transversely to the tibia in the coronal plane for reliable osteoclasis. The resultant fractures were usually transverse or short oblique, with rare comminution. The two medial and lateral support rods were retightened to properly align and reduce the fracture as judged by palpation of the subcutaneous bone and visual examination of the limb. The rats were returned to their cages for observation during recovery from anesthesia. Distractions of onehalf turn (0.25 mm) counterclockwise were performed twice daily without sedation using a 7/64-inch (2.78 mm) hex key.

Analytical Studies Cage activity, including ambulation, feeding, and handling behavior, was noted daily on standardized forms. At death, the rats were given lethal injections of Nembutal (sodium pentobarbital) and were examined for contractures of knees, ankles, and toes just prior to removal of the lengthened and contralateral tibiae. The tibiae were divided into two analytical protocols. In one protocol, the tibiae were fixed in 10% neutral buffered formalin for microradiography and decalcified histology. For the alternate protocol, macerated tibiae were fresh-frozen for sequential analysis by microradiography, gravimetrics, biomechanical testing, and wet ashing for calcium and phosphorus content. Radiodensity by video microscopy: Following gross examination (length, mass, and volume), radiographs of the specimens were made using one anteroposterior view to standardize the results and because the lateral view was obscured by the fixator support rods. We used X-OMAT film (Eastman Kodak, Rochester, NY, U.S.A.) in a Micro 50 closed-system radiography unit (Xerox, Pasadena, CA, U.S.A.) at 40 kV for 20 seconds to maximize bone detail while minimizing soft-tissue interference (8). Distraction gap radiodensities, using the contralateral tibia and adjacent cortex for standardization, were compared with the aid of video microscopy using

223

a Media Grabber 2.0 video capture board (Raster Ops, Santa Clara, CA, U.S.A.) and Image Analysis 1.49 (National Institutes of Health, Bethesda, MD, U.S.A.). The mineralized bone area within the distraction gap was measured by selecting and outlining regions with a radiodensity defined as equivalent to or greater than the cancellous bone surfaces of the adjacent host. Nonmineralized tissue regions were defined and measured when the radiodensity was less than the host cancellous bone surfaces. The distraction gap area was measured from proximal to distal cortices. The percentage of mineralized bone area within the distraction gap was calculated by dividing the mineralized bone area by the total gap area. The contralateral tibiae were used as controls for the host bone values. Hisrologyc Following radiography, the tibiae from the lengthened and contralateral limbs were decalcified in 5% formic acid (9,lO). The specimens were embedded in paraffin and cut into 6-7 pm longitudinal (coronal) sections on a microtome for staining (hematoxylin and eosin and Masson trichrome). The samples chosen for analysis were sections that represented an endosteal (central or near central gap) location. This was accomplished by choosing slides that, at both the proximal and distal ends, contained full-thickness cortices with an intact marrow space. In several specimens, sections were taken end-to-end from one peripheral cortex to the opposite cortex in 50 pm increments to test the reliability of our sampling technique. For quantitation of histology, the slides were videorecorded and analyzed by Image Analysis 1.49 under low-power magnification. Relative areas of tissue types (fibrous, bone. cartilage, and hematoma) were then measured. Biomechanical testing: The previously fresh-frozen macerated tibiae were thawed for testing to failure by three-point bending in the midsagittal plane (dorsal deflection at the center of the tibia) o n a Bionix 810/858 testing system (MTS, Minneapolis, MN, U.S.A.) at a constant rate of 0.25 Nisec. The load and deflection were measured on line with a Macintosh IIx computer. The strength was calculated as the maximum bending moment and ultimate load to failure. The bending stiffness was determined from the slope of the linear part of the curve. Chemical analysis: Following the strength analyses above, the samples were prepared for calcium and phosphorus analysis by wet ashing and were measured in the hospital's clinical laboratory using a serum chemistry kit (Dimension; DuPont, Wilmington, DE, U.S.A.).

Statistical Analyses In all experiments, animals of approximately equal weight and age were randomly assigned to groups. For these experiments, differences between group means were determined by the Student's t test.

RESULTS Histological Analysis As in previous work with the dog model, we identified five distinct zones of intramembranous bone formation in the gap between the host bone surfaces (1). A central radiolucent zone, termed the fibrous interzone, occupies approximately the central 30% of the gap. The fibrous interzone consists of fibroblastlike cells interspersed between collagen bundles that are organized parallel to the direction of distraction. Osteoid and osteoblastic cells are routinely absent from the fibrous interzone. The fibrous interzone is bounded, proximally and distally, by the primary mineralizatiodmatrix front, where nascent osteoid spicJ Orthop Res, Vol. 15, No. 2, 1997

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ules are first seen. Osteoblastic cells resting on the surface of the osteoid spicules become enveloped within the enlarging spicule. The spicule tips begin at diameters of 7-10 pm and expand to microcolumns of 150-200 pm toward each host bone surface. On cross section, the microcolumns alternate with thinwalled sinusoids and are devoid of haversian canals. The sinusoids extend from the medullary canal of each host bone surface to the ends of the developing trabeculae in the primary mineralization/matrix front zone. The primary mineralizatiodmatrix front zones are, therefore, bounded proximally and distally on the host bone sides by the microcolumn formation zones. The sections taken from one peripheral cortex to the opposite cortex in 50 pm increments demonstrated that intramembranous bone formation predominates throughout. In this model, the total area of cartilage islands (in relatively avascular environments) in the central intercortical gap was measured at 3-5%. Additional subperiosteal cartilage can usually be identified peripheral to the intercortical gap. Isolated areas of "chondroid" bone formation are seen adjacent to the cartilage islands. During the consolidation phase, after the gap has been bridged by the bone microcolumns, multinucleated osteoclasts appear in the newly forming marrow spaces at both the proximal and the distal gap ends. These osteoclasts are presumably involved in the remodeling of the new medullary cavity. Predominantly intramembranous bone formation occurred in all rats in the 0-day latency group (Fig. 2). In the rats in the 7-day latency group, both intramembranous and endochondral bone formation occurred during the first 2 weeks, and then intramembranous bone formation predominated from 3 to 6 weeks. SpecJ Orthop Res, Vol. 15, No. 2, 1997

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imens that were taken from the 7-day latency group at additional time points demonstrated that bridging occurred about 2 weeks later than in the 0-day latency group. This delay was attributed to the presence of endochondral bone formation within the gap.

Distraction Rate The effects on osteogenesis of rate of distraction (the total daily distraction distance) were investigated. The distraction rate of 0.5 mm/day was tolerated better by the animals, as evidenced by better feeding

FIG. 3. Representative radiographs of day 20 (left) and day 70 (right) distraction gaps.

RAT M O D E L OF DISTRACTION OSTEOGENESIS

and ambulation, easier handling behavior, and fewer contractures. Histological analysis suggested that more than half of the animals in the faster rate group (2.0 mm/day) had evidence of delayed union or nonunion, since the central fibrous interzones contained predominantly disorganized fibrocartilage.

Radiodensity The densities of the regenerate bone were measured to compare potential differences in osteogenesis between 0-day and 7-day latency periods. During the 20-day distraction period, a central radiolucent zone (corresponding to the histological fibrous interzone) was observed between areas of radiodense new bone arising from each host bone surface (Fig. 3). By day 70, the radiodensity of the central fibrous interzone equaled the proximal and distal primary mineralizatiodmatrix front-microcolumn formation zones in both groups (Fig. 3). The radiodensity of the distraction gaps on day 70 reached an average of 97% of control density in the 0-day latency group and 92% of control density in the 7-day latency group. Biomechanical Testing Strength analyses were performed on the consolidated tibiae from the study described above. All distracted tibiae from both latency groups failed by transverse or short oblique fracture through the center of the osteogenic area. All control tibiae failed by comminuted fracture, transversely or obliquely in the diaphysis, except one that occurred at the proximal metaphyseal-diaphyseal junction. No significant differences were observed between the 0 and 7-day latency groups in the ultimate load (31.7 compared with 28.4 N), stiffness (37.5 compared with 25.7 N/mm), or maximum bending moment (28.4 compared with 25.7 N cm); however, all mechanical parameters were significantly lower in the lengthened tibiae compared with the contralateral controls (load, 77-80 N; stiffness, 91-100 N/mm; and moment, 93-95 N cm).

Chemical Analyses The mineral content of the regenerate bone of both latency groups at day 70 averaged 151-176 pg of calcium and 61-82 pg of phosphorus per mg of dry bone, about 72-78% of the values for the contralateral tibia1 controls. The calcium-phosphorus ratio ranged from 2.15 to 2.16 in all specimens.These values are expected for hydroxyapatite.

DISCUSSION We have developed a rat model of distraction osteogenesis, analogous to the dog and rabbit models, that reliably results in zonal regions of predominantly intramembranous bone formation. The effects of dis-

225

traction rate and latency on the histology, strength, and mineral content of the regenerate bone were investigated. Although the faster rate achieved the lengthening goal sooner with additional time for consolidation, the slower rate (0.25 mm twice a day) produced more mineralized bone. Also, a distraction rate of 0.25 mm twice a day with a 0-day latency period optimized intramembranous bone production. By this protocol, rats regenerated a 25% increase in bone length. For these experiments, we employed a distraction period of 20 days with varying consolidation periods of as long as 50 days. Mineral analyses demonstrated that calcium and phosphorus levels in new bone reached 78% of those in the contralateral, unaffected tibiae by day 70. The mineral content was less than the overall radiodensity might suggest. This finding may reflect an overall increased bone volume with slightly subnormal mineral con tent. The bone strength analyses showed that rats, with this protocol and supplied with rat chow ad libitum, produced a solid bone bridge after 20 days of distraction and 50 days of consolidation. The mechanical qualities, when expressed as a percentage of the contralateral tibia, were equivalent to the levels reached in the dog model (15% lengthening) after 28 days of distraction and 84 days of consolidation (3). This bone bridge had less than 50% of normal bone strength, stiffness, and energy to failure, indicating that considerable remodeling has yet to take place. In all quadruped models tested, weight-bearing without bending of the distracted bone has been demonstrated. Histologically,the zonal intramembranous bone formation in the rat model is very similar to our prior studies in the dog and rabbit, as well as to biopsies from human patients undergoing distraction osteogenesis (2). It should be noted that, in these animal models, a distinction can be made concerning the nomenclature of the primary mineralization/matrix front zone depending on whether the analysis is radiographic or histologic (hematoxylin and eosin). Specifically, on radiographs the primary mineralization/matrix front zone designation represents the primary mineralization front, whereas histologically the primary mineralizatiodmatrix front zone designation represents the primary matrix (osteoid) front. This rat model should allow, by immunohistochemical and in situ hybridization techniques, in vivo comparison of direct bone formation (intramembranous ossification) with established in v i m models derived from rat calvaria, chicken bone, and rat and human bone marrow (6,ll). This model may enable the study of the effects of administration of local or systemic modulators, or both, on bone formed directly from the osteoblast cell lineage. To our knowledge, one other group has published a J Orthop Res, Vol. 15, No. 2, 1997

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rat model for limb lengthening (by callus distraction) that demonstrated primarily endochondral gap ossification (5). Despite a similar rate of distraction, the open osteotomy, the 5-day latency period, or the histological sampling protocol, or all of these, may account for the lack of organized intramembranous ossification reported in that study. We concluded that a rat model of distraction osteogenesis will be a useful analog to the human distraction osteogenesis process. We think that this model has several advantages over dog, rabbit, or sheep models. Among the advantages are size, expense, handling, a growing number of molecular probes, and a database on the physiologic and biochemical systems important to the process of intramembranous ossification. Acknowledgment: This work was supported by the United States Department of Agriculture Grant 9402890, Brooks Medical Research Fund, Arkansas Children’s Hospital, and the Departments of Orthopaedics and Pediatrics, University of Arkansas for Medical Sciences.

REFERENCES 1. Aronson J, Good B, Stewart C, Harrison B, Harp J: Preliminary studies of mineralization during distraction osteogenesis. Clin Orthop 250:43-49, 1990 2. Aronson J: Experimental and clinical experience with distraction osteogenesis. Cleft Palate Craniofac J 31:473-481, 1994 3. Aronson J: Experimental assessment of bone regenerate

J Orthop Res, Vol. IS, No. 2, 15’97

quality during distraction osteogenesis. In: Bone Formation and Repair, pp 441-463. Ed by CT Brighton, G E Friedlaender, and JM Lane. Rosemont, Illinois, American Academy of Orthopaedic Surgeons, 1994 4. Badger TM, Ronis MJJ, Lumpkin CK Jr, Valentine CR, Shahare MM, Irby D, Huang J, Mercado C, Thomas P, IngelmanSundberg M, Crouch J: Effects of chronic ethanol on growth hormone secretion and hepatic cytochrome P450 isozymes of the rat. J Pharm Exp Ther 264:438-447,1993 5. Kossmann T, Giebel G, Glombitza A: Rat model for limb lengthening by callus distraction. Res Exp Med (Berl) 193:1320,1993 6. Rickard DJ, Kassem M, Hefferan TE, Sarkar G, Spelsberg TC, Riggs B L Isolation and characterization of osteoblast precursor cells from human bone marrow. J Bone Miner Res 111312-324,1996 7. Ronis MJJ, Lumpkin CK Jr, Ingelman-Sundberg M, Badger TM: Effects of short-term ethanol and nutrition on the hepatic microsomal monooxygenase system in a model utilizing total enteral nutrition in the rat.Alcoho1 Clin Exp Res 15:693699, 1991 8. Skinner RA, Fromowitz FB: Fuxitron X-ray Unit in Histologic Evaluation of Breast Biopsies: Tech Sample HT-5. Chicago, ASCP Press, 1989 9. Skinner RA, Nicholas R: Preparing Superior Quality Slides of Orthopedic Tissue Using Previously Decalcified Material: Tech Sample HT-2. Chicago, ASCP Press, 1990 10. Skinner RA, Nicholas RW, Stewart CL, Vireday C: Resected allograft bone containing an implanted expanded polytetrafluoroethylene prosthetic ligament: comparison of paraffin, glycolmethacrylate, and Exakt grinding techniques. J Histotech 16:129-137,1993 11 Stein GS, Lian JB: Molecular mechanisms mediating proliferatioddifferentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 141424-442,1993

Rat model of distraction osteogenesis - Wiley Online Library

transverse plane and fixed under tension to slotted bolts on both sides of the proximal ring. Two needles were similarly placed above the ankle and attached to the distal ring. The tibia was thus secured by these four transosscous needles, two above and two below the mid-diaphyseal osteotomy site. The fixator (fabricated in ...

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