Journal of Orthopaedic Research 8769-775 Raven Press, Ltd., New York 0 1990 Orthopaedic Research Society
Effect of Direct Current Stimulation on Bone Growth After Distraction Epiphysiolysis of the Rabbit Tibia Peter M. van Roermund, *Anne Hoekstra, tWalter J. Visser, and SWillem Renooij Department of Orthopaedic Surgery, *Department of Nuclear Medicine, ?Clinical Research Group for Bone Metabolism, #Department of Surgery, University Hospital, State University of Utrecht, Utrecht, The Netherlands
Summary: The effect of direct current stimulation on bone formation during
limb lengthening was tested in a lower leg lengthening model in the rabbit. Limb lengthening was performed by distraction epiphysiolysis. A specially designed external distraction device was placed at the tibia. The distractor allowed 10 mm of lengthening in 4 weeks. Two weeks after starting the distraction, a platinum electrode was passed through the anterior cortex below the tibia1 tuberosity and advanced via the medullary cavity so that the tip rested in the elongated zone. Stimulation started at the time of placement of the electrodes and was continued for 3 weeks. The electrode in the elongated zone served as the cathode; the anode was placed subcutaneously. A 20 pA stimulus was selected. A control group received the same treatment without stimulation. Bone formation in the elongated zone was evaluated by histology and scintigraphy. The data from this experiment show that direct current stimulation in the early phase of a limb lengthening procedure had no effect on the extent of bone formation in the elongated zone. Key Words: Distraction epiphysiolysis-Bone scintigraphy-Electrostimulation-Osteogenesis-Limb lengthening-Rabbit .
Significant inequality in limb length in childhood is a major problem in orthopedic management. The method of limb lengthening widely accepted in the Western world (30,36) involves a diaphyseal osteotomy followed by bone grafting and external fixation. Many problems and complications of these lengthening procedures have been reported (9). In the last decades, good results were reported with distraction epiphysiolysis as a method of limb lengthening (1,5,11,12,14,21,2&28). Applying a gradually increasing distraction force between epiphysis and metaphysis will eventually result in fracturing of the cartilaginous epiphyseal
plate, which is the weakest site of the immature bone. Progressive continuous distraction increases the distance between epiphysis and metaphysis until the desired lengthening is achieved. Ossification of the hematoma in the fracture gap almost always occurs spontaneously, thus restoring the continuity of the lengthened bone. In spite of many advantages of this method, with remarkably minor and infrequent complications, the long healing time remains a problem for the young patient and his family. Full weight-bearing after a lengthening of 5 cm is usually allowed only after 8-9 months. Reducing the duration of the therapy by stimulating bone formation would be a significant breakthrough. Electrical stimulation of bone growth is an accepted alternative method for treatment of delayed bone union and congenital or acquired pseudarthrosis (2,3,6,8,10).
Received August 30, 1989; accepted February 28, 1990. Address correspondence and reprint requests to Dr. W. Renooij at Department of Surgery, Utrecht University Hospital Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
769
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The objective of this investigation was to study the effect of direct current stimulation on bone formation during distraction epiphysiolysis of the tibia of the rabbit. Bone formation in the elongated zone was evaluated with histology and scintigraphy . MATERIALS AND METHODS Distraction Procedure By means of a specially designed device (Fig. l), distraction was applied to the proximal epiphysis of the right tibia of female New Zealand white rabbits (each weighing about 3 kg). Under general anesthesia and oxytetracyclin prophylaxis, two Kirchner wires (1 mm diameter) were drilled through the proximal epiphysis with the aid
of a specially designed drill guide, two wires through the distal cortex of the tibia, and a fifth wire through the anterior cortex of the tibia1 tuberosity to prevent the epiphysis from being drawn dorsally after lysis. The wires were locked in blocks on two divided brass screw bars. The upper part of the bars had a left thread, and the lower part a right thread; both parts were connected inside a turnbuckle. A 360" turn of the buckles caused a 1 mm increase in length of the distractor. The bars were connected with two brass arcs, ventrally and dorsally located so that the animals could move knee and ankle freely. The weight of the device was 50 g. After placing the distractor, the position of the wires was checked by roentgenography. Distraction was performed by daily adjustment of the turnbuckles. A fracture of the growth plate usu-
FIG. 1. A: Schematic representation of the distraction device positioned at the rabbit tibia. B: The roentgenogram shows the distraction device, electrodes, and power pack in position.
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ELECTROSTIMULATION DURING DISTRACTION EPIPHYSIOL YSIS
ally occurred after 4 days. Progressive distraction, approximately 0.5 mm per day, increased the distance between epiphysis and metaphysis such that a separation of 5 mm was achieved in 2 weeks. On the 15th day after placing the distractor, a second operation was performed to introduce an electrode into the elongated zone. Under general anesthesia, an electrode wire was passed through a 1 mm drill hole in the anterior cortex just below the tuberosity and advanced via the medullary cavity into the elongated zone until the tip touched the epiphysis. A widening at the lead-electrode junction locked in the cortical drill hole. The electrode was subcutaneously secured with silk sutures. A power pack was pushed in a subcutaneous pocket in the groin. In the following 2 weeks, distraction was continued at the same rate as before. Thus, after 4 weeks, a total elongation of 10 mm was achieved and the bare tip of the electrode was located inside the elongated zone, in the distal one-half to two-thirds of the zone. The position of the electrode was checked by roentgenography (Fig. 1B). Elongation was measured as the distance between the proximal and distal pins, and the length of the tibia on weekly roentgenograms. At thc start of the experiment, the rabbits were 18 weeks old, which is near the end of their growth phase (19). The animals showed no marked inconvenience during the observation period. Two animals were excluded from the study because of improper electrode placement and one because of a fracture of the tibia. These animals were replaced. Characteristics of Electrostimulation The power supply consisted of a 6 V battery in combination with an electronic circuitry used to produce a constant direct current regardless of changes in resistance between the electrodes. Each circuit was preset to deliver a current of 20 ? 1 FA. The constant current circuitry is documented in detail by Janssen (22). The battery and circuitry were sealed in a capsule of hardened epoxy resin (Hysol, C8-W795, Dexter Corporation, Olean, NY, U.S.A.). The capsule was covered with silicone rubber (Micro-Measurements, Romulus, MI, U.S.A.). Electrodes were made of Teflon-coated 99.9% pure platinum wires, 0.5 mm in diameter (Drijfhout Edelmetaalbedrijven NV, Amsterdam, The Netherlands) with a bare tip of 10 mm. Plati-
771
num was selected because of its good biocompatibility and electrical characteristics (32,33). The connections of the leads to the electrodes were covered with silicone rubber. All electric equipment was sterilized with ethylene oxide gas. Stimulation was continued until 1 week after maximal elongation had been reached. Therefore, the total stimulation period was 3 weeks. The electrode in the elongated zone served as cathode. The anode was placed in the groin near the power pack. Control animals received the same treatment, but the capsule contained no battery. At the end of the experiment, the current supply was checked with an electrometer (Hewlett Packard Nederland, Amstelveen, The Netherlands, model 3465A) before disconnecting the leads. In none of the animals were deviant current intensities measured. The leads were disconnected. The electrodes were left in position. Bone Scintigraphy Procedure On the last day of the experiment, a scintigram was made of the hind legs 2 h after intramuscular administration of 175 MBq of [99"Tc] technetium methylene diphosphonate (99"Tc-MPD) as described before (34). The radioactivity was measured in regions of interest of 15 x 15 mm over the elongated zone and over the eorresponding area on the contralateral tibia. The uptake ratio (UR) of radioactivity was calculated as UR = ( U , - Ub)/(u, + ub),where U , is the radioactivity in the region over the elongated zone, and Ub the radioactivity in the region over the contralateral tibia. Histology The animals were killed with an overdose of pentobarbital. The hind legs were exarticulated, and the tibia grossly cleaned of muscle tissue and processed for histologic evaluation. The bones were stored in 80% ethanol and subsequently dehydrated and embedded in monomethylmethacrylate as described before (20). After complete polymerization 100 pm plane-parallel transverse sections were cut at 1 mm intervals with a low-speed Isomet saw (Buehler Ltd., Evanston, IL, U.S.A.) and further ground to a 50 Fm thickness. Thus, 9-10 undecalcified slides were obtained from the elongated zone
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of each tibia. The surface of the slides was stained with 0.15% light green (Merck 1315, Darmstadt, F.R.G.) in 1% acetic acid. The total area of bone within the perimeter of the elongated zone on these slides was histomorphometrically quantitated with an image-analyzing computer (Quantimet 720, Imanco, Metals Research Ltd., Melbourn Cambridge, England) and expressed as percentage of the surface area within the perimeter of the crosssections.
percentage of the cross-section of histologic slides filled with mineralized bone. Electrostimulation had no significant effect on bone formation. Table 2 presents the results of the scintigraphy measurements at the end of the experiment. There is no significant difference in uptake ratio of 99"Tc-MDP in the elongated zone of control and stimulated animals.
Statistics
The purpose of this report was to test the effect of direct current stimulation on bone formation in a tibia elongation model in the rabbit. Any possible way of stimulating bone formation after distraction epiphysiolysis is worth testing, because positive results shortening the long healing time would mean a major breakthrough in the treatment. In clinical practice, leg lengthening by distraction epiphysiolysis always involves an acute fracture. A fresh fracture of bone may be considered the optimal stimulus for bone repair and growth. Chances of observing an effect from an added external stimulus might be highest as early in the distraction phase as possible. Van Roermund et al. (35) reported that in this rabbit tibia elongation model, active net bone formation occurred from the start of the distraction until 3-4 weeks after reaching maximal elongation. In this experimental design, the electrode was introduced after 2 weeks of distraction when trauma
Differences between stimulated and control data were statistically evaluated using the Student's t test. Each group consisted of five animals. This number is sufficient to evaluate the statistical significance of the effect of electrostimulation specifically focussed on the relevance in clinical therapeutic practice. RESULTS
Bone healing after leg lengthening occurred spontaneously without complications. At the end of the experiment, the elongated zone was filled with a callus mass, both in control and stimulated series (Fig. 2). The extent of bone formation in the elongated zone is presented in Table 1. The data show the
DISCUSSION
FIG.2. Histologic slide of the center of the elongated zone of a control tibia. The black dot in the center is the electrode.
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TABLE 1. Results of histologic evaluation Percentage of tibia cross-section occupied by bone (mean 2 SEM) Control Stimulated
54 2 8 48 2 4
The percentage of bone per cross-section in the 9-10 histologic slides covering the length of the elongated zone was averaged in each animal. The means of the data obtained from five animals in each series are presented.
from fracturing of the growth plate might have subsided. In the 2 weeks of the second phase of lengthening, and 1 week beyond, stimulation was active. In this second phase of the distraction, de novo bone formation would occur in the further elongated zone proximal to the electrode, while at the level of the electrode tip a continued increase in already present callus mass might be expected. Figure 3 illustrates the position of the electrode tip. The results from histologic evaluation of the tibia specimen (Table 1) clearly show that direct current stimulation had no effect on bone formation in the elongated zone. Figure 4 shows a histologic slide of a stimulated tibia directly distal to the elongated zone. It is clear that newly formed bone in the medullary cavity is concentrated around the active electrode, which was not observed around the dummy electrode in the control series. Thus, although stimulation of bone formation could not be detected in the elongated zone, the electrical stimulus had caused bone formation near the electrode tip, which is in agreement with the work of others (7,13,16,18,23,29). In the elongated zone, the normal rate of bone healing is probably already optimal and therefore can not be further stimulated. The second technique to evaluate bone formation in the elongated zone was 99mTc-MDP scintigraphy. Apart from other factors (4,15,17,23,25,31), uptake of 99"Tc-MDP may reflect metabolic activity of osteogenesis. Results from control and stimulated animals were similar (Table 2), which corroborates the histologic data. As discussed before (34), trauma induced by fracturing of the growth TABLE 2. Results of 99"Tc-MDP scintigraphy Uptake ratio (mean 2 SEM) Control Stimulated
33.7 2 6.6 35.4 2 4.1
FIG. 3. Roentgenogram of a rabbit's tibia after re. ieval. The position of the electrode in the center of the elongated zone is clearly demonstrated. The bone present at the level of the electrode in the distal part of the elongated zone was deposited in the first phase of the distraction period and continued to be formed in the second phase, while bone in the region proximal to the electrode tip was derived from formation in the second distraction phase only.
plate and distraction forces exerted in the tissue largely determine the level of uptake of 99"Tc-MDP and may anyway have obscured any stimulatory effects on uptake of 99"Tc-MDP due to an increased osteogenic response. The results of this study clearly indicate that the present form of electrostimulation in the early phase of distraction epiphysiolysis of the rabbit tibia did not enhance the rate of bone formation. In clinical practice, the strength of the new bone in the elongated zone is the determining factor. The present data do not rule out that new bone on the stimulated side may be stronger than on the control side.
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FIG. 4. Histologic slide of a stimulated tibia just distal to the elongated zone. In the medullary canal, bone is concentrated around the negative electrode.
REFERENCES 1. Aldegheri R, Trivella G, Lavini F: Epiphyseal distraction. Chondrodiatasis. Clin Orthop 241: 117-127, 1989 2. Bassett CAL, Mitchell SN, Gaston SR: Treatment of ununited tibial diaphyseal fractures with pulsing electromagnetic fields. J Bone Joint Surg [Am] 6351 1-523, 1981 3. Becker RO, Spadaro JA, Marino AA: Clinical experiences with low intensity direct current stimulation of bone growth. Clin Orthop 124:75-83, 1977 4. Bell FG: Nuclear medicine and skeletal disease. Hosp Pract 7:49-60, 1972 5 . Bjerkheim J: Limb lengthening by physeal distraction. Acta Orthop Scand 6 0 140-142, 1989 6. Brighton CT: The treatment of non-unions with electricity. J Bone Joint Surg [Am] 63:847-851, 1981 7. Brighton CT, Friedenberg ZB, Black J, Esterhai JL, Mitchell JEI, Montique F: Electrically induced osteogenesis: relationship between charge, current density, and the amount of bone formed. Clin Orthop 161:122-132, 1981 8. Brighton CT, Friedenberg ZB, Mitchell EI, Booth RE: Treatment of nonunion with constant current. Clin Orthop 124:106-123, 1977 9. Coleman SS: Lower limb length discrepancy. In: Pediatric Orthopaedics, Vol. 2, ed by WW Love11 and RB Winter, Philadelphia, Lippincott, 1987, pp 805-880 10. Connolly JF: Selection, evaluation and indications for electrical stimulation of ununited fractures. Clin Orthop 161:3953, 1981 11. DeBastiani G, Aldegheri R, Renzi-Brivio L, Trivella G: Chondrodiastasis4ontrolled symmetrical distraction of the epiphyseal plate. Limb lengthening in children. J Bone Joint Surg [Br] 68:55&556, 1986 12. DeBastiani G, Aldegheri R, Renzi-Brivio L, Trivella G: Limb lengthening by distraction of the epiphyseal plate. A comparison of two techniques in the rabbit. J Bone Joint Surg [Br] 68545-549, 1986 13. Dymecki SM, Black J, Nord DS, Jones SB, Baranowski TJ, Brighton CT: Medullary osteogenesis with platinum cathodes. J Orthop Res 3:125-136, 1985 14. Fjeld TO, Steen H: Limb lengthening by low rate epiphyseal distraction. An experimental study in the caprine tibia. J Orthop Res 6:360-368, 1988 15. Francis MD, Ferguson DL, Tofe AJ, Bevan JA, Michaels
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SE: Comparative evaluation of three diphosphonates: in vitro adsorption (C-14 labeled) and in vivo osteogenic uptake (Tc-99m complexed). J Nucl Med 21:1185-1189, 1980 16. Friedenberg ZB, Zemsky LM, Pollis RP, Brighton CT: The response of non-traumatized bone to direct current. J Bone Joint Surg [Am] 56:1023-1030, 1974 17. Genant HK, Bautovich GJ, Singh M, Lathrop KA, Harper PV: Bone-seeking radionuclides: an in vivo study of factors affecting skeletal uptake. Radiology 113:373-382, 1974 18. Harris WH, Moyen BJ-L, Thrasher EL, Davis LA, Cobden RH, MacKenzie DA, Cywinski JK: Differential response to electrical stimulation: a distinction between induced osteogenesis in intact tibiae and the effect of fresh fracture defects in radii. Clin Orthop 124:31-40, 1977 19. Heikel HVA: On ossification and growth of certain bones of the rabbit; with a comparison of the skeletal age in the rabbit and in man. Acta Orthop Scand 29171-184, 1960 20. Hoogendoorn HA, Renooij W, Akkermans LMA, Visser WJ, Wittebol P: Long-term study of large ceramic implants (porous hydroxyapatite) in dog femora. Clin Orthop 187: 281-288, 1984 21. Ilizarov GA, Soybelman LM: Some clinical and experimental data concerning bloodless lengthening of lower extremities. Eksp Khir Anestheziol4:27-32, 1969 22. Janssen LWM: Electrical stimulation of bone tissue. Thesis, State University of Utrecht, Utrecht, The Netherlands, 1987 23. Lavender JP, Khan RAA, Hughes SPF: Blood flow and tracer uptake in normal and abnormal canine bone: comparisons with Sr-85 microspheres, Kr-8lm, and Tc-99m MDP. J Nucl Med 20:413418, 1979 24. Lagey CLRS, Roelofs JMM, Janssen LWM, Breedijk M, Lentferink RHF, Visser WJ, Akkermans LMA, Wittebol P, Renooij W: Electrical stimulation of bone growth with direct current. Clin Orthop 204:303-312, 1986 25. Marty R, Denney JD, McKamey MR, Rowley MJ: Bone trauma and related benign disease: assessment by bone scanning. Semin Nucl Med 6:107-120, 1976 26. Monticelli G, Spinelli R: Distraction epiphysiolysis as a method of limb lengthening. I. Experimental study. Clin Orthop 154:254261, 1981 27. Monticelli G, Spinelli R: Distraction epiphysiolysis as a method of limb lengthening. 111. Clinical applications. Clin Orthop 154:274285, 1981
ELECTROSTIMULATION DURING DISTRACTION EPIPHYSIOL YSIS 28. Monticelli G, Spinelli R, Bonucci E: Distraction epiphysiolysis as a method of limb lengthening. 11. Morphologic investigations. Clin Orthop 154:262-273, 1981 29. Moyen BJL, Lans DA, Thrasher EL, Harris WH: La stimulation Clectrique de I’ostCogCn&se.fitude de la cathode. Acta Orthop Belg 44:664-670, 1978 30. Paley D: Current techniques of limb lengthening. J Pediatr Orthop 8:73-92, 1988 31. Siege1 BA, Donovan RL, Alderson PO, Mack GR: Skeletal uptake of 99mTc-diphosphonate in relation to local bone blood flow. Radiology 120:121-123, 1976 32. Spadaro JA: Electrically enhanced osteogenesis at various metal cathodes. J Biomed Muter Res 16:861-873, 1982 33. Spadaro JA, Becker RO: Function of implanted cathodes in
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electrode-induced bone growth. Med Biol Eng Comput 17~769-775,1979 34. Van Roermund PM, Hoekstra A, Ter Haar Romeny BM, Renooij W: Bone healing during lower limb lengthening by distraction epiphysiolysis. J Nucl Med 29: 1259-1263, 1988 35. Van Roermund PM, Ter Haar Romeny BM, Schoonderwoert GJ, Brandt CJWM, Sijbrandij S, Renooij W: The use of computed tomography to quantitate bone formation after distraction epiphysiolysis in the rabbit. Skeletal Radio1 1652-56, 1987 36. Wagner H: Surgical lengthening or shortening of femur and tibia: technique and indications. In:Progress in Orthopaedic Surgery, Vol. 1, ed by DS Hungerford, Berlin, Springer, 1977, pp 71-94
J Orthop Res, Vol. 8, No.5 , 1990
Effect of Direct Current Stimulation on Bone Growth After Distraction Epiphysiolysis of the Rabbit Tibia Peter M. van Roermund, *Anne Hoekstra, tWalter J. Visser, and SWillem Renooij Department of Orthopaedic Surgery, *Department of Nuclear Medicine, ?Clinical Research Group for Bone Metabolism, #Department of Surgery, University Hospital, State University of Utrecht, Utrecht, The Netherlands
Summary: The effect of direct current stimulation on bone formation during
limb lengthening was tested in a lower leg lengthening model in the rabbit. Limb lengthening was performed by distraction epiphysiolysis. A specially designed external distraction device was placed at the tibia. The distractor allowed 10 mm of lengthening in 4 weeks. Two weeks after starting the distraction, a platinum electrode was passed through the anterior cortex below the tibia1 tuberosity and advanced via the medullary cavity so that the tip rested in the elongated zone. Stimulation started at the time of placement of the electrodes and was continued for 3 weeks. The electrode in the elongated zone served as the cathode; the anode was placed subcutaneously. A 20 pA stimulus was selected. A control group received the same treatment without stimulation. Bone formation in the elongated zone was evaluated by histology and scintigraphy. The data from this experiment show that direct current stimulation in the early phase of a limb lengthening procedure had no effect on the extent of bone formation in the elongated zone. Key Words: Distraction epiphysiolysis-Bone scintigraphy-Electrostimulation-Osteogenesis-Limb lengthening-Rabbit .
Significant inequality in limb length in childhood is a major problem in orthopedic management. The method of limb lengthening widely accepted in the Western world (30,36) involves a diaphyseal osteotomy followed by bone grafting and external fixation. Many problems and complications of these lengthening procedures have been reported (9). In the last decades, good results were reported with distraction epiphysiolysis as a method of limb lengthening (1,5,11,12,14,21,2&28). Applying a gradually increasing distraction force between epiphysis and metaphysis will eventually result in fracturing of the cartilaginous epiphyseal
plate, which is the weakest site of the immature bone. Progressive continuous distraction increases the distance between epiphysis and metaphysis until the desired lengthening is achieved. Ossification of the hematoma in the fracture gap almost always occurs spontaneously, thus restoring the continuity of the lengthened bone. In spite of many advantages of this method, with remarkably minor and infrequent complications, the long healing time remains a problem for the young patient and his family. Full weight-bearing after a lengthening of 5 cm is usually allowed only after 8-9 months. Reducing the duration of the therapy by stimulating bone formation would be a significant breakthrough. Electrical stimulation of bone growth is an accepted alternative method for treatment of delayed bone union and congenital or acquired pseudarthrosis (2,3,6,8,10).
Received August 30, 1989; accepted February 28, 1990. Address correspondence and reprint requests to Dr. W. Renooij at Department of Surgery, Utrecht University Hospital Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
769
770
P . M . VAN ROERMUND ET AL.
The objective of this investigation was to study the effect of direct current stimulation on bone formation during distraction epiphysiolysis of the tibia of the rabbit. Bone formation in the elongated zone was evaluated with histology and scintigraphy . MATERIALS AND METHODS Distraction Procedure By means of a specially designed device (Fig. l), distraction was applied to the proximal epiphysis of the right tibia of female New Zealand white rabbits (each weighing about 3 kg). Under general anesthesia and oxytetracyclin prophylaxis, two Kirchner wires (1 mm diameter) were drilled through the proximal epiphysis with the aid
of a specially designed drill guide, two wires through the distal cortex of the tibia, and a fifth wire through the anterior cortex of the tibia1 tuberosity to prevent the epiphysis from being drawn dorsally after lysis. The wires were locked in blocks on two divided brass screw bars. The upper part of the bars had a left thread, and the lower part a right thread; both parts were connected inside a turnbuckle. A 360" turn of the buckles caused a 1 mm increase in length of the distractor. The bars were connected with two brass arcs, ventrally and dorsally located so that the animals could move knee and ankle freely. The weight of the device was 50 g. After placing the distractor, the position of the wires was checked by roentgenography. Distraction was performed by daily adjustment of the turnbuckles. A fracture of the growth plate usu-
FIG. 1. A: Schematic representation of the distraction device positioned at the rabbit tibia. B: The roentgenogram shows the distraction device, electrodes, and power pack in position.
J Orthop Res, Vol. 8, No.5 , 1990
ELECTROSTIMULATION DURING DISTRACTION EPIPHYSIOL YSIS
ally occurred after 4 days. Progressive distraction, approximately 0.5 mm per day, increased the distance between epiphysis and metaphysis such that a separation of 5 mm was achieved in 2 weeks. On the 15th day after placing the distractor, a second operation was performed to introduce an electrode into the elongated zone. Under general anesthesia, an electrode wire was passed through a 1 mm drill hole in the anterior cortex just below the tuberosity and advanced via the medullary cavity into the elongated zone until the tip touched the epiphysis. A widening at the lead-electrode junction locked in the cortical drill hole. The electrode was subcutaneously secured with silk sutures. A power pack was pushed in a subcutaneous pocket in the groin. In the following 2 weeks, distraction was continued at the same rate as before. Thus, after 4 weeks, a total elongation of 10 mm was achieved and the bare tip of the electrode was located inside the elongated zone, in the distal one-half to two-thirds of the zone. The position of the electrode was checked by roentgenography (Fig. 1B). Elongation was measured as the distance between the proximal and distal pins, and the length of the tibia on weekly roentgenograms. At thc start of the experiment, the rabbits were 18 weeks old, which is near the end of their growth phase (19). The animals showed no marked inconvenience during the observation period. Two animals were excluded from the study because of improper electrode placement and one because of a fracture of the tibia. These animals were replaced. Characteristics of Electrostimulation The power supply consisted of a 6 V battery in combination with an electronic circuitry used to produce a constant direct current regardless of changes in resistance between the electrodes. Each circuit was preset to deliver a current of 20 ? 1 FA. The constant current circuitry is documented in detail by Janssen (22). The battery and circuitry were sealed in a capsule of hardened epoxy resin (Hysol, C8-W795, Dexter Corporation, Olean, NY, U.S.A.). The capsule was covered with silicone rubber (Micro-Measurements, Romulus, MI, U.S.A.). Electrodes were made of Teflon-coated 99.9% pure platinum wires, 0.5 mm in diameter (Drijfhout Edelmetaalbedrijven NV, Amsterdam, The Netherlands) with a bare tip of 10 mm. Plati-
771
num was selected because of its good biocompatibility and electrical characteristics (32,33). The connections of the leads to the electrodes were covered with silicone rubber. All electric equipment was sterilized with ethylene oxide gas. Stimulation was continued until 1 week after maximal elongation had been reached. Therefore, the total stimulation period was 3 weeks. The electrode in the elongated zone served as cathode. The anode was placed in the groin near the power pack. Control animals received the same treatment, but the capsule contained no battery. At the end of the experiment, the current supply was checked with an electrometer (Hewlett Packard Nederland, Amstelveen, The Netherlands, model 3465A) before disconnecting the leads. In none of the animals were deviant current intensities measured. The leads were disconnected. The electrodes were left in position. Bone Scintigraphy Procedure On the last day of the experiment, a scintigram was made of the hind legs 2 h after intramuscular administration of 175 MBq of [99"Tc] technetium methylene diphosphonate (99"Tc-MPD) as described before (34). The radioactivity was measured in regions of interest of 15 x 15 mm over the elongated zone and over the eorresponding area on the contralateral tibia. The uptake ratio (UR) of radioactivity was calculated as UR = ( U , - Ub)/(u, + ub),where U , is the radioactivity in the region over the elongated zone, and Ub the radioactivity in the region over the contralateral tibia. Histology The animals were killed with an overdose of pentobarbital. The hind legs were exarticulated, and the tibia grossly cleaned of muscle tissue and processed for histologic evaluation. The bones were stored in 80% ethanol and subsequently dehydrated and embedded in monomethylmethacrylate as described before (20). After complete polymerization 100 pm plane-parallel transverse sections were cut at 1 mm intervals with a low-speed Isomet saw (Buehler Ltd., Evanston, IL, U.S.A.) and further ground to a 50 Fm thickness. Thus, 9-10 undecalcified slides were obtained from the elongated zone
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of each tibia. The surface of the slides was stained with 0.15% light green (Merck 1315, Darmstadt, F.R.G.) in 1% acetic acid. The total area of bone within the perimeter of the elongated zone on these slides was histomorphometrically quantitated with an image-analyzing computer (Quantimet 720, Imanco, Metals Research Ltd., Melbourn Cambridge, England) and expressed as percentage of the surface area within the perimeter of the crosssections.
percentage of the cross-section of histologic slides filled with mineralized bone. Electrostimulation had no significant effect on bone formation. Table 2 presents the results of the scintigraphy measurements at the end of the experiment. There is no significant difference in uptake ratio of 99"Tc-MDP in the elongated zone of control and stimulated animals.
Statistics
The purpose of this report was to test the effect of direct current stimulation on bone formation in a tibia elongation model in the rabbit. Any possible way of stimulating bone formation after distraction epiphysiolysis is worth testing, because positive results shortening the long healing time would mean a major breakthrough in the treatment. In clinical practice, leg lengthening by distraction epiphysiolysis always involves an acute fracture. A fresh fracture of bone may be considered the optimal stimulus for bone repair and growth. Chances of observing an effect from an added external stimulus might be highest as early in the distraction phase as possible. Van Roermund et al. (35) reported that in this rabbit tibia elongation model, active net bone formation occurred from the start of the distraction until 3-4 weeks after reaching maximal elongation. In this experimental design, the electrode was introduced after 2 weeks of distraction when trauma
Differences between stimulated and control data were statistically evaluated using the Student's t test. Each group consisted of five animals. This number is sufficient to evaluate the statistical significance of the effect of electrostimulation specifically focussed on the relevance in clinical therapeutic practice. RESULTS
Bone healing after leg lengthening occurred spontaneously without complications. At the end of the experiment, the elongated zone was filled with a callus mass, both in control and stimulated series (Fig. 2). The extent of bone formation in the elongated zone is presented in Table 1. The data show the
DISCUSSION
FIG.2. Histologic slide of the center of the elongated zone of a control tibia. The black dot in the center is the electrode.
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TABLE 1. Results of histologic evaluation Percentage of tibia cross-section occupied by bone (mean 2 SEM) Control Stimulated
54 2 8 48 2 4
The percentage of bone per cross-section in the 9-10 histologic slides covering the length of the elongated zone was averaged in each animal. The means of the data obtained from five animals in each series are presented.
from fracturing of the growth plate might have subsided. In the 2 weeks of the second phase of lengthening, and 1 week beyond, stimulation was active. In this second phase of the distraction, de novo bone formation would occur in the further elongated zone proximal to the electrode, while at the level of the electrode tip a continued increase in already present callus mass might be expected. Figure 3 illustrates the position of the electrode tip. The results from histologic evaluation of the tibia specimen (Table 1) clearly show that direct current stimulation had no effect on bone formation in the elongated zone. Figure 4 shows a histologic slide of a stimulated tibia directly distal to the elongated zone. It is clear that newly formed bone in the medullary cavity is concentrated around the active electrode, which was not observed around the dummy electrode in the control series. Thus, although stimulation of bone formation could not be detected in the elongated zone, the electrical stimulus had caused bone formation near the electrode tip, which is in agreement with the work of others (7,13,16,18,23,29). In the elongated zone, the normal rate of bone healing is probably already optimal and therefore can not be further stimulated. The second technique to evaluate bone formation in the elongated zone was 99mTc-MDP scintigraphy. Apart from other factors (4,15,17,23,25,31), uptake of 99"Tc-MDP may reflect metabolic activity of osteogenesis. Results from control and stimulated animals were similar (Table 2), which corroborates the histologic data. As discussed before (34), trauma induced by fracturing of the growth TABLE 2. Results of 99"Tc-MDP scintigraphy Uptake ratio (mean 2 SEM) Control Stimulated
33.7 2 6.6 35.4 2 4.1
FIG. 3. Roentgenogram of a rabbit's tibia after re. ieval. The position of the electrode in the center of the elongated zone is clearly demonstrated. The bone present at the level of the electrode in the distal part of the elongated zone was deposited in the first phase of the distraction period and continued to be formed in the second phase, while bone in the region proximal to the electrode tip was derived from formation in the second distraction phase only.
plate and distraction forces exerted in the tissue largely determine the level of uptake of 99"Tc-MDP and may anyway have obscured any stimulatory effects on uptake of 99"Tc-MDP due to an increased osteogenic response. The results of this study clearly indicate that the present form of electrostimulation in the early phase of distraction epiphysiolysis of the rabbit tibia did not enhance the rate of bone formation. In clinical practice, the strength of the new bone in the elongated zone is the determining factor. The present data do not rule out that new bone on the stimulated side may be stronger than on the control side.
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FIG. 4. Histologic slide of a stimulated tibia just distal to the elongated zone. In the medullary canal, bone is concentrated around the negative electrode.
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