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Intermittent parathyroid hormone (1–34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats
ELSEVIER
Journal of Orthopaedic Research
Journal ol' Orthopaedic Research 22 (2004) 4 7 2 4 7 8
www.elsevier.com1locatelorthres
Intermittent parathyroid hormone (1-34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats C. Seebach a,b, R. Skripitz
a,c,
T.T. Andreassen
d,
P. Aspenberg
'' Orthopedic Depurrnient .f'Luntl Utiiiw,yity Ho.spitul, SE-22185 Lund,
a,e,*
Sioeden
Orthopedic Uniorrsity Hospital Frierlrichslleinl, 060590 FrankfurilM., Gerrnuny Orthopedic Depurtment, Universirj, Hmpitul Eppendwf; 020246 Humburg, Germun?. Depurtriient uf Connective Tissue Biology, Instilute u f Anutorriy, University qf Aarhus. DK-8000 Aurhus, Deninurk Orthopedic Depurtnient of Linktiping Uniuer.sitj.. SE-58185 Linkiiping, Sweden Received 25 October 2002; accepted 21 August 2003
Abstract
Distraction osteogenesis is used both for leg lengthening and for bone transportation in the treatment of fractures and nonunions. The main problem with this method is that the time until full recovery may be up to a year, partly because of the time needed for the new formed bone to consolidate and become strong enough for weight bearing. We have studied whether intermittent parathyroid hormone (PTH( 1-34)) could accelerate the consolidation of new formed bone after distraction osteogenesis in rats. Forty-seven, 3-months-old male Sprague-Dawley rats underwent lengthening of the right femur using an external fixator. After a middiaphyseal osteotomy and a 7-day latency period, the callus was distracted during 10 days, with a distraction rate of 0.25 mm twice a day. The consolidation time was either 20 days or 40 days after distraction was completed. A dose of 60 pg of human PTH(I-34)/kg body weightlinjection or vehicle was given every second day beginning 30 days before the rats were killed. Both femura of each rat were subjected to mechanical testing and dual-energy X-ray absorptiometry. Blinded histological examination was done for the distracted femura. In the 20 days consolidation experiment, PTH( 1-34) increased ultimate load (56'%)),stiffness (1 17'%1),total regenerate callus volume (58'%1),callus BMC (24%) and histologic bone density (35%) compared to untreated distraction osteogenesis specimens. In the 40 days consolidation experiment, PTH( 1-34) increased ultimate load (54'%1),stiffness (55'%), callus BMC (33%) and histologic bone density (23%))compared to untreated distraction osteogenesis specimens, Total regenerate callus volume was unchanged. The contralateral femur also became stronger, stiffer and denser under PTH( 1-34) treatment, but to a lesser degree. PTH(1-34) might become useful to shorten the consolidation time after distraction osteogenesis in humans. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Kc.?.ivorcis: Parathyroid hormone; Bone regeneration; Distraction osteogenesis; Rat
Introduction
The anabolic effect of intermittent parathyroid hormone (PTH( 1-34)) administration on both cortical and cancellous bone has been established in a number of animal studies [7]. The PTH( I-34)-induced increase bone mass results in an enhanced mechanical strength of * Corresponding author. Address: Department of Orthopedics. Department of Neuroscience & Locomotion, Faculty of Health Science. Linkoping University Hospital, SE-58185 Linkoping, Sweden. Tel.: +46-13-22-4166: fax: +46-13-22-4503. E-mail addrixs: [email protected] (P, Aspenberg).
the bones [6]. Recent experiments have demonstrated that PTH(1-34) treatment is able to increase the mechanical strength and callus formation in normal healing fractures [1,2,8]. Also in delayed fracture healing, PTH(1-34) has been found to induce an anabolic effect and a concomitant enhanced fracture strength [5, lo]. Distraction osteogenesis is widely used clinically both for leg lengthening and for the treatment of nonunion fractures. The procedure is characterized by bone regeneration between bone surfaces which are separated by gradual distraction [3,4,9,11,15]. First, the bone is separated by osteotomy, and the fragments are kept together allowing initial healing to bridge the osteotomy
0736-0266/$ - see front matter 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.10164 .orthres.2003.08.018
C. Sechuch et ul. I Journul
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gap. A slow progressive distraction is then performed by means of an external fixation device. The required surgery is minimally invasive, and the method is being used to an increasing extent. For some indications, distraction osteogenesis has replaced difficult and resourceconsuming operations, such as vascularized autografts or bone allografting. An important problem with this method, however, is the time needed for the new formed bone in the distraction gap to consolidate and become strong enough for weight bearing. This can take up to a year. In order to shorten the consolidation period, a systemic treatment which enhances strength and density of the distracted callus would be valuable. PTH( 1-34) treatment seems to be relevant in this context because this treatment enhances callus density and mechanical strength of healing fractures. We have therefore investigated how PTH( 1-34) treatment influences strength and callus density in a rat model for distraction osteogenesis.
Materials and methods Animals
Forty-seven male Sprague-Dawley rats (weight range 31 1 4 2 2 g. M&B, LI. Skensved, Denmark) were used. The animals were fed a standard laboratory diet ad libidum. The rats were housed single in cages (height 20 cm) in temperature controlled rooms (22 “ C )having a 12-h light cycle. Distraction decicc.
The self-constructed external distraction device was made to hold four transfixation pins. The threaded part of the pins had a diameter of 1 mm, a length of 7 mm and was self-tapping. The rest of the pin had a diameter of 2 mm and a length of 24 mm (Fig. I).
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mixture of Streptomycin (0.125 mg) and Benzylpenicillin (0.1 mg) was injected intramuscularly before surgery and then once a day for 3 days. In order t o fix the monolateral external fixator to the anterolateral aspect of the right femur. the entire right leg of the rat was shaved, cleaned and prepared with chlorhexidin solution. A sterile draping was used. A lateral longitudinal incision over the femur was made under aseptic conditions. The fascia was cut and the muscles separated between the quadriceps femoris and the hamstrings. The bone cortex was drilled using a 0.8 mm bit and four self-tapping pins were placed at right angles t o the surface of the femoral shaft. The pins were then clamped to the external fixation device. An osteotomy was made midshaft between the second and the third pin using an oscillating saw (blade thickness 0.5 mm). The wound was closed with continuous subcutaneous stiches using a 4/0-monofilament nylon suture. Distraction uird tsrcitinei?lprotocol
Two separate groups of rats, with consolidation periods of 20 and 40 days. respectively, were included. For each consolidation period, the rats were randomly divided into a vehicle-injected group and a PTH( I--34)-injected group (human PTH( 1-34), Bachem. Bubendorf, Switzerland, dissolved in 0.5 M saline with 2% heat inactivated rat serum). The distraction started 7 days postoperatively with a distraction rate of 0.5 mm per day (2 times 0.25 mm with a minimum of 6 h in between). The distraction continued for 10 days, followed by consolidation periods of 20 and 40 days, respectively. The PTH( 1-34) treatment (60 pgl kg body weight) was given subcutaneously every second day, starting 30 days before the animals were killed. The animals were weighed once a week. and the dose was adjusted to body weight. At the end of the experiment. the rats were killed with an overdose of pentobarbital (150 mg/kg intraperitoneally) and weighed. Both femura were dissected free and the distraction device was removed. Blinded with respect to treatment. all pins were checked for pin fixation and only in cases where all four pins had to be twisted in order to be removed, the rat was included. Fourteen animals were excluded: Eleven with pin loosening obviously because of pin-tract infection; (seven controls, four PTH(1-34) treated) one PTH(1-34) rat with a broken femur, one PTH(1-34) rat with a grossly unstable osteotomy for unknown reasons. and one control rat that was found dead at day 10 for unknown reasons. Data from the mechanical testing of one specimen were lost due to a technical error. The experiment was approved by the Regional Animal Ethics Committee.
Surgicul psoceciure.\
Evaluation
General anesthesia with 0.6-0.7 ml of a mixture of pentobarbital (15 mglml) and diazepam (2.5 mglml) was given intraperitoneally. A
Totul rtyyrier’ute callu.s rolunie and dinierisions The total volumes of both the distracted and the intact femur were gauged using Archimedes’ principle [l]. Total regenerate volume, which includes both external callus and length gained over the course of distraction (5 mm), was calculated as the volume of the distracted femur minus the volume of the intact femur. The external mediallateral and anterior-posterior diameters were measured at the middle of the distraction callus using a sliding caliper. Also. total femoral length was gauged (top of femur head to distal edge of medial condyle). In the opposite intact femur. the corresponding diameters and length were measured. .Mechunical testing
Fig. I , An external distraction device in the femur of a rat.
Immediately after harvesting and measurement of the external dimensions. the mechanical properties of the distracted callus was measured by a destructive three point bending procedure using a materials testing machine (200 R, DDL, Eden Prairie MN. USA) linked to a computer (Dimension T600r. Dell, Rahem, Limerick; software MTEST Windows). The femur was placed on two rounded bars 19.5 mm apart. so that the distracted part was half-way between the bars. The bending until failure was performed by lowering a third bar onto the distracted tissue, using a constant deflection speed of 2 mm/min. Load and deflection were recorded continuously by transducers coupled to measuring bridges. Ultimate load, stiffness, apparent peak stress and apparent modulus of elasticity were calculated by the
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computer software program, with the assumption that the transverse area of the callus was homogenous and elliptic. Duul-energy X-ruy uhsorptiometry ( D E X A )
The DEXA analysis was done after mechanical testing. This was not carried out first, because of a concern that the freezing and thawing would have an effect on the mechanical properties. After mechanical testing, the two ends of the tested femura were fixed with surgical tape in their original position, moistened in Ringer’s solution and frozen at -30 “C for later measurement of the bone mineral content (BMC) by dual-energy X-ray absorptiometry (DEXA). The femura were placed with the posterior surface downward and scanned by DEXA using a line spacing of 0.31 1 mm, a point resolution of 0.31 1 mm and the regional high resolution analysis program for small animals (QDR4500A; Hologic, Inc., Waltham, MA, USA). BMC of the whole bone in the distracted and in the intact femur were also measured. The BMC around the new formed bone was measured using a region of interest that was a 7.4-mm-long diaphyseal segment (3.7 mm proximal and 3.7 mm distal t o the middle of the distraction callus). BMD was calculated as BMCkarea. Cu1hr.s hi.~io~izorphonietr~.
was only performed on the entire material. Results under subheadings “20 days consolidation” and “40 days consolidation” are descriptive and not formally tested for statistical significance, due to the small sample size. Instead, 95Y1 confidence intervals of untransformed data are used for description in Tables 1-3.
Results
Over all, in the new formed bone, PTH(1-34) increased the ultimate load (p = 0.01; log transformed p = 0.0007), stiffness 0, = 0.01, log transformed p = 0.009), BMC of the whole distracted femur (p = 0.001), BMD of the distracted area (p = O.OOOl), histological bone density 0, = 0.0001) and bone surface density (p = 0.0001). Ultimate load and stiffness increased dramatically over time (p = 0.0001, Fig. 2). Results are summarized in Tables 1-3. Twenty duys consolidation
After DEXA analysis, the femura were fixed in formalin and decalcified. A section of the decalcified specimens was taken parallel to the long axis of the femur and stained with haematoxylin and eosin. Histological and histomorphometric assessments were performed with blinded specimens examined in random order. The Merz grid was used for point counting in the distracted tissue to measure histologic bone density [13]. The area for measurement (3.2 mm’) was chosen in the callus next to the cortex, at the surface of the osteotomy cut. Bone density was expressed as the percentage of points covering bone tissue in relation to the total number of points. Furthermore we counted bone intersections (IS) with the lines of Merz grid. These give evidence about the thickness and quantity of the bone trabeculae. Bone density (BVITV) and surface density (BS) were calculated according to Merz. Trabecular thickness (TbTh) was calculated as 2 .(BV/BS). Slut isticul unu1j.si.s
Two-way ANOVA was used to test the hypothesis that PTH( 1-34) would increase the consolidation of the distracted callus. For ultimate load and stiffness, the variance appeared to differ between groups. This could be corrected by logarithmic transformation. Hypothesis testing
PTH( 1-34) increased the ultimate load and the stiffness of the distracted bone by more than 50‘% N o major difference was found for apparent peak stress or apparent modulus of elasticity. The intact contralateral femur became slightly stronger and denser, but to a lesser degree (10% and 5% increase in ultimate load and stiffness). The total regenerate callus volume and the transverse area were larger in the PTH( 1-34) group. The BMD in the distracted callus was increased by 24% in the PTH( 1-34) group, and there was less increase in the intact femur (5%). The histological analyses showed an increase in bone density (35%) and a decrease in bone surface density (19%), in the PTH(1-34) treated group. There was no change in trabecular thickness.
Table 1 Callus parameters of distracted femur after 20 days of consolidation
95%)CI limits for difference
Mean (SD) Control, n
=7
PTH 60 pg/kg/injection, n
Mechanical analysis Ultimate load (N) Stiffness (N/mm) Apparent peak stress (MPa) Apparent modulus of elasticity (MPd)
25 ( 5 ) 17 (6) 4 (1) 29 (9)
39 (13) 37 (30) 5 (1) 39 (27)
External callus dimensions Anterior -posterior (mm) Medial-lateral (mm)
6.5 (0.8) 7.0 (1.1)
7.0 (0.5) 8.3 (0.5)
’
=8
4.2 -2.0 -0.7 -10.1
24.0 40.6 1.6 29.9
-0.2 0.4
1.2 2.2
Transverse area (mm’) 36 (8) 45 (5) 2.8 Total regenerate callus volume (mm3) 172 (54) 272 (51) 50 Femur DXA-BMC (mg) 474 (52) 565 (50) 40 Callus DXA-BMD (mg/cm2) 258 (25) 320 (17) 40 Callus DXA-BMC (mg) 153 (28) 190 (34) 5.6 Bone density (%I) 48 (8) 65 (9) 8.2 Surface density (mm’/mm’) 10.8 ( 1 . 1 ) 8.7 (1.1) -3.2 Trabecular thickness (mm) 0.19 (0.26) 0.15 (0.03) -0.2 PTH(1-34) (60 pg/kg) was given every second day during the 10 days of distraction and the following 20 days of consolidation. DEXA, dual-energy X-ray absorptiometry; BMC, bone mineral content; BMD. bone mineral density.
16.8 150
I40 80 68.4 25.8 -1.0 0.2
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Tdbk 2 Callus parameters of distracted femur after 40 days of consolidation Mean (SD), 17
=9
95% CI limits for difference
Controls
PTH( 1-34) 60 pg/kg/injection
Mechanical analysis Ultimate load ( N ) Stiffness (N/mm) Apparent peak stress (MPa) Apparent modulus of elasticity (MPa)
52 (18) 107 (45) 15 (5) 421 (308)
80 (34) 166 (57) 20 ( 1 1 ) 535 (400)
2.8 10.5 -2.5 -222.8
External callus dimensions Anterior-posterior (mm) Medial-lateral (mm)
5.4 (0.7) 6.3 (0.6)
5.7 (1.0) 6.6 ( I .O)
-0.4 -0.5
1.1 I.0
-3.4 -100 10 0 4.4 8.0 -5.1 0.1
10.4 90 140 60 91.6 19.4 -2.2 0.2
Transverse area (mm') Total regenerate callus volume (mm') Femur DXA-BMC (mg) Callus DXA-BMD (mglcm') Callus DXA-BMC (mg) Bone density ('%) Surface density (mm'/mm') Trabecular thickness (mm)
27 (5) 30 (9) 202 (66) 199 (131) 483 (61) 557 (81) 292 (27) 323 (30) 145 (27) 193 (61) 62 ( 6 ) 76 ( 6 ) 9.4 ( I .4) 5.8 (1.7) 0.14 (0.02) 0.3 (0.13) PTH(I-34) (60 pg/kg) was given every second day during the last 30 days of consolidation. DEXA, dual-energy X-ray absorptiometry: BMC, bone mineral content; BMD, bone mineral density.
53.8 107.9 13.6 451.8
Table 3 Mechanical analysis, dimensions, BMC and BMD of contralateral intact femur after 20 and 40 days of consolidation Mean fSD)
95% CI limits for difference
Controls
PTH( 1-34) 60 pg/kg/injection
20 duys oJ consolidution Ultimate load ( N ) Stiffness (N/mm)
n=7 118 (12) 187 (17)
n Z 8
130 (11) 197 (13)
-0.2 -5.3
23.2 26.3
Ext. middiaphyseal dimensions Anterior-posterior (mm) Medial-lateral (mm)
3.7 (0.2) 4.5 (0.2)
3.7 (0.2) 4.5 (0.2)
-0.2 -0.2
0.2 0.2
Transverse area (mm') Femur DXA-BMC (mg) Diaphyseal DXA-BMD' (mg/cm') Diaphyseal DXA-BMC' (mg)
13.1 (1.3) 327 (21) 203 ( 10) 83 (9)
13.1 (1.1) 359 (23) 214 (10) 89 ( 6 )
-1.3 10 0 -1.9
1.1 50 20 13.8
40 duys of consoliduiion Ultimate load (N) Stiffness (N/mm)
I1 =
9 140 (16) 261 (51)
17
296 (57)
5.0 --16.0
47.9 86.3
3.7 (0.2) 4.4 (0.2)
3.8 (0.1) 4.5 (0.2)
0 -0.1
0.3 0.2
Ext. middiaphyseal dimensions Anterior-posterior (mm) Medial-lateral (mm)
=9
166 (28)
-0.2 12.8 (1.1) 13.5 (0.8) 10 363 (39) 416 (48) -30 250 (19) 242 (28) -17.7 81 (19) 80 (17) At both consolidation periods, PTH(I-34) (60 pglkg) was given every second day during the last 30 days of the experiment. DEXA, dual-energy X-ray absorptiometry; BMC, bone mineral content: BMD, bone mineral density. 'Measured using a segment. Transverse area (mm') Femur DXA-BMC (mg) Diaphyseal DXA-BMD' (mglcm') Diaphyseal DXA-BMC' (mg) . .
Forty duys consolidation PTH( 1-34) increased the ultimate load and the stiffness of the distracted bone by more than 50'341.No major
I .6 90 10 15.7 7.4-mm-high
difference was found for apparent peak stress or apparent modulus of elasticity with PTH( 1-34), The intact contralateral femur became slightly stronger and stiffer, but to a lesser degree. No difference between PTH( 1-34)
C. Seehuch et u1. I Journul oJ Orthi pieilic Rrwurcll 22 (2004) 472-478
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I
I
h
z
v
100-
0
J
--
io
20
(4
40
40
time (d)
300
'E .
h
200
cocbo 100
0 (B)
20
20
40 time (d)
40
Fig. 2. Ultimate load in N (A) and stiffness in N/mm (B) of the distraction callus after either 20 or 40 days consolidation time with PTH (60 pg/kg) or vehicle treatment.
treated rats and control was found in callus dimensions or total regenerate callus volume. The callus BMD was 11% higher and the callus BMC was 33% higher in the PTH(1-34) group. No difference was found in BMD in the contralateral, intact femur. The histological analyses showed an increase in bone density (23'%)),a decrease in bone surface density (380/0), and at this time also a large increase in trabecular thickness (1 14'%1)in the distracted callus with PTH( 1-34) treatment. The trabeculae had become fewer and thicker with an increase of the bone mass in the area next to the cortex (Fig. 3). Weight gain was not affected by PTH( 1-34) (data not shown).
Discussion
Only few experiments have investigated the influence of intermittent PTH( 1-34) treatment on fracture heal-
ing, including callus formation and mechanical strength development, in animal models. Our current rat model of distraction osteogenesis for femoral lengthening reliably results in zonal regions of predominantly intramembranous bone. Although our distraction model is different from other experimental models of fracture healing, our findings confirm previous fracture and bone chamber studies, that PTH( 1-34) increases the mechanical strength as well as the amount of new mineralized bone [2,8,16,17]. Further, we have previously shown that the effect of PTH(1-34) is clearly more dramatic in newly forming bone, like a callus, than in bone that is merely undergoing normal remodelling [16]. We chose to compare data from distracted calluses between treatment groups directly. However, if each mechanical parameter is instead expressed as a percentage of the unoperated contralateral leg, the results are essentially the same, although the significance level is less. Thus, still at 40 days, the ultimate load was 38% of the contralateral femur in the controls and 48% in the PTH( 1-34) treated group. The ultimate load depends less on the amount of load-bearing material than on callus dimensions and the arrangement and dimension of each trabecula [14]. We found that PTH(I- 34) treated animals were able to increase their total regenerate callus volume when treatment was initiated while distraction was on-going, whereas no difference on total regenerate callus volume was observed when treatment was administered during the consolidation phase only. At that time point, the outer dimensions of the callus appear to have been determinated already. The absence of a difference in callus volume at 40 days may also be due to the more advanced stage of remodelling with more mature and thicker trabeculae in the PTH(1-34) treated group. It seems that the callus with an early PTH(1-34) treatment becomes stronger, because of an increase of total regenerate callus volume (Table 1). The callus with a later PTH( 1-34) treatment becomes stronger, because of improved arrangement and dimension of each trabecula (Table 2 and Fig. 3). However, because we did not do a group treated with PTH( 1-34) from the distraction and for 40 days consolidation, the differences between the 20 and the 40 days groups should be interpreted with caution. In normal well adapted rat fractures, PTH( 1-34) treatment during the first weeks of healing increases callus volume; however, to achieve this effect higher doses of PTH(1-34) than used in the present experiment are needed [2,7,8]. In ovariectomized rats with delayed fracture healing, PTH( 1-34) treatment given daily in a dose similar to the one used in this experiment has been shown to increase both mechanical strength and callus volume [lo]. Also, in glucocorticoid treated rabbits with delayed fracture healing, a PTH analogue has been
411
Fig. 3 . Histology after 40 days consolidation of a long axis section of the rat femur. (A) PTH treated specimen shows an increase in trabecular density and thickness in the area next to the cortex. (B) Framed area from A . ( C ) Vehicle treated specimen with a larger amount. butthinner trabeculae. (D) Framed area from C (H&E staining. bar length 1 mm).
shown to augment mechanical strength development and callus formation [ 5 ] . We also could see an anabolic effect of PTH( 1-34) at the intact, contralateral site, but to a lesser degree than in the distraction callus. It seems that PTH(1-34) has a stronger effect on bone where a repair response is activated [16]. When starting to develop this distraction model some years ago we saw many pin infections leading to loosening. The incidence of pin-tract infection in this study has been reduced by improving the surgical techniques, giving antibiotics, and intensive postoperative care. Considering the difficulties to achieve this, we are surprised that pin-tract infections are not reported in other publications. This is a profound issue for distraction studies in rats. Pin-tract infections decrease the stability of the construct, leading to small motions within the distraction gap that may inhibit bony bridging. In a previous unpublished study, we used the same protocol as in the 20 days group of this study, but with a less meticulous operation technique and postoperative wound care. Almost all of these rats had pin-tract infection. In consequence to the ensuing instability, there
was often no continuous bone bridge across the distraction gap, but instead a pseudarthrosis PTH( 1-34) did not seem to improve repair in this situation. PTH(134) increases bone matrix synthesis and prevents osteoblast apoptosis [12], but does not induce de novo bone formation like e.g. BMPs. Therefore, it is unlikely that PTH would be useful in treatment of an unstable pseudarthrosis. This study suggests that PTH( 1-34) might be useful as a stimulator of bone formation in order to improve fracture stability while consolidation is occurring.
Acknowledgements We thank Mats Christensson for help with design and manufacturing of the distraction devices, Inger Martensson and Carina Forslund for technical assistance in Lund, and Dr. Thorsten Hennings for help with the DEXA measurement at the University hospital in Frankfurt am Main. This study was financially supported by the Swedish Medical Research Council (project 2031)
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C. Seebach et al. I Journal of Orthopaedic Research 22 (2004) 472478
and Deutsche Forschungsgemeinschaft.The experiments were performed in Lund, Sweden. References [l] Andreassen TT, Ejersted C, Oxlund H. Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures. J Bone Miner Res 1999;14:960-8. [2] Andreassen TT, Fledelius C, Ejersted C, Oxlund H. Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone. Acta Orthop Scand 2001;72:304-7. [3] Aronson J. Limb-lengthening, skeletal reconstruction and bone transport with the Ilizarov method. J Bone Joint Surg [Am] 1997;79:1243-58. [4] Aronson J, Shen XC, Skinner RA, et al. Rat model of distraction osteogenesis. J Orthop Res 1997;15:2214. [5] Bostrom MP, Gamradt SC, Asnis P, et al. Parathyroid hormonerelated protein analog RS-66271 is an effective therapy for impaired bone healing in rabbits on corticosteroid therapy. Bone 2000;26:43742. [6] Ejersted C , Andreassen TT, Oxlund H, et al. Human parathyroid hormone (1-34) and (1-84) increase the mechanical strength and thickness of cortical bone in rats. J Bone Miner Res 1993;9:1097101. [7] Hock JM, Gera I, Fonseca J, Raisz LG. Human parathyroid hormone (1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122:2899-904.
[S] Holzer G, Majeska RJ, Lundy MW, et al. Parathyroid hormone enhances fracture healing. A preliminary report. Clin Orthop 1999;366:25&63. [9] Ilizarov GA. Clinical application of the tension-stress effect for limb lengthening. Clin Orthop 1990;250:8-26. [lo] Jahng JS, Kim HW. Effect of intermittent administration of parathyroid hormone on fracture healing in ovariectomized rats. Orthopedics 2000;23: 1089-94. [ll] Jazrawi LM, Majeska RJ, Klein ML, Kagel E, Stromberg L, Einhorn TA. Bone and cartilage formation in an experimental model of distraction osteogenesis. J Orthop Trauma 1998;12: 111-6. [12] Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 1999;10443946. [13] Merz WA, Schenk RK. Quantitative structural analysis of human cancellous bone. Acta Anat 1970;75:54-66. [14] Pauwels F. Uber die Verteilung der Spongiosadichte im coxalen Femurende und ihre Bedeutung fur die Lehre vom funktionellen Bau des Knochens. Morph Jb 1954;95:35-54. [15] Sat0 M, Yasui N, Nakase T, et al. Expression of bone matrix proteins mRNA during distraction osteogenesis. J Bone Miner Res 1998;13:1221-31. [16] Skripitz R, Andreassen TT, Aspenberg P. Strong effect of PTH(134) on regenerating bone: a time sequence study in rats. Acta Orthop Scand 2000;71:619-24. [I71 Skripitz R, Andreassen TT, Aspenberg P. Parathyroid hormone (1-34) increases the density of rat cancellous bone in a bone chamber. J Bone Joint Surg [Br] 2000;82:13841.
Journal of Orthopaedic Research
Journal ol' Orthopaedic Research 22 (2004) 4 7 2 4 7 8
www.elsevier.com1locatelorthres
Intermittent parathyroid hormone (1-34) enhances mechanical strength and density of new bone after distraction osteogenesis in rats C. Seebach a,b, R. Skripitz
a,c,
T.T. Andreassen
d,
P. Aspenberg
'' Orthopedic Depurrnient .f'Luntl Utiiiw,yity Ho.spitul, SE-22185 Lund,
a,e,*
Sioeden
Orthopedic Uniorrsity Hospital Frierlrichslleinl, 060590 FrankfurilM., Gerrnuny Orthopedic Depurtment, Universirj, Hmpitul Eppendwf; 020246 Humburg, Germun?. Depurtriient uf Connective Tissue Biology, Instilute u f Anutorriy, University qf Aarhus. DK-8000 Aurhus, Deninurk Orthopedic Depurtnient of Linktiping Uniuer.sitj.. SE-58185 Linkiiping, Sweden Received 25 October 2002; accepted 21 August 2003
Abstract
Distraction osteogenesis is used both for leg lengthening and for bone transportation in the treatment of fractures and nonunions. The main problem with this method is that the time until full recovery may be up to a year, partly because of the time needed for the new formed bone to consolidate and become strong enough for weight bearing. We have studied whether intermittent parathyroid hormone (PTH( 1-34)) could accelerate the consolidation of new formed bone after distraction osteogenesis in rats. Forty-seven, 3-months-old male Sprague-Dawley rats underwent lengthening of the right femur using an external fixator. After a middiaphyseal osteotomy and a 7-day latency period, the callus was distracted during 10 days, with a distraction rate of 0.25 mm twice a day. The consolidation time was either 20 days or 40 days after distraction was completed. A dose of 60 pg of human PTH(I-34)/kg body weightlinjection or vehicle was given every second day beginning 30 days before the rats were killed. Both femura of each rat were subjected to mechanical testing and dual-energy X-ray absorptiometry. Blinded histological examination was done for the distracted femura. In the 20 days consolidation experiment, PTH( 1-34) increased ultimate load (56'%)),stiffness (1 17'%1),total regenerate callus volume (58'%1),callus BMC (24%) and histologic bone density (35%) compared to untreated distraction osteogenesis specimens. In the 40 days consolidation experiment, PTH( 1-34) increased ultimate load (54'%1),stiffness (55'%), callus BMC (33%) and histologic bone density (23%))compared to untreated distraction osteogenesis specimens, Total regenerate callus volume was unchanged. The contralateral femur also became stronger, stiffer and denser under PTH( 1-34) treatment, but to a lesser degree. PTH(1-34) might become useful to shorten the consolidation time after distraction osteogenesis in humans. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Kc.?.ivorcis: Parathyroid hormone; Bone regeneration; Distraction osteogenesis; Rat
Introduction
The anabolic effect of intermittent parathyroid hormone (PTH( 1-34)) administration on both cortical and cancellous bone has been established in a number of animal studies [7]. The PTH( I-34)-induced increase bone mass results in an enhanced mechanical strength of * Corresponding author. Address: Department of Orthopedics. Department of Neuroscience & Locomotion, Faculty of Health Science. Linkoping University Hospital, SE-58185 Linkoping, Sweden. Tel.: +46-13-22-4166: fax: +46-13-22-4503. E-mail addrixs: [email protected] (P, Aspenberg).
the bones [6]. Recent experiments have demonstrated that PTH(1-34) treatment is able to increase the mechanical strength and callus formation in normal healing fractures [1,2,8]. Also in delayed fracture healing, PTH(1-34) has been found to induce an anabolic effect and a concomitant enhanced fracture strength [5, lo]. Distraction osteogenesis is widely used clinically both for leg lengthening and for the treatment of nonunion fractures. The procedure is characterized by bone regeneration between bone surfaces which are separated by gradual distraction [3,4,9,11,15]. First, the bone is separated by osteotomy, and the fragments are kept together allowing initial healing to bridge the osteotomy
0736-0266/$ - see front matter 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved doi: 10.10164 .orthres.2003.08.018
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gap. A slow progressive distraction is then performed by means of an external fixation device. The required surgery is minimally invasive, and the method is being used to an increasing extent. For some indications, distraction osteogenesis has replaced difficult and resourceconsuming operations, such as vascularized autografts or bone allografting. An important problem with this method, however, is the time needed for the new formed bone in the distraction gap to consolidate and become strong enough for weight bearing. This can take up to a year. In order to shorten the consolidation period, a systemic treatment which enhances strength and density of the distracted callus would be valuable. PTH( 1-34) treatment seems to be relevant in this context because this treatment enhances callus density and mechanical strength of healing fractures. We have therefore investigated how PTH( 1-34) treatment influences strength and callus density in a rat model for distraction osteogenesis.
Materials and methods Animals
Forty-seven male Sprague-Dawley rats (weight range 31 1 4 2 2 g. M&B, LI. Skensved, Denmark) were used. The animals were fed a standard laboratory diet ad libidum. The rats were housed single in cages (height 20 cm) in temperature controlled rooms (22 “ C )having a 12-h light cycle. Distraction decicc.
The self-constructed external distraction device was made to hold four transfixation pins. The threaded part of the pins had a diameter of 1 mm, a length of 7 mm and was self-tapping. The rest of the pin had a diameter of 2 mm and a length of 24 mm (Fig. I).
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mixture of Streptomycin (0.125 mg) and Benzylpenicillin (0.1 mg) was injected intramuscularly before surgery and then once a day for 3 days. In order t o fix the monolateral external fixator to the anterolateral aspect of the right femur. the entire right leg of the rat was shaved, cleaned and prepared with chlorhexidin solution. A sterile draping was used. A lateral longitudinal incision over the femur was made under aseptic conditions. The fascia was cut and the muscles separated between the quadriceps femoris and the hamstrings. The bone cortex was drilled using a 0.8 mm bit and four self-tapping pins were placed at right angles t o the surface of the femoral shaft. The pins were then clamped to the external fixation device. An osteotomy was made midshaft between the second and the third pin using an oscillating saw (blade thickness 0.5 mm). The wound was closed with continuous subcutaneous stiches using a 4/0-monofilament nylon suture. Distraction uird tsrcitinei?lprotocol
Two separate groups of rats, with consolidation periods of 20 and 40 days. respectively, were included. For each consolidation period, the rats were randomly divided into a vehicle-injected group and a PTH( I--34)-injected group (human PTH( 1-34), Bachem. Bubendorf, Switzerland, dissolved in 0.5 M saline with 2% heat inactivated rat serum). The distraction started 7 days postoperatively with a distraction rate of 0.5 mm per day (2 times 0.25 mm with a minimum of 6 h in between). The distraction continued for 10 days, followed by consolidation periods of 20 and 40 days, respectively. The PTH( 1-34) treatment (60 pgl kg body weight) was given subcutaneously every second day, starting 30 days before the animals were killed. The animals were weighed once a week. and the dose was adjusted to body weight. At the end of the experiment. the rats were killed with an overdose of pentobarbital (150 mg/kg intraperitoneally) and weighed. Both femura were dissected free and the distraction device was removed. Blinded with respect to treatment. all pins were checked for pin fixation and only in cases where all four pins had to be twisted in order to be removed, the rat was included. Fourteen animals were excluded: Eleven with pin loosening obviously because of pin-tract infection; (seven controls, four PTH(1-34) treated) one PTH(1-34) rat with a broken femur, one PTH(1-34) rat with a grossly unstable osteotomy for unknown reasons. and one control rat that was found dead at day 10 for unknown reasons. Data from the mechanical testing of one specimen were lost due to a technical error. The experiment was approved by the Regional Animal Ethics Committee.
Surgicul psoceciure.\
Evaluation
General anesthesia with 0.6-0.7 ml of a mixture of pentobarbital (15 mglml) and diazepam (2.5 mglml) was given intraperitoneally. A
Totul rtyyrier’ute callu.s rolunie and dinierisions The total volumes of both the distracted and the intact femur were gauged using Archimedes’ principle [l]. Total regenerate volume, which includes both external callus and length gained over the course of distraction (5 mm), was calculated as the volume of the distracted femur minus the volume of the intact femur. The external mediallateral and anterior-posterior diameters were measured at the middle of the distraction callus using a sliding caliper. Also. total femoral length was gauged (top of femur head to distal edge of medial condyle). In the opposite intact femur. the corresponding diameters and length were measured. .Mechunical testing
Fig. I , An external distraction device in the femur of a rat.
Immediately after harvesting and measurement of the external dimensions. the mechanical properties of the distracted callus was measured by a destructive three point bending procedure using a materials testing machine (200 R, DDL, Eden Prairie MN. USA) linked to a computer (Dimension T600r. Dell, Rahem, Limerick; software MTEST Windows). The femur was placed on two rounded bars 19.5 mm apart. so that the distracted part was half-way between the bars. The bending until failure was performed by lowering a third bar onto the distracted tissue, using a constant deflection speed of 2 mm/min. Load and deflection were recorded continuously by transducers coupled to measuring bridges. Ultimate load, stiffness, apparent peak stress and apparent modulus of elasticity were calculated by the
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computer software program, with the assumption that the transverse area of the callus was homogenous and elliptic. Duul-energy X-ruy uhsorptiometry ( D E X A )
The DEXA analysis was done after mechanical testing. This was not carried out first, because of a concern that the freezing and thawing would have an effect on the mechanical properties. After mechanical testing, the two ends of the tested femura were fixed with surgical tape in their original position, moistened in Ringer’s solution and frozen at -30 “C for later measurement of the bone mineral content (BMC) by dual-energy X-ray absorptiometry (DEXA). The femura were placed with the posterior surface downward and scanned by DEXA using a line spacing of 0.31 1 mm, a point resolution of 0.31 1 mm and the regional high resolution analysis program for small animals (QDR4500A; Hologic, Inc., Waltham, MA, USA). BMC of the whole bone in the distracted and in the intact femur were also measured. The BMC around the new formed bone was measured using a region of interest that was a 7.4-mm-long diaphyseal segment (3.7 mm proximal and 3.7 mm distal t o the middle of the distraction callus). BMD was calculated as BMCkarea. Cu1hr.s hi.~io~izorphonietr~.
was only performed on the entire material. Results under subheadings “20 days consolidation” and “40 days consolidation” are descriptive and not formally tested for statistical significance, due to the small sample size. Instead, 95Y1 confidence intervals of untransformed data are used for description in Tables 1-3.
Results
Over all, in the new formed bone, PTH(1-34) increased the ultimate load (p = 0.01; log transformed p = 0.0007), stiffness 0, = 0.01, log transformed p = 0.009), BMC of the whole distracted femur (p = 0.001), BMD of the distracted area (p = O.OOOl), histological bone density 0, = 0.0001) and bone surface density (p = 0.0001). Ultimate load and stiffness increased dramatically over time (p = 0.0001, Fig. 2). Results are summarized in Tables 1-3. Twenty duys consolidation
After DEXA analysis, the femura were fixed in formalin and decalcified. A section of the decalcified specimens was taken parallel to the long axis of the femur and stained with haematoxylin and eosin. Histological and histomorphometric assessments were performed with blinded specimens examined in random order. The Merz grid was used for point counting in the distracted tissue to measure histologic bone density [13]. The area for measurement (3.2 mm’) was chosen in the callus next to the cortex, at the surface of the osteotomy cut. Bone density was expressed as the percentage of points covering bone tissue in relation to the total number of points. Furthermore we counted bone intersections (IS) with the lines of Merz grid. These give evidence about the thickness and quantity of the bone trabeculae. Bone density (BVITV) and surface density (BS) were calculated according to Merz. Trabecular thickness (TbTh) was calculated as 2 .(BV/BS). Slut isticul unu1j.si.s
Two-way ANOVA was used to test the hypothesis that PTH( 1-34) would increase the consolidation of the distracted callus. For ultimate load and stiffness, the variance appeared to differ between groups. This could be corrected by logarithmic transformation. Hypothesis testing
PTH( 1-34) increased the ultimate load and the stiffness of the distracted bone by more than 50‘% N o major difference was found for apparent peak stress or apparent modulus of elasticity. The intact contralateral femur became slightly stronger and denser, but to a lesser degree (10% and 5% increase in ultimate load and stiffness). The total regenerate callus volume and the transverse area were larger in the PTH( 1-34) group. The BMD in the distracted callus was increased by 24% in the PTH( 1-34) group, and there was less increase in the intact femur (5%). The histological analyses showed an increase in bone density (35%) and a decrease in bone surface density (19%), in the PTH(1-34) treated group. There was no change in trabecular thickness.
Table 1 Callus parameters of distracted femur after 20 days of consolidation
95%)CI limits for difference
Mean (SD) Control, n
=7
PTH 60 pg/kg/injection, n
Mechanical analysis Ultimate load (N) Stiffness (N/mm) Apparent peak stress (MPa) Apparent modulus of elasticity (MPd)
25 ( 5 ) 17 (6) 4 (1) 29 (9)
39 (13) 37 (30) 5 (1) 39 (27)
External callus dimensions Anterior -posterior (mm) Medial-lateral (mm)
6.5 (0.8) 7.0 (1.1)
7.0 (0.5) 8.3 (0.5)
’
=8
4.2 -2.0 -0.7 -10.1
24.0 40.6 1.6 29.9
-0.2 0.4
1.2 2.2
Transverse area (mm’) 36 (8) 45 (5) 2.8 Total regenerate callus volume (mm3) 172 (54) 272 (51) 50 Femur DXA-BMC (mg) 474 (52) 565 (50) 40 Callus DXA-BMD (mg/cm2) 258 (25) 320 (17) 40 Callus DXA-BMC (mg) 153 (28) 190 (34) 5.6 Bone density (%I) 48 (8) 65 (9) 8.2 Surface density (mm’/mm’) 10.8 ( 1 . 1 ) 8.7 (1.1) -3.2 Trabecular thickness (mm) 0.19 (0.26) 0.15 (0.03) -0.2 PTH(1-34) (60 pg/kg) was given every second day during the 10 days of distraction and the following 20 days of consolidation. DEXA, dual-energy X-ray absorptiometry; BMC, bone mineral content; BMD. bone mineral density.
16.8 150
I40 80 68.4 25.8 -1.0 0.2
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Tdbk 2 Callus parameters of distracted femur after 40 days of consolidation Mean (SD), 17
=9
95% CI limits for difference
Controls
PTH( 1-34) 60 pg/kg/injection
Mechanical analysis Ultimate load ( N ) Stiffness (N/mm) Apparent peak stress (MPa) Apparent modulus of elasticity (MPa)
52 (18) 107 (45) 15 (5) 421 (308)
80 (34) 166 (57) 20 ( 1 1 ) 535 (400)
2.8 10.5 -2.5 -222.8
External callus dimensions Anterior-posterior (mm) Medial-lateral (mm)
5.4 (0.7) 6.3 (0.6)
5.7 (1.0) 6.6 ( I .O)
-0.4 -0.5
1.1 I.0
-3.4 -100 10 0 4.4 8.0 -5.1 0.1
10.4 90 140 60 91.6 19.4 -2.2 0.2
Transverse area (mm') Total regenerate callus volume (mm') Femur DXA-BMC (mg) Callus DXA-BMD (mglcm') Callus DXA-BMC (mg) Bone density ('%) Surface density (mm'/mm') Trabecular thickness (mm)
27 (5) 30 (9) 202 (66) 199 (131) 483 (61) 557 (81) 292 (27) 323 (30) 145 (27) 193 (61) 62 ( 6 ) 76 ( 6 ) 9.4 ( I .4) 5.8 (1.7) 0.14 (0.02) 0.3 (0.13) PTH(I-34) (60 pg/kg) was given every second day during the last 30 days of consolidation. DEXA, dual-energy X-ray absorptiometry: BMC, bone mineral content; BMD, bone mineral density.
53.8 107.9 13.6 451.8
Table 3 Mechanical analysis, dimensions, BMC and BMD of contralateral intact femur after 20 and 40 days of consolidation Mean fSD)
95% CI limits for difference
Controls
PTH( 1-34) 60 pg/kg/injection
20 duys oJ consolidution Ultimate load ( N ) Stiffness (N/mm)
n=7 118 (12) 187 (17)
n Z 8
130 (11) 197 (13)
-0.2 -5.3
23.2 26.3
Ext. middiaphyseal dimensions Anterior-posterior (mm) Medial-lateral (mm)
3.7 (0.2) 4.5 (0.2)
3.7 (0.2) 4.5 (0.2)
-0.2 -0.2
0.2 0.2
Transverse area (mm') Femur DXA-BMC (mg) Diaphyseal DXA-BMD' (mg/cm') Diaphyseal DXA-BMC' (mg)
13.1 (1.3) 327 (21) 203 ( 10) 83 (9)
13.1 (1.1) 359 (23) 214 (10) 89 ( 6 )
-1.3 10 0 -1.9
1.1 50 20 13.8
40 duys of consoliduiion Ultimate load (N) Stiffness (N/mm)
I1 =
9 140 (16) 261 (51)
17
296 (57)
5.0 --16.0
47.9 86.3
3.7 (0.2) 4.4 (0.2)
3.8 (0.1) 4.5 (0.2)
0 -0.1
0.3 0.2
Ext. middiaphyseal dimensions Anterior-posterior (mm) Medial-lateral (mm)
=9
166 (28)
-0.2 12.8 (1.1) 13.5 (0.8) 10 363 (39) 416 (48) -30 250 (19) 242 (28) -17.7 81 (19) 80 (17) At both consolidation periods, PTH(I-34) (60 pglkg) was given every second day during the last 30 days of the experiment. DEXA, dual-energy X-ray absorptiometry; BMC, bone mineral content: BMD, bone mineral density. 'Measured using a segment. Transverse area (mm') Femur DXA-BMC (mg) Diaphyseal DXA-BMD' (mglcm') Diaphyseal DXA-BMC' (mg) . .
Forty duys consolidation PTH( 1-34) increased the ultimate load and the stiffness of the distracted bone by more than 50'341.No major
I .6 90 10 15.7 7.4-mm-high
difference was found for apparent peak stress or apparent modulus of elasticity with PTH( 1-34), The intact contralateral femur became slightly stronger and stiffer, but to a lesser degree. No difference between PTH( 1-34)
C. Seehuch et u1. I Journul oJ Orthi pieilic Rrwurcll 22 (2004) 472-478
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I
I
h
z
v
100-
0
J
--
io
20
(4
40
40
time (d)
300
'E .
h
200
cocbo 100
0 (B)
20
20
40 time (d)
40
Fig. 2. Ultimate load in N (A) and stiffness in N/mm (B) of the distraction callus after either 20 or 40 days consolidation time with PTH (60 pg/kg) or vehicle treatment.
treated rats and control was found in callus dimensions or total regenerate callus volume. The callus BMD was 11% higher and the callus BMC was 33% higher in the PTH(1-34) group. No difference was found in BMD in the contralateral, intact femur. The histological analyses showed an increase in bone density (23'%)),a decrease in bone surface density (380/0), and at this time also a large increase in trabecular thickness (1 14'%1)in the distracted callus with PTH( 1-34) treatment. The trabeculae had become fewer and thicker with an increase of the bone mass in the area next to the cortex (Fig. 3). Weight gain was not affected by PTH( 1-34) (data not shown).
Discussion
Only few experiments have investigated the influence of intermittent PTH( 1-34) treatment on fracture heal-
ing, including callus formation and mechanical strength development, in animal models. Our current rat model of distraction osteogenesis for femoral lengthening reliably results in zonal regions of predominantly intramembranous bone. Although our distraction model is different from other experimental models of fracture healing, our findings confirm previous fracture and bone chamber studies, that PTH( 1-34) increases the mechanical strength as well as the amount of new mineralized bone [2,8,16,17]. Further, we have previously shown that the effect of PTH(1-34) is clearly more dramatic in newly forming bone, like a callus, than in bone that is merely undergoing normal remodelling [16]. We chose to compare data from distracted calluses between treatment groups directly. However, if each mechanical parameter is instead expressed as a percentage of the unoperated contralateral leg, the results are essentially the same, although the significance level is less. Thus, still at 40 days, the ultimate load was 38% of the contralateral femur in the controls and 48% in the PTH( 1-34) treated group. The ultimate load depends less on the amount of load-bearing material than on callus dimensions and the arrangement and dimension of each trabecula [14]. We found that PTH(I- 34) treated animals were able to increase their total regenerate callus volume when treatment was initiated while distraction was on-going, whereas no difference on total regenerate callus volume was observed when treatment was administered during the consolidation phase only. At that time point, the outer dimensions of the callus appear to have been determinated already. The absence of a difference in callus volume at 40 days may also be due to the more advanced stage of remodelling with more mature and thicker trabeculae in the PTH(1-34) treated group. It seems that the callus with an early PTH(1-34) treatment becomes stronger, because of an increase of total regenerate callus volume (Table 1). The callus with a later PTH( 1-34) treatment becomes stronger, because of improved arrangement and dimension of each trabecula (Table 2 and Fig. 3). However, because we did not do a group treated with PTH( 1-34) from the distraction and for 40 days consolidation, the differences between the 20 and the 40 days groups should be interpreted with caution. In normal well adapted rat fractures, PTH( 1-34) treatment during the first weeks of healing increases callus volume; however, to achieve this effect higher doses of PTH(1-34) than used in the present experiment are needed [2,7,8]. In ovariectomized rats with delayed fracture healing, PTH( 1-34) treatment given daily in a dose similar to the one used in this experiment has been shown to increase both mechanical strength and callus volume [lo]. Also, in glucocorticoid treated rabbits with delayed fracture healing, a PTH analogue has been
411
Fig. 3 . Histology after 40 days consolidation of a long axis section of the rat femur. (A) PTH treated specimen shows an increase in trabecular density and thickness in the area next to the cortex. (B) Framed area from A . ( C ) Vehicle treated specimen with a larger amount. butthinner trabeculae. (D) Framed area from C (H&E staining. bar length 1 mm).
shown to augment mechanical strength development and callus formation [ 5 ] . We also could see an anabolic effect of PTH( 1-34) at the intact, contralateral site, but to a lesser degree than in the distraction callus. It seems that PTH(1-34) has a stronger effect on bone where a repair response is activated [16]. When starting to develop this distraction model some years ago we saw many pin infections leading to loosening. The incidence of pin-tract infection in this study has been reduced by improving the surgical techniques, giving antibiotics, and intensive postoperative care. Considering the difficulties to achieve this, we are surprised that pin-tract infections are not reported in other publications. This is a profound issue for distraction studies in rats. Pin-tract infections decrease the stability of the construct, leading to small motions within the distraction gap that may inhibit bony bridging. In a previous unpublished study, we used the same protocol as in the 20 days group of this study, but with a less meticulous operation technique and postoperative wound care. Almost all of these rats had pin-tract infection. In consequence to the ensuing instability, there
was often no continuous bone bridge across the distraction gap, but instead a pseudarthrosis PTH( 1-34) did not seem to improve repair in this situation. PTH(134) increases bone matrix synthesis and prevents osteoblast apoptosis [12], but does not induce de novo bone formation like e.g. BMPs. Therefore, it is unlikely that PTH would be useful in treatment of an unstable pseudarthrosis. This study suggests that PTH( 1-34) might be useful as a stimulator of bone formation in order to improve fracture stability while consolidation is occurring.
Acknowledgements We thank Mats Christensson for help with design and manufacturing of the distraction devices, Inger Martensson and Carina Forslund for technical assistance in Lund, and Dr. Thorsten Hennings for help with the DEXA measurement at the University hospital in Frankfurt am Main. This study was financially supported by the Swedish Medical Research Council (project 2031)
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and Deutsche Forschungsgemeinschaft.The experiments were performed in Lund, Sweden. References [l] Andreassen TT, Ejersted C, Oxlund H. Intermittent parathyroid hormone (1-34) treatment increases callus formation and mechanical strength of healing rat fractures. J Bone Miner Res 1999;14:960-8. [2] Andreassen TT, Fledelius C, Ejersted C, Oxlund H. Increases in callus formation and mechanical strength of healing fractures in old rats treated with parathyroid hormone. Acta Orthop Scand 2001;72:304-7. [3] Aronson J. Limb-lengthening, skeletal reconstruction and bone transport with the Ilizarov method. J Bone Joint Surg [Am] 1997;79:1243-58. [4] Aronson J, Shen XC, Skinner RA, et al. Rat model of distraction osteogenesis. J Orthop Res 1997;15:2214. [5] Bostrom MP, Gamradt SC, Asnis P, et al. Parathyroid hormonerelated protein analog RS-66271 is an effective therapy for impaired bone healing in rabbits on corticosteroid therapy. Bone 2000;26:43742. [6] Ejersted C , Andreassen TT, Oxlund H, et al. Human parathyroid hormone (1-34) and (1-84) increase the mechanical strength and thickness of cortical bone in rats. J Bone Miner Res 1993;9:1097101. [7] Hock JM, Gera I, Fonseca J, Raisz LG. Human parathyroid hormone (1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122:2899-904.
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