Archives of Orthopaedic and Trauma Surgery Springer-Verlag 2002

Arch Orthop Trauma Surg (2002) 122: 279-282

Original Article

Bone turnover during distraction osteogenesis in an experimental sheep model

H. Windhagen1, F. Thorey1 , F. Witte1, C. Hurschler1, O. Maciejewski1, D. Linnenberg1 and

Abstract. Biochemical serum markers of osteoblastic activity and collagen turnover were measured in a sheep model of distraction osteogenesis. Significant increases of the bone formation marker osteocalcin were found during the first part of the consolidation phase and peaked at a time point equalling the distraction phase. Collagen turnover parameters pyridinoline and the specific type I collagen marker desoxypyridinoline consistently increased during the distraction and consolidation phases. While pyridinoline peaked at a time point similar to osteocalcin, desoxypyridinoline increased to a later stage in consolidation, indicating continuous turnover of bone-specific type I collagen. The results indicate a characteristic pattern of osteoblast cellular activation during distraction osteogenesis with possible consequences for the timing of treatment. Keywords. Osteocalcin - Distraction osteogenesis - Desoxypyridinoline - Pyridinoline Serum marker - Bone turnover

Introduction
Distraction osteogenesis is a technique capable of generating amounts of bone by gradual distraction of osteotomized bone ends. The method is now widely accepted for the treatment of shortened extremities, bony defects from tumour and trauma, and bone axis correction. Introduced by Codivilla in 1905 and Bier in 1923 [1], the method has been greatly refined by Ilizarov [6] and is now accepted worldwide. After an initial latency period following the osteotomy and stabilization, a distraction of between 1 and 1.5 mm per day is performed. After reaching the final extent, the lesion is allowed to consolidate. The histology and ultrastructure of the regenerating tissue have been well studied, but the cellular and molecular events associated with this process are still unknown. Information about the cellular and molecular processes of distraction osteogenesis may have a significant clinical impact as it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. This is especially important as the long time needed for full consolidation during distraction osteogenesis is the major problem connected with the acceptance of this method.

The molecular events governing endosteal bone development and repair have been extensively described and provide an important background for the investigation during distraction osteogenesis. While these studies have implicated a number of regulatory growth factors and extracellular matrix proteins, little is known about the time points of maximum cellular activation and activation of different tissue regeneration mechanisms during the distraction and consolidation phases. To assess the time points of cellular activation, the aims of this study were (1) to quantify the development of osteoblastic function and activity and (2) to assess the collagen turnover during distraction and consolidation. Osteoblastic activity can be measured by the calcium-binding protein osteocalcin. Its precise function in bone metabolism has not yet been clarified, but suggested functions include the formation of bone matrix and mediation of bone resorption [5, 12]. Osteocalcin is synthesized solely by osteoblasts and is incorporated into the extracellular matrix of bone. However, a fraction of the newly synthesized intact osteocalcin is released into the circulation, where it can be measured by radioimmunoassay. Due to the good correlation between serum osteocalcin levels and histomorphometric indices of bone formation [2, 9], the serum level of osteocalcin directly reflects the relative activity of the osteoblasts. Collagen turnover can be measured by pyridinium cross-links and their higher molecular derivatives, which are presently considered to be the most specific markers of bone resorption and collagen turnover. The pyridinium derivatives pyridinoline (PYD) and desoxypyridinoline (DPD) are formed as cross-links during the maturation of collagen, and are thus responsible for the stability of the extracellular collagen. During bone resorption these matured matrix collagens are decomposed proteolytically, leading to among other things a release of the cross-linked components into the circulation. Since there is no further use for these complex structures within the body, and they cannot be metabolized, PYD and DPD are excreted in the urine, where they can be measured by high performance liquid chromatography (HPLC). The specific questions to be answered by this study were how levels of urinary pyridinoline and desoxypyridinoline and serum levels of osteocalcin would develop during distraction osteogenesis. Addressing these specific questions, biochemical serum markers were measured during a distraction osteogenesis performed in a sheep model.

Materials and methods

The right tibiae of 14 sheep were osteotomized at the mid-diaphysis and stabilized with an external, double, half-ring fixator. All animal experiments were conducted with the permission of the local animal protection authorities and according to US and German animal welfare laws. After a latency period, the tibiae were first distracted at 1.25 mm per day (two distraction time points) for 20 days and then allowed to consolidate for 50 days (Fig. 1). During the treatment period all animals were allowed full use of the osteotomized leg. The animals were killed after 74 days. Blood and urine samples were collected during the entire treatment period on a weekly basis. Blood was obtained from punction of the jugular vein. Urine was obtained using a

special external urine collector. Blood was centrifuged at 5000 rpm, serum separated and stored at -70C until analysis. Urinary pyridinoline (PYD) and desoxypyridinoline (DPD) were measured by HPLC. Reconstitution of the urine standard and control was done with 3 ml and 5 ml distilled water, respectively, under protection from light. Urine specimens were hydrolysed with 150 l of 12 N HCl for each sample at 100C over 16 h. The HPLC procedure was then run with the Bio-Rad Clinical HPLC System using a pyridinium-crosslinks analytical cartridge. Pyridinoline and desoxypyridinoline peaks were detected by a fluorescence detector at excitation levels of =295 nm and =400 nm, respectively. PYD and DPD concentrations were calculated using a multiplying factor computed from the PYD /DPD concentration of the standard divided by the PYD/DPD peak height of the standard. Concentrations of pyridinoline and desoxypyridinoline were related to urinary creatinine. In our protocol, the intra- and interassay coefficients of variation were 8.0% (low probe) and 5.3% (high probe), and 11.8% and 5.7%, respectively. Intact osteocalcin was measured by a competitive radioimmunoassay using the coated tube technique (OSCA test, Brahms, Berlin). This test is based on the pronciple of competition of osteocalcin from the sheep serum samples with a fragment analogue acting as tracer, 125I-osteocalcin for the antigen-binding sites of a highly specific antibody immobilized on the inner surface of the tube. The concentration of the tracer and the concentration of the antibody are constant in all the tubes with one assay. Thus, the remaining variable parameter is the concentration of unlabelled osteocalcin in the sample. A higher concentration of osteocalcin in the sample leads to reduced binding of 125 I-labelled osteocalcin to the antibody. Thus, the amount of radioactivity bound to the tube is inversely proportional to the osteocalcin concentration of the tested sample. For osteocalcin content measurements 50-l samples and standards were pipetted into special tubes, 250 l of tracer were added, and the tubes incubated for 24 h at 5C. The coated tubes were then washed 5 times with 2 ml washing solution and the radioactivity measured over 60 s counting time. Results were calculated using a computer-assisted analysis of OSCA test osteocalcin. In our protocol, the functional assay sensitivity (lowest concentration in which the intra-assay coefficient of variation is <10.0% and the interassay coefficient of variation is <20%) was 1.8 ng/ml.

Statistical analysis
A two-way analysis of variance (ANOVA) was used to demonstrate time differences between the biochemical markers. Markers were also expressed as percentage change in order to allow the interpretation and comparison of the time-related changes of osteoblastic and osteolytic markers.

Results
Urinary and serum markers of bone turnover varied significantly during the course of distraction osteogenesis. Pyridinoline increased consistently up to 88% of its initial value during the 20-day distraction phase and during the consolidation phas,e until leveling at 450 nmol/mmol creatinine (Fig. 2A). Desoxypyridinoline exhibited a pattern

of increasing concentration up to day 50 followed by a period of decreasing concentration. The average concentration increased up to 42% of its initial value. The variability of the desoxypyridinoline data was much higher than that of the pyridinoline data (Fig. 2B). Osteocalcin increased slowly during the distraction phase. During the consolidation phase it increased dramatically up to 110% by day 44. The concentration remained consistent until death (Fig. 2C). The highest changes were recorded for osteocalcin, followed by pyridinoline and desoxypyridinoline.

Discussion
The goal of this study was to measure longitudinal changes of the biochemical markers of bone turnover pyridinoline, desoxypyridionline and osteocalcin, during distraction osteogenesis. As a whole, both markers of bone resorption and bone formation increased during distraction and during the first part of the consolidation phase, indicating that both bone formation and collagen turnover occurs during distraction osteogenesis. Osteocalcin concentration, as the sole marker of bone formation, increased by only 15% during the distraction phase, but by more than 100% during the consolidation phase. As this protein is synthesized by osteoblasts, the relative activation of osteoblasts during distraction osteogenesis can be estimated; thus, osteoblasts are increasingly activated during distraction and consolidation and peak at a consolidation time that in this study was equivalent to the total amout of distraction time. The concentration of pyridinoline rose in a manner similar to osteocalcin during distraction and consolidation, but in a more linear fashion. The interpretation of this parameter is difficult, as it is not a marker specific for type I collagen, and changes in other connective tissues during distraction osteogenesis may influence this parameter. For instance, muscle and tendon lengthening can be reflected by this parameter. Nonetheless, the average rate of increase in pyridinoline concentration continued from distraction through consolidation.Thus, collagen turnover in the maturating callus tissue could be increasingly activated. Changes in the biochemical markers of bone formation have been reported by several authors [3, 4, 10, 11, 13]. Emami et al. found increased osteocalcin levels after nailed tibial shaft fractures peaking between 4 and 10 weeks and thereafter remaining constant over 1 year. Further time points were not assessed. Ohishi et al. measured N-terminal mid-fragment osteocalcin during 24 weeks in patients with femoral neck fractures, trochanteric fractures and spine fractures and found still elevated levels at this time point. Different values were obtained for different locations. Similarly, Obrant reported increasing serum osteocalcin (serum bone GLA-protein) in an almost linear function over 60 days. The increase was interpreted as paralleling the metabolic response to fracture in histological studies [10]. Although no direct comparisons can be made between distraction osteogenesis in sheep species and human fracture healing,

similarities in activation of bone formation are obvious. In the presented study the maximum osteocalcin was observed at a time point where the distraction phase equalled the consolidation phase. The measured increase in osteoblastic activity by osteocalcin agrees with the measurements of bone alkaline phosphatase (ALP) performed by Leung et al. [8]. In that study of distraction osteogenesis, a steady increase in ALP was measured until consolidation had lasted just as long as the distraction time, similarly to the osteoblastic marker osteocalcin in the present study. This supports the hypothesis that migration and activation of osteoblasts takes a considerable time during consolidation until a steady state has been reached. The detected collagen turnover observed through the increase in DPD level closely mimics the histological differentiation observed during distraction osteogenesis. After resorption of the initial haematoma and after capillary growth into the osteotomy gap, there occurs a high development of vascularized collagen tissue. In contrast to fracture healing where expression of collagen type II is observed, in distraction osteogenesis increased amounts of collagen type I are produced, indicating the development of mature bone matrix. [7]. The results of this study suggest a steady increase of collagen I turnover peaking in the first part of consolidation phase. The increase in pyridinoline concentration can be explained in part by collagen turnover during bone healing but also by collagen metabolism occurring with the muscle lengthening that parallels bone lengthening. In contrast to DPD, PYD reflects changes in collagens I, II and III, and is thus less specific in the detection of bone healing metabolism. Ohishi et al. [11] described a characteristic DPD and PYD increase immediately following surgery that was explained as a reaction to osteonecrosis and tissue necrosis. In the present study, such an increase was not detected, indicating either that only minimum tissue trauma was inflicted by the initial surgery, or that specific differences in collagen turnover response occur between different species. Osteocalcin exhibits differences in its molecular structure between humans and sheep. In humans, the protein weigh 5000 Da and contains 49 amino acids. Sheep osteocalcin is different in the five base positions: 3, 4, 5, 9, 10. Moreover, the sheep osteocalcin is vitamin C dependent. To address these differences, a sheep antibody binding specifically to sheep osteocalcin was used in this study, thus enhancing the overall sensitivity and precision. The specific test used for osteocalcin analysis in this study was somewhat limited as only intact osteocalcin was measured, and intact osteocalcin fragments were ignored. In conclusion, a characteristic activity of osteoblastic function and collagen turnover was observed during distraction osteogenesis. The data may be used in the future to help determine the timing of treatments, for example to optimize the stimulation of osteoblast migration and differentiation. Further studies will be required to address the late phase of bone remodelling in distraction osteogenesis.