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Early changes in prostaglandins precede bone formation in a rabbit model of heterotopic ossification
Bone 38 (2006) 322 – 332 www.elsevier.com/locate/bone
Early changes in prostaglandins precede bone formation in a rabbit model of heterotopic ossification Craig S. Bartlett a , Bruce E. Rapuano b , Dean G. Lorich c , Timothy Wu d , Richard C. Anderson e , Emre Tomin f , John F. Hsu g , Joseph M. Lane h , David L. Helfet i,⁎ a
Department of Orthopaedic Surgery, University of Vermont Medical School, Burlington, VT 05401, USA b Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA c Jacobi Medical Center, 1400 Pelham Parkway South, Bronx, NY 10461, USA d Downstate Medical Center, 6303 13th Avenue, Brooklyn, NY 11219, USA e Department of Orthopaedic Surgery, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 10021, USA f Department of Orthopaedic Surgery, Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA g New York College of Osteopathic Medicine, PO Box 8000, Old Westbury, NY 11568-8000, USA h Department of Orthopaedic Surgery, Weill Medical College of Cornell University, Hospital for Special Surgery, New York, NY 10021, USA i Orthopaedic Trauma Service, Department of Orthopaedic Surgery, Weill Medical College of Cornell University/Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA Received 25 October 2004; revised 22 August 2005; accepted 23 August 2005 Available online 12 October 2005
Abstract We have tested the hypothesis that the formation of heterotopic ossification (HO) in a rabbit model is correlated with a local increase in specific prostaglandins that may modulate mechanisms of ossification. Rabbits were sacrificed at 1 to 21 days following the daily forcible flexion of immobilized knees. The extraction and analysis of prostaglandins (PG) E2, F2alpha, D2, 6-keto-F1alpha, and thromboxane B2 in vastus intermedius muscles of manipulated legs revealed increases compared to control hindlimbs for all five prostaglandins, albeit of differing magnitude. The earliest increase was observed for PGF2alpha after 24 h (to 2.6-fold of control) with peak levels observed at day ten (185-fold of control). PGE2 was increased above control from 2 to 21 days following manipulation, with a peak level of 33-fold of control after 10 days. In a separate arm of the study, the role of PGE2 was investigated through the use of pharmacological antagonist of the PGE2 receptors and one of its second messengers, cAMP. Rabbits were preadministered the PGE2/PGD receptor antagonist AH 6809 or the cAMP antagonist Rp-cAMP prior to undergoing the regimen of limb immobilization and passive exercise. Both AH 6809 and Rp-cAMP were found to prevent the later development of radiographically documented heterotopic ossification in 15 out of 16 animals, thus identifying prostaglandins as being required for the development of ectopic bone. In this latter group, all but one pharmacologically treated animal showed an absence of HO at 3, 4, 5, or 6 weeks. These findings suggest an obligate cascade of prostaglandins for HO that offers the potential for novel prophylactic therapies, including those that target receptors for specific prostaglandins. © 2005 Elsevier Inc. All rights reserved. Keywords: Heterotopic ossification; Myositis ossificans; Prostaglandins
Introduction Heterotopic ossification (HO) is the formation of mature trabecular bone in sites where it is not normally present. HO initially manifests as highly metabolically active woven bone in the soft tissue [1], which matures into lamellar bone, ⁎ Corresponding author. Fax: +1 212 717 4340. E-mail address: [email protected] (D.L. Helfet). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
containing osteoblasts and osteoclasts, often with true marrow [2–4]. HO is of particular interest to the orthopedic surgeon due to increased induction after total hip arthroplasty [2,5–10] and trauma, including neurological injuries [2,10–12], burns [2,10,13,14], and the open reduction and internal fixation of acetabular fractures [11,15–21] (Fig. 1). Although its incidence varies widely, HO is typically seen in 20% to 30% of these cases [5,6,9,10,16–20,22]. Its grade, severity, and functional loss appears to be associated with surgical approaches that
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Fig. 1. Time-dependent changes in muscle content of PGF2alpha during development of HO. Animals were sacrificed at 1, 2, 4, 7, 10, 14, or 21 days following commencement of forcible manipulation of right limbs, the vastus intermedius muscle was removed from the right (HO) and unmanipulated left (control) legs, and prostaglandins were extracted from the distal one-third of each muscle and assayed as described in Materials and methods. PGF2alpha content expressed as ng prostaglandin per mg protein of extracted muscle tissue is presented as means ± SE for unmanipulated (control) and manipulated (HO) legs at each time point. Paired Wilcoxon (Z value) comparing muscle PGF2alpha levels in HO to those measured in control muscles (obtained at the corresponding time of sacrifice) was conducted to determine whether the effects of manipulation/trauma on prostaglandin synthesis were statistically significant at each time point. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
require significant periosteal and muscle stripping, especially the hip abductor musculature, which result in pericapsular hematomas [23–25]. This suggests that trauma is an important factor in the development of HO in the orthopedic clinical setting. Many prophylactic regimens have been proposed and applied for HO, including radiation [18,19] and pharmacological therapy with non-steroidal anti-inflammatory drugs (NSAIDs) [5,7,8,15,19]. Non-steroidal inflammatory drugs are variably effective when used prophylactically [26] and have demonstrated the capacity to delay fracture healing, inhibit bone remodeling [27–32], and decrease ingrowth into porous arthroplasty components [33,34]. However, adverse drug reactions to these drugs, including gastrointestinal bleeding and renal toxicity, prevent 20% to 37% of patients from completing treatment [24,35,36]. Furthermore, the therapeutic window for radiation has usually passed by the time gastrointestinal intolerance is recognized. Neither is radiation therapy a panacea as it carries the theoretical risk of malignancy, genetic alterations, and gonadal effects [18,37,38]. Despite the limited effectiveness of prophylactic regimens, there has been little research performed to evaluate the mechanism by which NSAIDs prevent HO. This is, in part, due to the fact that the biochemical pathways leading to its formation have yet to be elucidated. The usefulness of NSAID'S in HO, although inconsistent [26], suggests that prostaglandins, which are local mediators of inflammation and bone remodeling, may be involved in this process. Importantly, time-dependent changes in the profile of tissue prostaglandins, including those of PGE2, PGF2alpha, thromboxane B2, and 6-keto-PGF1alpha, have been observed during endochondral ossification [42], suggesting that individual prostaglandins may serve specific roles in normal
bone formation as well as in HO. In the Michelsson rabbit model [43] we have adapted in the present study, muscle trauma and the resulting inflammatory repair process probably constitute an essential event in the pathophysiological mechanism of HO. Therefore, an investigation into the putative role(s) for prostaglandins in the ectopic ossification observed in this animal model is warranted. Prostaglandins and thromboxanes, i.e. the prostanoids, which are cyclooxygenase metabolites of arachidonic acid, exert a wide range of actions which are mediated by specific cellular receptors. The classes of receptors which have been identified include the DP, EP, FP, IP, and TP receptors for PGD, PGE, PGF, PGI (prostacyclin), and thromboxane (A and B2), respectively. EP receptors are subdivided into four subtypes, EP1, EP2, EP3 and EP4, which are encoded for by different genes and respond differently to various agonists and antagonists [44]. PGE2 is the major prostanoid secreted by osteoblastic cells [45]. A number of reports have established that the administration of type E prostaglandins has an overall anabolic effect in bone when applied systemically [46,47] or locally [48,49]. PGE2 has also
Table 1 Stimulatory effects of prostaglandin receptors on cAMP and bone formation PG receptor
cAMP formation
Bone formation
DP EP1 EP2 EP4 F(1-alpha)P F(2-alpha)P IP TP
Yes [53] No [54] Yes [54,55] Yes [55] No [56] No [57] Yes [58,59] No [60–62]
No [63] No [54] Yes [54,64,65] Yes [66–70] – No [63,68–70] No [63] –
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been shown to increase intracellular levels of cAMP in bonederived cells [50–52]. Furthermore, other findings have demonstrated that receptors for PGD2, prostacyclin, and PGE2 are the only prostanoid receptors that work through cAMP (Table 1). Since, of these three prostaglandins, only the receptors for PGE2 have been shown to transduce anabolic effects in bone (Table 1), it is appropriate to focus on the role of cAMP signaling through EP receptors in the mechanism of traumatic HO. In addition, the potential involvement of various specific prostaglandin molecular species in the process of ectopic bone formation also needs to be clarified. Therefore, in the current study, we have measured PGE2, PGF2alpha, PGD2, thromboxane B2, and 6-keto-PGF1alpha levels in soft tissue sites destined for heterotopic bone formation in a rabbit model to identify a specific prostaglandin cascade that precedes the formation of heterotopic ossification in a rabbit model. The role of receptors for specific prostaglandins was also investigated by blocking HO through the in vivo administration of pharmacological antagonists, including the compound 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), an EP1, DP, and EP2 receptor antagonist [74–76], and the cAMP antagonist Rp-cAMP [77]. Materials and methods Materials AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) was obtained as a generous gift from Glaxo Wellcome Medicines Research (Hertfordshire, UK). Adenosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMP), was obtained from Biolog-Life Science Institute (La Jolla, CA). Alzet osmotic minipumps (model 2ML2) were supplied by the ALZA Corporation (Palo Alto, CA).
Animals The rabbit HO model of Michelsson et al. [39–41,44] was utilized after approval by an institutional animal care and use committee (IACUC). Numerous authors have reported that Michelsson's model reliably induces HO, which ultimately forms lamellar bone within 1 month [26,78,79]. Michelsson's rabbit model was chosen over the rat model proposed by Brown et al. [80] which produces limited bone and appears to have little to do with how heterotopic bone is induced in humans. The right leg of thirty-nine New Zealand rabbits was forcibly manipulated repeatedly from full extension to full flexion for 2 to 3 min daily then immobilized in a bivalved long-leg cast. Range of motion, stiffness, and the animal's response to manipulation were recorded. Groups of rabbits were sacrificed at 1, 2, 4, 7, 10, 14, or 21 days after the commencement of manipulation. Five or six rabbits were present in each group except for the 3week group (n = 3). For each time point, unmanipulated legs served as paired controls for the corresponding contralateral manipulated limbs for all subsequent biochemical analyses. The time periods chosen are consistent with previous findings that the definitive event which drives the system towards heterotopic bone formation occurs within the first 2 weeks [26].
Radiographic and analyses Plain and high-resolution (Faxitron) radiographs were obtained of both hindlimbs at the time of sacrifice to identify any calcification. Both manipulated and unmanipulated (control) legs were dissected and inspected, gross specimens obtained, and the vastus intermedius muscles removed, and prostaglandin levels assayed. All remaining tissues were stored in a −80°C refrigeration unit. Prostaglandins were extracted from the distal one-third of the vastus intermedius muscle, noted in preliminary experiments to be the area of maximal bone
formation. Muscles were ground using a Spex 6700 freezer mill (Spex Industries, Inc., Edison, NJ) then placed in a buffer and homogenized on ice by hand in a Tenbroeck tissue grinder. Aliquots were then taken for both prostaglandin and protein assays.
Administration of prostaglandin receptor and cyclic AMP antagonists Alzet osmotic minipumps were implanted subcutaneously in the lateral thigh of each manipulated limb to effect a local delivery of each pharmacological antagonist to the vastus intermedius. These osmotic pumps are small implantable drug reservoirs that deliver a controlled dose of medication continuously over a controlled period of time. The only surgical procedure for implantation involved the placement of the Alzet pumps in the subcutaneous tissue of the thigh, with a delivery tube inserted underneath or into the vastus intermedius muscle. Due to the short duration of the procedure, intubation and inhalation anesthetic was not required. Anesthesia with ketamine, acetylpromazine, and xylazine was sufficient. The leg was sterilely prepped and draped from the hip to the ankle. The lateral and anterior thigh was clipped. A lateral incision 2–3 cm in length was made, and the iliotibial band was divided in line with the incision. The tube from the Alzet pump was then placed underneath or into the vastus intermedius muscle, thus delivering the antagonists directly to the site of HO formation. The pump was then placed in the subcutaneous space of the lateral thigh. The subcutaneous tissue was then closed over the pump using dissolvable suture, and the skin was closed using 3/0 nylon suture. The hindlimb was then dressed in a sterile dressing. Drugs were delivered during the first 14 days of manipulation (at 1 ml/ week) at which point the pumps were surgically removed. Plain radiographs were obtained of the manipulated hindlimbs at 3, 4, 5, and 6 weeks. Rabbits were killed immediately before the 6-week radiograph was obtained. The endpoint measured was simply the formation of HO, which was measured radiographically. A dosage of 1 mg/week of either AH 6809 (an EP1, EP2, and DP receptor antagonist) [74–76] or Rp-cAMP (cAMP antagonist) [77] was delivered directly to the vastus intermedius (of 8 rabbits for each antagonist). At this dosage, it is expected that target concentrations of 3.5 μg/ml (10 μM) and 11 μg/ml (30 μM) were reached or exceeded for AH 6809 and Rp-cAMP respectively, within 1/2 to 2 h, respectively, at a constant delivery rate of 0.1 μg/ml/min. This is based on an average wet weight of 1 g and volume of extracellular fluid of 1 ml for each rabbit vastus intermedius. Moreover, since none of these drugs is effectively metabolized unless through hepatic detoxification (which should be minimal with local application at a site between the femur periosteum and the vastus intermedius), it is expected that concentrations which have been shown to antagonize PGE2 or cAMP were easily attainable. It has been shown that prostaglandin E1, which has effects and potency similar to PGE2 at the type EP2 receptor in bone cells [52], could be delivered locally to periosteal and subperiosteal sites via Alzet pumps to stimulate only local bone growth in vivo [48].
Prostaglandin analyses Prostaglandins were extracted following the method of Powell [81]. Ground muscle homogenate was mixed with 15% ethanol. After vortexing and centrifugation, the supernatants were acidified with 1 N HCL. A standard extraction process was then performed, utilizing SEP-PAK cartridges (Water Associates, Milford, MA). In a stepwise fashion, the fatty acids, leukotrienes, polar, and neutral lipids were eluted with ethanol and petroleum ether; methyl formate was then employed to elute prostaglandins. Methyl formate eluants were evaporated under a stream of liquid nitrogen at a bath temperature of not more than 60°C. After re-suspension in enzyme immunoassay (EIA) buffer, EIA kits (Cayman Chemical, Ann Arbor, MI) were used to determine the levels of prostaglandin E2, D2, 6-keto-PGF1, F2alpha, and thromboxane B2 in muscle tissue extracts at levels as low as 10 pg/ml. Protein assays were also performed using BioRad protein dye reagent (BioRad Laboratories, Hercules, CA) to determine prostaglandin concentrations per milligram of muscle protein. All chemicals used were of reagent grade and were obtained from Fisher Scientific (Springfield, NJ).
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Fig. 2. Time-dependent changes in muscle content of PGE2 during development of HO. PGE2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
Statistical analyses Paired Wilcoxon (data from manipulated limb were paired with that from contralateral limb for each animal) was performed. Only statistical differences associated with a Z value of 0.05 or less were reported as significant.
Results Prostaglandin assays Following the lipid extraction of vastus intermedius muscles removed from manipulated and control limbs after sacrifice, statistically significant increases were measured for all five prostaglandins assayed. Peak concentrations of each prostaglandin expressed in ng per mg protein were reached at different time points. The earliest and some of the most dramatic increases
in muscles from manipulated compared to control legs were observed for PGF2alpha (Fig. 1). Statistically elevated within 1 day of the first manipulation (Z = 0.008), PGF2alpha reached a peak that measured 185-fold (1.39 ± 0.6 ng/mg protein; mean ± SE) that of control at day ten (Z = 0.005) (Fig. 1). Although not statistically elevated above control levels until 48 h following the first manipulation, peak PGE2 levels (Fig. 2) were greater than those of PGF2alpha (Fig. 1). PGE2 levels were increased maximally to 33.4-fold that of control at day ten (Z = 0.005) (Fig. 2). These early changes in prostaglandins preceded the development of HO by several days since early calcification was observed on both plain and Faxitron radiographs by day 10 (data not shown). Less dramatic increases were observed for 6-keto-PGF1alpha and thromboxane B2; these levels did not rise significantly above control levels until 4 and 10 days, respectively, following the initial limb manipulation (Figs.
Fig. 3. Time-dependent changes in muscle content of thromboxane B2 during development of HO. Thromboxane B2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
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Fig. 4. Time-dependent changes in muscle content of 6-keto-PGF1alpha during development of HO. 6-keto-PGF1alpha levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 4–6 rabbits for 1–14 days and 3 rabbits for 21 days.
3 and 4). Statistically significant increases in PGD2 levels were not observed until 10 days following the initial manipulation (Fig. 5). At this time point, PGD2 levels (0.16 ± 0.22 ng/mg protein; mean ± SE) (Fig. 5) were 20-fold less than that of PGE2 (3.4 ± 4.3 ng/mg protein; mean ± SE) (Fig. 2). When an antagonist of either PGE2/PGD receptors or cAMP was administered in vivo via Alzet minipumps for 14 days following surgery (8 rabbits per drug group), heterotopic ossification failed to develop in all but 1 animal. After a 2-week period during which time drugs were completely delivered (demonstrated by size reduction of internal pumping chamber documented radiographically), no adverse effects of pump implantation were observed (even when pumps were left in for an additional several days after the complete release of drug) other than a moderate degree of scar tissue that had formed around the pump. Control (non-manipulated) rabbits (3 rabbits in this arm of the study) or rabbits receiving either AH 6809 or Rp-cAMP underwent a regimen of 3 weeks of daily hindlimb
manipulation and were either sacrificed at this point or sacrificed 3 weeks later. After sacrifice at 3 weeks, hematoma formation could be observed throughout the quadriceps musculature of manipulated limbs of control rabbits or rabbits administered AH 6809 or Rp-cAMP. After sacrifice at 3 or 6 weeks, the quadriceps in the manipulated limbs of rabbits in the control, AH 6809, or Rp-cAMP groups also appeared edematous, darker, thicker, and less elastic compared to quadriceps muscles in non-manipulated limbs. Faxitron radiographs of the femurs of manipulated hindlimbs showed calcification in the region of the vastus intermedius for all of the control rabbits (Fig. 6). In contrast, no calcification was observed at 3 weeks (Fig. 7) or 6 weeks (Fig. 8) for eight rabbits receiving AH 6809 or for seven rabbits receiving Rp-cAMP (Figs. 9 and 10, respectively). In one rabbit administered RpcAMP, small islands of mineralization were found in the vastus intermedius muscle at the time of sacrifice and gross examination of the dissected quadriceps 6 weeks after initiation
Fig. 5. Time-dependent changes in muscle content of PGD2 during development of HO. PGD2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 3–6.
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Fig. 6. Faxitron radiograph of femur of control rabbit (one of three rabbits) in drug antagonist arm of study after 3 weeks of daily hindlimb manipulation showing calcification in the region of the right vastus intermedius (left limb was not manipulated).
of the immobilization/passive exercise regimen. Nevertheless, both AH 6809 and Rp-cAMP were found to prevent the development of radiographically or macroscopically documented (at 3, 4, 5, and 6 weeks) heterotopic ossification in 15 out of 16 animals, thus identifying PGE2 or PGD as being obligate for the development of ectopic bone.
Discussion In this study, using a rabbit model of heterotopic ossification, we have demonstrated that multiple species of prostaglandins are elevated in soft tissue (quadriceps muscle) immediately prior to and during the development of HO in that tissue. To our
Fig. 7. Three-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the prostaglandin receptor antagonist AH 6809 and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
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Fig. 8. Six-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the prostaglandin receptor antagonist AH 6809 and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
knowledge, this is the first study to demonstrate that elevations in prostaglandin levels are present sequentially with HO and are temporally correlated with the process of heterotopic bone formation in a model of HO. Marked increases in PGE2 and PGF2alpha were observed within 24 to 48 h of the experimental induction of HO which preceded ossification by several days. Therefore, this suggests that, to be effective, prophylaxis with NSAIDs or prostaglandin receptor antagonists must begin almost immediately after the initial tissue injury in traumatic HO since prostaglandins probably become elevated within 24 h of this event.
PGE2 is likely to play a more important role in the development of HO in the rabbit model than the other prostaglandins studied for three reasons. Firstly, unlike PGE2, none of the other prostaglandins manifests direct anabolic effects in bone (see Table 1). Secondly, PGD2, 6-ketoPGF1alpha, and thromboxane B2 all manifested slower (requiring 4–10 days of manipulation) and less dramatic increases in their levels compared to PGE2. Thirdly, since early calcification was observed on both plain and Faxitron radiographs by day 10 (data not shown), the earliest elevations in prostaglandin synthesis are likely to be the most relevant to
Fig. 9. Three-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the cAMP antagonist RpcAMP and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
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Fig. 10. Six-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the cAMP antagonist Rp-cAMP and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
the mechanism of HO. Prostaglandin levels 7–10 days or more following the initial limb manipulation could also be irrelevant. In contrast to PGD2, 6-keto-PGF1alpha, and thromboxane B2, PGF2alpha levels were significantly increased within 24 h of the initial limb manipulation. A statistically significant increase was not observed for PGE2 until 24 h later. Therefore, PGF2alpha could indirectly promote HO by stimulating PGE2 synthesis, which has been previously demonstrated in bone and bone cell cultures [71–73,82,83]. Early elevations in PGE2 or PGF2alpha could also stimulate later increases in the synthesis of PGD2, 6-keto-PGF1alpha, and thromboxane B2, although the contributions of the latter three prostaglandins to the observed ossification are unclear. Since 6-keto-PGF1alpha and thromboxane B2 cause circulatory actions [84–86] which could amplify the degree of muscle trauma caused by limb manipulation, these prostaglandins might be able to indirectly stimulate ectopic bone formation. Nevertheless, findings that (a) thromboxane B2 was not significantly elevated above control until day 10 and that (b) the EP1, DP, and EP2 receptor antagonist AH 6809 [74–76] completely inhibited HO suggest that the contributions of either of 6-keto-PGF1alpha and thromboxane B2 to HO in this model are minimal. The ability of AH 6809 to block HO in this traumatic animal model implicates role(s) for EP and/or DP receptors in the pathophysiological mechanism. A number of reports have studied the potential involvement of EP1, EP2, and EP4 receptors in osteogenic mechanisms. The activation of EP2 receptors with selective pharmacological agonists has been shown to heal canine long bone segmental and fracture model defects [64,65], suggesting that EP2 is a major contributor to PGE2's local anabolic bone activity [64]. Another study using knockout mice for EP1 or EP2 receptors showed that EP2 receptors stimulate cAMP formation and have a major influence on the biomechanical properties of bone [54]. In contrast, EP1
receptors are coupled with Ca2+ mobilization, not cAMP formation, and have a minimal influence on skeletal strength or size in EP1 knockout mice [54]. Using such knockout models, EP4 receptors have been shown to play a role in bone resorption [64,87]. Other findings suggest that the activation of cAMP by both EP2 and EP4 receptors may mediate the effects of PGE2 on the differentiation of mesenchymal cells into chondrocytes [55]. It has been shown that an EP4 antagonist suppressed the increase in the formation of rat trabecular bone volume [66] and that an EP4 agonist enhanced BMP-2-induced ectopic bone formation in a mouse model [67], suggesting an anabolic role for EP4 in bone. However, another study showed that an EP4 agonist failed to increase the total bone volume of regenerating rat femoral bone but instead increased cortical bone healing by upregulating local bone turnover [88]. Other reports have shown that EP4 receptors increase bone mass and fracture healing [68], stimulate cortical bone formation [69], and regulate remodeling by increasing de novo bone formation [70]. Therefore, the studies discussed above have demonstrated roles for both EP4 and EP2 receptors in normal osteogenesis. Importantly, we have shown in this study that the compound AH 6809, an EP1, DP, and EP2 receptor antagonist [74–76], which has no demonstrated effects on EP4 receptors, completely blocked HO in the rabbit model. While it is certainly possible that EP4 receptors help regulate HO, our findings suggest that EP2 receptors are more directly involved in the mechanism of ectopic osteogenesis. The parallel findings that both the cAMP antagonist and the EP1, DP, and EP2 receptor antagonist AH 6809 [74–76] completely blocked HO formation suggest that EP2 receptors (which are coupled to cAMP formation [54,55]) play an important role in traumatic HO for the following three reasons. Firstly, neither EP1 receptors (which are not coupled to cAMP
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formation; [54]) nor DP receptors have been shown to regulate bone formation or skeletal development using receptor knockout models [54,63], whereas EP2 receptors have [54]. Secondly, we have shown that statistically significant increases in PGD2 levels were not observed until 10 days following the initial manipulation (Fig. 5). As discussed above, radiographic evidence of ectopic calcification had already been obtained by day 10. Thirdly, the levels of PGD2 at this time point were 20× lower (Fig. 5) than that of PGE2 (Fig. 2). Therefore, neither EP1 or DP receptors are likely to be as important as EP2 receptors for the development of traumatic HO. Nevertheless, potential roles of these two classes of receptors in the mechanism of HO cannot be ruled out due to their antagonism by AH 6809. In addition, the significance of PGF2alpha, PGF1alpha, and thromboxane B2 in the traumatic form of HO has yet to be elucidated. However, the findings in the current study together with those in the literature suggest that EP2 receptors are necessary, if not sufficient, for ectopic bone development following tissue injury. In summary, the findings in this study indicate an obligate role for prostaglandins in heterotopic ossification and also suggest that novel and safer therapies that target the receptors for prostaglandins, especially PGE2, may hold promise for the prophylactic therapy of HO. Conclusions Heterotopic bone formation is a significant problem, which challenges the orthopedic surgeon, particularly in the fields of trauma and arthroplasty. A greater understanding of the factors, which are involved in this process, and their mechanisms of action will improve both prevention and treatment. This may involve novel or synergistic combinations of anti-inflammatory drugs and/or adjuvant radiotherapy. However, in order to develop more effective and less toxic preventive and therapeutic regimens, the biochemical mediators of heterotopic bone formation must be identified. Our data provide preliminary support for the proposed inflammatory basis of heterotopic bone formation and the role of prostaglandins in that process. Prostaglandin levels are statistically elevated in a heterotopic ossification model early on its development. Moreover, pharmacological agents which interfere with the activation of specific cellular receptors for prostaglandins block the development of HO, suggesting that prostaglandins are essential to the pathophysiological mechanism. The evaluation and analysis of other possible mediators of heterotopic bone formation, including bone morphogenetic protein, cytokines, and other molecules, may be required in the future to truly understand the etiology of HO. However, the morbidity associated with standard prophylactic therapies can potentially be avoided or lessened through the clinical use of antagonists for prostaglandin receptors. Acknowledgments This research has been supported, in part, by a grant from the AO/ASIF foundation (AO/ASIF Research Project 96-H55/98H47). We gratefully acknowledge the advice and assistance of
Dr. Stephen Doty, Dr. Peter Torzilli, Dr. Corey Brayton, Dr. Adele Boskey, Darlene Grillo, Craig Klinger, and Rena Frantzis.
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Early changes in prostaglandins precede bone formation in a rabbit model of heterotopic ossification Craig S. Bartlett a , Bruce E. Rapuano b , Dean G. Lorich c , Timothy Wu d , Richard C. Anderson e , Emre Tomin f , John F. Hsu g , Joseph M. Lane h , David L. Helfet i,⁎ a
Department of Orthopaedic Surgery, University of Vermont Medical School, Burlington, VT 05401, USA b Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA c Jacobi Medical Center, 1400 Pelham Parkway South, Bronx, NY 10461, USA d Downstate Medical Center, 6303 13th Avenue, Brooklyn, NY 11219, USA e Department of Orthopaedic Surgery, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA 10021, USA f Department of Orthopaedic Surgery, Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA g New York College of Osteopathic Medicine, PO Box 8000, Old Westbury, NY 11568-8000, USA h Department of Orthopaedic Surgery, Weill Medical College of Cornell University, Hospital for Special Surgery, New York, NY 10021, USA i Orthopaedic Trauma Service, Department of Orthopaedic Surgery, Weill Medical College of Cornell University/Hospital for Special Surgery, 535 E. 70th Street, New York, NY 10021, USA Received 25 October 2004; revised 22 August 2005; accepted 23 August 2005 Available online 12 October 2005
Abstract We have tested the hypothesis that the formation of heterotopic ossification (HO) in a rabbit model is correlated with a local increase in specific prostaglandins that may modulate mechanisms of ossification. Rabbits were sacrificed at 1 to 21 days following the daily forcible flexion of immobilized knees. The extraction and analysis of prostaglandins (PG) E2, F2alpha, D2, 6-keto-F1alpha, and thromboxane B2 in vastus intermedius muscles of manipulated legs revealed increases compared to control hindlimbs for all five prostaglandins, albeit of differing magnitude. The earliest increase was observed for PGF2alpha after 24 h (to 2.6-fold of control) with peak levels observed at day ten (185-fold of control). PGE2 was increased above control from 2 to 21 days following manipulation, with a peak level of 33-fold of control after 10 days. In a separate arm of the study, the role of PGE2 was investigated through the use of pharmacological antagonist of the PGE2 receptors and one of its second messengers, cAMP. Rabbits were preadministered the PGE2/PGD receptor antagonist AH 6809 or the cAMP antagonist Rp-cAMP prior to undergoing the regimen of limb immobilization and passive exercise. Both AH 6809 and Rp-cAMP were found to prevent the later development of radiographically documented heterotopic ossification in 15 out of 16 animals, thus identifying prostaglandins as being required for the development of ectopic bone. In this latter group, all but one pharmacologically treated animal showed an absence of HO at 3, 4, 5, or 6 weeks. These findings suggest an obligate cascade of prostaglandins for HO that offers the potential for novel prophylactic therapies, including those that target receptors for specific prostaglandins. © 2005 Elsevier Inc. All rights reserved. Keywords: Heterotopic ossification; Myositis ossificans; Prostaglandins
Introduction Heterotopic ossification (HO) is the formation of mature trabecular bone in sites where it is not normally present. HO initially manifests as highly metabolically active woven bone in the soft tissue [1], which matures into lamellar bone, ⁎ Corresponding author. Fax: +1 212 717 4340. E-mail address: [email protected] (D.L. Helfet). 8756-3282/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
containing osteoblasts and osteoclasts, often with true marrow [2–4]. HO is of particular interest to the orthopedic surgeon due to increased induction after total hip arthroplasty [2,5–10] and trauma, including neurological injuries [2,10–12], burns [2,10,13,14], and the open reduction and internal fixation of acetabular fractures [11,15–21] (Fig. 1). Although its incidence varies widely, HO is typically seen in 20% to 30% of these cases [5,6,9,10,16–20,22]. Its grade, severity, and functional loss appears to be associated with surgical approaches that
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Fig. 1. Time-dependent changes in muscle content of PGF2alpha during development of HO. Animals were sacrificed at 1, 2, 4, 7, 10, 14, or 21 days following commencement of forcible manipulation of right limbs, the vastus intermedius muscle was removed from the right (HO) and unmanipulated left (control) legs, and prostaglandins were extracted from the distal one-third of each muscle and assayed as described in Materials and methods. PGF2alpha content expressed as ng prostaglandin per mg protein of extracted muscle tissue is presented as means ± SE for unmanipulated (control) and manipulated (HO) legs at each time point. Paired Wilcoxon (Z value) comparing muscle PGF2alpha levels in HO to those measured in control muscles (obtained at the corresponding time of sacrifice) was conducted to determine whether the effects of manipulation/trauma on prostaglandin synthesis were statistically significant at each time point. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
require significant periosteal and muscle stripping, especially the hip abductor musculature, which result in pericapsular hematomas [23–25]. This suggests that trauma is an important factor in the development of HO in the orthopedic clinical setting. Many prophylactic regimens have been proposed and applied for HO, including radiation [18,19] and pharmacological therapy with non-steroidal anti-inflammatory drugs (NSAIDs) [5,7,8,15,19]. Non-steroidal inflammatory drugs are variably effective when used prophylactically [26] and have demonstrated the capacity to delay fracture healing, inhibit bone remodeling [27–32], and decrease ingrowth into porous arthroplasty components [33,34]. However, adverse drug reactions to these drugs, including gastrointestinal bleeding and renal toxicity, prevent 20% to 37% of patients from completing treatment [24,35,36]. Furthermore, the therapeutic window for radiation has usually passed by the time gastrointestinal intolerance is recognized. Neither is radiation therapy a panacea as it carries the theoretical risk of malignancy, genetic alterations, and gonadal effects [18,37,38]. Despite the limited effectiveness of prophylactic regimens, there has been little research performed to evaluate the mechanism by which NSAIDs prevent HO. This is, in part, due to the fact that the biochemical pathways leading to its formation have yet to be elucidated. The usefulness of NSAID'S in HO, although inconsistent [26], suggests that prostaglandins, which are local mediators of inflammation and bone remodeling, may be involved in this process. Importantly, time-dependent changes in the profile of tissue prostaglandins, including those of PGE2, PGF2alpha, thromboxane B2, and 6-keto-PGF1alpha, have been observed during endochondral ossification [42], suggesting that individual prostaglandins may serve specific roles in normal
bone formation as well as in HO. In the Michelsson rabbit model [43] we have adapted in the present study, muscle trauma and the resulting inflammatory repair process probably constitute an essential event in the pathophysiological mechanism of HO. Therefore, an investigation into the putative role(s) for prostaglandins in the ectopic ossification observed in this animal model is warranted. Prostaglandins and thromboxanes, i.e. the prostanoids, which are cyclooxygenase metabolites of arachidonic acid, exert a wide range of actions which are mediated by specific cellular receptors. The classes of receptors which have been identified include the DP, EP, FP, IP, and TP receptors for PGD, PGE, PGF, PGI (prostacyclin), and thromboxane (A and B2), respectively. EP receptors are subdivided into four subtypes, EP1, EP2, EP3 and EP4, which are encoded for by different genes and respond differently to various agonists and antagonists [44]. PGE2 is the major prostanoid secreted by osteoblastic cells [45]. A number of reports have established that the administration of type E prostaglandins has an overall anabolic effect in bone when applied systemically [46,47] or locally [48,49]. PGE2 has also
Table 1 Stimulatory effects of prostaglandin receptors on cAMP and bone formation PG receptor
cAMP formation
Bone formation
DP EP1 EP2 EP4 F(1-alpha)P F(2-alpha)P IP TP
Yes [53] No [54] Yes [54,55] Yes [55] No [56] No [57] Yes [58,59] No [60–62]
No [63] No [54] Yes [54,64,65] Yes [66–70] – No [63,68–70] No [63] –
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been shown to increase intracellular levels of cAMP in bonederived cells [50–52]. Furthermore, other findings have demonstrated that receptors for PGD2, prostacyclin, and PGE2 are the only prostanoid receptors that work through cAMP (Table 1). Since, of these three prostaglandins, only the receptors for PGE2 have been shown to transduce anabolic effects in bone (Table 1), it is appropriate to focus on the role of cAMP signaling through EP receptors in the mechanism of traumatic HO. In addition, the potential involvement of various specific prostaglandin molecular species in the process of ectopic bone formation also needs to be clarified. Therefore, in the current study, we have measured PGE2, PGF2alpha, PGD2, thromboxane B2, and 6-keto-PGF1alpha levels in soft tissue sites destined for heterotopic bone formation in a rabbit model to identify a specific prostaglandin cascade that precedes the formation of heterotopic ossification in a rabbit model. The role of receptors for specific prostaglandins was also investigated by blocking HO through the in vivo administration of pharmacological antagonists, including the compound 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), an EP1, DP, and EP2 receptor antagonist [74–76], and the cAMP antagonist Rp-cAMP [77]. Materials and methods Materials AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) was obtained as a generous gift from Glaxo Wellcome Medicines Research (Hertfordshire, UK). Adenosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-cAMP), was obtained from Biolog-Life Science Institute (La Jolla, CA). Alzet osmotic minipumps (model 2ML2) were supplied by the ALZA Corporation (Palo Alto, CA).
Animals The rabbit HO model of Michelsson et al. [39–41,44] was utilized after approval by an institutional animal care and use committee (IACUC). Numerous authors have reported that Michelsson's model reliably induces HO, which ultimately forms lamellar bone within 1 month [26,78,79]. Michelsson's rabbit model was chosen over the rat model proposed by Brown et al. [80] which produces limited bone and appears to have little to do with how heterotopic bone is induced in humans. The right leg of thirty-nine New Zealand rabbits was forcibly manipulated repeatedly from full extension to full flexion for 2 to 3 min daily then immobilized in a bivalved long-leg cast. Range of motion, stiffness, and the animal's response to manipulation were recorded. Groups of rabbits were sacrificed at 1, 2, 4, 7, 10, 14, or 21 days after the commencement of manipulation. Five or six rabbits were present in each group except for the 3week group (n = 3). For each time point, unmanipulated legs served as paired controls for the corresponding contralateral manipulated limbs for all subsequent biochemical analyses. The time periods chosen are consistent with previous findings that the definitive event which drives the system towards heterotopic bone formation occurs within the first 2 weeks [26].
Radiographic and analyses Plain and high-resolution (Faxitron) radiographs were obtained of both hindlimbs at the time of sacrifice to identify any calcification. Both manipulated and unmanipulated (control) legs were dissected and inspected, gross specimens obtained, and the vastus intermedius muscles removed, and prostaglandin levels assayed. All remaining tissues were stored in a −80°C refrigeration unit. Prostaglandins were extracted from the distal one-third of the vastus intermedius muscle, noted in preliminary experiments to be the area of maximal bone
formation. Muscles were ground using a Spex 6700 freezer mill (Spex Industries, Inc., Edison, NJ) then placed in a buffer and homogenized on ice by hand in a Tenbroeck tissue grinder. Aliquots were then taken for both prostaglandin and protein assays.
Administration of prostaglandin receptor and cyclic AMP antagonists Alzet osmotic minipumps were implanted subcutaneously in the lateral thigh of each manipulated limb to effect a local delivery of each pharmacological antagonist to the vastus intermedius. These osmotic pumps are small implantable drug reservoirs that deliver a controlled dose of medication continuously over a controlled period of time. The only surgical procedure for implantation involved the placement of the Alzet pumps in the subcutaneous tissue of the thigh, with a delivery tube inserted underneath or into the vastus intermedius muscle. Due to the short duration of the procedure, intubation and inhalation anesthetic was not required. Anesthesia with ketamine, acetylpromazine, and xylazine was sufficient. The leg was sterilely prepped and draped from the hip to the ankle. The lateral and anterior thigh was clipped. A lateral incision 2–3 cm in length was made, and the iliotibial band was divided in line with the incision. The tube from the Alzet pump was then placed underneath or into the vastus intermedius muscle, thus delivering the antagonists directly to the site of HO formation. The pump was then placed in the subcutaneous space of the lateral thigh. The subcutaneous tissue was then closed over the pump using dissolvable suture, and the skin was closed using 3/0 nylon suture. The hindlimb was then dressed in a sterile dressing. Drugs were delivered during the first 14 days of manipulation (at 1 ml/ week) at which point the pumps were surgically removed. Plain radiographs were obtained of the manipulated hindlimbs at 3, 4, 5, and 6 weeks. Rabbits were killed immediately before the 6-week radiograph was obtained. The endpoint measured was simply the formation of HO, which was measured radiographically. A dosage of 1 mg/week of either AH 6809 (an EP1, EP2, and DP receptor antagonist) [74–76] or Rp-cAMP (cAMP antagonist) [77] was delivered directly to the vastus intermedius (of 8 rabbits for each antagonist). At this dosage, it is expected that target concentrations of 3.5 μg/ml (10 μM) and 11 μg/ml (30 μM) were reached or exceeded for AH 6809 and Rp-cAMP respectively, within 1/2 to 2 h, respectively, at a constant delivery rate of 0.1 μg/ml/min. This is based on an average wet weight of 1 g and volume of extracellular fluid of 1 ml for each rabbit vastus intermedius. Moreover, since none of these drugs is effectively metabolized unless through hepatic detoxification (which should be minimal with local application at a site between the femur periosteum and the vastus intermedius), it is expected that concentrations which have been shown to antagonize PGE2 or cAMP were easily attainable. It has been shown that prostaglandin E1, which has effects and potency similar to PGE2 at the type EP2 receptor in bone cells [52], could be delivered locally to periosteal and subperiosteal sites via Alzet pumps to stimulate only local bone growth in vivo [48].
Prostaglandin analyses Prostaglandins were extracted following the method of Powell [81]. Ground muscle homogenate was mixed with 15% ethanol. After vortexing and centrifugation, the supernatants were acidified with 1 N HCL. A standard extraction process was then performed, utilizing SEP-PAK cartridges (Water Associates, Milford, MA). In a stepwise fashion, the fatty acids, leukotrienes, polar, and neutral lipids were eluted with ethanol and petroleum ether; methyl formate was then employed to elute prostaglandins. Methyl formate eluants were evaporated under a stream of liquid nitrogen at a bath temperature of not more than 60°C. After re-suspension in enzyme immunoassay (EIA) buffer, EIA kits (Cayman Chemical, Ann Arbor, MI) were used to determine the levels of prostaglandin E2, D2, 6-keto-PGF1, F2alpha, and thromboxane B2 in muscle tissue extracts at levels as low as 10 pg/ml. Protein assays were also performed using BioRad protein dye reagent (BioRad Laboratories, Hercules, CA) to determine prostaglandin concentrations per milligram of muscle protein. All chemicals used were of reagent grade and were obtained from Fisher Scientific (Springfield, NJ).
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Fig. 2. Time-dependent changes in muscle content of PGE2 during development of HO. PGE2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
Statistical analyses Paired Wilcoxon (data from manipulated limb were paired with that from contralateral limb for each animal) was performed. Only statistical differences associated with a Z value of 0.05 or less were reported as significant.
Results Prostaglandin assays Following the lipid extraction of vastus intermedius muscles removed from manipulated and control limbs after sacrifice, statistically significant increases were measured for all five prostaglandins assayed. Peak concentrations of each prostaglandin expressed in ng per mg protein were reached at different time points. The earliest and some of the most dramatic increases
in muscles from manipulated compared to control legs were observed for PGF2alpha (Fig. 1). Statistically elevated within 1 day of the first manipulation (Z = 0.008), PGF2alpha reached a peak that measured 185-fold (1.39 ± 0.6 ng/mg protein; mean ± SE) that of control at day ten (Z = 0.005) (Fig. 1). Although not statistically elevated above control levels until 48 h following the first manipulation, peak PGE2 levels (Fig. 2) were greater than those of PGF2alpha (Fig. 1). PGE2 levels were increased maximally to 33.4-fold that of control at day ten (Z = 0.005) (Fig. 2). These early changes in prostaglandins preceded the development of HO by several days since early calcification was observed on both plain and Faxitron radiographs by day 10 (data not shown). Less dramatic increases were observed for 6-keto-PGF1alpha and thromboxane B2; these levels did not rise significantly above control levels until 4 and 10 days, respectively, following the initial limb manipulation (Figs.
Fig. 3. Time-dependent changes in muscle content of thromboxane B2 during development of HO. Thromboxane B2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 5–6 rabbits for 1–14 days and 3 rabbits for 21 days.
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Fig. 4. Time-dependent changes in muscle content of 6-keto-PGF1alpha during development of HO. 6-keto-PGF1alpha levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 4–6 rabbits for 1–14 days and 3 rabbits for 21 days.
3 and 4). Statistically significant increases in PGD2 levels were not observed until 10 days following the initial manipulation (Fig. 5). At this time point, PGD2 levels (0.16 ± 0.22 ng/mg protein; mean ± SE) (Fig. 5) were 20-fold less than that of PGE2 (3.4 ± 4.3 ng/mg protein; mean ± SE) (Fig. 2). When an antagonist of either PGE2/PGD receptors or cAMP was administered in vivo via Alzet minipumps for 14 days following surgery (8 rabbits per drug group), heterotopic ossification failed to develop in all but 1 animal. After a 2-week period during which time drugs were completely delivered (demonstrated by size reduction of internal pumping chamber documented radiographically), no adverse effects of pump implantation were observed (even when pumps were left in for an additional several days after the complete release of drug) other than a moderate degree of scar tissue that had formed around the pump. Control (non-manipulated) rabbits (3 rabbits in this arm of the study) or rabbits receiving either AH 6809 or Rp-cAMP underwent a regimen of 3 weeks of daily hindlimb
manipulation and were either sacrificed at this point or sacrificed 3 weeks later. After sacrifice at 3 weeks, hematoma formation could be observed throughout the quadriceps musculature of manipulated limbs of control rabbits or rabbits administered AH 6809 or Rp-cAMP. After sacrifice at 3 or 6 weeks, the quadriceps in the manipulated limbs of rabbits in the control, AH 6809, or Rp-cAMP groups also appeared edematous, darker, thicker, and less elastic compared to quadriceps muscles in non-manipulated limbs. Faxitron radiographs of the femurs of manipulated hindlimbs showed calcification in the region of the vastus intermedius for all of the control rabbits (Fig. 6). In contrast, no calcification was observed at 3 weeks (Fig. 7) or 6 weeks (Fig. 8) for eight rabbits receiving AH 6809 or for seven rabbits receiving Rp-cAMP (Figs. 9 and 10, respectively). In one rabbit administered RpcAMP, small islands of mineralization were found in the vastus intermedius muscle at the time of sacrifice and gross examination of the dissected quadriceps 6 weeks after initiation
Fig. 5. Time-dependent changes in muscle content of PGD2 during development of HO. PGD2 levels were measured in the vastus intermedius muscles in unmanipulated and manipulated limbs at various times following commencement of manipulation as described in Fig. 1. Tests of statistical significance were performed, and results are presented as described in Fig. 1. (n) = 3–6.
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Fig. 6. Faxitron radiograph of femur of control rabbit (one of three rabbits) in drug antagonist arm of study after 3 weeks of daily hindlimb manipulation showing calcification in the region of the right vastus intermedius (left limb was not manipulated).
of the immobilization/passive exercise regimen. Nevertheless, both AH 6809 and Rp-cAMP were found to prevent the development of radiographically or macroscopically documented (at 3, 4, 5, and 6 weeks) heterotopic ossification in 15 out of 16 animals, thus identifying PGE2 or PGD as being obligate for the development of ectopic bone.
Discussion In this study, using a rabbit model of heterotopic ossification, we have demonstrated that multiple species of prostaglandins are elevated in soft tissue (quadriceps muscle) immediately prior to and during the development of HO in that tissue. To our
Fig. 7. Three-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the prostaglandin receptor antagonist AH 6809 and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
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Fig. 8. Six-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the prostaglandin receptor antagonist AH 6809 and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
knowledge, this is the first study to demonstrate that elevations in prostaglandin levels are present sequentially with HO and are temporally correlated with the process of heterotopic bone formation in a model of HO. Marked increases in PGE2 and PGF2alpha were observed within 24 to 48 h of the experimental induction of HO which preceded ossification by several days. Therefore, this suggests that, to be effective, prophylaxis with NSAIDs or prostaglandin receptor antagonists must begin almost immediately after the initial tissue injury in traumatic HO since prostaglandins probably become elevated within 24 h of this event.
PGE2 is likely to play a more important role in the development of HO in the rabbit model than the other prostaglandins studied for three reasons. Firstly, unlike PGE2, none of the other prostaglandins manifests direct anabolic effects in bone (see Table 1). Secondly, PGD2, 6-ketoPGF1alpha, and thromboxane B2 all manifested slower (requiring 4–10 days of manipulation) and less dramatic increases in their levels compared to PGE2. Thirdly, since early calcification was observed on both plain and Faxitron radiographs by day 10 (data not shown), the earliest elevations in prostaglandin synthesis are likely to be the most relevant to
Fig. 9. Three-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the cAMP antagonist RpcAMP and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
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Fig. 10. Six-week post-surgery Faxitron radiograph of right femurs of two different rabbits (A and B) following local administration of the cAMP antagonist Rp-cAMP and 3 weeks of daily hindlimb manipulation showing no calcification in the region of the vastus intermedius.
the mechanism of HO. Prostaglandin levels 7–10 days or more following the initial limb manipulation could also be irrelevant. In contrast to PGD2, 6-keto-PGF1alpha, and thromboxane B2, PGF2alpha levels were significantly increased within 24 h of the initial limb manipulation. A statistically significant increase was not observed for PGE2 until 24 h later. Therefore, PGF2alpha could indirectly promote HO by stimulating PGE2 synthesis, which has been previously demonstrated in bone and bone cell cultures [71–73,82,83]. Early elevations in PGE2 or PGF2alpha could also stimulate later increases in the synthesis of PGD2, 6-keto-PGF1alpha, and thromboxane B2, although the contributions of the latter three prostaglandins to the observed ossification are unclear. Since 6-keto-PGF1alpha and thromboxane B2 cause circulatory actions [84–86] which could amplify the degree of muscle trauma caused by limb manipulation, these prostaglandins might be able to indirectly stimulate ectopic bone formation. Nevertheless, findings that (a) thromboxane B2 was not significantly elevated above control until day 10 and that (b) the EP1, DP, and EP2 receptor antagonist AH 6809 [74–76] completely inhibited HO suggest that the contributions of either of 6-keto-PGF1alpha and thromboxane B2 to HO in this model are minimal. The ability of AH 6809 to block HO in this traumatic animal model implicates role(s) for EP and/or DP receptors in the pathophysiological mechanism. A number of reports have studied the potential involvement of EP1, EP2, and EP4 receptors in osteogenic mechanisms. The activation of EP2 receptors with selective pharmacological agonists has been shown to heal canine long bone segmental and fracture model defects [64,65], suggesting that EP2 is a major contributor to PGE2's local anabolic bone activity [64]. Another study using knockout mice for EP1 or EP2 receptors showed that EP2 receptors stimulate cAMP formation and have a major influence on the biomechanical properties of bone [54]. In contrast, EP1
receptors are coupled with Ca2+ mobilization, not cAMP formation, and have a minimal influence on skeletal strength or size in EP1 knockout mice [54]. Using such knockout models, EP4 receptors have been shown to play a role in bone resorption [64,87]. Other findings suggest that the activation of cAMP by both EP2 and EP4 receptors may mediate the effects of PGE2 on the differentiation of mesenchymal cells into chondrocytes [55]. It has been shown that an EP4 antagonist suppressed the increase in the formation of rat trabecular bone volume [66] and that an EP4 agonist enhanced BMP-2-induced ectopic bone formation in a mouse model [67], suggesting an anabolic role for EP4 in bone. However, another study showed that an EP4 agonist failed to increase the total bone volume of regenerating rat femoral bone but instead increased cortical bone healing by upregulating local bone turnover [88]. Other reports have shown that EP4 receptors increase bone mass and fracture healing [68], stimulate cortical bone formation [69], and regulate remodeling by increasing de novo bone formation [70]. Therefore, the studies discussed above have demonstrated roles for both EP4 and EP2 receptors in normal osteogenesis. Importantly, we have shown in this study that the compound AH 6809, an EP1, DP, and EP2 receptor antagonist [74–76], which has no demonstrated effects on EP4 receptors, completely blocked HO in the rabbit model. While it is certainly possible that EP4 receptors help regulate HO, our findings suggest that EP2 receptors are more directly involved in the mechanism of ectopic osteogenesis. The parallel findings that both the cAMP antagonist and the EP1, DP, and EP2 receptor antagonist AH 6809 [74–76] completely blocked HO formation suggest that EP2 receptors (which are coupled to cAMP formation [54,55]) play an important role in traumatic HO for the following three reasons. Firstly, neither EP1 receptors (which are not coupled to cAMP
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formation; [54]) nor DP receptors have been shown to regulate bone formation or skeletal development using receptor knockout models [54,63], whereas EP2 receptors have [54]. Secondly, we have shown that statistically significant increases in PGD2 levels were not observed until 10 days following the initial manipulation (Fig. 5). As discussed above, radiographic evidence of ectopic calcification had already been obtained by day 10. Thirdly, the levels of PGD2 at this time point were 20× lower (Fig. 5) than that of PGE2 (Fig. 2). Therefore, neither EP1 or DP receptors are likely to be as important as EP2 receptors for the development of traumatic HO. Nevertheless, potential roles of these two classes of receptors in the mechanism of HO cannot be ruled out due to their antagonism by AH 6809. In addition, the significance of PGF2alpha, PGF1alpha, and thromboxane B2 in the traumatic form of HO has yet to be elucidated. However, the findings in the current study together with those in the literature suggest that EP2 receptors are necessary, if not sufficient, for ectopic bone development following tissue injury. In summary, the findings in this study indicate an obligate role for prostaglandins in heterotopic ossification and also suggest that novel and safer therapies that target the receptors for prostaglandins, especially PGE2, may hold promise for the prophylactic therapy of HO. Conclusions Heterotopic bone formation is a significant problem, which challenges the orthopedic surgeon, particularly in the fields of trauma and arthroplasty. A greater understanding of the factors, which are involved in this process, and their mechanisms of action will improve both prevention and treatment. This may involve novel or synergistic combinations of anti-inflammatory drugs and/or adjuvant radiotherapy. However, in order to develop more effective and less toxic preventive and therapeutic regimens, the biochemical mediators of heterotopic bone formation must be identified. Our data provide preliminary support for the proposed inflammatory basis of heterotopic bone formation and the role of prostaglandins in that process. Prostaglandin levels are statistically elevated in a heterotopic ossification model early on its development. Moreover, pharmacological agents which interfere with the activation of specific cellular receptors for prostaglandins block the development of HO, suggesting that prostaglandins are essential to the pathophysiological mechanism. The evaluation and analysis of other possible mediators of heterotopic bone formation, including bone morphogenetic protein, cytokines, and other molecules, may be required in the future to truly understand the etiology of HO. However, the morbidity associated with standard prophylactic therapies can potentially be avoided or lessened through the clinical use of antagonists for prostaglandin receptors. Acknowledgments This research has been supported, in part, by a grant from the AO/ASIF foundation (AO/ASIF Research Project 96-H55/98H47). We gratefully acknowledge the advice and assistance of
Dr. Stephen Doty, Dr. Peter Torzilli, Dr. Corey Brayton, Dr. Adele Boskey, Darlene Grillo, Craig Klinger, and Rena Frantzis.
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