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Effect of tenoxicam on fracture healing in rat tibiae
Injury, Int. J. Care Injured 34 (2003) 85–94
Effect of tenoxicam on fracture healing in rat tibiae Vincenzo Giordano a,∗ , Marcos Giordano a , Irocy G. Knackfuss a , Mara Ibis R. Apfel b , Renato Das C. Gomes a a
Departamento de Ortopedia e Traumatologia, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil b Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcˆ antara Gomes, Centro Biomédico, Universidade Estadual do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Abstract Background: Nonsteroidal anti-inflammatory drugs have been implicated in the development of delayed unions and nonunion after fractures in animal models. Previous investigations have identified two important factors as determinants of delayed fracture healing: early drug administration and a dose-dependent effect. Objective: The purpose of this investigation was to study the effect of tenoxicam, a nonsteroidal anti-inflammatory drug, on the fracture healing process in rat tibiae. Methods: Fifty-eight Wistar rats were randomly divided in four groups (I, II, III, and IV). Group I (control group, n = 12) was given 0.1 ml saline solution per day intramuscularly. Groups II (n = 12), III (n = 12), and IV (n = 12) were administered 10 mg per kg per day of tenoxicam intramuscularly. Administration of substances was begun on a week before to 48 h after the fracturing procedure and continued during the entire experiment. Callus formation was studied histologically and histomorphologically, using light microscopy. In addition, a histologic grading based on the morphologic stage of fracture healing was carried out at 4 weeks, according to the criteria proposed by Allen et al. Results: There was a significant difference in treatment effect between Group I (saline solution) and Groups II, III, and IV (tenoxicam) (P = 0.07). Histologically and histomorphologically, there were qualitative and quantitative delay in callus formation at all tenoxicam groups. This was more pronounced the earlier the nonsteroidal anti-inflammatory drug was started, although no significant difference could be detected between Groups II, III, and IV (P > (α = 10%)). Four weeks after fracture, Group I (n = 3) showed complete osseous union, Groups II (n = 3) and III (n = 3), complete cartilaginous union, and Group IV (n = 3), incomplete osseous union, according to Allen et al. By using this rating scale, the difference between control and drug-treated groups was statistically significant (P < 0.1). Conclusion: Under studied conditions, this investigation shows that administration of tenoxicam intramuscularly delays fracture healing process in rat tibiae. These results suggest the hypothesis that early drug administration may delay bone healing after experimental fractures in animals, although it could not be detected statistically significant. © 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Bone is a highly specialised tissue of the skeletal system. The metabolic bone response after a fracture constitutes an ordered, well-differentiated sequence of events leading to the healing of the injured tissue in a manner very similar to its prior architecture [1–4]. However, multiple, interrelated factors can unfavourably affect this process, resulting in delayed union and nonunion [5–9]. In that context, studies performed in animals [10–22] suggest that use of nonsteroidal anti-inflammatory drugs (NSAID) delays osteogenesis post-fracture. In addition, NSAID have been shown to ∗ Corresponding author. Present address: Rua Aristides Esp´ınola 11/301, Leblon, Rio de Janeiro, RJ 22440-050, Brazil. E-mail address: [email protected] (V. Giordano).
prevent ectopic ossification after total hip arthroplasty and acetabular reconstruction in adult humans [23–27]. The NSAID are potent analgesic, antipyretic, and antiinflammatory substances, which decrease a great part of the inflammatory events elicited by any trauma [28,29]. The main effect of these drugs is the inhibition of prostaglandin G/H synthase, an important enzyme involved in prostaglandin synthesis [30]. It is hypothesised that inhibition of the biosynthesis of these autacoids could prevent the mechanisms associated with healing of injured tissues [17–30]. Although, the role of the prostaglandins (PG) on bone formation is not yet completely defined, many authors stress its function of bone resorption [31,32]. Keller et al. [15] demonstrated that during bone healing process osteoclasts and osteoblasts are intimately linked. Those authors suggested that resorption of fracture edges probably enhances
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pluripotential cells differentiation, ensuring rapid restoration of skeletal function. Dekel et al. [33] found high concentrations of PG on muscles surrounding tibia fractures in rabbits. Thus, they reinforced the importance of this class of compounds upon the mechanism of skeletal repair. Subsequent investigations on bone healing process in animals showed that the administration of different NSAID was associated with impaired healing, supporting the contention that PG are directly related to bone repair. Despite several studies conducted in animals to determine the role of NSAID on fracture repair, we believe that no one presented a uniform design of research. For example, the beginning and duration of drug administration, and the time of animals’ sacrifice were usually not given appropriate importance. Furthermore, indomethacin was largely studied, however, to our knowledge, such a study has not been performed using tenoxicam. Although, the NSAID mechanism of action seems to be basically the same (inhibition of PG biosynthesis), it has been shown repeatedly that there is a strong correlation between (1) drug pH and tissue resorption, and (2) PK/PD relationship and inflammatory response [28]. In the current investigation we studied the effect of tenoxicam on fracture healing process in rat tibiae. 2. Material and methods 2.1. Animal preparation Fifty-eight male Wistar rats (Rattus norvegicus albinus), with median body weight of 100 g, were used in the experiment. Animals were randomly divided in four groups. A fracture in the middle third of the left tibia was produced manually in every rat under ether anaesthesia by a three-point bending technique [6,7]. The fractures were not stabilised and the animals were housed in appropriate cages. Unprotected weight bearing was allowed as soon as the animals recovered from anaesthesia. Tap water and rodent chow were provided ad libitum. Group I (n = 12) was given 0.1 ml saline solution per day intramuscularly and served as control. Groups II (n = 12), III (n = 12), and IV (n = 12) were administered 10 mg per kg per day of tenoxicam 20 mg intramuscularly (0.1 ml per day). Administration of the substances was initiated from a week before to 48 h after the fracturing procedure and was continued during the entire experiment. Groups I and III were begun the substances the day of the fractures. Group II was begun the nonsteroidal anti-inflammatory drug 7 days before the fractures. Group IV was begun the nonsteroidal anti-inflammatory drug 2 days after the fractures. Three animals of each group were sacrificed at 3 days and at the end of the first, second, and fourth weeks post-fracture. The rats were anaesthetised with ether and sacrificed by cervical dislocation. This method has been recommended by the European convention for the protection of vertebrate
animals used for experimental and other scientific purposes [35]. 2.2. Tissue preparation The fractured extremities were knees disarticulated. The callus tissues were dissected free from the soft tissues, immediately collected, and fixed in 10% paraformaldehyde 10% for 5 days. Histological analysis was performed using light microscopy. After fixation, the fragments were decalcified in 5% nitric acid for 5 days, dehydrated in a graded ethanol series, and embedded in paraffin. Five micrometer sections were prepared, placed on slides, and stained with hematoxylin and eosin, picromallory, and alcian blue pH 2.5. This method has been described by Bancroft and Cook [36]. Histomorphological analysis was performed at the end of the study protocol (4 weeks post-fracture). Three rats of each groups had the histologic sections examined in random sequence without knowledge of the treatment regimen and the degree of fracture repair was determined using a five point scale proposed by Allen et al. [10]. According to this score, a grade four is applied if there is a complete bony union, a grade three represents an incomplete bony union (presence of a small amount of cartilage in the callus), a grade two represents a complete cartilaginous union (well-formed plate of hyaline cartilage uniting the fragments), a grade one represents an incomplete cartilaginous union (retention of fibrous elements in the cartilaginous plate), and a grade zero indicates the formation of a pseudoarthrosis (most severe form of arrest in fracture repair). 2.3. Drug The drug used in this experiment was tenoxicam 20 mg intramuscular. This nonsteroidal anti-inflammatory drug is an oxicam derivative, a class of enolitic acids [37]. Tenoxicam is about 99% protein-bound in plasma, achieving peak concentrations about 20 h after administration [38]. 2.4. Statistics The histomorphological analysis based on bone fracture repair scores described by Allen et al. [10] allowed a statistical analysis for overall comparison among the four groups experimentally designed (Kruskall–Wallis test), with α = 0.1 level of significance. Comparison between control and drug-treated groups was done using the Mann–Whitney test, with a level of significance α = 1% [39]. Although, the sample size for the histomorphological comparisons appears small (n = 3), a retrospective power analysis demonstrates that the P values obtained with a level of significance α = 10% are valid with a power of greater than 80% (β < 0.2).
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Fig. 1. Photomicrograph of 3-day-old fracture of the tibia in a rat of Group I (control). (A) Fractured bony ends surrounded by a very abundant fibroblastic tissue with small areas of chondrogenesis and newly formed bone; areas of myositis and haemorrhage were observed. Original magnification 20×, Picro-Mallory staining. (B) Groups of red cells coloured in orange between plasma and fragments of muscle tissue. Original magnification 400×, Picro-Mallory staining. (C) Old bone with some osteocytes surrounded by extra-cellular matrix showing the cement line and a large number of osteoblasts synthesising new bone. Original magnification 400×, H&E staining.
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Fig. 2. Photomicrograph of 1-week-old fracture of the tibia in a rat of Group I (control). Large fibrocartilaginous callus with intense proliferation of chondroblasts and chondrocytes surrounding one of the fracture extremities. Note newly formed bone on the left upper corner of the picture. Original magnification 100×, H&E staining.
3. Results 3.1. Histological sections Fracture healing was assessed histologically in 48 rats. By gross observation, the bone ends were typically apposed resulting in a shortened, bayoneted position of the bone. These findings, however, did not represent technical problems during the histological sections.
At day 3, histological analysis evidenced similar aspects in all groups. Bony edges showed osteocytes surrounded by matrix with blue lines. Areas of chondrogenesis were beginning in a very abundant fibroblastic tissue (Fig. 1). Small fragments of muscle tissue and areas of myositis, and areas of localised haemorrhage were observed. By 1 week post-fracture, Group I demonstrated a very cellular fibrocartilaginous tissue with a small amount of collagen fibres between the bone ends (Fig. 2), Groups II and
Fig. 3. Photomicrograph of 1-week-old fracture of the tibia in a rat of Group IV (treated with tenoxicam 10 mg per kg per day). Endochondral and intramembranous newly formed primary bone with small areas of hyaline cartilage (chondrogenesis en foci) surrounded by a thick periosteum laterally to one of the body extremities. Original magnification 100×, Picro-Mallory staining.
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III presented a predominantly fibrous callus with few chondrocytes on each side of the fracture line, and Group IV demonstrated a fibrocartilaginous tissue with areas of hyaline cartilage cells between the bone ends (chondrogenesis en foci) (Fig. 3). By 2 weeks post-fracture, Group I showed extensive areas of endochondral and intramembranous bone formation in the callus, with a small amount of fibrocartilage between the bone ends. Bone union was detectable at the periphery of the callus (Fig. 4). By this time, Groups II and III evidenced a predominantly cartilaginous callus between the bone ends and a very thick periosteum at the periphery (Fig. 5). Group IV demonstrated areas of endochondral and intramembra-
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nous ossification surrounded by fibrocartilaginous tissue in the fracture callus. These observations indicated that a subset of cells at the fracture site had begun to differentiate along an osteogenic lineage in Groups I and IV (more evident in control animals) but not in Groups II and III. By 4 weeks post-fracture, Group I evidenced complete fracture site healing, with irregularly formed trabeculae of woven bone and no signs of remodelling (Fig. 6). By this stage, Groups II and III demonstrated an extensive zone of fibrous tissue and fibrocartilage remaining at the centre of the callus (Fig. 7). On an adjacent tissue section, it was noted that the superficial layer of periosteal callus was ossified, although, the fracture was not bridged. Group IV showed
Fig. 4. Photomicrograph of 2-week-old fracture of the tibia in a rat of Group I (control). (A) Endochondral and intramembranous newly formed primary bone with fragments of cartilage in the connective tissue remaining between the bone ends. Note the thick periosteum. Original magnification 40×, Picro-Mallory staining. (B) Bone union was detectable at the periphery of the callus. Original magnification 100×, Picro-Mallory staining.
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Fig. 5. Photomicrograph of 2-week-old fracture of the tibia in a rat of Group III (treated with tenoxicam 10 mg per kg per day). Small area of primary new bone in the fracture line. A large band of fibrocartilaginous callus is still observed between the extremities of the fractured bone. Note the very thin periosteum at the periphery. Original magnification 40×, H&E staining.
different behaviour among the animals. Two rats demonstrated some fibrocartilage present between an abundant osteoid tissue in the callus, although at the periphery bony union was complete (Fig. 8). In one animal there was a very large amount of fibrous-connective tissue and woven bone present between the fractured ends, suggesting the aspect of a delayed bone healing process. 3.2. Histomorphological sections Four weeks post-fracture, the histomorphological analysis was the following (according to Allen et al. [10]): Group Group Group Group
I: grade four (n = 3); II: grade two (n = 3); III: grade two (n = 3); IV: grade three (n = 3) and grade one (n = 1).
3.3. Statistics By using the Kruskall–Wallis test, since P = 0.07 (α = 0.1), there was a statistically significant difference among the results of the four groups. When the Mann–Whitney test was used to compare the differences between Group I and Groups II, III, and IV, there were statistically significant differences in all of the samples (P ≤ (α = 10%)). Thus, the null hypothesis H0 was rejected: there is a statistically significant delay on fracture healing process in rat tibiae at all drug-treated groups compared to those animals at control group. However, no significant difference could be detected between groups II and III, II and IV, and III and IV (P > 0.1).
4. Discussion NSAID administered to different animals with experimental fracture delay the bone healing process [10–22]. In addition, similar effect was attributed to these drugs in the prevention of heterotopic ossification after total hip arthroplasty and acetabular reconstruction in humans [23–27]. This may be partly explained by the fact that callus formation occurs in a manner very similar to heterotopic bone formation, both processes beginning with an inflammatory reaction which is followed by bone formation [22]. Although, the mechanism of action of the NSAID on osteogenesis remains uncertain, Altman et al. [11] observed that the time between fracture and drug administration, and the frequency of drug administration may influence bone formation. Furthermore, Törnkvist et al. [22] contend that once endochondral ossification takes place histogenesis becomes unlikely to exogenous factors, as NSAID. It means that bone healing possibly can be impaired during osteoprogenitor cells differentiation. Both pH and PK/PD relationship of NSAID could be involved during the inflammatory response phase. It is very well established in the literature the inhibitory effects of NSAID on exsudate PG, serum thromboxane, leukotriene B4 (LTB4), and platelet activating factor (PAF) [28,34,41,42]. Tool and Verhoeven [42] observed a marked inhibitory effect on PAF synthesis and LTB4 production after nimesulide administration. As a matter of fact, swelling and acute inflammation are reduced after NSAID administration. This leads to a substantial reduction in bone resorption and may be one reason for the decrease of cellular differentiation [12,15,21,24,27,34,40–42]. Although, it was not the purpose of the current study to investigate the factors
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Fig. 6. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group I (control). (A) Endochondral and intramembranous newly formed bone bridging the fracture line. Original magnification 100×, H&E staining. (B) Periphery of the fracture gap with abundant callus between one of the fractured extremities and the periosteum. Original magnification 100×, H&E staining.
affecting tenoxicam pharmacokinetic and pharmacodynamic parameters, we demonstrated that tenoxicam delays fracture healing in rat tibiae. Bone union appeared in control group after 4 weeks post-fracture, however, delayed union occurred in all tenoxicam-treated animals at this time. Statistical analysis based on histomorphological score [10] showed a significant difference among the results of the four studied groups (P = 0.07). When we compared the results between control and drug-treated groups, there were significant differences in all of the matched samples (P < (α = 0.1%)). Considering tenoxicam-treated animals, we observed that the histological and histomorphological analysis showed a slight difference among the groups. However, despite these findings no statistically significant difference could be
demonstrated between groups II and III, II–IV, and III and IV by using the Mann–Whitney test (P > (α = 10%)). The relation between NSAID and delayed bone union is supported by other animal experiments. However, to our knowledge, such a study has not been performed using tenoxicam. In addition, we believe that no one presented a reliable experimental design. We paid particular attention to this fact. In this study we used equivalent groups achieved by randomisation. Stratified randomisation assures unbiased assignment of experimental subjects to the groups, assuring the initial equivalence of such groups [43]. We created three tenoxicam-treated groups, differing from the beginning of the drug administration. Insel [29] observed that serum concentrations bigger than 5–6 g/ml of piroxicam (another
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Fig. 7. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group II (treated with tenoxicam 10 mg per kg per day). (A) Extensive zone of fibrocartilaginous callus with vessels resulting in a complete cartilaginous bridge across the fracture line. Small areas of new bone. Original magnification 40×, Picro-Mallory staining. (B) Callus containing mainly chondrocytes bridging bony ends. Original magnification 100×, H&E staining.
oxicam derivative) give a satisfactory anti-inflammatory response; if we can extrapolate this data to tenoxicam, these serum levels are expected after a mean of 7–10 days of continuous drug administration [37]. Nonsteroidal anti-inflammatory doses were calculated based on study of Strub et al. [44]. These authors demonstrated that 10 mg/kg of tenoxicam reduce effectively the acute edema induced by kaolin injection in rats. The animals were killed in accordance to previous study of Udupa and Prasad [45], except for the remodelling phase. Although, Sudmann and Bang [20] have shown that indomethacin significantly inhibited haversian canal remodelling in rabbits, several preliminary studies have suggested no effect in bone remodelling associated with NSAID [16,27]. Otherwise, we preferred to
observe callus formation by 3 days post-fracture. Several authors who have shown maximal mitotic activity and excellent vascular response to fracture at this time support this [46–48]. Keller et al. [16] demonstrated almost complete inhibition of PG after 3 days of treatment with indomethacin, however, they were unable to show any effect of this drug during bone remodelling. Finally, the results were analysed qualitatively, quantitatively, and statistically. One of the weaknesses in our study was the small number of specimens. However, we feel that the use of randomisation in such a small population helps to achieve a balanced representation. Furthermore, because the differences between control and drug-treated groups were so great, we were able to achieve statistical significance with a small number of
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Fig. 8. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group IV (treated with tenoxicam 10 mg per kg per day). Bone callus with small zones of cartilage bridging the bony extremities. Original magnification 100×, H&E staining.
animals. The probability of a type-II error was retrospectively quantified and an adequate power was detected for the samples (β < 0.2). Other weaknesses included the absence of radiographic and mechanical investigation of the callus. Most authors agree that mechanical studies are the best way to evaluate bone healing process, however, we could not perform these studies for economical reasons. On the other hand, we decided to not use simple radiographic study because it appears to be inaccurate for evaluating fracture healing, as previously demonstrated in the study of Giordano [6]. Unexpected finding occurred in one animal of Group IV by 4 weeks post-fracture. A small zone of bone formation had formed, suggesting an extremely delayed bone repair. Excessive movement has been associated with the formation of a cartilage callus [49]. It is possible that our nonstabilised tibial fracture rat model could be a potential cause of delayed callus formation and healing. The molecular aspects of healing in stabilised and nonstabilised fractures in a mouse model have been recently studied by Le et al. [50]. In a very elegant experiment these authors demonstrated that mechanical influences regulate cartilage and bone formation during fracture repair. However, they did not observe that stabilised fracture calluses matured faster than nonstabilised fracture calluses based on histological and molecular analyses. By 28 days post-fracture all animals showed the fracture site completely healed [50]. Therefore, we could not definitively explain this observation in the animal of Group IV. Animal constitutional characteristics, like malnutrition or chronic illnesses, which may impair the process of bone healing [8], although, these possibilities remain purely speculative. In summary, the data presented herein demonstrate that tenoxicam administered intramuscularly for 4 weeks to Wistar rats significantly delays the bone healing process after
experimental diaphyseal tibia fracture. These findings suggest the hypothesis that early drug administration may delay bone healing, although, it could not be detected statistically significant.
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Effect of tenoxicam on fracture healing in rat tibiae Vincenzo Giordano a,∗ , Marcos Giordano a , Irocy G. Knackfuss a , Mara Ibis R. Apfel b , Renato Das C. Gomes a a
Departamento de Ortopedia e Traumatologia, Faculdade de Medicina, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil b Departamento de Histologia e Embriologia, Instituto de Biologia Roberto Alcˆ antara Gomes, Centro Biomédico, Universidade Estadual do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Abstract Background: Nonsteroidal anti-inflammatory drugs have been implicated in the development of delayed unions and nonunion after fractures in animal models. Previous investigations have identified two important factors as determinants of delayed fracture healing: early drug administration and a dose-dependent effect. Objective: The purpose of this investigation was to study the effect of tenoxicam, a nonsteroidal anti-inflammatory drug, on the fracture healing process in rat tibiae. Methods: Fifty-eight Wistar rats were randomly divided in four groups (I, II, III, and IV). Group I (control group, n = 12) was given 0.1 ml saline solution per day intramuscularly. Groups II (n = 12), III (n = 12), and IV (n = 12) were administered 10 mg per kg per day of tenoxicam intramuscularly. Administration of substances was begun on a week before to 48 h after the fracturing procedure and continued during the entire experiment. Callus formation was studied histologically and histomorphologically, using light microscopy. In addition, a histologic grading based on the morphologic stage of fracture healing was carried out at 4 weeks, according to the criteria proposed by Allen et al. Results: There was a significant difference in treatment effect between Group I (saline solution) and Groups II, III, and IV (tenoxicam) (P = 0.07). Histologically and histomorphologically, there were qualitative and quantitative delay in callus formation at all tenoxicam groups. This was more pronounced the earlier the nonsteroidal anti-inflammatory drug was started, although no significant difference could be detected between Groups II, III, and IV (P > (α = 10%)). Four weeks after fracture, Group I (n = 3) showed complete osseous union, Groups II (n = 3) and III (n = 3), complete cartilaginous union, and Group IV (n = 3), incomplete osseous union, according to Allen et al. By using this rating scale, the difference between control and drug-treated groups was statistically significant (P < 0.1). Conclusion: Under studied conditions, this investigation shows that administration of tenoxicam intramuscularly delays fracture healing process in rat tibiae. These results suggest the hypothesis that early drug administration may delay bone healing after experimental fractures in animals, although it could not be detected statistically significant. © 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction Bone is a highly specialised tissue of the skeletal system. The metabolic bone response after a fracture constitutes an ordered, well-differentiated sequence of events leading to the healing of the injured tissue in a manner very similar to its prior architecture [1–4]. However, multiple, interrelated factors can unfavourably affect this process, resulting in delayed union and nonunion [5–9]. In that context, studies performed in animals [10–22] suggest that use of nonsteroidal anti-inflammatory drugs (NSAID) delays osteogenesis post-fracture. In addition, NSAID have been shown to ∗ Corresponding author. Present address: Rua Aristides Esp´ınola 11/301, Leblon, Rio de Janeiro, RJ 22440-050, Brazil. E-mail address: [email protected] (V. Giordano).
prevent ectopic ossification after total hip arthroplasty and acetabular reconstruction in adult humans [23–27]. The NSAID are potent analgesic, antipyretic, and antiinflammatory substances, which decrease a great part of the inflammatory events elicited by any trauma [28,29]. The main effect of these drugs is the inhibition of prostaglandin G/H synthase, an important enzyme involved in prostaglandin synthesis [30]. It is hypothesised that inhibition of the biosynthesis of these autacoids could prevent the mechanisms associated with healing of injured tissues [17–30]. Although, the role of the prostaglandins (PG) on bone formation is not yet completely defined, many authors stress its function of bone resorption [31,32]. Keller et al. [15] demonstrated that during bone healing process osteoclasts and osteoblasts are intimately linked. Those authors suggested that resorption of fracture edges probably enhances
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pluripotential cells differentiation, ensuring rapid restoration of skeletal function. Dekel et al. [33] found high concentrations of PG on muscles surrounding tibia fractures in rabbits. Thus, they reinforced the importance of this class of compounds upon the mechanism of skeletal repair. Subsequent investigations on bone healing process in animals showed that the administration of different NSAID was associated with impaired healing, supporting the contention that PG are directly related to bone repair. Despite several studies conducted in animals to determine the role of NSAID on fracture repair, we believe that no one presented a uniform design of research. For example, the beginning and duration of drug administration, and the time of animals’ sacrifice were usually not given appropriate importance. Furthermore, indomethacin was largely studied, however, to our knowledge, such a study has not been performed using tenoxicam. Although, the NSAID mechanism of action seems to be basically the same (inhibition of PG biosynthesis), it has been shown repeatedly that there is a strong correlation between (1) drug pH and tissue resorption, and (2) PK/PD relationship and inflammatory response [28]. In the current investigation we studied the effect of tenoxicam on fracture healing process in rat tibiae. 2. Material and methods 2.1. Animal preparation Fifty-eight male Wistar rats (Rattus norvegicus albinus), with median body weight of 100 g, were used in the experiment. Animals were randomly divided in four groups. A fracture in the middle third of the left tibia was produced manually in every rat under ether anaesthesia by a three-point bending technique [6,7]. The fractures were not stabilised and the animals were housed in appropriate cages. Unprotected weight bearing was allowed as soon as the animals recovered from anaesthesia. Tap water and rodent chow were provided ad libitum. Group I (n = 12) was given 0.1 ml saline solution per day intramuscularly and served as control. Groups II (n = 12), III (n = 12), and IV (n = 12) were administered 10 mg per kg per day of tenoxicam 20 mg intramuscularly (0.1 ml per day). Administration of the substances was initiated from a week before to 48 h after the fracturing procedure and was continued during the entire experiment. Groups I and III were begun the substances the day of the fractures. Group II was begun the nonsteroidal anti-inflammatory drug 7 days before the fractures. Group IV was begun the nonsteroidal anti-inflammatory drug 2 days after the fractures. Three animals of each group were sacrificed at 3 days and at the end of the first, second, and fourth weeks post-fracture. The rats were anaesthetised with ether and sacrificed by cervical dislocation. This method has been recommended by the European convention for the protection of vertebrate
animals used for experimental and other scientific purposes [35]. 2.2. Tissue preparation The fractured extremities were knees disarticulated. The callus tissues were dissected free from the soft tissues, immediately collected, and fixed in 10% paraformaldehyde 10% for 5 days. Histological analysis was performed using light microscopy. After fixation, the fragments were decalcified in 5% nitric acid for 5 days, dehydrated in a graded ethanol series, and embedded in paraffin. Five micrometer sections were prepared, placed on slides, and stained with hematoxylin and eosin, picromallory, and alcian blue pH 2.5. This method has been described by Bancroft and Cook [36]. Histomorphological analysis was performed at the end of the study protocol (4 weeks post-fracture). Three rats of each groups had the histologic sections examined in random sequence without knowledge of the treatment regimen and the degree of fracture repair was determined using a five point scale proposed by Allen et al. [10]. According to this score, a grade four is applied if there is a complete bony union, a grade three represents an incomplete bony union (presence of a small amount of cartilage in the callus), a grade two represents a complete cartilaginous union (well-formed plate of hyaline cartilage uniting the fragments), a grade one represents an incomplete cartilaginous union (retention of fibrous elements in the cartilaginous plate), and a grade zero indicates the formation of a pseudoarthrosis (most severe form of arrest in fracture repair). 2.3. Drug The drug used in this experiment was tenoxicam 20 mg intramuscular. This nonsteroidal anti-inflammatory drug is an oxicam derivative, a class of enolitic acids [37]. Tenoxicam is about 99% protein-bound in plasma, achieving peak concentrations about 20 h after administration [38]. 2.4. Statistics The histomorphological analysis based on bone fracture repair scores described by Allen et al. [10] allowed a statistical analysis for overall comparison among the four groups experimentally designed (Kruskall–Wallis test), with α = 0.1 level of significance. Comparison between control and drug-treated groups was done using the Mann–Whitney test, with a level of significance α = 1% [39]. Although, the sample size for the histomorphological comparisons appears small (n = 3), a retrospective power analysis demonstrates that the P values obtained with a level of significance α = 10% are valid with a power of greater than 80% (β < 0.2).
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Fig. 1. Photomicrograph of 3-day-old fracture of the tibia in a rat of Group I (control). (A) Fractured bony ends surrounded by a very abundant fibroblastic tissue with small areas of chondrogenesis and newly formed bone; areas of myositis and haemorrhage were observed. Original magnification 20×, Picro-Mallory staining. (B) Groups of red cells coloured in orange between plasma and fragments of muscle tissue. Original magnification 400×, Picro-Mallory staining. (C) Old bone with some osteocytes surrounded by extra-cellular matrix showing the cement line and a large number of osteoblasts synthesising new bone. Original magnification 400×, H&E staining.
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Fig. 2. Photomicrograph of 1-week-old fracture of the tibia in a rat of Group I (control). Large fibrocartilaginous callus with intense proliferation of chondroblasts and chondrocytes surrounding one of the fracture extremities. Note newly formed bone on the left upper corner of the picture. Original magnification 100×, H&E staining.
3. Results 3.1. Histological sections Fracture healing was assessed histologically in 48 rats. By gross observation, the bone ends were typically apposed resulting in a shortened, bayoneted position of the bone. These findings, however, did not represent technical problems during the histological sections.
At day 3, histological analysis evidenced similar aspects in all groups. Bony edges showed osteocytes surrounded by matrix with blue lines. Areas of chondrogenesis were beginning in a very abundant fibroblastic tissue (Fig. 1). Small fragments of muscle tissue and areas of myositis, and areas of localised haemorrhage were observed. By 1 week post-fracture, Group I demonstrated a very cellular fibrocartilaginous tissue with a small amount of collagen fibres between the bone ends (Fig. 2), Groups II and
Fig. 3. Photomicrograph of 1-week-old fracture of the tibia in a rat of Group IV (treated with tenoxicam 10 mg per kg per day). Endochondral and intramembranous newly formed primary bone with small areas of hyaline cartilage (chondrogenesis en foci) surrounded by a thick periosteum laterally to one of the body extremities. Original magnification 100×, Picro-Mallory staining.
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III presented a predominantly fibrous callus with few chondrocytes on each side of the fracture line, and Group IV demonstrated a fibrocartilaginous tissue with areas of hyaline cartilage cells between the bone ends (chondrogenesis en foci) (Fig. 3). By 2 weeks post-fracture, Group I showed extensive areas of endochondral and intramembranous bone formation in the callus, with a small amount of fibrocartilage between the bone ends. Bone union was detectable at the periphery of the callus (Fig. 4). By this time, Groups II and III evidenced a predominantly cartilaginous callus between the bone ends and a very thick periosteum at the periphery (Fig. 5). Group IV demonstrated areas of endochondral and intramembra-
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nous ossification surrounded by fibrocartilaginous tissue in the fracture callus. These observations indicated that a subset of cells at the fracture site had begun to differentiate along an osteogenic lineage in Groups I and IV (more evident in control animals) but not in Groups II and III. By 4 weeks post-fracture, Group I evidenced complete fracture site healing, with irregularly formed trabeculae of woven bone and no signs of remodelling (Fig. 6). By this stage, Groups II and III demonstrated an extensive zone of fibrous tissue and fibrocartilage remaining at the centre of the callus (Fig. 7). On an adjacent tissue section, it was noted that the superficial layer of periosteal callus was ossified, although, the fracture was not bridged. Group IV showed
Fig. 4. Photomicrograph of 2-week-old fracture of the tibia in a rat of Group I (control). (A) Endochondral and intramembranous newly formed primary bone with fragments of cartilage in the connective tissue remaining between the bone ends. Note the thick periosteum. Original magnification 40×, Picro-Mallory staining. (B) Bone union was detectable at the periphery of the callus. Original magnification 100×, Picro-Mallory staining.
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Fig. 5. Photomicrograph of 2-week-old fracture of the tibia in a rat of Group III (treated with tenoxicam 10 mg per kg per day). Small area of primary new bone in the fracture line. A large band of fibrocartilaginous callus is still observed between the extremities of the fractured bone. Note the very thin periosteum at the periphery. Original magnification 40×, H&E staining.
different behaviour among the animals. Two rats demonstrated some fibrocartilage present between an abundant osteoid tissue in the callus, although at the periphery bony union was complete (Fig. 8). In one animal there was a very large amount of fibrous-connective tissue and woven bone present between the fractured ends, suggesting the aspect of a delayed bone healing process. 3.2. Histomorphological sections Four weeks post-fracture, the histomorphological analysis was the following (according to Allen et al. [10]): Group Group Group Group
I: grade four (n = 3); II: grade two (n = 3); III: grade two (n = 3); IV: grade three (n = 3) and grade one (n = 1).
3.3. Statistics By using the Kruskall–Wallis test, since P = 0.07 (α = 0.1), there was a statistically significant difference among the results of the four groups. When the Mann–Whitney test was used to compare the differences between Group I and Groups II, III, and IV, there were statistically significant differences in all of the samples (P ≤ (α = 10%)). Thus, the null hypothesis H0 was rejected: there is a statistically significant delay on fracture healing process in rat tibiae at all drug-treated groups compared to those animals at control group. However, no significant difference could be detected between groups II and III, II and IV, and III and IV (P > 0.1).
4. Discussion NSAID administered to different animals with experimental fracture delay the bone healing process [10–22]. In addition, similar effect was attributed to these drugs in the prevention of heterotopic ossification after total hip arthroplasty and acetabular reconstruction in humans [23–27]. This may be partly explained by the fact that callus formation occurs in a manner very similar to heterotopic bone formation, both processes beginning with an inflammatory reaction which is followed by bone formation [22]. Although, the mechanism of action of the NSAID on osteogenesis remains uncertain, Altman et al. [11] observed that the time between fracture and drug administration, and the frequency of drug administration may influence bone formation. Furthermore, Törnkvist et al. [22] contend that once endochondral ossification takes place histogenesis becomes unlikely to exogenous factors, as NSAID. It means that bone healing possibly can be impaired during osteoprogenitor cells differentiation. Both pH and PK/PD relationship of NSAID could be involved during the inflammatory response phase. It is very well established in the literature the inhibitory effects of NSAID on exsudate PG, serum thromboxane, leukotriene B4 (LTB4), and platelet activating factor (PAF) [28,34,41,42]. Tool and Verhoeven [42] observed a marked inhibitory effect on PAF synthesis and LTB4 production after nimesulide administration. As a matter of fact, swelling and acute inflammation are reduced after NSAID administration. This leads to a substantial reduction in bone resorption and may be one reason for the decrease of cellular differentiation [12,15,21,24,27,34,40–42]. Although, it was not the purpose of the current study to investigate the factors
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Fig. 6. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group I (control). (A) Endochondral and intramembranous newly formed bone bridging the fracture line. Original magnification 100×, H&E staining. (B) Periphery of the fracture gap with abundant callus between one of the fractured extremities and the periosteum. Original magnification 100×, H&E staining.
affecting tenoxicam pharmacokinetic and pharmacodynamic parameters, we demonstrated that tenoxicam delays fracture healing in rat tibiae. Bone union appeared in control group after 4 weeks post-fracture, however, delayed union occurred in all tenoxicam-treated animals at this time. Statistical analysis based on histomorphological score [10] showed a significant difference among the results of the four studied groups (P = 0.07). When we compared the results between control and drug-treated groups, there were significant differences in all of the matched samples (P < (α = 0.1%)). Considering tenoxicam-treated animals, we observed that the histological and histomorphological analysis showed a slight difference among the groups. However, despite these findings no statistically significant difference could be
demonstrated between groups II and III, II–IV, and III and IV by using the Mann–Whitney test (P > (α = 10%)). The relation between NSAID and delayed bone union is supported by other animal experiments. However, to our knowledge, such a study has not been performed using tenoxicam. In addition, we believe that no one presented a reliable experimental design. We paid particular attention to this fact. In this study we used equivalent groups achieved by randomisation. Stratified randomisation assures unbiased assignment of experimental subjects to the groups, assuring the initial equivalence of such groups [43]. We created three tenoxicam-treated groups, differing from the beginning of the drug administration. Insel [29] observed that serum concentrations bigger than 5–6 g/ml of piroxicam (another
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Fig. 7. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group II (treated with tenoxicam 10 mg per kg per day). (A) Extensive zone of fibrocartilaginous callus with vessels resulting in a complete cartilaginous bridge across the fracture line. Small areas of new bone. Original magnification 40×, Picro-Mallory staining. (B) Callus containing mainly chondrocytes bridging bony ends. Original magnification 100×, H&E staining.
oxicam derivative) give a satisfactory anti-inflammatory response; if we can extrapolate this data to tenoxicam, these serum levels are expected after a mean of 7–10 days of continuous drug administration [37]. Nonsteroidal anti-inflammatory doses were calculated based on study of Strub et al. [44]. These authors demonstrated that 10 mg/kg of tenoxicam reduce effectively the acute edema induced by kaolin injection in rats. The animals were killed in accordance to previous study of Udupa and Prasad [45], except for the remodelling phase. Although, Sudmann and Bang [20] have shown that indomethacin significantly inhibited haversian canal remodelling in rabbits, several preliminary studies have suggested no effect in bone remodelling associated with NSAID [16,27]. Otherwise, we preferred to
observe callus formation by 3 days post-fracture. Several authors who have shown maximal mitotic activity and excellent vascular response to fracture at this time support this [46–48]. Keller et al. [16] demonstrated almost complete inhibition of PG after 3 days of treatment with indomethacin, however, they were unable to show any effect of this drug during bone remodelling. Finally, the results were analysed qualitatively, quantitatively, and statistically. One of the weaknesses in our study was the small number of specimens. However, we feel that the use of randomisation in such a small population helps to achieve a balanced representation. Furthermore, because the differences between control and drug-treated groups were so great, we were able to achieve statistical significance with a small number of
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Fig. 8. Photomicrograph of 4-week-old fracture of the tibia in a rat of Group IV (treated with tenoxicam 10 mg per kg per day). Bone callus with small zones of cartilage bridging the bony extremities. Original magnification 100×, H&E staining.
animals. The probability of a type-II error was retrospectively quantified and an adequate power was detected for the samples (β < 0.2). Other weaknesses included the absence of radiographic and mechanical investigation of the callus. Most authors agree that mechanical studies are the best way to evaluate bone healing process, however, we could not perform these studies for economical reasons. On the other hand, we decided to not use simple radiographic study because it appears to be inaccurate for evaluating fracture healing, as previously demonstrated in the study of Giordano [6]. Unexpected finding occurred in one animal of Group IV by 4 weeks post-fracture. A small zone of bone formation had formed, suggesting an extremely delayed bone repair. Excessive movement has been associated with the formation of a cartilage callus [49]. It is possible that our nonstabilised tibial fracture rat model could be a potential cause of delayed callus formation and healing. The molecular aspects of healing in stabilised and nonstabilised fractures in a mouse model have been recently studied by Le et al. [50]. In a very elegant experiment these authors demonstrated that mechanical influences regulate cartilage and bone formation during fracture repair. However, they did not observe that stabilised fracture calluses matured faster than nonstabilised fracture calluses based on histological and molecular analyses. By 28 days post-fracture all animals showed the fracture site completely healed [50]. Therefore, we could not definitively explain this observation in the animal of Group IV. Animal constitutional characteristics, like malnutrition or chronic illnesses, which may impair the process of bone healing [8], although, these possibilities remain purely speculative. In summary, the data presented herein demonstrate that tenoxicam administered intramuscularly for 4 weeks to Wistar rats significantly delays the bone healing process after
experimental diaphyseal tibia fracture. These findings suggest the hypothesis that early drug administration may delay bone healing, although, it could not be detected statistically significant.
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