Clinical and biomechanical analysis of the effect of diclofenac sodium in tibial fracture healing in rats

Abstracts

Os AINH (Antiinflamatórios não hormonais) são agentes utilizados na prática clínica que interferem no processo inflamatório pela inibição da síntese de prostaglandinas e tromboxanos. Alguns trabalhos experimentais investigaram sua ação no processo de consolidação de fraturas, por meio de estudos clínicos e histológicos, sendo escassas as análises biomecânicas. Nesse estudo foram utilizados 20 ratos da linhagem Wistar, divididos aleatoriamente em dois grupos iguais: grupo A (controle) e grupo B (tratado com diclofenaco sódico). Em ambos os grupos foram realizadas fraturas abertas, após perfuração, na tíbia direita. A administração da droga foi via intramuscular, dose única diária, por 28 dias. Os animais foram pesados semanalmente. Após o sacrifício as tíbias foram dissecadas, pesadas e submetidas a ensaio biomecânico de flexão analisando-se carga máxima, deformação e coeficiente de rigidez. Observou-se que no grupo tratado com AINH não houve aumento do peso corpóreo a partir da segunda semana e as tíbias fraturadas foram mais pesadas. Neste grupo o calo ósseo suportou menor carga máxima, apresentando maior deformação e menor coeficiente de rigidez. Nos animais tratados, o osso não fraturado também se mostrou menos rígido. Concluiu-se, nas condições estudadas, que o DS alterou o processo de consolidação e o metabolismo ósseo, levando a retardo na maturação do calo e menor rigidez do osso intacto, respectivamente.

Antiinflamatório; Consolidação da fratura; Ratos

ORIGINAL ARTICLE

Clinical and biomechanical analysis of the effect of diclofenac sodium in tibial fracture healing in rats

Sérgio Swain Müller^I; Emílio Carlos Curcelli^II; Trajano Sardenberg^II; Alexandre Zuccon^III; José Luiz De Crudis Júnior^III; Carlos Roberto Padovani^IV

^IAssistant Professor PHD at the Department of Surgery and Orthopedics, FMB - UNESP

^IIAssistant Professor at the Department of Surgery and Orthopedics, FMB -UNESP

^IIIEx-Resident at the Service of Orthopedics and Traumatology, FMB-UNESP

^IVChairman of the Biostatistical Department - UNESP

^{Correspondence}

SUMMARY

The antinflammatories are agents utilized on clinical practice that interfere on inflammatory process by synthesis inhibition of prostaglandin and tromboxanes. Some experimental studies investigated their action on the fractures consolidation process, through clinical and histological studies, as the biomechanical analyses are scarce. In this study, 20 (twenty) Wistar pedigree rats were used, aleatory divided into two groups: A group (control) and B group (treated with diclofenac). In both groups open fractures were made through perforation on right tibia. The drug administration was done intramuscularly in a single daily dose, during 28 (twenty-eight) days. The animals were weekly weighed. After the sacrifice the tibias were dissected, weighed and submitted to a biomechanical analysis of flexion and the maximum load, deformation and rigidity coefficient were measured. It was remarkable that the treated group with antinflammatory did not present body weight gain after the second week and the broken tibias were heavier. In this group the osseous callous supported less maximum load, presenting larger deformation and smaller rigidity coefficient. In the treated group, the non-fractured bone was also less rigid. Concluding, at this studied conditions, the diclofenac can interfere on the consolidation process and on the osseous metabolism, promoting a delay on the callous maturation and a smaller rigidity of the intact bone, respectively.

Keywords: Antiinflammatory; Fracture healing; Rats.

INTRODUCTION

Fracture repair potential depends upon several factors, including those related to the patient, fracture site, and treatment.

Treatment aims to enhance fracture repair by improving biological and mechanical conditions.

Patient and fracture characteristics restrict healing. However, treatment may have an effect upon cure^[3].

In contrast with other tissues and organs, bone tissue is characterized by a singular repair potential, biological and mechanical repair being possible ^[7].

Different factors can positively or negatively affect the organization and outcome of fracture repair. Growth factors, demineralized matrix, bone marrow cells ^[3] .

Healing of musculoskeletal tissues is a tissular response consisting of three phases that do not occur separately, namely, inflammation, repair, and remodeling.

Inflammatory response, therefore, is essencial for repair. Tissues would not heal in the absence of inflammation ^{[5, 28]}.

Nonsteroidal antiinflammatory drugs (NSAIDs) are often used in clinical practice in patients with trauma, fractures, following surgery, etc because of their analgesic effect.

NSAIDs have antiinflammatory, analgesic, and antipyretic effects. Most of these agentes can inhibit platelet function.

Their pharmacological effects are due to cyclooxygenase inhibition. This enzyme modulates the synthesis of some inflammation mediators, such as prostaglandins ^{[2, 9]}.

Several NSAIDs are used today. Diclofenac sodium (DS), a commonly used NSAID, is derived from phenylacetic acid and indicated in the management of acute and chronic painful conditions, postoperative and posttraumatic inflammation, because it relieves pain and decreases inflammatory reaction and edema ^{[16, 27]}.

The majority of antiinflammatory effects of DS are due to inhibition of prostaglandin synthesis. DS is a potent inhibitor of cyclooxygenase in vivo and in vitro. It decreases the synthesis of prostaglandins and also of thromboxane and prostacyclin ^{[18, 26, 27]}.

Several authors have described delayed fracture repair in tibias in histological studies in NSAIDs-treated rats ^{[1, 12]}.

Biomechanical analysis of bone callus has been considered one of the best methods to evaluate fracture repair ^{[6, 8]}.

Biomechanical properties have been studied in few experimental studies evaluating fracture repair and NSAIDs use.

The routine use of NSAIDs in patients with acute trauma has led us to carry out the present experimental study using one of the most widely used antiinflamatory agents in the current clinical practice, diclofenac sodium.

MATERIAL AND METHODS

Twenty clinically healthy Wistar rats (Rattus norvegicus albinus) aged 60 days were used. Mean weight was 253 g. Two groups of 10 animals each were defined by draw. Group A was used as control and Group B included diclofenac-treated rats.

The experimental study had six sequential phases: adaptation; fracture; group formation; maintenance and treatment; sacrifice; and mechanical assay.

Animals were submitted to clinical examination, identification, weighing and confined in cages in a dry and ventilated area. Cages were cleaned daily. Temperature was controlled. Animals were given water and food ad libitum.

Following 7-day adaptation animals were examined, weighed, and anesthetized with sodium pentobarbital at the dose of 30 mg/kg given intraperitoneally ^[25].

Under aseptical conditions a perforation was drilled at the mean point of the right tibia using an electric dril with a 1 mm-drill and pedal-controlled rotation.

After perforation drill was removed and fracture was manually completed ^[14]. Groups were defined by draw following fracture. Animals were then confined in a cage.

They were examined daily and weighed at 7-day intervals and on the sacrifice day. Free motion of the fractured limb was allowed, immobilization being not used. Intramuscular diclofenac sodium (10 mg/kg/day) was given 12 h following fracture and every 24 h thereafter.

All animals were submitted to general physical examination, examination of the fractured limb (as for fracture focus mobility) and weighing 28 days after fracture. They were sacrificed by decapitation and both tibias were removed.

Clinical and biomechanical analyses of fractured and non-fractured tibias were carried out. Clinical analysis included weekly weight, behavior, eventual complications, such as infection, hematoma, or gastrointestinal disorders.

A longitudinal incision was made on the anterior face of the limb for tibial removal by proximal and distal desarticulation.

Fibula and muscular, capsular, tendinous structures were completely removed. (Figure 1).

Only bone tissue remained in the specimen. Tibias were then weighed with high-precision analytical scales (4 decimals) of 110-gm capacity. Specimens were stored in plastic bags at -20 °C (a domestic refrigerator was used).

On the day when assays were carried out specimens were thawed at room temperature and wrapped in moist drapes.

A three-point flexion assay was carried out with an electromechanical-type universal assay machine (EMIC, DL 10000) under controlled temperature (20^o C). System precision is ± (0,018 + F 3700) KN, according to NBR 6674 specifications (ABNT, 1981).

The specimen with the convex side facing upward was placed on two supports equally distant from the proximal and distal tibial ends. No fixation was used and distance corresponded to 2/3 of bone length. A blunt knife (2 mm) fixed to the assay machine applied the load on the equidistant point until the specimen was broken ^{[8, 24]} (speed: 30 mm/min)(Figure 2).

Values of maximum load and deformation upon breakage as well as the load-deformation graph are included in the final assay report. The proportionality coefficient (K) or rigidity coefficient was calculated for each diagram.

Body weight, bone weight, maximum load, deformation, and rigidity coefficient were compared by multivariate analysis of mean profiles and non-parametric ANOVA for repeated measurements in two independent groups ^{[15, 20]}.

Statistical analysis adopted a significance level of 5%.

RESULTS

3.1 Clinical Evaluation

Diclofenac-treated animals developed gastrointestinal disorders (diarrhea) during the first five days. Their body weight gain was smaller from the second week on, as compared to controls.

All rats progressively supported the limb following tibial fracture.

Clinical evaluation of tibias has shown mobility of bone callus in diclofenac-treated rats.

Animal and tibia weight data are shown in Tables 1, 2, 3, and Figure 3.

3.2 Mechanical Properties

Mechanical property data are shown in Tables 4, 5, and ⁶ and load-deformation diagrams for fractured and non-fractured bones are shown in Figures 4 and 5.

DISCUSSION

In the present experimental study rats were used as experimental animals, mainly because they are small and cheap ^[21] and develop fast bone repair with or with treatment ^[19] (approximately 28 days for tibial fracture). In addition, they are often used in models of fracture repair, among other advantages.

Nonsteroidal antiinflammatory drugs (NSAIDs) can lead to changes in bone metabolism and fracture repair, probably because they affect the inflammatory phase of healing ^[17].

By inhibiting cyclooxygenases they decrease the synthesis of inflammatory mediators, including prostaglandings responsible for chemotaxis durign the first phase of fracture healing, thus reducing the amount of cells in the fracture site and reabsorption of necrotic tissue and possibly modifying the amount of repair cells essential for bone callus formation ^[4].

Diclofenac sodium (DS) was chosen because it is one of the most widely used NSAIDs in clinical practice and has been studied in laboratory animals.

Therapeutical dosage recommended for rats ranges from 2 to 10 mg/kg/day. It can be given intramuscularly, intraperitoneally, and subcutaneously ^[11];

In the present study, the maximal therapeutical dose of DS ^[4] (10 mg/kg/day) was used for 28 days (estimated time required for a fracture to heal).

The expected maximal effect was studied throughout fracture repair.

We chose the experimental model of fracture following open perforation because this technique has been used in similar studies in our institution, allows one to control the fracture site, and requires less force ^{[14, 19]}

The biomechanical analysis of the fracture callus has been considered by several authors a technique of bone repair evaluation ^{[6, 8]}.

In diclofenac-treated animals body weight gain was not seen from the second week on. This finding can be related to the DS effect upon protein synthesis inhibition ^[10] as well as to diarrhea seen in DS-treated animals. This side effect has been described in literature when doses higher than 10 mg/kg/day have been used ^[11].

As for the mean weight of fractured tibias, greater values were found in DS-treated animals, which could be explained by greater tissue formation due to probable callus instability ^[22].

Biochemical analysis evaluated maximum load, absolute deformation, and rigidity coefficient.

Tibial specimens from DS-treated rats supported significantly lower load before bone callus rupture and showed greater deformation and lower rigidity coefficients than control animals. NSAIDs-induced decreased prostaglandin and thromboxane synthesis probably affected neoangiogenesis, less oxygen being supplied to mesenchymal cells. Under these conditions there would be a tendency to differentiation into chondroblasts and fibroblast responsible for extracellular matrix synthesis with no calcium affinity. Therefore, an immature and less mineralized bone callus would develop ^[1].

The evaluation of mechanical properties of non-fractured tibias showed lower rigidity coefficients in DS-treated rats, a finding that could be explained by the NSAID effect upon not only fracture repair, but also upon bone homeostasis so as to stimulate reabsorptive action of osteoclasts ^[13].

In a biomechanical study of femoral fracture repair in indomethacin-treated rats Ro et al. ^[23] showed less resistant bone callus.

Allen et al. ^[1] histologically studied regenerated bone in indomethacin- and aspirin-treated rats and observed a higher percentage of cartilaginous union in these animals, thus confirming delayed fracture healing.

Keller et al. ^[17] observed blood flow in bone callus in indomethacin-treated rabbits and carried out a biomechanical evaluation.

Less blood was supplied to bone callus during the first phases of bone repair in treated animals, as compared to animals. Another finding was decreased maximum load

In a recent study Giordano et al. ^[12] histologically confirmed Keller's findigns. They also confirmed delayed ossification of bone callus in tenoxicam-treated rats.

Clinical Considerations

Literature reports and the present study show evidence of a possible deleterious effect of NSAIDs upon bone healing.

Therefore, NSAIDs should be carefully indicated in patients with fracture only for short-term therapy and only if trauma-related symptoms require their use. Patients should be instructed to avoid self-medication.

CONCLUSIONS

Under study conditions the following has been observed in rats:

• DS inhibited body weight gain;

• The weight of fractured bone was greater in treated ani mals;

• DS reduced the rigidity coefficient in non-fractured bones;

• DS affected mechanical properties of bone callus, decreasing maximum load and rigidity and increasing deformation.

These findings confirm delayed bone repair in diclofenac-treated animals.

REFERÊNCIAS BIBLIOGRÁFICAS

Trabalho recebido em04/02/2003.

Aprovado em 25/07/2004

Work performed at the Department of Surgery and Orthopedics of the Medicine School of (UNESP), Paulista State University "Júlio de Mesquita Filho".

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Publication Dates

• Publication in this collection
  01 Mar 2005
• Date of issue
  Dec 2004

History

• Accepted
  25 July 2004
• Received
  04 Feb 2003