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Isotretinoin effect on the repair of bone defects – A study in rat calvaria
Journal of Cranio-Maxillo-Facial Surgery 41 (2013) 581e585
Contents lists available at SciVerse ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Isotretinoin effect on the repair of bone defects e A study in rat calvaria Henrique T.R. de Oliveira a, Roberta D. Bergoli b, Wâneza D.B. Hirsch b, Otacílio L. Chagas Jr. c, *, Cláiton Heitz d, Daniela N. Silva b, e a
Dentistry Department, Fundação para Reabilitação das Deformidades Cranio Faciais (FUNDEF), Lajeado, RS, Brazil Oral and Maxillofacial Surgery Post-Graduate Program, School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil c Department of Oral and Maxillofacial Surgery and Maxillofacial Prosthodontics, Bone Repair Research Group, School of Dentistry, Universidade Federal de Pelotas (UFPEL), Pelotas, RS, Brazil d Surgery Department, School of Dentistry, PUCRS, Porto Alegre, RS, Brazil e Universidade Federal do Espírito Santo (UFES), Vitória, ES, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 August 2012 Accepted 26 November 2012
Background: Isotretinoin is a vitamin A derivative, indicated for the treatment of patients with severe acne, which shows several side effects on bone metabolism. Objective: This study analyzed the process of bone repair in rats receiving 7.5 mg/kg/day of oral isotretinoin. Methods: Thirty-three male albino Wistar rats, at approximately 60 days of age, were randomly assigned to control (n ¼ 15) and experimental (n ¼ 18) groups. Only the experimental group underwent oral isotretinoin therapy. In both groups, a 2-mm cavity was established in the calvarium of each animal. The animals were euthanize 21, 28 and 90 days postoperatively. The parietal bone was removed and the surgical specimens underwent histological examination. Computed histomorphometry allowed the measurement of the total area of bone defects and the proportion of newly formed bone at the different observation time points. Results: In the experimental group, the results, expressed as mean percentage of newly formed bone, were: 25.37% (9.14) at day 21; 41.78% (7.00) at day 28; and 57.51% (11.62) at day 90. In the control group, the results were: 17.10% (9.23) at day 21; 34.42% (7.70) at day 28; and 48.49% (16.40) at day 90. Conclusion: These results enabled us to conclude that isotretinoin promoted acceleration in the process of new bone formation in rat calvaria, although this increase was not statistically significant. Ó 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bone and Bones Vitamin A Hypervitaminosis A Isotretinoin
1. Introduction Isotretinoin, or 13-cis-retinoic acid, is a vitamin A derivative currently used as the drug of choice in the treatment of severe acne that is unresponsive to the usual antimicrobial therapy. Since the introduction of the drug, in September 1982, isotretinoin has become an important therapeutic agent for dermatologists, who should, therefore, be aware of its side effects (Azulay et al., 1985). Excessive retinoid intake may cause unpleasant and even worrying acute or chronic clinical alterations, which can vary from individual to individual in a dose-related fashion. Side effects are similar to * Corresponding author. R. Santa Cruz, 1948 A/504 e Centro, Pelotas, RS, CEP: 96015-710, Brazil. Tel./fax: þ55 53 32224305. E-mail address: [email protected] (O.L. Chagas).
those observed with hypervitaminosis A, including dry lips as well as more severe alterations, such as reduced bone mineral density e increasing the risk of fracture, liver lesions, inhibition of bone growth, in addition to laboratory alterations, such as an increase in cholesterol and triglycerides, and alterations in liver enzymes and alkaline phosphatase (Azulay et al., 1985; Sampaio and Pimentel, 1985). Concerning bone tissue, some reports have shown that excessive retinoid intake has site-specific effects, such as a reduction in bone density of 9% at the Ward triangle, hyperostotic changes or calcification of tendons and ligaments, premature closure of epiphyses (Leachman et al., 1999; DiGiovanna et al., 2004), increased bone resorption, increase in the number and size of osteoclasts, decrease in osteoid surface, and deterioration of cartilage (Frankel et al., 1986). However, the effects on cranial bones have yet to be determined.
1010-5182/$ e see front matter Ó 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2012.11.030
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Taking into consideration that acne vulgaris is the most common skin disorder among adolescents and adults, that isotretinoin is the drug of choice in the treatment of severe acne, and that isotretinoin-related craniofacial alterations remain unclear, patients undergoing this therapy have become an object of great concern among professionals in the area of Oral and Maxillofacial Surgery and Trauma, since bone tissue is one of the main tissues approached in several branches of this specialty. This study evaluated the effects of daily administration of isotretinoin on the repair of bone defects made in rat calvaria, using a therapeutic dose corresponding to that used for skin disorders such as severe acne in humans.
the outer and inner cortex of the calvarium, with no meningeal lesion. The periosteum, subcutaneous tissue and skin were then sutured with 4e0 mononylon simple running suture. For postoperative pain management, the animals received analgesia with paracetamol (80 mg/kg) intramuscularly on the first three postoperative days. The animals were euthanized 21, 28 and 90 days after the surgical procedure, through continuous inhalation in an isoflurane chamber.
2. Materials and methods
An osteotomy was performed in the right parietal bone, with a tronco-conical drill (702), at low drilling speed, under continuous irrigation with saline solution, at a distance of at least 4 mm from the cavity, completed with the use of a straight chisel, aiming to minimize possible damage to the bone due to drilling friction. The complete operated area was removed and surgical specimens were then prepared. The specimens obtained were placed into labeled glass containers containing 10% buffered formalin solution. After 48-h fixation, the specimens were decalcified in 5% nitric acid solution, changed daily, for 2e4 days, according to bone thickness. After decalcification, a 6-mm thick section was obtained from the central region of the defect of each specimen, at the greater diameter (2 mm), and stained with hematoxylineeosin (HE).
This study was approved by the Research Ethics Committee of the School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), and the Ethics Committee for the Care and Use of Animals e PUCRS. Thirty-three male albino rats (Rattus novergicus albinus, Wistar), at approximately 60 days of age and mean weight of 250 g, were used in the study. The animals were randomly assigned to two groups: Control group e 15 rats without isotretinoin administration were divided into three subgroups (five rats per group) according to the time point for observation of bone repair: 21, 28 and 90 postoperative days; Experimental group e 18 rats receiving daily isotretinoin for 30 days prior to the surgical procedure were divided into three subgroups (six rats per group) according to the same observation time points used for the control group, with continuous isotretinoin administration until euthanasia. The animals in the experimental group were weighed before treatment and then weekly for drug dosage adjustment. 2.1. Oral administration of isotretinoin Isotretinoin, available as a powder, was diluted in sunflower oil to produce oral suspensions. Isotretinoin powder and sunflower oil were placed in dark glass containers and stored in a refrigerator until their use in the experiment. Each suspension was prepared immediately before administration due to isotretinoin high sensitivity to air, heat, and light, especially as a suspension. Suspensions were prepared by injecting sunflower oil, using an adjustable dosage syringe, into a container with powder isotretinoin until the desired concentration of 7.5 mg/kg body weight was provided (Ferguson et al., 2005), shaking the container for a better dilution of the content. Isotretinoin was administered orally, by the gavage procedure, using a 2-mL syringe and a metal cannula suitable for this purpose. 2.2. Surgical procedure The rats were anaesthetized intraperitoneally with 50 mg/kg 10% ketamine hydrochloride (0.05 mL/100 g) and 5 mg/kg xylazine hydrochloride (0.025 mL/100 g). Trichotomy was performed in the calvarium in the region between the ear auricles. Surgical access was established by means of a linear coronal cutaneous incision (skin, subcutaneous tissue and periosteum) of approximately 1.5 cm between the ears. A cavity was made in the right parietal bone, lateral to the median sagittal suture, using a lowtorque electric motor, with 2-mm spherical drills, corresponding to the size of the defects produced. Perforations were made under abundant irrigation with 0.9% saline solution, with a rupture of
2.3. Preparation of samples
2.4. Image capture procedure and histomorphometry Histological slides underwent microscopic examination using a computer-assisted image processing and analysis system (Image J software, version 1.41, Media Cybernetics Inc., Bethesda, MD). The microscope image was captured with a fixed-focus camera (SonyCCD-Iris Color Video Camera, model DXX-107a, Sony, Tokyo, Japan) attached to a PC-based workstation (1.8 GHz processor, 128 MB RAM, 40 GB hard drive, Compaq Computer Corporation, Houston, TX), at 40 magnification. The images were then transformed into an electrical analog signal and transmitted to the computer screen, where the image was digitized, constituting a set of pixels (1 pixel ¼ 6.5 mm). After the images were saved as JPEG files (a total of 33 histological slides corresponding to all control and experimental groups), they were submitted to histomorphometry. Using the image processing and analysis software, we could measure the total area of the defect and the area of new bone formation by moving the cursor over the image to draw its outline, thus resulting in the proportion of newly formed bone at the different observation time points (Fig. 1). The values obtained from each newly formed trabecular bone specimen were transferred to a table, in which total bone formation was recorded and calculated for each slide analyzed. All these values were entered on definitive Excel spreadsheets (Microsoft Corporation, Redmond, WA) and submitted to statistical analysis using the SPSS software (Statistical Package for the Social Sciences, version 11.5; SPSS Inc., Chicago, IL). After image acquisition, the outline of the area of bone defect and areas of newly formed bone was drawn with the mouse. The value of these areas, as mm2, was quantified using the image processing and analysis software, the proportion of newly formed bone being calculated as follows: area of newly formed bone/total area of defect. The nonparametric ManneWhitney test was used to compare the percentage of new bone formation between experimental and control groups (intergroup test). The nonparametric Kruskale Wallis test was used to compare observation time points (21, 28 and 90 postoperative days) within the same group (intragroup test:
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was filled with new bone. In the control group, values were lower: at day 21, we observed 17.10% of new bone formation; at day 28, 34.42%; and at day 90, 48.49%. The results of the nonparametric ManneWhitney test revealed that there were no statistically significant differences between groups at any observation time point. The results of the nonparametric KruskaleWallis test revealed statistically significant differences between time points for both study groups (Table 2). We can observe that: Isotretinoin group: All time points are different from each other, day 21 showing the lowest values, followed by day 28, and, finally, the highest values being observed at day 90. Control group: Day 21 shows values significantly lower than those at day 90. Day 28 did not differ among groups.
Fig. 1. Image J software, version 1.41, used to measure the area of defect outlined in yellow. Results are shown in the upper right box of the image.
experimental or control group). The results were considered statistically significant at a 5% level of significance (p 0.05). Statistical analysis was performed using the SPSS software, version 10.0 (SPSS Inc., Chicago, IL). 3. Results Table 1 shows the percentage of bone formation when comparing experimental vs. control groups, at the respective observation time points. The highest percentages of new bone formation were found in the experimental groups (isotretinoin). In the experimental group, at day 21 there was 25.37% of newly formed bone; at day 28, 41.78%; and at day 90, 57.51% of the cavity
Table 1 Comparison of the percentage of new bone formation between study groups at each observation time point. Time point
n
Percentage of new bone formation (%) Mean
Z
p
4.0 9.3 15.2
13.135
0.001
3.8 8.8 11.4
7.460
0.024
Standard deviation
Mean rank
Isotretinoin group 21 days 6 25.4A 28 days 6 41.8B 90 days 6 57.5C
9.1 7.0 11.6
Control group 21 days 5 28 days 5 90 days 5
9.2 7.7 16.4
17.1A 34.4AB 48.5B
Means followed by the same letter do not differ from each other. p 0.05.
Table 2 Comparison of the percentage of new bone formation between observation time points in each study group. Group
n
Percentage of new bone formation (%) Mean
Standard deviation
Z
p
Mean rank
Time point: 21 days Isotretinoin 6 25.37 Control 5 17.10
9.14 9.23
7.33 4.40
1.461
0.177
Time point: 28 days Isotretinoin 6 41.78 Control 5 34.42
7.00 7.70
7.50 4.20
1.643
0.126
Time point: 90 days Isotretinoin 6 57.51 Control 5 48.49
11.62 16.40
6.83 5.00
0.913
0.429
p 0.05.
New bone formation was higher in the isotretinoin than in the control group at all time points, although this difference was not statistically significant. 4. Discussion Isotretinoin is a synthetic metabolic product of vitamin A, which has been widely prescribed for the treatment of disorders of cornification, severe cystic acne, severe recalcitrant nodular acne, patients with xeroderma pigmentosum, skin cancer chemoprevention, the nevoid basal cell carcinoma syndrome, recipients of organ or bone marrow transplantation, multiple basal and squamous cell carcinomas, and diseases with abnormal keratinization (Valentic et al., 1983; Azulay et al., 1985; Frankel et al., 1986; Mclane, 2001; Fleischer et al., 2003; Goldsmith et al., 2004; Campbell and DiGiovanna, 2006). Despite the numerous benefits and indications of this drug, several side effects may be associated with its application, such as liver damage, elevated triglycerides and cholesterol, hypercalcemia, anemia, increase in the number of platelets, and decrease in the number of leukocytes or erythrocytes (Azulay et al., 1985), mainly those of the musculoskeletal system (Kilcoyne et al., 1986; Carey et al., 1988; Ellis and Kent, 2001; Feskanich et al., 2002; Johansson et al., 2002; Kawahara et al., 2002; Promislow et al., 2002; Michaëlsson et al., 2003; DiGiovanna et al., 2004). High-dose intake of vitamin A and its derivatives has been related to opposing changes in bone metabolism, causing bone resorption e through a significant increase in osteoclasts and decrease in osteoid tissue e, hyperostotic changes or calcification of tendons and ligaments, and premature closure of epiphyses (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989; Leachman et al., 1999; DiGiovanna et al., 2004). Since bone tissue is one of the main tissues approached in the area of Oral and Maxillofacial Surgery and Trauma, through exodontia, implantation, orthognathic surgery, trauma, among others, it is of utmost importance that the surgeon can identify bone alterations resulting from isotretinoin use, as well as understand how such alterations occur. To date, the mechanisms of isotretinoin action on bone metabolism remain unclear (Margolis et al., 1996; Rohde and DeLuca, 2003). Comparing bone defects between animals that were given isotretinoin and controls, we observed that, at day 21, there was greater new bone formation at the experimental cavities. At this observation time point, mean new bone formation was 25.37% in the experimental group compared to 17.10% in the control group. At day 28, the isotretinoin group continued to have higher values, 41.78% compared to 34.42% in the control group. At the last time point, day 90, the isotretinoin group obtained values of 57.51%, which were higher than those in the control group, 48.49%
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(Table 1). We observed that the numerical values of area, through histomorphometric analysis, showed higher mean percentages of new bone formation in the isotretinoin group than in the control group (but without statistical significance). These results corroborate the studies by Kamm (1982) and Bérgoli et al. (2011), who described that animals fed excess synthetic isotretinoin have accelerated bone repair, however, we observed that in both groups bone repair was not complete at day 90. Similarly, in the study by Turnbull and Freeman (1974), 2-mm defects did not heal during this same observation period. Therefore, based on these results, we could consider that a 2-mm defect in rat calvaria may be termed a critical size defect, since the defects did not heal completely within 12 weeks, as established by Schmitz and Hollinger (1986). It is important to highlight that no material was inserted into the cavity. For those pieces of research which make use of graft materials in rat calvarium, critical defects having a larger diameter are recommended (Frederik et al., 2012; Zanchetta et al. (2012). Establishing the ideal moment to approach the bone tissue in patients using isotretinoin is essential for a rehabilitation prognosis. Several studies have shown that the side effects of this drug are dose- and time-dependent, and may remain after drug withdrawal (Azulay et al., 1985; Frankel et al., 1986; DiGiovanna, 2001; Ellis and Kent, 2001; Campbell and DiGiovanna, 2006). Therefore, it is necessary to identify whether there were alterations in the bone tissue that will be the target of surgical procedures, even after isotretinoin withdrawal, and whether such alterations may restrict, contraindicate or postpone the intervention. In this study, animals were administered 7.5 mg/kg/day of isotretinoin, minimum dose equivalent to the standard dose administered to humans in the treatment of severe acne (1 mg/kg/day) (Frankel et al., 1986; Ferguson et al., 2005). Animals in the experimental group received the drug for an initial period of 30 days before bone cavities were made, remaining on this drug until the observation time points (21, 28 and 90 days), to simulate surgical intervention during isotretinoin therapy. Patients receiving high doses (3e4 mg/kg) of isotretinoin have shown both hyperostosis (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989) and accelerated bone mineral resorption (Valentic et al., 1983), which has been less evident or without effect with the recommended (standard) dose of 1 mg/kg/day (DiGiovanna et al., 2004). The same dose, administered in fractionated doses (2/day), may reduce the side effects (Ellis and Kent, 2001). There are reports that short-term isotretinoin administration has no side effects on the skeleton, specifically on bone mineral density (Milstone et al., 2005), but in the long term (2e6 years) alterations may be marked, through the occurrence of hyperostosis (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989; DiGiovanna et al., 2004), pathological changes in bone tissue resulting from alterations in calcium metabolism, and increased risk of fractures (Frankel et al., 1986; Johansson et al., 2002; Kawahara et al., 2002). In this study, bone alterations were observed, with accelerated bone repair in the defects made in rat calvaria, after ingestion of isotretinoin, even using the standard therapeutic dose for treatment of severe cystic acne. In this study, the lack of statistical significance in the proportion of new bone formation between the experimental and control groups suggests that isotretinoin does not interfere with bone repair. However, although not statistically significant, descriptive microscopic analysis revealed an increase in the percentage of new bone formation at all observation time points in the animals receiving oral isotretinoin. This increase in new bone formation represents acceleration in the process of bone repair in the calvarium. Under the same experimental conditions, Bérgoli et al. (2011) concluded, after descriptive microscopic analysis, that
there was acceleration in the process of alveolar repair in rat incisors after exodontia. It is believed that the mechanism of acceleration of new bone formation (in the calvarium or dental alveolus) is similar to that occurring in hyperostosis, calcification of ligaments, or premature closure of epiphyses, since in all cases there seems to be a higher local metabolic demand. It is worth mentioning that this study quantified new bone formation in cavities previously made in the calvaria alone, where the repair process requires higher local and systemic metabolism. Other bone sites were not analyzed to detect possible resorption, hyperostosis or other alterations. It remains unknown whether changes in bone tissue occur alone, sequentially or simultaneously; thus, further investigation is warranted to better understand this process. It is of paramount importance that professionals have detailed knowledge of isotretinoin effects in cases of grafting, implantation, fractures, orthognathic surgery, orthodontic movement, and periodontal treatment, in which bone metabolism and bone tissue repair are necessary to achieve treatment success. We suggest that, based on this study, further investigations are conducted in the craniofacial region, examining repair in other facial bones and using other periods of isotretinoin administration and dosage. 5. Conclusion These results enabled us to conclude that isotretinoin promoted acceleration in the process of new bone formation in rat calvaria, although this increase was not statistically significant. Conflicts of interest The authors report no conflicts of interest related to this study. References Azulay DR, Abulafia LA, Nery JAC, Sodré CT: Tecido de granulação exuberante e Efeito colateral da terapêutica com isotretinoína. An Bras Dermatol 60: 179e 182, 1985 Bérgoli RD, et al: Isotretinoin effect on alveolar repair after exodontia e a study in rats. Oral Maxillofac Surg 15(2): 85e92, 2011 Campbell RM, DiGiovanna JJ: Skin cancer chemoprevention with systemic retinoids: an adjunct in the management of selected high-risk patients. Dermatol Ther 19: 306e314, 2006 Carey BM, Parkin GJ, Cunliffe WJ, Pritlove J: Skeletal toxicity with isotretinoin therapy: a clinico-radiological evaluation. Br J Dermatol 119: 609e614, 1988 DiGiovanna JJ, Langman CB, Tschen EH, Jones T, Menter A, Lowe NJ, et al: Effect of a single course of isotretinoin therapy on bone mineral density in adolescent patients with severe, recalcitrant, nodular acne. J Am Acad Dermatol 51: 709e 717, 2004 DiGiovanna JJ: Isotretinoin effects on bone. J Am Acad Dermatol 45: S176eS182, 2001 Ellis CN, Kent KJ: Uses and complications of isotretinoin therapy. J Am Acad Dermatol 45: S150eS157, 2001 Ferguson SA, Cisneros FJ, Gough B, Hanig JP, Berry KJ: Chronic oral treatment with 13-cis-retinoic acid (isotretinoin) or all-trans-retinoic acid does not alter depression-like behaviors in rats. Toxicol Sci 87: 451e459, 2005 Feskanich D, Singh V, Willett WC, Colditz GA: Vitamin A intake and hip fractures among postmenopausal women. JAMA 287: 47e54, 2002 Fleischer Jr AB, Simpson JK, McMichael A, Feldman SR: Are there racial and sex differences in the use of oral isotretinoin for acne management in the United States? J Am Acad Dermatol 49: 662e666, 2003 Frankel TL, Seshadri MS, McDowall DB, Cornish CJ: Hypervitaminosis A and calcium-regulating hormones in the rat. J Nutr 116: 578e587, 1986 Frederik ZN, Natsuko K, Satoshi K, Kenji S, et al: Bone regeneration with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J Craniomaxillofac Surg 40(3): 287e291, 2012 Goldsmith LA, Bolognia JL, Callen JP, Chen SC, Feldman SR, Lim HW, et al: American Academy of Dermatology Consensus Conference* on the safe and optimal use of isotretinoin: summary and recommendations. J Am Acad Dermatol 50: 900e 906, 2004 Johansson S, Lind PM, Hakansson H, Oxlund H, Orberg J, Melhus H: Subclinical hypervitaminosis A causes fragile bones in rats. Bone 31: 685e689, 2002 Kamm JJ: Toxicology, carcinogenicity, and teratogenicity of some orally administered retinoids. J Am Acad Dermatol 6: 652e659, 1982 Kawahara TN, Krueger DC, Engelke JA, Harke JM, Binkley NC: Short-term vitamin A supplementation does not affect bone turnover in man. J Nutr 132: 1169e1172, 2002
H.T.R. de Oliveira et al. / Journal of Cranio-Maxillo-Facial Surgery 41 (2013) 581e585 Kilcoyne RF, Cope R, Cunningham W: Minimal spinal hyperostosis with low-dose isotretinoin therapy. Invest Radiol 21: 41e44, 1986 Leachman SA, Insogna KL, Katz L, Ellison A, Milstone LM: Bone densities in patients receiving isotretinoin for cystic acne. Arch Dermatol 135: 961e965, 1999 Margolis DJ, Attie M, James J: Effects of isotretinoin on bone mineralization during routine therapy with isotretinoin for acne vulgaris. Arch Dermatol 132: 769e 774, 1996 Mclane J: Analysis of common side effects of isotretinoin. J Am Acad Dermatol 45: S188eS194, 2001 Michaëlsson K, Lithell H, Vessby B, Melhus H: Serum retinol levels and the risk of fracture. N Engl J Med 348: 287e294, 2003 Milstone LM, Insogna KL, Leachman SA: Isotretinoin does have an adverse effect on bone mineral density. J Am Acad Dermatol 53: 181, 2005 Promislow JH, Goodman-Gruen D, Slymen DJ, Barrett-Connor E: Retinol intake and bone mineral density in the elderly: the Rancho Bernardo Study. J Bone Miner Res 17: 1349e1358, 2002
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Rohde CM, DeLuca H: Bone resorption activity of all-trans retinoic acid is independent of vitamin D in rats. J Nutr 133: 777e783, 2003 Sampaio SAP, Pimentel ERA: Isotretinoína no tratamento da acne vulgar. An Bras Dermatol 60: 349e352, 1985 Schmitz JP, Hollinger JO: The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res 205: 299e308, 1986 Török L, Galuska L, Kása M, Kádár L: Bone-scintigraphic examinations in patients treated with retinoids: a prospective study. Br J Dermatol 120: 31e36, 1989 Turnbull RS, Freeman E: Use of wounds in the parietal bone of the rat for evaluating bone marrow for grafting into periodontal defects. J Periodont Res 9: 39e43, 1974 Valentic JP, Elias AN, Weinstein GD: Hypercalcemia associated with oral isotretinoin in the treatment of severe acne. JAMA 250: 1899e1900, 1983 Zanchetta P, Nicole L, Arnaud U, Pascale M: Mixture of hyaluronic acid, chondroitin 6 sulphate and dermatan sulphate used to completely regenerate bone in rat critical size defect model. J Craniomaxillofac Surg 40: 783e787, 2012
Contents lists available at SciVerse ScienceDirect
Journal of Cranio-Maxillo-Facial Surgery journal homepage: www.jcmfs.com
Isotretinoin effect on the repair of bone defects e A study in rat calvaria Henrique T.R. de Oliveira a, Roberta D. Bergoli b, Wâneza D.B. Hirsch b, Otacílio L. Chagas Jr. c, *, Cláiton Heitz d, Daniela N. Silva b, e a
Dentistry Department, Fundação para Reabilitação das Deformidades Cranio Faciais (FUNDEF), Lajeado, RS, Brazil Oral and Maxillofacial Surgery Post-Graduate Program, School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil c Department of Oral and Maxillofacial Surgery and Maxillofacial Prosthodontics, Bone Repair Research Group, School of Dentistry, Universidade Federal de Pelotas (UFPEL), Pelotas, RS, Brazil d Surgery Department, School of Dentistry, PUCRS, Porto Alegre, RS, Brazil e Universidade Federal do Espírito Santo (UFES), Vitória, ES, Brazil b
a r t i c l e i n f o
a b s t r a c t
Article history: Paper received 29 August 2012 Accepted 26 November 2012
Background: Isotretinoin is a vitamin A derivative, indicated for the treatment of patients with severe acne, which shows several side effects on bone metabolism. Objective: This study analyzed the process of bone repair in rats receiving 7.5 mg/kg/day of oral isotretinoin. Methods: Thirty-three male albino Wistar rats, at approximately 60 days of age, were randomly assigned to control (n ¼ 15) and experimental (n ¼ 18) groups. Only the experimental group underwent oral isotretinoin therapy. In both groups, a 2-mm cavity was established in the calvarium of each animal. The animals were euthanize 21, 28 and 90 days postoperatively. The parietal bone was removed and the surgical specimens underwent histological examination. Computed histomorphometry allowed the measurement of the total area of bone defects and the proportion of newly formed bone at the different observation time points. Results: In the experimental group, the results, expressed as mean percentage of newly formed bone, were: 25.37% (9.14) at day 21; 41.78% (7.00) at day 28; and 57.51% (11.62) at day 90. In the control group, the results were: 17.10% (9.23) at day 21; 34.42% (7.70) at day 28; and 48.49% (16.40) at day 90. Conclusion: These results enabled us to conclude that isotretinoin promoted acceleration in the process of new bone formation in rat calvaria, although this increase was not statistically significant. Ó 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.
Keywords: Bone and Bones Vitamin A Hypervitaminosis A Isotretinoin
1. Introduction Isotretinoin, or 13-cis-retinoic acid, is a vitamin A derivative currently used as the drug of choice in the treatment of severe acne that is unresponsive to the usual antimicrobial therapy. Since the introduction of the drug, in September 1982, isotretinoin has become an important therapeutic agent for dermatologists, who should, therefore, be aware of its side effects (Azulay et al., 1985). Excessive retinoid intake may cause unpleasant and even worrying acute or chronic clinical alterations, which can vary from individual to individual in a dose-related fashion. Side effects are similar to * Corresponding author. R. Santa Cruz, 1948 A/504 e Centro, Pelotas, RS, CEP: 96015-710, Brazil. Tel./fax: þ55 53 32224305. E-mail address: [email protected] (O.L. Chagas).
those observed with hypervitaminosis A, including dry lips as well as more severe alterations, such as reduced bone mineral density e increasing the risk of fracture, liver lesions, inhibition of bone growth, in addition to laboratory alterations, such as an increase in cholesterol and triglycerides, and alterations in liver enzymes and alkaline phosphatase (Azulay et al., 1985; Sampaio and Pimentel, 1985). Concerning bone tissue, some reports have shown that excessive retinoid intake has site-specific effects, such as a reduction in bone density of 9% at the Ward triangle, hyperostotic changes or calcification of tendons and ligaments, premature closure of epiphyses (Leachman et al., 1999; DiGiovanna et al., 2004), increased bone resorption, increase in the number and size of osteoclasts, decrease in osteoid surface, and deterioration of cartilage (Frankel et al., 1986). However, the effects on cranial bones have yet to be determined.
1010-5182/$ e see front matter Ó 2012 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jcms.2012.11.030
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Taking into consideration that acne vulgaris is the most common skin disorder among adolescents and adults, that isotretinoin is the drug of choice in the treatment of severe acne, and that isotretinoin-related craniofacial alterations remain unclear, patients undergoing this therapy have become an object of great concern among professionals in the area of Oral and Maxillofacial Surgery and Trauma, since bone tissue is one of the main tissues approached in several branches of this specialty. This study evaluated the effects of daily administration of isotretinoin on the repair of bone defects made in rat calvaria, using a therapeutic dose corresponding to that used for skin disorders such as severe acne in humans.
the outer and inner cortex of the calvarium, with no meningeal lesion. The periosteum, subcutaneous tissue and skin were then sutured with 4e0 mononylon simple running suture. For postoperative pain management, the animals received analgesia with paracetamol (80 mg/kg) intramuscularly on the first three postoperative days. The animals were euthanized 21, 28 and 90 days after the surgical procedure, through continuous inhalation in an isoflurane chamber.
2. Materials and methods
An osteotomy was performed in the right parietal bone, with a tronco-conical drill (702), at low drilling speed, under continuous irrigation with saline solution, at a distance of at least 4 mm from the cavity, completed with the use of a straight chisel, aiming to minimize possible damage to the bone due to drilling friction. The complete operated area was removed and surgical specimens were then prepared. The specimens obtained were placed into labeled glass containers containing 10% buffered formalin solution. After 48-h fixation, the specimens were decalcified in 5% nitric acid solution, changed daily, for 2e4 days, according to bone thickness. After decalcification, a 6-mm thick section was obtained from the central region of the defect of each specimen, at the greater diameter (2 mm), and stained with hematoxylineeosin (HE).
This study was approved by the Research Ethics Committee of the School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), and the Ethics Committee for the Care and Use of Animals e PUCRS. Thirty-three male albino rats (Rattus novergicus albinus, Wistar), at approximately 60 days of age and mean weight of 250 g, were used in the study. The animals were randomly assigned to two groups: Control group e 15 rats without isotretinoin administration were divided into three subgroups (five rats per group) according to the time point for observation of bone repair: 21, 28 and 90 postoperative days; Experimental group e 18 rats receiving daily isotretinoin for 30 days prior to the surgical procedure were divided into three subgroups (six rats per group) according to the same observation time points used for the control group, with continuous isotretinoin administration until euthanasia. The animals in the experimental group were weighed before treatment and then weekly for drug dosage adjustment. 2.1. Oral administration of isotretinoin Isotretinoin, available as a powder, was diluted in sunflower oil to produce oral suspensions. Isotretinoin powder and sunflower oil were placed in dark glass containers and stored in a refrigerator until their use in the experiment. Each suspension was prepared immediately before administration due to isotretinoin high sensitivity to air, heat, and light, especially as a suspension. Suspensions were prepared by injecting sunflower oil, using an adjustable dosage syringe, into a container with powder isotretinoin until the desired concentration of 7.5 mg/kg body weight was provided (Ferguson et al., 2005), shaking the container for a better dilution of the content. Isotretinoin was administered orally, by the gavage procedure, using a 2-mL syringe and a metal cannula suitable for this purpose. 2.2. Surgical procedure The rats were anaesthetized intraperitoneally with 50 mg/kg 10% ketamine hydrochloride (0.05 mL/100 g) and 5 mg/kg xylazine hydrochloride (0.025 mL/100 g). Trichotomy was performed in the calvarium in the region between the ear auricles. Surgical access was established by means of a linear coronal cutaneous incision (skin, subcutaneous tissue and periosteum) of approximately 1.5 cm between the ears. A cavity was made in the right parietal bone, lateral to the median sagittal suture, using a lowtorque electric motor, with 2-mm spherical drills, corresponding to the size of the defects produced. Perforations were made under abundant irrigation with 0.9% saline solution, with a rupture of
2.3. Preparation of samples
2.4. Image capture procedure and histomorphometry Histological slides underwent microscopic examination using a computer-assisted image processing and analysis system (Image J software, version 1.41, Media Cybernetics Inc., Bethesda, MD). The microscope image was captured with a fixed-focus camera (SonyCCD-Iris Color Video Camera, model DXX-107a, Sony, Tokyo, Japan) attached to a PC-based workstation (1.8 GHz processor, 128 MB RAM, 40 GB hard drive, Compaq Computer Corporation, Houston, TX), at 40 magnification. The images were then transformed into an electrical analog signal and transmitted to the computer screen, where the image was digitized, constituting a set of pixels (1 pixel ¼ 6.5 mm). After the images were saved as JPEG files (a total of 33 histological slides corresponding to all control and experimental groups), they were submitted to histomorphometry. Using the image processing and analysis software, we could measure the total area of the defect and the area of new bone formation by moving the cursor over the image to draw its outline, thus resulting in the proportion of newly formed bone at the different observation time points (Fig. 1). The values obtained from each newly formed trabecular bone specimen were transferred to a table, in which total bone formation was recorded and calculated for each slide analyzed. All these values were entered on definitive Excel spreadsheets (Microsoft Corporation, Redmond, WA) and submitted to statistical analysis using the SPSS software (Statistical Package for the Social Sciences, version 11.5; SPSS Inc., Chicago, IL). After image acquisition, the outline of the area of bone defect and areas of newly formed bone was drawn with the mouse. The value of these areas, as mm2, was quantified using the image processing and analysis software, the proportion of newly formed bone being calculated as follows: area of newly formed bone/total area of defect. The nonparametric ManneWhitney test was used to compare the percentage of new bone formation between experimental and control groups (intergroup test). The nonparametric Kruskale Wallis test was used to compare observation time points (21, 28 and 90 postoperative days) within the same group (intragroup test:
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was filled with new bone. In the control group, values were lower: at day 21, we observed 17.10% of new bone formation; at day 28, 34.42%; and at day 90, 48.49%. The results of the nonparametric ManneWhitney test revealed that there were no statistically significant differences between groups at any observation time point. The results of the nonparametric KruskaleWallis test revealed statistically significant differences between time points for both study groups (Table 2). We can observe that: Isotretinoin group: All time points are different from each other, day 21 showing the lowest values, followed by day 28, and, finally, the highest values being observed at day 90. Control group: Day 21 shows values significantly lower than those at day 90. Day 28 did not differ among groups.
Fig. 1. Image J software, version 1.41, used to measure the area of defect outlined in yellow. Results are shown in the upper right box of the image.
experimental or control group). The results were considered statistically significant at a 5% level of significance (p 0.05). Statistical analysis was performed using the SPSS software, version 10.0 (SPSS Inc., Chicago, IL). 3. Results Table 1 shows the percentage of bone formation when comparing experimental vs. control groups, at the respective observation time points. The highest percentages of new bone formation were found in the experimental groups (isotretinoin). In the experimental group, at day 21 there was 25.37% of newly formed bone; at day 28, 41.78%; and at day 90, 57.51% of the cavity
Table 1 Comparison of the percentage of new bone formation between study groups at each observation time point. Time point
n
Percentage of new bone formation (%) Mean
Z
p
4.0 9.3 15.2
13.135
0.001
3.8 8.8 11.4
7.460
0.024
Standard deviation
Mean rank
Isotretinoin group 21 days 6 25.4A 28 days 6 41.8B 90 days 6 57.5C
9.1 7.0 11.6
Control group 21 days 5 28 days 5 90 days 5
9.2 7.7 16.4
17.1A 34.4AB 48.5B
Means followed by the same letter do not differ from each other. p 0.05.
Table 2 Comparison of the percentage of new bone formation between observation time points in each study group. Group
n
Percentage of new bone formation (%) Mean
Standard deviation
Z
p
Mean rank
Time point: 21 days Isotretinoin 6 25.37 Control 5 17.10
9.14 9.23
7.33 4.40
1.461
0.177
Time point: 28 days Isotretinoin 6 41.78 Control 5 34.42
7.00 7.70
7.50 4.20
1.643
0.126
Time point: 90 days Isotretinoin 6 57.51 Control 5 48.49
11.62 16.40
6.83 5.00
0.913
0.429
p 0.05.
New bone formation was higher in the isotretinoin than in the control group at all time points, although this difference was not statistically significant. 4. Discussion Isotretinoin is a synthetic metabolic product of vitamin A, which has been widely prescribed for the treatment of disorders of cornification, severe cystic acne, severe recalcitrant nodular acne, patients with xeroderma pigmentosum, skin cancer chemoprevention, the nevoid basal cell carcinoma syndrome, recipients of organ or bone marrow transplantation, multiple basal and squamous cell carcinomas, and diseases with abnormal keratinization (Valentic et al., 1983; Azulay et al., 1985; Frankel et al., 1986; Mclane, 2001; Fleischer et al., 2003; Goldsmith et al., 2004; Campbell and DiGiovanna, 2006). Despite the numerous benefits and indications of this drug, several side effects may be associated with its application, such as liver damage, elevated triglycerides and cholesterol, hypercalcemia, anemia, increase in the number of platelets, and decrease in the number of leukocytes or erythrocytes (Azulay et al., 1985), mainly those of the musculoskeletal system (Kilcoyne et al., 1986; Carey et al., 1988; Ellis and Kent, 2001; Feskanich et al., 2002; Johansson et al., 2002; Kawahara et al., 2002; Promislow et al., 2002; Michaëlsson et al., 2003; DiGiovanna et al., 2004). High-dose intake of vitamin A and its derivatives has been related to opposing changes in bone metabolism, causing bone resorption e through a significant increase in osteoclasts and decrease in osteoid tissue e, hyperostotic changes or calcification of tendons and ligaments, and premature closure of epiphyses (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989; Leachman et al., 1999; DiGiovanna et al., 2004). Since bone tissue is one of the main tissues approached in the area of Oral and Maxillofacial Surgery and Trauma, through exodontia, implantation, orthognathic surgery, trauma, among others, it is of utmost importance that the surgeon can identify bone alterations resulting from isotretinoin use, as well as understand how such alterations occur. To date, the mechanisms of isotretinoin action on bone metabolism remain unclear (Margolis et al., 1996; Rohde and DeLuca, 2003). Comparing bone defects between animals that were given isotretinoin and controls, we observed that, at day 21, there was greater new bone formation at the experimental cavities. At this observation time point, mean new bone formation was 25.37% in the experimental group compared to 17.10% in the control group. At day 28, the isotretinoin group continued to have higher values, 41.78% compared to 34.42% in the control group. At the last time point, day 90, the isotretinoin group obtained values of 57.51%, which were higher than those in the control group, 48.49%
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(Table 1). We observed that the numerical values of area, through histomorphometric analysis, showed higher mean percentages of new bone formation in the isotretinoin group than in the control group (but without statistical significance). These results corroborate the studies by Kamm (1982) and Bérgoli et al. (2011), who described that animals fed excess synthetic isotretinoin have accelerated bone repair, however, we observed that in both groups bone repair was not complete at day 90. Similarly, in the study by Turnbull and Freeman (1974), 2-mm defects did not heal during this same observation period. Therefore, based on these results, we could consider that a 2-mm defect in rat calvaria may be termed a critical size defect, since the defects did not heal completely within 12 weeks, as established by Schmitz and Hollinger (1986). It is important to highlight that no material was inserted into the cavity. For those pieces of research which make use of graft materials in rat calvarium, critical defects having a larger diameter are recommended (Frederik et al., 2012; Zanchetta et al. (2012). Establishing the ideal moment to approach the bone tissue in patients using isotretinoin is essential for a rehabilitation prognosis. Several studies have shown that the side effects of this drug are dose- and time-dependent, and may remain after drug withdrawal (Azulay et al., 1985; Frankel et al., 1986; DiGiovanna, 2001; Ellis and Kent, 2001; Campbell and DiGiovanna, 2006). Therefore, it is necessary to identify whether there were alterations in the bone tissue that will be the target of surgical procedures, even after isotretinoin withdrawal, and whether such alterations may restrict, contraindicate or postpone the intervention. In this study, animals were administered 7.5 mg/kg/day of isotretinoin, minimum dose equivalent to the standard dose administered to humans in the treatment of severe acne (1 mg/kg/day) (Frankel et al., 1986; Ferguson et al., 2005). Animals in the experimental group received the drug for an initial period of 30 days before bone cavities were made, remaining on this drug until the observation time points (21, 28 and 90 days), to simulate surgical intervention during isotretinoin therapy. Patients receiving high doses (3e4 mg/kg) of isotretinoin have shown both hyperostosis (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989) and accelerated bone mineral resorption (Valentic et al., 1983), which has been less evident or without effect with the recommended (standard) dose of 1 mg/kg/day (DiGiovanna et al., 2004). The same dose, administered in fractionated doses (2/day), may reduce the side effects (Ellis and Kent, 2001). There are reports that short-term isotretinoin administration has no side effects on the skeleton, specifically on bone mineral density (Milstone et al., 2005), but in the long term (2e6 years) alterations may be marked, through the occurrence of hyperostosis (Kilcoyne et al., 1986; Carey et al., 1988; Török et al., 1989; DiGiovanna et al., 2004), pathological changes in bone tissue resulting from alterations in calcium metabolism, and increased risk of fractures (Frankel et al., 1986; Johansson et al., 2002; Kawahara et al., 2002). In this study, bone alterations were observed, with accelerated bone repair in the defects made in rat calvaria, after ingestion of isotretinoin, even using the standard therapeutic dose for treatment of severe cystic acne. In this study, the lack of statistical significance in the proportion of new bone formation between the experimental and control groups suggests that isotretinoin does not interfere with bone repair. However, although not statistically significant, descriptive microscopic analysis revealed an increase in the percentage of new bone formation at all observation time points in the animals receiving oral isotretinoin. This increase in new bone formation represents acceleration in the process of bone repair in the calvarium. Under the same experimental conditions, Bérgoli et al. (2011) concluded, after descriptive microscopic analysis, that
there was acceleration in the process of alveolar repair in rat incisors after exodontia. It is believed that the mechanism of acceleration of new bone formation (in the calvarium or dental alveolus) is similar to that occurring in hyperostosis, calcification of ligaments, or premature closure of epiphyses, since in all cases there seems to be a higher local metabolic demand. It is worth mentioning that this study quantified new bone formation in cavities previously made in the calvaria alone, where the repair process requires higher local and systemic metabolism. Other bone sites were not analyzed to detect possible resorption, hyperostosis or other alterations. It remains unknown whether changes in bone tissue occur alone, sequentially or simultaneously; thus, further investigation is warranted to better understand this process. It is of paramount importance that professionals have detailed knowledge of isotretinoin effects in cases of grafting, implantation, fractures, orthognathic surgery, orthodontic movement, and periodontal treatment, in which bone metabolism and bone tissue repair are necessary to achieve treatment success. We suggest that, based on this study, further investigations are conducted in the craniofacial region, examining repair in other facial bones and using other periods of isotretinoin administration and dosage. 5. Conclusion These results enabled us to conclude that isotretinoin promoted acceleration in the process of new bone formation in rat calvaria, although this increase was not statistically significant. Conflicts of interest The authors report no conflicts of interest related to this study. References Azulay DR, Abulafia LA, Nery JAC, Sodré CT: Tecido de granulação exuberante e Efeito colateral da terapêutica com isotretinoína. An Bras Dermatol 60: 179e 182, 1985 Bérgoli RD, et al: Isotretinoin effect on alveolar repair after exodontia e a study in rats. Oral Maxillofac Surg 15(2): 85e92, 2011 Campbell RM, DiGiovanna JJ: Skin cancer chemoprevention with systemic retinoids: an adjunct in the management of selected high-risk patients. Dermatol Ther 19: 306e314, 2006 Carey BM, Parkin GJ, Cunliffe WJ, Pritlove J: Skeletal toxicity with isotretinoin therapy: a clinico-radiological evaluation. Br J Dermatol 119: 609e614, 1988 DiGiovanna JJ, Langman CB, Tschen EH, Jones T, Menter A, Lowe NJ, et al: Effect of a single course of isotretinoin therapy on bone mineral density in adolescent patients with severe, recalcitrant, nodular acne. J Am Acad Dermatol 51: 709e 717, 2004 DiGiovanna JJ: Isotretinoin effects on bone. J Am Acad Dermatol 45: S176eS182, 2001 Ellis CN, Kent KJ: Uses and complications of isotretinoin therapy. J Am Acad Dermatol 45: S150eS157, 2001 Ferguson SA, Cisneros FJ, Gough B, Hanig JP, Berry KJ: Chronic oral treatment with 13-cis-retinoic acid (isotretinoin) or all-trans-retinoic acid does not alter depression-like behaviors in rats. Toxicol Sci 87: 451e459, 2005 Feskanich D, Singh V, Willett WC, Colditz GA: Vitamin A intake and hip fractures among postmenopausal women. JAMA 287: 47e54, 2002 Fleischer Jr AB, Simpson JK, McMichael A, Feldman SR: Are there racial and sex differences in the use of oral isotretinoin for acne management in the United States? J Am Acad Dermatol 49: 662e666, 2003 Frankel TL, Seshadri MS, McDowall DB, Cornish CJ: Hypervitaminosis A and calcium-regulating hormones in the rat. J Nutr 116: 578e587, 1986 Frederik ZN, Natsuko K, Satoshi K, Kenji S, et al: Bone regeneration with BMP-2 and hydroxyapatite in critical-size calvarial defects in rats. J Craniomaxillofac Surg 40(3): 287e291, 2012 Goldsmith LA, Bolognia JL, Callen JP, Chen SC, Feldman SR, Lim HW, et al: American Academy of Dermatology Consensus Conference* on the safe and optimal use of isotretinoin: summary and recommendations. J Am Acad Dermatol 50: 900e 906, 2004 Johansson S, Lind PM, Hakansson H, Oxlund H, Orberg J, Melhus H: Subclinical hypervitaminosis A causes fragile bones in rats. Bone 31: 685e689, 2002 Kamm JJ: Toxicology, carcinogenicity, and teratogenicity of some orally administered retinoids. J Am Acad Dermatol 6: 652e659, 1982 Kawahara TN, Krueger DC, Engelke JA, Harke JM, Binkley NC: Short-term vitamin A supplementation does not affect bone turnover in man. J Nutr 132: 1169e1172, 2002
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