Wound healing is a dynamic process requiring the involvement of soluble mediators, blood cells, extracellular matrix, and parenchymal cells. Wound healing has three phases of inflammation, tissue formation and remodeling, which overlap one another. ¹ Many chemical mediators are involved in the phases of inflammation and healing; nitric oxide (NO) is among these mediators. NO can show pro- or anti-inflammatory properties depending on its concentration, the potential to produce toxic derivatives such as proxy nitrite and the location of the pathological process. ²

NO is a free gas radical. NO synthase (NOS) affects the L-arginine and results in the production of NO. Three isoforms of NOS are available namely endothelial and neural constitutive NOS (cNOS), and inducible NOS (iNOS). Cells containing cNOS produce small amounts of NO immediately and transiently in response to factors that increase the intracellular level of free calcium; whereas, cells expressing iNOS produce large amounts of NO over a longer period after a several-hour lag phase as long as the inducing enzyme is present. ³ NO is an important signaling molecule in bone, which is produced in response to different stimuli such as the pro-inflammatory cytokines, mechanical tension, and sexual hormones. ⁴ NO mediates the function of bone cells and the process of bone remodeling. ⁴ , ⁵

NOS inhibitors decrease the bone mass via creating an imbalance between bone resorption (BR) and bone formation. ⁶ Evidence shows that N-Nitroarginine methyl ester (L-NAME), a NOS inhibitor, inhibits the process of bone formation, which is induced by mechanical factors. This indicates that NO is a primary mediator in increasing the bone mass via mechanical stimulation. ⁷ , ⁸ High concentrations of NO inhibit BR by suppressing the production of osteoclasts and their activity; whereas, lower concentrations of NO reinforce bone loss cytokines and are necessary for normal function of osteoclasts. Moreover, the growth and differentiation of osteoblasts are inhibited by higher concentrations of NO. Lower concentrations of NO produced by cNOS enzyme may mediate the proliferation and normal activity of osteoblasts. ⁴ Inhibiting the synthesis of NO in the wound decreases the accumulation of collagen and subsequently, the mechanical strength of the wound. ⁹ , ¹⁰ Moreover, it was demonstrated that local application of NO-releasing polymers enhanced wound healing. ¹¹

Aminoguanidine (AG) was introduced in 1992 as one of the first selective iNOS inhibitors. ¹² AG is fifty times more effective in inhibiting the enzymatic activity of iNOS than inhibiting endothelial and neural isoforms. ¹³ Farhad et al. ¹⁴ in their study on the role of NO in the progression of the inflammatory process in the periapical tissue in a cat model demonstrated that systemic administration of a selective iNOS inhibitor (AG) significantly decreased the severity of inflammation in the periapical lesions induced in the periapical region of canine teeth. Moreover, Farhad et al. ¹⁵ reported that systemic administration of AG significantly increased the healing score of induced periapical lesions in canine teeth in a cat model.

Bone defects may develop under different clinical conditions such as endodontic periapical and maxillofacial surgeries. In such cases, bone tissue must be regenerated as soon as possible. Numerous studies have evaluated the effect of NO or systemic inhibition of its synthesis on inflammation and wound healing. However, the obtained results are conflicting, and since no previous study has assessed the local effect of the selective iNOS inhibitor on the bone healing score, the current experiment was designed. This study aimed to assess the local effect of selective inhibition of iNOS by AG on bone healing score in rats.

Animal model

A rat model was used to assess the bone healing rate. The sample size was calculated to be 12 in each group considering the minimum number of animals required for obtaining a statistically significant difference among groups. The study protocol was approved by the Ethics Committee of Isfahan University of Medical Sciences (393029). Thirty-six male Wistar rats aged between 12 and 16 weeks and weighing between 300 and 400 g were selected. The study was conducted in the animal lab of Torabinejad Research Center at Isfahan University of Medical Sciences Isfahan, Iran. The rats were kept in separate cages under standard controlled temperature, and humidity conditions and they had free access to food and water.

Aminoguanidine and placebo gel preparation

To provide gradual and controlled release of gel, carboxymethyl cellulose polymer was used as a base. To prepare the gel, 70 g of 4% carboxymethyl cellulose polymer was dissolved in pure water heated up to 60°C to obtain a homogenous gel; 40 g of this gel was used as the placebo gel.

AG crystals are water soluble. To prepare 20% gel, 6 g of AG powder was dissolved in 2 mL of pure water and added to 30 g of gel. A homogeneous 20% gel was prepared as such. Since AG is sensitive to air and moisture, all the preparation steps of the AG gel were performed under neutral gas environment.

Surgical procedure

Anesthesia was induced by administration of 10% ketamine (Alfasan, Woerden, Holland) at a dose of 50 mg/kg and 2% acepromazine (Alfasan, Woerden, Holland) at a dose of 0.5 mg/kg. After the induction of anesthesia, distal of the left femur was shaved and scrubbed with alcohol, betadine, and chlorhexidine three times. A 1 cm incision was made in a direct lateral approach to expose the external surface of the lateral condyle of the left femur. Fascia was retracted, and the distal femur was approached through the anterior and posterior compartment muscles. A 5 mm × 5 mm defect was created in the distal condyle of the femur using an HM 141F 050 round bur (Meisinge, Dusseldorf, Germany) and S 11 model low-speed handpiece (W and H, Burmoos, Austria). The defect was created at the center of the femoral condyle in such a way that the bony walls around the defect were preserved. The surgical site was constantly irrigated with sterile saline during drilling to prevent over-heating. Hemorrhage from the defect site was controlled by constant local pressure Figure 1. Rats were randomly divided into three groups of 12 namely AG, placebo and control. Bone defects remained empty in the control group. In the placebo group, the placebo gel was applied to defects and in AG group, bone defects were filled with AG gel. Surgical wound was closed in two layers (periosteum-muscle and cutaneous layers). Closure of the muscle helps maintain the material in place. The muscle layer was sutured using 4-0 vicryl sutures (C.G Co.I.R, Iran) while skin closure was carried out using 4-0 nylon sutures (C.G Co.I.R, Iran). Chloramphenicol antibiotic was sprayed on the suture sites. Animals recovered from anesthesia and 2.5 mg/kg flunixin meglumine (Razak Laboratories from active material supplied by Nobrook, Ireland) was injected subcutaneously every 12 h postoperatively for 3 days for pain relief. To prevent infection, 15 mg/kg cefazolin (Dana, Tabriz, Iran) was injected subcutaneously every 12 h for 3 days. At 8 weeks, the rats were sacrificed using Halothane for histological and histomorphometric analyses. Bone and soft tissue were detached from the defect site. Specimens were fixed by immersion in 10% neutral buffered formalin for 1 day at room temperature and then demineralized by 1 week immersion in 7% nitric acid. Specimens were dehydrated and embedded in paraffin blocks. Sections were made perpendicular to the long axis of the defects, and the specimens were stained with hematoxylin and eosin according to the standard protocol. Figure 1

A 5 mm × 5 mm defect was created in the distal condyle of the femur (arrow).

Histological assessment

Analysis of histological data for bone healing at 8 weeks Table 2revealed that the healing score in AG group was significantly higher than that in the control and placebo groups (P = 0.036). No significant difference was noted between control and placebo groups (P = 0.680), but there were significant differences between control and AG groups (P = 0.038) and placebo and AG groups (P = 0.021) Figure 2. The mean healing score was 2.67 ± 0.49 in the control group, 2.58 ± 0.515 in the placebo group, and 3.17 ± 0.577 in the AG group. The histological pattern of healing scores is shown in Figure 3.{Table 2} Figure 2

The effect of aminoguanidine (AG) on healing score. The results showed a significant difference in the healing of bone in aminoguanidine group and the two other groups. * P= 0.038, ** P= 0.021.

This study showed that local application of AG into the bone defects accelerated bone healing. It appears that inhibition of NO synthesis at the initial phases of inflammation accelerates the process of healing in the following phases. A number of studies have evaluated the function of NO in the inflammatory process of the oral mucosa, ¹⁶ periodontal tissue, ¹⁷ , ¹⁸ dental pulp ¹⁹ and periapical region. ²⁰ Reactive oxygen and nitrogen species such as NO are abundantly found at the sites of inflammation and healing, but their function has yet to be fully understood. Considering the fact that the function of NO can affect different phases of healing, better understanding of the role of NO in pathophysiology of the healing process of bone defects can be useful for future pharmacological interventions. Thus, to better understand, the complex process of bone healing, the current animal study was conducted aiming to assess the role of NO in the process of bone healing.

The iNOS is not normally expressed in healthy noninflamed tissues. Some previous studies have demonstrated the pro-inflammatory role of NO in the pulp and periapical tissues. Kawanishi et al. ¹⁹ stated that NO might be responsible for infiltration of immune cells in pulpitis. da Silva et al., Law et al., and Fan et al. indicated higher concentrations of NOS enzyme in inflamed tissues compared to noninflamed sites. ² , ²¹ , ²² In an induced periapical lesion in a rat model, the NOS released from the macrophages, which had been stimulated by lipopolysaccharides, induced apoptosis of macrophages and osteoblasts and enhanced the progression of periapical lesions. ²⁰ It is not known whether NO is a nonspecific host defence mediator in the primary phase of healing or is a more specific controlling signal for successful completion of the healing process. ²³

In addition to the inflammatory phase, the effect of NO and AG on other components of the healing phases must be taken into account. Fibroblasts are the main cells in the healing phase. Fibroblasts present in the wound produce more NO than normal tissue fibroblasts. ²⁴ NO enhances the migration and proliferation of fibroblasts. ²⁴ Wang et al. ²⁵ showed that AG can be beneficial for growth and proliferation of fibroblasts. It appears that AG stops the cell division or prevents the entry of cells from the M or S phase into the G0 phase of cell cycle. The results of Wang et al. support our findings.

Blood supply is a critical factor influencing the healing of fractures ²⁶ and considering the role of NO in angiogenesis and vasodilation; it may be specifically important in bone healing. NO stimulates the proliferation of endothelial cells, protects them from apoptosis and mediates the production of vascular endothelial growth factor. ²⁷ NO produced by endothelial NOS (eNOS) has both pro- and anti-inflammatory properties. Under physiological conditions, NO released from the endothelium regulates vascular tonicity and maintains the vessels' patency through preventing platelet accumulation and decreasing the expression of adhesion molecules. ²⁸ Corbett et al. ²⁹ demonstrated that in the primary phase of fracture healing, the expression of eNOS in cortical blood vessels and osteoblasts reaches its maximum level. As the process of healing progresses, this expression returns to its baseline value. High levels of NO and maximum intensity of vascular reaction in the primary phase of healing are probably responsible for the increased blood flow during this period. ²⁹ , ³⁰ The correlation of angiogenesis and wound healing has yet to be confirmed. Increased blood flow in the inflammatory phase of healing results in delivery of higher levels of inflammatory mediators to the site which delay the process of healing. In contrast, angiogenesis seems necessary in the proliferative phase of healing. ³¹

The role of NO in wound healing is complex and multifactorial. Baldik et al. ³² showed that local administration of single dose bovine serum albumin containing NO along with demineralized bone matrix-induced new bone formation by 62% higher than that by bone matrix alone in femoral bone defects in rats at 10 weeks postoperatively. This finding was in contrast to our obtained results; which may be due to the different functions of NO at different concentrations. They also demonstrated that oral administration of AG enhanced defect filling. Giardino et al. ³³ explained that the positive role of AG in bone healing may be attributed to the protection against adverse and destructive effects of excessive NO production. Paul-Clark et al. ³⁴ in their study on a rat model evaluated the effect of direct and indirect administration of NOS inhibitors on the intensity of inflammation. In acute inflammation, delivery of NOS inhibitors directly into the site of inflammation aggravated the inflammatory response and prolonged the process of healing. Several mechanisms may explain the more severe inflammatory response. The inhibition of NO production increases the level of histamine, leukotriene B4, cytokine-induced neutrophil chemoattractant and free oxygen radicals, which indicates the protective role of local production of NO in the process of inflammation. On the other hand, systemic administration of NO inhibitors decreases inflammation. ³⁴ It appears that NOS inhibitors have different effects on the intensity of inflammation, depending on their method of administration. In the absence of acute inflammation, the use of local NO inhibitors did not increase the infiltration of inflammatory cells. This result was in line with our findings. In addition to the method of the administration of NO inhibitors, the prescribed dosage can also affect the intensity of inflammation and rate of healing. Leitão et al. ¹⁸ reported that 5 and 10 mg/kg doses of AG significantly decreased BR in rat experimental models with artificially induced periodontal lesions; whereas, 100 mg/kg dose of AG did not inhibit alveolar bone loss or local inflammatory changes. These results may be attributed to the fact that high concentrations of AG inhibit physiologic NOS. To achieve optimal effects, administration of NOS inhibitors at low doses may be more efficacious than the use of high doses. ³⁵ Farhad et al. ¹⁴ in their study on a cat model evaluated the effect of systemic administration of AG on the severity of inflammation and demonstrated that AG significantly decreased the severity of inflammation in periapical lesions. In another study, Farhad et al. ¹⁵ assessed the effect of systemic administration of AG on healing rate of periapical tissues and showed that selective NO inhibitor enhanced the healing of periapical lesions. The first phase of healing is based on an inflammatory process. Thus, enhanced recovery of periapical lesions in their study may be attributed to the suppression of local inflammation due to the inhibition of NOS in the periapical region. The results of the afore-mentioned two studies. ¹⁴ , ¹⁵ were in accordance with our findings. This indicates that the local application of AG may have the same desired effects as systemic administration of iNOS inhibitors without the unwanted systemic side effects.

Nonspecific NOS inhibitors such as L-NAME interfere with eNOS and neuronal NOS, which have important physiological functions. ³⁶ Thus, selective iNOS inhibitors are ideal for the alleviation of inflammatory responses with no adverse effects on physiological reactions. Considering the wide spectrum of biological functions of NO, its inhibition may bring about numerous systemic effects. Thus, in the clinical application of NOS inhibitors, local administration must be preferred to systemic administration. For this reason, the current study was designed to assess the process of healing with the local application of AG.

Commercially, available AG is supplied in the form of crystals. Thus, for application into bone defects, it requires a carrier for easy handling. On the other hand, if this material is applied to the bone defect alone, it is quickly washed out of the area by the tissue fluids and would only exert a short-term effect. To maintain the material in the desired location for a longer period, a slow-release gel base is required. AG is soluble in carboxymethyl cellulose polymer gel. To ensure the neutral state of this gel and that it has no effect on healing, the base gel alone (without AG) was applied to bone defects in the placebo group. The AG gel used in the current study gradually releases AG within 2 weeks. The expression of iNOS is the highest in the primary phase of acute inflammation ³⁷ and over time, the activity of iNOS probably decreases due to the elimination of inflammatory responses or cytokine signals. ³⁸ Thus, it can be assumed that the gel used in this study releases AG at the target site for a sufficient period.

This study presented evidence about the role of NO in the process of bone healing. The obtained results supported the local application of selective iNOS inhibitors such as AG for enhancement of bone healing. However, AG has yet to be used in the clinical setting and requires further investigations. AG may be suitable for future use in periapical surgeries involving bone defects. Future studies are required to investigate the local application of AG at lower concentrations to determine the minimum concentration of AG gel capable of significantly enhancing bone healing.

Acknowledgments

The authors would like to thank Dr. Abolfazl Aslani for the technical support and Dr. Mohammad Reza Maracy for statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or nonfinancial in this article.