Introduction

Medication-related osteonecrosis of the jaw (MRONJ) is a progressive condition with no spontaneous resolution that has garnered increasing attention in recent years and affects especially patients with metabolic bone conditions, such as osteoporosis and bone metastases [1, 2]. This adverse effect was initially related to bisphosphonates; however, cases reported of osteonecrosis associated with denosumab, angiogenic drugs, monoclonal antibodies (MABs), tyrosine kinase inhibitors (mTOR), selective estrogen receptor modulators (SERMs) and immunosuppressants have been reported [3]. An increase in the number of infusions and prolonged administrations of antiresorptive drugs results in MRONJ increased risk that reaches 8.4% in oncological patients [4], especially during the use of Zoledronic acid and Denosumab [5].

Treatment for MRONJ lesions remains a challenge due to its pathophysiology, which is not yet fully known. Both conservative and surgical treatments have been proposed depending on clinical disease degree and MRONJ clinical classification [6]. However, most of the time, treatment is integrated. It involves using antibiotics, local irrigation with chlorhexidine solution, local debridement, and segmental resection in cases of non-response to surgery or conservative procedures [6, 7]. Surgical procedures consist of superficial bone debridement or more radical interventions such as whole resection of the bone with or without reconstruction [8].

Different therapies have been reported as adjuvants or isolated treatments for MRONJ [9], such as pentoxifylline and tocopherol (vitamin E), ozone therapy, hyperbaric oxygen therapy, photobiomodulation (PBM) therapy, platelet-rich plasma (PRP) therapy, platelet-rich fibrin (PRF) application of recombinant human bone morphogenetic proteins (rhBMPs) [10], and antimicrobial photodynamic therapy (aPDT) [11,12,13,14].

Most studies of MRONJ treatments look for a complete response showing at least non-exposed bone [10, 11, 15, 16]. However, even reaching the closure of the mucosa on the top of the lesion, the bone regeneration to the treatments is also of relevance. Thus, in this scenario of several different treatment modalities for MRONJ lesions, we decided to study bone response to these treatments, which would be best analyzed in animal model. Moreover, in the literature comparisons were most done between treatment versus control, which makes difficult the choice of treatment modality. Therefore, this study aimed to compare the bone response of MRONJ lesions induced in rats to different treatments (PBM, aPDT, and PRF) combined with conventional bone debridement.

Materials and methods

This project was approved by the Animal Use Ethics Committee [blinded]. For the in vivo study, 30 six-week-old male Wistar rats, 160–222 g in weight received dietary supplement (Special Diet RC) feed and ad libitum water. In the postoperative period, the bran diet was always served to avoid the retention of larger grains in operated regions and to facilitate the ingestion and digestion of food. The experiments were conducted following the ethical recommendations of the Guide for Caring and Use of Laboratory for Animals [17] and The Animal Research: Reporting In vivo Experiments (ARRIVE) 2.0 guidelines [18].

Experimental model of induced MRONJ

All animals received intraperitoneally 0.06 mg/kg zoledronate (ZLN; Zometa®, Novartis Pharma, Basel, Switzerland) [19] once every seven days, being the first injection on the first day of treatment and the others until euthanasia. Twenty-one days after the first ZLN application the animals were submitted to extraction of the upper second molar of left or right side randomly defined. Fourteen days after tooth extraction the MRONJ lesions were randomly allocated in one of the 5 experimental groups (n = 6 animals in each group). These experimental groups were, as follows: negative control (NC): animals that had no clinical treatment for MRONJ; Positive control (PC): only surgical treatment (debridement); surgical treatment and application of aPDT (aPDT); surgical treatment and PBM therapy application (red and infra-red) (PBM); surgical treatment and PRF. The animals were euthanized 7 or 28 days after lesion treatment (n = 3 per group per period). The specimens obtained from the animals were submitted to MicroCT analysis to verify the bone volume, number, and thickness of bone trabeculae data at both times.

Surgical procedures

After 21 days of ZLN application, MRONJ was randomly induced by extraction of the maxillary second molars. To perform the surgery procedure, it was necessary to use the MC-M22 surgical microscope. After weighing, the animals were anesthetized with an intramuscular injection of Ketamine hydrochloride (50 mg/kg; Ketalar, Cristália, Itapira, SP, Brazil) associated with Diazepan (5 mg/kg; Valium, Cristália). Thereafter they received intraperitoneally 50 mg/ml Tramadol Hydrochloride (Tramadon®, Cristália, Sao Paulo, Brazil). Then, animals were fixed on and positioned with mouth openers made with orthodontic wire, followed by antisepsis of the surgical site with 0.12% chlorhexidine digluconate. All extractions were performed by the same researcher, with similar duration, material, and techniques. In case of root fractures or problems that made it impossible to tooth complete removal, the animal was not considered in the study.

Treatments

Debridement (all groups except NC)

Bone debridement surgeries were performed under general anesthesia following the same protocol previously mentioned in all animals except those of the negative control group (NC) where after MRONJ lesion induction no further treatment was done. Even so, all animals in this group were anesthetized and received analgesia along with other groups of animals. During the surgical procedure of the other groups (PC, aPDT, PBM, and PRF), oral and perioral disinfection, mouth opening, incision over the affected region, mucoperiosteal detachment, removal of all necrotic tissue was performed. Bone tissue was cured until bleeding, and the surgical local was irrigated with 0.9% saline. All animals received sutures with vicryl 6 − 0 (Ethicon®), and each group received further treatment, except the PC group (positive control) which received debridement treatment only.

Antimicrobial photodynamic therapy (aPDT)

For the aPDT treatment, an Indium-gallium aluminum phosphide diode laser (InGaAlP) (DMC®, São Carlos, SP, Brazil) was used after the placement of the photosensitizer, which was the methylene blue (MB) solution at 0.01% (Chimiolux, DMC, Brazil). The MB solution comes in syringes, and it was applied into the lesion in amount enough to fill the whole cavity. The pre-irradiation time was 5 min in contact with the area to be treated and then irradiation was applied continuously, punctually, and in contact. The parameters used were: 660 nm (red), 100 mW, spot size of 0.028 cm2, 3.57 W/cm2, 40 s, 142.8 J/cm2 e 4 J [20]. Figure 1 illustrates the aPDT procedures.

Fig. 1
figure 1

Illustrative photographs of the aPDT procedures. (A): MRONJ lesions after debridement (B): placement of the MB solution inside the lesion site (C): removal of the excessive dye by aspiration (D): Laser irradiation

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Photobiomodulation (PBM)

The PBM treatment was done with the same Indium-gallium aluminum phosphide diode laser used for aPDT. The PBM therapy included both red and infrared laser. For PBM application the same laser equipment used for aPDT was used in continuous, punctual, and in contact mode of operation. The parameters used were: 660 nm (red), 40 mW, spot size of 0.028 cm2, 1.43 W/cm2, 6 s, 8.6 J/cm2 e 0.24 J for red and 780 nm (infrared), 40 mW, 0.04 cm2, 1 W/cm2, 3 s, 3 J/cm2 e 0.12 J for infra-red [16, 21, 22]. Both wavelengths were applied simultaneously to stimulate dept (bone) and superficial (soft) tissues at once.

Three irradiations of each type were performed at 48-hour intervals. The first irradiation was performed immediately after debridement and suture and then at 48 and 96 h. For these last irradiations, the animals were lightly sedated with inhalation anesthetic (Isoforine®, Cristália, Itapira, São Paulo). The irradiations were made punctually (2 points/lesion) and in contact with the alveolus mucosa by buccal and palatine (Fig. 2).

Fig. 2
figure 2

Illustrative photographs of the PBM (A-B): Laser irradiation (2 points/lesion)

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The power of laser equipment was measured before and after all irradiation sessions, with the aid of a power meter (Laser Check, MM Optics Ltda, São Carlos, SP, Brazil). The emission value was programmed for the study parameter through this meter, not by the value on the equipment control panel. All laser procedures were performed following all safety standards NBR / IEC 601.2.22 and IEC 60825-1 / 2001-8.

Platelet-rich fibrin

The protocol for obtaining the PRF had a tail-warm water warming sequence (30 s in the microwave at full power) for vasodilation. Then a superficial puncture with a 15 C scalpel in the ventral region between the proximal and middle tail was performed, maintaining pressure on the distal portion to stimulate bleeding. Blood collection was performed with a sterile disposable pipette and transferred to an Eppendorf without exceeding one minute to prevent coagulation without the need for anticoagulants. The Eppendorf was placed in the microcentrifuge (Mini-Spin-Eppendorf, Germany) which was adjusted for centrifugation for 12 min at 3,000 revolutions per minute, and the PRF was obtained immediately after the procedure and placed in the animal socket (adapted from Ghanaati et al., 2014) [23]. (Fig. 3).

Fig. 3
figure 3

Illustrative photographs of the PRF procedures (A): Tail warm-water water (B-C): Microcentrifugation of blood punctured from the region between the proximal and middle tail (D-E): PRF obtained after the microcentrifugation (F): PRF in the animal socket

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Euthanasia

Animals were euthanized at seven and 28 days after MRONJ treatment. The maxilla was immediately removed with surgical scissors, carefully dissecting the soft tissue so as not to manipulate the bone defect region. Afterwards, the pieces were fixed in 10% formaldehyde solution for 24 h and after this period, kept in 70% alcohol until they were taken for MicroCT analysis.

Computerized microtomography (MicroCT)

High-resolution MicroCT includes histomorphometric analysis that can provide additional information on the variation of hard tissue radiopacity, as well as being a method that enables 3D analysis of tissue density and volume. The experimental times for this analysis were 7 and 28 days. The dissected jaws were taken to the high-resolution MicroCT (SkyScan 1176; Bruker microCT, Kontich, Belgium) located in the [blinded]. Digitalization followed the parameters adapted from Romão et al., 2015 described in Table 1 [24].

After scanning the images were reconstructed. Data were evaluated using appropriate software (CTAnalyser®, SkyScan, Antwerp, Belgium) for trabecular microstructure analysis from the region of interest (ROI) in binary images. For each socket where bone tissue volume formed was measured, the (bone volume (BV), the ROI was the area delimited by the distal root of the first molar and the mesial root of the third molar in the sagittal plane [25, 26].

The maximum gray scale threshold was 99 and the minimum was 55. With this threshold set, the program (Adaptive, CTAnalyser® and SkyScan) analyzed the following bone morphometric data: number, thickness, and separation of bone trabeculae, as well as relative bone formation through BV analysis.

Data analysis

All results were submitted to a statistical analysis program. As the data were parametric, the two-way ANOVA analysis of variance was applied, complemented by the Tukey test, with the significance level adopted at 5% (p ≤ 0.05).

Results

Fourteen days after tooth extraction it was clinically possible to observe changes in the mucosa at the area of extraction. Thus, after confirmation of bone necrosis, the animals were treated according to the treatments stipulated by groups, and all survived until the end of the experiments.

Conventional treatment considered as bone debridement in PC (positive control) group resulted in positive results in all observed parameters; however, adjuvant treatments, such as PBM and PRF improved bone regeneration. In the comparison between groups, the PRF group was superior in all the analyzed parameters and had an advance in the regeneration process already observed at 7 days. At 28 days, the PRF group also showed greater results in the three parameters analyzed when compared to the other groups (Fig. 4).

Fig. 4
figure 4

Illustrative microCT images of all groups at 7 and 28 days. The graphic of bone volume shows a significant decrease in the negative control (*). The highest BV was observed in both the PBM and PRF at 28 days (&) (p = 0.02). The smallest BV was observed at 28 days in NC (a) (p = 0.04). The other groups showed similar BV between themselves and when compared to control groups in both experimental times. The graphic of number of trabeculae shows that at 7 days the number was significantly higher in PRF compared to all other groups (*) (p = 0.03). At 28 days, the NC had the smallest number of trabeculae (a) (p = 0.02). At 7 days, the PRF had thicker trabeculae than all the other groups (p = 0.04) (*). At 28 days, PC and PRF showed thicker trabeculae than NC (p = 0.02) (a)

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Bone volume (BV)

In all groups, the bone volume (BV) showed a tendency to increase at 28 days when compared to 7 days, except in the negative control group (NC). In the negative control, the MRONJ lesion progressed, with increased bone loss (p = 0.04). The highest BV was observed in both the PBM and PRF at 28 days (p = 0.02). The other groups showed similar BV between themselves and when compared to control groups in both experimental times.

Trabeculae number

The trabeculae number at 7 days was significantly higher in PRF compared to all other groups (p = 0.03). At 28 days, NC presented significantly lesser trabeculae than all other groups (p = 0.02).

Trabeculae thickness

There was no change in trabeculae thickness between 7 and 28 days inside the experimental groups (p = 0.05). At 7 days, the PRF had thicker trabeculae than all the other groups (p = 0.04). At 28 days, PC and PRF showed thicker trabeculae than NC (p = 0.02).

Discussion

In recent decades the American Association of Oral and Maxillofacial Surgeons (AAOMS) has made efforts to establish a consensus on treating MRONJ. Nevertheless, there remains a lack of agreement regarding managing different stages of this lesion [6, 10]. Surgical therapy is reported to be a highly successful treatment for all stages of MRONJ; however, non-operative therapies are studied, especially for patients with comorbidities that make surgery impractical, and as a complement to bone debridement/resection, to increase the treatment effectiveness [6]. The PRF as an adjuvant therapy improved bone regeneration, superior to other treatment modalities, showing bone volume, trabeculae number, and thickness greater than isolated debridement.

The treatments for MRONJ focus on controlling pain, infections, and the progression of necrotic areas [27], in conjunction, in many cases, with bone reconstruction, necessary to recover basic functions and esthetic [6, 28]. Despite this, the success rate for conservative treatment is below 50% and surgical treatment ranges from 60 to 80% [29], therefore, more effective treatments are sought that are capable of reducing the inhibitory effects of antiresorptive medications and accelerating bone and soft tissue regeneration [27], to this end, is necessary to induce tissue regeneration through cellular chemotaxis, proliferation and differentiation, angiogenesis, and deposition of new extracellular matrix [30].

Among adjuvant therapies, PRF is being studied and showing promising preclinical evidence promoting the epithelialization of wounds [27, 31]. This therapy consists of an autologous matrix scaffold comprising a fibrin matrix, cytokines, platelets, leukocytes, and circulating stem cells [32, 33]. In addition to macrophages, involved in the process of resorption and osteogenesis [34]. PRF has also been demonstrated to promote bone regeneration by releasing cytokines and growth factors such as transforming growth factor-1 (TGF-β1), vascular endothelial growth factor (VEGF), bone morphogenetic protein-1 (BMP-1), platelet-derived growth factors (PDGFs), and insulin-like growth factors (IGFs) [34].

Additionally, clinical studies already demonstrated the possibility of PRF acting as a barrier membrane between alveolar bone, accelerating the closure of bone exposure [35], causing resolution of lesions [36], in addition to preventing the recurrence of MRONJ [37], including in association with bone debridement [32, 38]. This is associated with the microenvironment conducive to osteogenesis associated with the three-dimensional structure of PRF, which directs cell migration and capture of cytokines, in addition to the increase in fibronectin available in the PRF [34]. A clinical trial has also shown that this treatment could potentially prevent MRONJ [15].

In fact, in our study, the treatment with debridement and PRF of lesions induced greater bone volume and trabecular number and thickness than the isolated surgery in 7 days. This is especially relevant due to the delay in initial healing caused by bisphosphonates. This is demonstrated in preclinical studies, where a prominent decrease in the number of osteoclasts was observed 3–7 days after tooth extraction under the administration of bisphosphonates [39], in addition to a decrease in bone volume and vascularization [26, 39]. The improvement in initial healing by PRF is also supported by clinical studies, which show a reduction in postoperative pain 1–3 days after dental extractions in 66.6% of cases, soft tissue healing in 75% after 7 days, and a decrease in dimensional bone loss after 8–15 days [40].

On the other hand, adjuvant PRF with bone debridement is few studied. Jamalpour et al. observed that PRF (surgery + advanced platelet-rich fibrine/ leukocyte platelet-rich fibrine) reduced the areas of bone exposure, healing of extra and intraoral fistulas, mucosal healing, and improvement in bone remodeling in rats, however, only evaluating clinical, radiographic, and histological parameters [27]. To the best of our knowledge, this is the first study that compared 3 adjunctive therapies with parameters of bone volume, the number, and thickness of trabeculae in MRONJ treatment using micro-CT, which promotes 3D analysis of bone response [41].

PBM therapy uses a low-level laser that results in the improvement of cell metabolism, induction of synthesis of growth factors, increased migratory activity, and survival among other cellular activities of importance to bone regeneration [27, 42,43,44,45,46]. Furthermore, this therapy can improve bone healing [47] and the microarchitecture of new bone after tooth extraction [48, 49] also has been used in the post-surgical period [14].

Preclinical studies demonstrated that PBM decreased inflammatory cell counts on the alveolar bone of rats submitted to bisphosphonate-induced osteonecrosis of the maxillaries, indicating relief from post-extraction inflammation [50]. In the present study, we observed that combining bone debridement with PBM resulted in a higher bone volume compared to the untreated group. Furthermore, a significant increase in this parameter was observed within 28 days, distinguishing it from the other tested groups. Similarly, a study of photobiomodulation in bone regeneration of critical size defects in rabbits, demonstrated from micro-CT a significantly higher new formation at 28 days [51]. In the literature and our previous studies [22, 24, 47, 49]. PBM has shown improvement in bone regeneration not only in terms of bone volume but also in the number and distribution of trabeculae in sound and diseased bone. For this reason, we were expecting superior results in the PBM group; however, this group showed results similar to PRF only when the bone volume was analyzed but not in the other parameters studied. We believe that the double irradiation could be the cause of it, once in the other studies only an infrared laser was applied. Further studies comparing double irradiation with single wavelength irradiation should be done to clarify this point.

When associated with a photosensitizer, aPDT offers the benefit of antimicrobial action without the risk of inducing bacterial resistance or other advanced effects [52, 53]. Preclinical studies show that this therapy can prevent the occurrence of MRONJ [12, 54]. For this, the rats were subjected to post-extraction aPDT post-extraction surgery, using methylene or toluidine blue [12, 54], thus, a lower percentage of non-vital bone tissue and better repair tissue was observed [12, 54].

Clinical studies also demonstrate the effectiveness of aPDT in preventing MRONJ, reporting follow-up of 6 months, in addition to regression of lesions. aPDT was performed pre-surgery until decreased signs of inflammation, local infection, and pain, in addition to association with antibiotic therapy during severe infections. Following bone debridement, aPDT was repeated and continued every week until the resolution of the lesion [14]. This was different from our method, where aPDT was performed after debridement, and an improvement of all parameters was observed, however, it was not possible to identify a statistically significant difference. Therefore, it is important to comprehend the best type and concentration of the photosensitizer, irradiation parameters and protocol, including exposure time and energy released for the treatment of MRONJ, all of which significantly impact treatment success [54].

Our results reinforce that the absence of treatment resulted in the progression of the MRONJ lesion, highlighting the risk of progression to extensive lesions that cause extreme loss of facial structures. This is relevant, especially for asymptomatic patients, considered at-risk patients [6], emphasizing the importance of monitoring during the use of drugs with MRONJ potential, also due to the persistent and recurrent infections [55]. Overall, our results indicate that positive responses of the bone of MRONJ-induced lesions were obtained after PBM and PRF treatments. These treatments seemed to speed up the process of bone neoformation in MRONJ lesions, especially PRF, which improved bone volume, trabeculae number, and thickness. Further evaluation in randomized clinical trials is warranted.

Table 1 MicroCT scan parameters
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