Zoledronic acid (ZA) is an intravenous bisphosphonate (BP) with potent pharmacological action and affinity for bones, especially in the jaws, where high bone metabolism has been observed (Lawson et al., 2010). This drug has been recommended for treatment of osteoporosis, Paget's disease (bone), giant cell bone tumors, tumor-induced hypercalcemia, prevention of bone metastases from malignancies, and control of multiple myeloma (Nicolatou-Galitis et al., 2019).

Among all BPs, ZA has the highest binding affinity to bone tissue, and it is commonly associated with medication-related osteonecrosis of the jaw (MRONJ) (Bullock et al., 2021, Granate-Marques et al., 2019, Ruggiero et al., 2022). This pathological condition can be defined as current or previous treatment with antiresorptive agents (such as BP) alone or in combination with immune modulators or antiangiogenic medications and exposed bone or bone that can be probed through an intraoral or extraoral fistula(e) in the maxillofacial region for more than eight weeks, with no history of radiation therapy to the jaws or metastatic disease to the jaws (Ruggiero et al., 2022). Although the first case of BP-related MRONJ was reported in 2003 (Marx, 2003), the pathogenesis of this condition remains unclear.

The main risk factors involved in the development of MRONJ include the type of BP used (potency and route of administration), duration of treatment, local factors (infection or inflammation presence), dentoalveolar surgery (tooth extraction or dental implant placement), and systemic comorbidities (Gelazius et al., 2018, Ruggiero et al., 2022). Thus, the indication of osseointegrated dental implant placement in patients under ZA regimen remains controversial because intravenous administration of BP may increase a patient’s chances of developing MRONJ triggered by implant placement (Gelazius et al., 2018, Sher et al., 2021). Moreover, it was previously shown that patients with osseointegrated dental implants that start intravenous BP therapy have increased risk of developing MRONJ (Giovannacci et al., 2016).

Accordingly, patients with a history of BP regimen, regardless of whether it is used for osteoporosis or bone malignancy management, present a risk of MRONJ developing after dental implant placement (Sher et al., 2021). Furthermore, no biomarkers are validated for clinical decision-making, which means that none bone turnover markers can predict MRONJ risk (Ruggiero et al., 2022).

Although the effect of ZA on osteoclasts has been well-established, its effects on other cell types have not yet been fully elucidated. Studies have shown that this drug causes cytotoxic effects in osteoblasts, decreasing the mineralization capacity of such cells, which are directly involved in bone matrix synthesis and mineralization processes, as well as inhibitory effects on osteoblast differentiation, which may favor the development of MRONJ (Basso et al., 2013, Patntirapong et al., 2012). The role of soft tissues in the pathogenesis of MRONJ has also been assessed, since the main features of this condition are the loss of soft-tissue covering and bone exposure (Reid et al., 2007). Thus, soft-tissue toxicity by BP has been indicated as one possible factor capable of favoring MRONJ.

In addition, investigations have demonstrated that ZA could enhance matrix metalloproteinases (MMPs) levels by gingival fibroblasts in a dental implant surface in vitro model (Basso et al., 2021, Pansani et al., 2021), in alveolar bone in an in vivo model (Basi et al., 2011), and in saliva from patients who developed MRONJ (Thumbigere-Math et al., 2015). Hence, increased synthesis and activity of MMPs may contribute to MRONJ establishment in patients under ZA therapy. MMPs are enzymes responsible for degradation and turnover of most extracellular matrix (ECM) components, presenting the ability to cleave fibrillar collagens in normal physiological conditions; however, in pathological conditions, such as periodontal diseases, the imbalance between MMPs and their tissue inhibitors (TIMPs) could enhance ECM degradation and tissue destruction (Paiva & Granjeiro, 2017). In periodontal tissue, TIMPs are not sufficient to down-regulate the pathologically elevated MMPs (Ingman et al., 1996); thus, different strategies for MMPs regulation are being investigated, such as the use of flavonoids.

Flavonoids are bioactive compounds that comprise an important class of low-molecular-weight plant secondary metabolites, presenting a polyphenolic structure and widely occurring in fruits, vegetables, herbs, beverages, spices, and oils (Ramesh et al., 2021). Among the different flavonoids, the potential of naringenin (NA) and proanthocyanidins (PA) for regulating MMPs has been demonstrated in pre-clinical models (Cardoso et al., 2022, Chang et al., 2017, Deziel et al., 2010, Fan et al., 2017, Nawrot-Hadzik et al., 2021; Villas Boas SB, 2021), as has their potential for promoting increased cell viability and modulation of pro-inflammatory mediators by gingival fibroblasts and oral keratinocytes (Cardoso et al., 2022; Villas Boas SB, 2021). NA and PA are found in different natural sources, and depending on their origin, cultivation conditions, climate, and extraction protocol, these flavonoids may have different formulations, resulting in variable therapeutic effects, which might result in the absence of standardized results (Oroian & Escriche, 2015). Thus, flavonoids whose synthetic variables can be commercially obtained, like NA, are interesting candidates for the use in biological analyses.

Therefore, the aim of this study was to assess the individual effects of pre-treatment with natural PA, from grape seed extract, and synthetic NA on the synthesis of MMP-2 (gelatinase A), MMP-9 (gelatinase B), TIMP-1, TIMP-2, and gelatinolytic activity of MMPs by human gingival fibroblasts and osteoblasts exposed to ZA in a dental implant surface in vitro model.