Emerging therapeutic strategies targeting bone signaling pathways in periodontitis

Abstract

Periodontitis is a multifactorial immune‐mediated disease exacerbated by dysregulated alveolar bone homeostasis. Timely intervention is crucial for disease management to prevent tooth loss. To successfully manage periodontitis, it is imperative to understand the cellular and molecular mechanisms involved in its pathogenesis to develop novel treatment modalities. Non‐surgical periodontal therapy (NSPT) such as subgingival instrumentation/debridement has been the underlying treatment strategy over the past decades. However, new NSPT approaches that target key signaling pathways regulating alveolar bone homeostasis have shown positive clinical outcomes. This narrative review aims to discuss endogenous bone homeostasis mechanisms impaired in periodontitis and highlight the clinical outcomes of preventive periodontal therapy to avoid invasive periodontal therapies. Although the anti‐resorptive therapeutic adjuncts have demonstrated beneficial outcomes, adverse events have been reported. Diverse immunomodulatory therapies targeting the osteoblast/osteoclast (OB/OC) axis have shown promising outcomes in vivo. Future controlled randomized clinical trials (RCT) would help clinicians and patients in the selection of novel preventing therapies targeting key molecules to effectively treat or prevent periodontitis.

Anti‐resorptive, immunomodulatory, and emerging therapeutic approaches to treat periodontal disease and promote resolution of inflammation.

1. INTRODUCTION

Periodontitis is a chronic, osteolytic disease characterized by the loss of supporting tissues of the dentition including the periodontal ligament, cementum, and alveolar bone (the periodontium). ¹ , ² , ³ Epidemiologic studies indicate that periodontitis is one of the most common inflammatory non‐communicable diseases (NCDs) and it is reported to be the sixth most prevalent disease condition in the world with at least 10% of the global adult population suffering from the severe form of periodontitis. ⁴ , ⁵ , ⁶ , ⁷ , ⁸ This multifactorial disease is caused by the impaired crosstalk between the host and the microbial biofilm, promoting an environment that favors bone resorption. ⁹ , ¹⁰ , ¹¹ , ¹² The well‐known pathogenic components of oral biofilm are the gram‐negative anaerobic species Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) that alter the resident commensal population important in maintaining gingival tissue homeostasis. ¹³ , ¹⁴ The chronic inflammation due to uncontrolled expression of virulence factors, viz. potent osteoclast stimulators such as Pg‐encoded Arg and Lys‐gingipain cysteine proteinases and Aa‐derived heat shock proteins, accelerates bone resorption and stimulates inflammation by fibroblast‐induced IL6 resulting in an imbalance in osteoblasts (OB; bone‐forming cells) and osteoclasts (OC; bone‐resorbing cells) activity impairing alveolar bone homeostasis. ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ Figure 1 describes the anatomic differences between healthy and inflamed gingiva. It is noteworthy that although periodontitis is generally considered an aging‐associated disease, young populations (≤35 years of age) are also experiencing this chronic disease with greater frequency. ¹⁹ , ²⁰ Furthermore, it is well reported that periodontitis is associated with other chronic diseases such as rheumatoid arthritis, neurologic disorders, cardiovascular disease, preterm low birth weight, diabetes, and cancer. ²¹ , ²² , ²³ Thus, new directions of periodontal research and novel treatment strategies are urgently needed. This narrative review aims to provide an update on the current concepts regarding dysregulated cellular and molecular mechanisms associated with the OB/OC imbalance impairing alveolar bone homeostasis in periodontitis and to provide an update of new preventive and regenerative therapeutic approaches.

FIGURE 1.

The anatomic structure of the periodontium in healthy and inflamed gingiva. Healthy gingiva is firmly attached to the tooth surface. The periodontal ligament, cementum, gingiva, and alveolar bone remain intact without impairment in their anatomic structure. However, in inflamed gingiva, periodontal pocket formation is the cardinal sign of periodontitis due to the dysbiosis caused by pathogenic biofilm. Thereafter, an overreactive immune‐inflammatory response is observed by the recruitment of neutrophils in the periodontal pocket along with an infiltration and proliferation of myeloid and lymphocyte cells ultimately leading to clinical manifestations of redness, edema, and bleeding.

2. OVERT IMMUNE RESPONSES TRIGGER PERIODONTAL PATHOPHYSIOLOGY

Alveolar bone homeostasis is regulated by the coupled crosstalk of OB/OC wherein the rate of bone formation and resorption are in equilibrium. Therefore, overall bone mass does not change. ¹⁶ , ²⁴ , ²⁵ , ²⁶ In periodontitis, the impaired OB/OC balance occurs due to overt immune activity against the dysbiotic microbiome and shifts the equilibrium of bone metabolism toward pro‐resorption. ¹⁶ , ²⁴ , ²⁵ , ²⁶ The prolonged exposure to virulence factors from oral biofilm components triggers an exuberant immune response which initiates molecular and cellular pathways activating alveolar bone loss. ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² Figure 2 shows the activation of uncontrolled innate immune responses that occur in periodontitis. Clinical manifestations of mucosal inflammation of gingival tissues are evident such as edema, redness, and bleeding due to the impaired intrinsic molecular mechanisms that control periodontal pathology. ³³ , ³⁴ , ³⁵ Dysbiosis is not the only cause of periodontitis. Diverse non‐modifiable risk factors including age, gender, genetic/epigenetic networks (polymorphisms in immune pathway‐related genes, e.g., IL‐1A, IL‐1B, IL‐6, IL‐10, MMP‐3, MMP‐9) are significantly associated with the risk of developing periodontitis. Modifiable factors such as systemic inflammatory diseases (obesity, diabetes, and others), diet, environmental, and behavioral factors (e.g., smoking and stress) can further exacerbate the disease progression, severity, or the outcomes of periodontal treatments. ³⁶ , ³⁷

FIGURE 2.

Uncontrolled activation of innate immune response occurs in periodontitis. (1) Oral biofilm composed of periodontal pathogens induce oral dysbiosis. (2) Virulence factors released by dysbiotic bacteria such as, gingipain proteases (Porphyromonas gingivalis) and heat shock protein (Aggregatibacter actinomycetemcomitans) (3) recruit neutrophils to the inflamed gingiva resulting in uncontrolled pro‐inflammatory cytokine released by keratinocytes, dendritic cells, and fibroblasts, (4) inducing an overreactive innate immune response due to (5) the increased release of pro‐inflammatory cytokines and metalloproteinases (MMPs) by macrophages and (6) overactivation of CD4+T cells, involved in the polarization of T‐helper subtypes and B cells. (7) The uncontrolled increase in levels of pro‐inflammatory cytokines, MMPs and upregulation of TH1 and TH17 promote bone resorptive cells and induction of OC.

A well‐functioning periodontal homeostatic mechanism requires an efficient system of bone homeostasis supported by periodontal ligament cells, gingival fibroblasts, junctional epithelial cells, keratinocytes, neutrophils, macrophages (Mφ), dendritic cells (DC), and lymphocytes for the maintenance of a healthy periodontium ³² , ³³ , ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² (Figure 2). However, in inflamed gingiva, dysbiosis‐induced metalloproteinase (MMP) activity accelerates collagen degradation in gingival connective tissue, junctional epithelium, and periodontal ligament leading to periodontal pocket formation accompanied by a significant increase in inflammatory cell infiltration. ³⁴ , ³⁵ , ⁴³ , ⁴⁴ Neutrophils located in the gingival sulcus are the first line of defense secreting α‐defensins to clear the colonizing periodontopathogenic bacteria. ⁴⁵ , ⁴⁶ , ⁴⁷ , ⁴⁸ Neutrophils phagocytose bacteria and contain copious amounts of reactive oxygen species (ROS) and antimicrobial agents stored in the granules driving pathogen clearance by intracellular killing. ⁴⁹ , ⁵⁰ In addition to these killing mechanisms, neutrophils deploy neutrophil extracellular traps (NETs), a network of extracellular fibers comprised of granule proteins and chromatin, aimed at preventing bacterial adhesion and invasion of host tissues. ⁵¹ , ⁵² NETs can be found within the purulent gingival crevicular exudate and play a vital role in the clearance of periodontal pathogens by trapping dispersed subgingival plaque. ⁵³ In response to dysbiosis, neutrophils show delayed apoptotic activity with impaired antibacterial function and uncontrolled immune response. ⁴⁵ , ⁴⁶ , ⁴⁷ In addition, keratinocytes release pro‐inflammatory mediators such as β‐defensins while fibroblasts, DC and keratinocytes secrete proinflammatory cytokines such as IL‐1β (Interleukin‐1 Beta), IL‐6 (Interleukin 6), IL‐23 (Interleukin 23), IL‐8 (Interleukin 8), and TNFα (Tumor Necrosis Factor Alpha) promoting recruitment of neutrophils, Mφ, and lymphocytes to the site of infection. This vicious cycle of autoinflammatory signal amplification, in turn, stimulates inflammatory Mφ to secrete copious amounts of pro‐inflammatory cytokines and MMPs that activate osteoclastogenesis and collagen degradation during disease progression. ⁵⁴ , ⁵⁵ , ⁵⁶ , ⁵⁷ , ⁵⁸ DC perform a dual role as both phagocytes and antigen‐presenting cells (APCs). APCs activate CD4+ T cells (CD4 T lymphocytes or “helper T cells”) to various pathogenic effector phenotypes. ⁵⁹ Cheng et al. ⁶⁰ demonstrated that uncontrolled upregulation of Th1 (Type 1 T helper) and Th17 (Type 17 T helper) cell pathways also promote the induction of OC, giving rise to areas of future research. Further, monocytic myeloid‐derived suppressor cells (M‐MDSCs) are positively associated with T cell immunosuppressive and osteoclastogenic activity in the context of periodontal health and disease. ⁶¹ A high percentage of M‐MDSCs express macrophage colony‐stimulating factor (M‐CSF) receptor (c‐fms). In turn, this c‐fms+ subpopulation is considered an osteoclast precursor and in the presence of RANKL (Receptor activator of Nuclear Factor Kappa Ligand) and MCSF, this subpopulation can differentiate into osteoclasts and promote bone loss in periodontitis. ⁶²

In summary, overt immune responses due to the upregulated pro‐inflammatory status drive impaired OB/OC crosstalk exacerbating periodontitis.

3. PERTURBATION OF OSTEOBLAST–OSTEOCLAST CROSSTALK OCCURS IN PERIODONTITIS

OB are of mesenchymal origin and their activation requires a sequential activity of transcription factors ¹⁷ , ⁶³ , ⁶⁴ , ⁶⁵ (Figure 3; Box 1‐dark yellow color). For instance, Runx2 (osteoblast‐specific factor/core binding factor) gene activates the expression of osteocalcin (OCN), bone sialoprotein (BSP), osteopontin (OPN), and collagen synthesis to ultimately activate the expression of bone‐related proteins such as bone morphogenetic protein (BMP‐2) and transforming growth factor beta (TGF‐β). ¹⁷ , ⁶³ In conjunction with Runx2, osterix (OSX) plays a key role in periodontal tissue remodeling, including differentiation and formation of OB and cementoblasts. OSX also regulates the expression of OCN, osteonectin (ON) and BSP, genes that regulate OB activity, collagen binding, and ECM calcification. ⁶⁶ , ⁶⁷ Transcription factor Krüppel‐Like Factor 4 (KLF4) promotes dentinogenesis and odontoblastic differentiation by modulating the TGF‐β signaling pathway, while Forkhead box protein O1 (FoxO1) has been reported as a crucial mediator in bone homeostasis by providing a favorable environment for OB proliferation and differentiation ⁶⁸ (Figure 3; Box 2‐Dark blue color). Conversely, Wnt acts on OB precursor cells to promote their differentiation into mature OB through the β‐Catenin‐dependent canonical pathway. However, the observed dysregulation of the Wnt/β‐Catenin pathway is a critical aspect during the progression of periodontitis ⁶⁹ , ⁷⁰ , ⁷¹ (Figure 3; Box 3‐brown color). In addition to TFs, non‐protein signaling molecules like nitric oxide (NO; a free radical mediator) and prostaglandin E2 (PGE2; a lipid mediator) also contribute to bone metabolism by regulating RANKL pathway ⁷² (Figure 3; Box 4‐red color). The biological effects of NO on bone metabolism vary based on concentration. For instance, low NO may support osteoblast bone formation and OC‐mediated bone remodeling, while at higher concentrations, NO inhibits OC formation and bone resorption. ⁷³ RANKL activates inducible nitric oxide synthase (iNOS) to enhance NO levels in OC thereby inhibiting osteoclastogenesis. ⁷⁴ PGE2 induces RANKL expression in OB in an autocrine and paracrine manner via activation of the prostaglandin E2 receptor 4 (EP4), exerting regulatory action on angiogenesis and vascular permeability thus modulating bone metabolism. ⁷⁵

FIGURE 3.

Uncoupling mechanism between osteoclasts and osteoblasts in periodontitis. The downregulation of OPG promotes an enhanced interaction of RANK and RANKL resulting in OC differentiation and activity. Pro‐inflammatory cytokines (IL‐1, IL‐11), MMPs, hormones (PTH), vitamin D3, inflammatory lipids (PGE2) can also promote OC differentiation and bone resorption. In addition, OB/OC communication by EFNB2‐EFNB4, FAS–FASL, and NRP1‐SEMA3A are disrupted, promoting altered alveolar bone homeostasis in periodontitis.

OB exhibit autocrine and paracrine activity that regulates activation of osteogenic as well as osteoclastic factors. Indeed, BMPs and TGF‐β control autocrine activity by promoting differentiation and activation of OB (Figure 3; Box 5‐green color), while colony‐stimulating factor‐1 (CSF‐1), granulocyte colony‐stimulating factor (G‐CSF), basic fibroblast growth factor (basic FGF), insulin‐like growth factor (IGF), and RANKL control paracrine activity involved in OC differentiation and activation ¹⁷ , ⁷⁶ , ⁷⁷ (Figure 3; Box 6‐dark pink color). In periodontitis, the autocrine function of OB is inhibited favoring differentiation and activation of OC due to OB‐associated paracrine function. ⁷⁸ , ⁷⁹

OC are multi‐nucleated, bone‐resorbing cells of myeloid origin. Activation of OC is regulated by the expression of IL‐1β, IL‐6, IL‐23, TNFα, and specific MMPs secreted by Mφ (Figure 3; Box 7‐ gray color). This facilitates the interaction of RANKL (expressed on OB) with its receptor RANK (Receptor of Nuclear Factor‐kappa) (expressed on OC precursors), promoting the differentiation and activation of mature OC ⁸⁰ , ⁸¹ , ⁸² , ⁸³ (Figure 3; Box 8‐gold color). In brief, the binding of M‐CSF to its receptor, c‐Fms, induces the transcription factor c‐Fos, whereas the binding of RANKL to its receptor, RANK, leads to the recruitment of TNF‐receptor‐associated factor 6 (TRAF6), the main adapter molecule of RANK. TRAF6 activates nuclear factor κB (NF‐κB) and mitogen‐activated kinases (MAPKs) including c‐Jun N‐terminal kinase (JNK). In turn, JNK activates the transcription factor c‐Jun. RANKL/RANK also induces c‐Fos to form activator protein 1 (AP‐1), a heterodimeric transcription factor, which along with c‐Jun. AP‐1 and NF‐κB then induce the nuclear factor of activated T cells 1 (NFATc1), a master transcription factor that regulates OC differentiation. NFATc1 works with other transcription factors, such as AP‐1, PU.1, and microphthalmia‐associated transcription factor (MITF) to induce various osteoclast‐specific genes. ⁸⁴ Conversely, expression of osteoprogeterin (OPG), a RANKL decoy that prevents differentiation and activation of OC, is downregulated in periodontitis. This favors interaction between RANKL and RANK receptor to promote OC differentiation and maturation. Similarly, OPN and ON expression are downregulated in periodontitis suggesting reduced OB cell activity. ⁸⁴ , ⁸⁵ , ⁸⁶ , ⁸⁷ Furthermore, parathyroid hormone (PTH) is secreted by the parathyroid glands and contributes to the maintenance of calcium levels in the blood (Figure 3; Box 9‐ Light yellow color). The upregulation of PTH or continuous treatment with PTH has been associated with an increase in OC number and activity due to an altered RANKL/OPG ratio. ⁸⁸ Vitamin D3 possesses anti‐inflammatory properties and plays an essential role in calcium regulation and bone metabolism. In periodontitis, downregulation of vitamin D3 is associated with an increase in OC‐associated bone resorption. ⁸⁹ Prostaglandin (PG) E2 (PGE‐2) and IL‐1β are important bone remodeling mediators. However, their upregulation stimulates the production of MMPs in myeloid cells and oral epithelial cells which in turn, induces RANKL expression in OB, promoting the differentiation and activation of OC via RANKL/RANK interaction. ⁹⁰ , ⁹¹ Paradoxically, IL‐11 exerts pro‐inflammatory effects by triggering periodontitis in the earlier stages of the disease but in the advanced stages of disease, an anti‐inflammatory role for this molecule has been observed. Indeed, in a dog model of periodontitis, treatment with IL‐11 resulted in the resolution of periodontal inflammation by downregulating the expression of pro‐inflammatory cytokines such as IL‐6, IL‐8, and TNFα. ⁹²

Synchronized communication between OB/OC is essential for bone homeostasis ²¹ , ²² (Figure 3; Box 10‐Light blue color). Ephrin2‐Ephrin4 (EFNB2‐EPHB4), FAS–FASL, and NRP1‐SEMA3A (Neuropilin‐1‐Semaphorin 3A) are the key molecules that fine‐tune OB/OC crosstalk. ⁹³ , ⁹⁴ EFNB2 and EPHB4 are expressed on the cell surface of OB and OC, respectively, and are bidirectionally involved in bone remodeling. OB differentiation is promoted by EFNB2‐mediated activation of EPHB4. However, the reverse signaling EPHB4‐mediated activation of EFNB2 suppresses OC differentiation. ⁹³ The efficient crosstalk between OC‐expressed FAS and OB‐expressed FASL plays an integral role in regulating apoptosis in FAS‐expressing cells, and their dysregulation is associated with tumorigenesis and auto‐immune diseases. Estrogens trigger the upregulation of FASL expression in OB resulting in apoptotic pre‐OC. However, in estrogen‐deficient mice, the downregulation of FASL expression resulted in reduced OC apoptosis associated with increased levels of bone resorption. ⁹⁴ NRP‐1‐SEMA3A expressed in OC and OB, respectively, has a role in alveolar bone homeostasis. Indeed, their interaction inhibits RANKL‐induced OC differentiation and promotes OB differentiation through the Wnt/β‐catenin pathway. ⁹⁵ Notch signaling pathways and their regulators (Notch1‐3) are key factors in periodontitis. ⁹⁶ , ⁹⁷ In healthy gingiva, Notch signaling controls OC differentiation and bone‐resorbing activity by targeting OC precursors and/or acting directly on OB by activating their differentiation. NOTCH1 inhibits OC through the RBPJk (recombination signal binding protein for immunoglobulin kappa J region) canonical pathway, and by enhancing OPG levels and Wnt signaling in OB and osteocytes (Figure 3; Box 11‐Light pink color). NOTCH2 induces OC through NF‐κB and HES1‐dependent (hairy and enhancer of split 1) mechanisms and by increasing RANKL in OB, while NOTCH3 promotes OC by enhancing RANKL levels in OB and osteocytes ⁹⁸ (Figure 3; Box 12‐purple color). In periodontitis, a significant increase in NOTCH2 (exerting pro‐OC function), TNF‐α, IL‐17, and RANKL was observed, while Notch1 and Jagged1 levels were decreased compared to healthy controls. Higher levels of TNF‐α and RANKL contribute to increased alveolar bone resorption by increasing the expression of RANK in OC precursors and RANKL expression in OB. ⁹⁶ , ⁹⁷ In summary, impaired crosstalk between OB and OC impacts bone homeostasis due to dysfunctional OB proliferation, differentiation, and survival. ²¹ , ²² , ²⁵

Uncontrolled bone resorption by OC occurs in periodontitis (Figure 3; Box 13‐orange color). In brief, the first step of bone resorption is the acidification of bone matrix composed of more than 90% type I collagen (osteoid) and hydroxyapatite (bone mineral composed of calcium and phosphate crystals). ⁹⁶ , ⁹⁷ , ⁹⁸ During the bone resorption process, OC forms unique cytoskeletal structures termed the sealing zone (SZ) and ruffled border (RB) that are essential to the bone resorption process. ⁹⁶ Once the SZ is attached to bone surface, vacuolar H⁺‐adenosine triphosphatase (H + ‐ATPase) located on the RB membrane secrete H⁺ to acidify the resorption lacuna formed underneath the RB. ⁷⁴ , ⁹⁶ , ⁹⁸ To counter the acidic pH driven by H⁺‐ATPase, the chloride voltage‐gated channel 7 (CLCN7), also located in the RB, secretes Cl⁻ to maintain electroneutrality. ⁹⁶ , ⁹⁷ , ⁹⁸ Thereafter, cathepsin k (CTSK), a member of the papain superfamily of cysteine protease, degrades the organic component of bone. ⁹⁶ , ⁹⁷ , ⁹⁸ Along with CTSK, MMPs, DC‐specific transmembrane protein (DC‐STAMP), tartrate‐resistant acid phosphatase (TRAP), and OC‐associated receptor (OSCAR) regulate the final steps of bone degradation. ⁹⁶ , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ Degraded products generated during this process are endocytosed from resorption lacuna and released through the apical membrane of the OC. ⁹⁶ , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ Once the bone resorption process is completed, apoptotic signals are activated in OC or a new bone resorption cycle ensues. ⁹⁶ , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ In periodontitis, impaired OC activation triggers additional and irreversible alveolar bone resorption cycles due to the overt dysbiosis‐driven immune response. ¹³ , ¹⁴ , ¹⁵ , ⁴³ In summary, the understanding of immunopathology and osteobiology of periodontitis is essential for developing novel treatment protocols.

4. THE EVOLUTION OF PERIODONTITIS TREATMENT FROM CONVENTIONAL TREATMENTS TO CURRENT NON‐SURGICAL PERIODONTAL THERAPY (NSPT)

Alveolar bone loss is a hallmark of periodontitis, and its prevention is a key clinical challenge in successful management of the disease. ¹ , ² , ³ , ⁷ , ⁸ , ⁹ , ²⁴ , ²⁵ Current treatment strategies focus on controlling adverse systemic health factors (ex. poor glycemic control for diabetics), behavior modification (smoking cessation, establishing a good oral hygiene habit, etc.), and mechanical plaque biofilm and calculus debridement. In advanced stages of disease, periodontal surgery is employed; however, the discussion of which is beyond the scope of this article. Excellent reviews have been published on the subject. ¹⁰¹ Unfortunately, these approaches do not always restore bone homeostasis and often result in limited success. ¹⁰² Recurrence of the disease is also common. Therefore, the development of novel therapeutic approaches is imperative to prevent overt periodontal inflammation and the cascade of events leading to periodontitis.

4.1. Treatment protocols to treat periodontitis

Importantly, mechanical debridement (scaling and root planing (SRP), “deep cleaning”) with or without the use of adjunctive antimicrobials such as systemic antibiotics (amoxicillin, metronidazole, azithromycin, and doxycycline) or topical (sulcular) application of antimicrobial agents (minocycline microspheres and chlorhexidine chip) is a viable therapeutic approach aimed at eliminating etiological factors of disease and initiating resolution of periodontal inflammation. ¹⁰³ , ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶ , ¹⁰⁷ , ¹⁰⁸ , ¹⁰⁹ , ¹¹⁰ Randomized clinical trials have indicated that treatment with amoxicillin and metronidazole adjuncts to SRP demonstrates a significant improvement in clinical outcomes. ¹¹¹ , ¹¹² , ¹¹³ Unfortunately, non‐surgical therapeutic approaches have not changed significantly over the last 2–3 decades and depend on the removal of etiological factors in a closed environment relying on the tactical feel of calculus, rather than visualization, which can be challenging. In advanced cases, periodontal surgery using resective or reconstructive approaches is indicated. ¹⁰⁰ , ¹⁰¹ In brief, resective periodontal surgery is based on the principles of surgical exposure, removal of etiological agents, recontouring of affected alveolar bone to create physiologic bony contours, and removal of excessive soft periodontal tissue from periodontal pockets. ¹¹⁴ Although this approach recreates a more favorable periodontal environment, it does not result in a gain of clinical attachment level (CAL), so a gingival recession is common. Alternatively, reconstructive (regenerative) periodontal surgery utilizes bone graft materials (autogenous, allograft, xenograft, alloplastic), membranes (resorbable, non‐resorbable), and/or biomimetics (PDGF‐BB, amelogenins, etc.) to regenerate damaged periodontal tissues and CAL. ¹¹⁵ Unfortunately, regenerative periodontal therapies are not always successful for advanced cases of disease, and specific periodontal treatment planning is required for different individuals. Therefore, new therapeutic strategies are needed to prevent or restore bone alveolar homeostasis at the earlier stages of the disease without the need for periodontal surgery. This shift toward prevention or restoration of alveolar bone homeostasis in periodontitis will unravel new research avenues in the field.

4.2. Overview of alternative non‐surgical therapeutic strategies for periodontitis

Concerns regarding antibiotic resistance have promoted new research and clinical investigation of alternative periodontal treatment strategies. Indeed, novel therapeutic approaches are oriented toward the identification of key molecular targets whose inhibition or activation results in reduced periodontal inflammation and return to physiologic alveolar bone homeostasis. New therapeutic adjuncts to SRP have been proposed as alternate treatment strategies to target inflammatory mediators and bone‐resorbing osteoclasts and are discussed below.

4.2.1. Bisphosphonates

Bisphosphonates are chemically stable derivatives of inorganic pyrophosphate (PPi) with anti‐resorptive properties that impair OC differentiation and activity thus inhibiting bone resorption ¹¹⁶ , ¹¹⁷ , ¹¹⁸ (Figure 4A). Bisphosphonates target mature OC by inhibiting farnesyl diphosphate synthase (FPP) impairing OC function and driving apoptosis. ¹¹⁶ Commonly used bisphosphonates to treat osteolytic diseases such as osteoporosis include zoledronate and alendronate. ¹¹⁶ , ¹¹⁷ , ¹¹⁸ Although oral and topical treatment with bisphosphonates has achieved successful outcomes in periodontitis patients, adverse clinical events have been noted. Osteonecrosis of the jaw (ONJ) is a result of prolonged use of bisphosphonates promoting an area of exposed alveolar bone, delayed wound healing, and necrosis of affected bone. ¹¹⁹ Thus, the risks and benefits of periodontal surgery should be carefully considered in these patients. In a rat model of ligature‐induced periodontitis (LIP), treatment with alendronate (2.5 mg/kg) resulted in reduced OC activity along with reduced alveolar bone loss but after 21 days of treatment, ONJ manifestations in the maxilla region were indicative of adverse biological effects of prolonged drug treatment. ¹²⁰ Conversely, another LIP study in rats treated with alendronate resulted in beneficial clinical outcomes exhibiting reduced inflammation and robust tissue repair. ¹²¹ To support preclinical observation, numerous clinical trials have been published over the last decade. Bisphosphonates as adjuncts to SRP have demonstrated expected clinical outcomes for short‐term periodontitis therapy, but concerns regarding ONJ limit their long‐term clinical use. ¹²⁰ Rocha et al. (2001), evaluated the effect of alendronate (10 mg/daily) for 6 months in a short‐term randomized controlled pilot trial with patients diagnosed for diabetes and established periodontitis. This RCT showed an improvement in the alveolar bone loss by decreasing the cementoenamel junction (CEJ) to alveolar bone crest distance. No metabolic parameter changes were noted at any time during the duration of the RCT. ¹²² In line with this, an RCT in postmenopausal women (without hormone replacement therapy) tested the efficacy of alendronate (10 mg/day) on periodontal disease. After 6 months of treatment with alendronate, the bone turnover ratio was improved in postmenopausal women with established periodontitis by reducing the BOP and gingival bleeding without any observed changes in the hormone levels. ¹²³ Lane et al. (2005) evaluated the efficacy of bisphosphonate therapy as an adjunct to SRP and 3‐month periodontal maintenance therapy in patients with moderate‐to‐severe chronic periodontitis. After 1 year, the SRP + bisphosphonate group demonstrated greater clinical improvements in BOP, CAL, and probing depth (PD) compared with the SRP alone. ¹²⁴ Graziani et al. (2009) observed similar clinical outcomes (BOP, CAL, and PD) in an RTC testing the efficacy of the bisphosphonates and SRP in patients with moderate‐to‐severe chronic periodontitis. This RCT indicated that bisphosphonates may be an appropriate adjunctive treatment by preserving the periodontal bone mass. ¹²⁵ Sharma et al. (2012) tested the efficacy of local delivery of 1% alendronate gel as an adjunct to SRP in chronic periodontitis patients for 6 months. The RCT results showed that local delivery of 1% alendronate into the gingival sulcus of diseased sites led to a significant decrease in PD and BOP and a gain in CAL compared to the placebo group. ¹²⁶ Similarly, patients with chronic periodontitis and type 2 diabetes (T2D) treated with local delivery of a 1% alendronate gel as an adjunct to SRP showed improvements in the bone mass‐related parameters (decreased PD, BOP, and gain in CAL). ¹²⁷ While the results presented above seem promising, a systematic review by Alwithanani ¹²⁸ compiled the RCTs mentioned above and concluded that although bisphosphonate therapy is a useful adjunct to periodontal treatment, the practical application of these agents in clinical practice is tempered by the possibility of developing ONJ. Furthermore, these drugs are not metabolized but are either excreted renally or deposited within bone and remain in the body for decades. The longest duration of placebo‐controlled clinical trials is 6 years and subjects in these observational studies have used the newer amino‐bisphosphonates for 10 year. Of note, there is no known method of removing these agents from bone tissues. As such, these medications exhibit profound effects on bone physiology that are not well recognized, along with uncertain long‐term consequences. ¹²⁹ Further studies detailing the safety and efficacy of bisphosphonate therapy are required before clinical recommendations can be made.

FIGURE 4.

Anti‐resorptive non‐surgical therapy adjuncts to scaling root and planing. The anti‐resorptive drugs: (A) Bisphosphonates, (B) OPG‐Fc and RANKL inhibitors, and (C) Strontium Ranelate, adjuncts to SRP inhibit OC differentiation and activity, driving a reduced bone resorption. However, some adverse clinical events have been observed due to long‐term administration of these drugs. ONJ is observed in long‐term treatment with bisphosphonates, OPG‐Fc, and RANKL inhibitors. To date, no ONJ cases have been reported during SR treatment.

4.2.2. OPG‐Fc and RANKL Inhibitors

OPG and RANKL are key molecules regulating the differentiation and activation of mature OC. ⁸¹ , ⁸² , ⁸³ , ⁸⁴ , ⁸⁵ , ⁸⁶ As mentioned above, the OPG/RANK/RANKL axis is impaired in periodontitis, driving a shift from OPG/RANKL to RANK/RANKL interaction and promotion of OC differentiation and maturation. ⁸¹ , ⁸² , ⁸³ , ⁸⁴ , ⁸⁵ , ⁸⁶ The discovery of these molecules and their crosstalk have spurred the development of new therapeutic strategies targeting periodontitis in both animal models and human clinical trials. ¹³⁰ , ¹³¹ , ¹³² , ¹³³ , ¹³⁴ , ¹³⁵ Recent in vivo studies have revealed the therapeutic potential of OPG. Teng et al. ¹³⁰ observed significant inhibition of alveolar bone resorption by targeting OPG. Indeed, mice inoculated with the periodontal pathogen A. actinomycetemcomitans and treated with OPG showed reduced alveolar bone loss associated with a decreased number and activity of mature OC ¹³⁰ (Figure 4B; upper panel). Denosumab, an anti‐RANKL monoclonal antibody, has been tested to treat osteoporosis and metastatic bone cancer ¹³¹ (Figure 4B; lower panel). It inhibits RANKL by mimicking OPG expressed in OB and regulates differentiation and activation of OC. ¹² , ¹³³ In periodontitis, bone turnover is disrupted due to overt bone resorption by OC. ¹⁶ , ²⁴ , ²⁵ , ²⁶ Denosumab treatment restores bone turnover similar to that observed with bisphosphonate therapy; however, shorter half‐life of Denosumab (generally 6 months or less) provides a pharmacological window that allows for safer surgical procedures, such as dental extractions or flap surgeries, compared to the long‐lasting effects of bisphosphonates, which can complicate such treatments. ¹³² However, similar to bisphosphonate therapy, adverse events such as denosumab‐related ONJ (DRONJ) have been reported. ¹³³ , ¹³⁴ A retrospective study showed that DRONJ is related to many risk factors such as invasive dental procedures (tooth extraction or dental implant surgery), poor oral hygiene, corticosteroids, smoking, anemia, diabetes, or renal failure. Identification of these risk factors in periodontitis patients may be a diagnostic marker for DRONJ. ¹³⁵ Perhaps, such drugs can be used as personalized dental treatments after further study.

4.2.3. Strontium ranelate

Another recent antiresorptive drug used in osteoporotic menopausal women is strontium ranelate (SR), composed of strontium salt of ranelic acid. SR is a dual‐action bone agent with concomitant antiresorptive and osteoanabolic properties which represents an advantage over bisphosphonates ¹³⁶ , ¹³⁷ , ¹³⁸ , ¹³⁹ , ¹⁴⁰ , ¹⁴¹ , ¹⁴² (Figure 4C). In brief, one of the mechanisms of action results from the interaction of strontium cations (Sr²⁺) with extracellular calcium‐sensing receptors (CaSRs) on mature OC‐inducing apoptosis. Concurrently, SR impacts CaSRs expression on OB leading to the activation of bone‐forming signaling pathways, such as TGFβ, BMPs, etc., driving the proliferation of new OB. ¹³⁹ Additionally, SR increases the expression of OPG and concomitantly reduces RANKL expression in OB. ¹³⁷ Karakan et al. (2017) tested three different doses of SR (300, 625, and 900 mg/kg) administered daily by oral gavage in a rat model of periodontitis for 11 days after ligature placement. Interestingly, SR treatment decreased bone loss, which was attributed to reduced numbers of OC and a concomitant increase in OB. ¹⁴⁰ Despite the contrasting effects, SR is a potent inhibitor of OC as observed by reduced alveolar bone loss in experimental animal models of periodontitis. ¹⁴⁰ , ¹⁴¹ , ¹⁴² Controlled clinical studies in humans is required before SR can be used as a therapeutic adjunct.

4.2.4. CTSK inhibitors

Oral antiresorptive therapies have yielded new approaches targeting impaired host immunity in periodontitis. ¹⁴³ CTSK is a member of the papain‐like cysteine protease family playing a role in the innate immune response and regulating OC‐mediated bone resorption. ¹⁴⁴ After OC activation, CTSK is released into the resorption lacuna leading to bone resorption. ⁹⁶ , ⁹⁷ , ⁹⁸ CTSK degrades type I collagen fibers (90% of the bone organic matrix) and cleaves and activates MMP9 leading to type II collagen degradation. ⁹⁶ , ⁹⁷ , ⁹⁸ CTSK is recognized as an important bone resorption marker and potential therapeutic target. ¹⁴⁴ CSTK inhibitors are among the most promising immunomodulatory drugs with the potential to restore alveolar bone homeostasis in periodontitis. ¹⁴⁴ , ¹⁴⁵ Inhibition of CTSK regulates bone resorption by inhibiting OC activity rather than reducing OC viability and without affecting OB activity, thereby restoring alveolar bone turnover. ¹⁴⁶ Numerous CTSK inhibitors are currently undergoing human clinical trials for the treatment of multiple diseases including osteoporosis, autoimmune disorders, metabolic diseases, and others. However, adverse events such as cardiovascular and cerebrovascular accidents have been reported ¹⁴⁶ , ¹⁴⁷ (Figure 5). In experimental periodontitis, the use of CTSK inhibitors has shown beneficial outcomes. ¹⁴⁸ , ¹⁴⁹ , ¹⁵⁰ , ¹⁵¹

FIGURE 5.

Use of Cathepsin K (CTSK) inhibitors to prevent or treat periodontitis. Balicatib, Odanacatib, adeno‐associated virus‐sh‐CTSK (AAV‐sh‐CTSK), BML‐224 and CsinCPl‐2 are therapeutic strategies targeting CTSK to treat periodontitis. Balicatib and Odanacatib have been tested to treat osteoporosis with insightful clinical outcomes reducing bone resorption. However, osteonecrosis on the jaw (ONJ) cases has been reported in Balicatib treatment and cardiovascular and cerebrovascular events have been observed in Odanacatib treatment. Odanacatib treatment in a mice model of periodontitis has resulted in positive outcomes by targeting innate immune signaling pathways such as TLR4, 5, and 9 pathways. Other experimental drugs targeting CTSK, such as AVV‐sh‐CTSK, BML‐224, and CsinCPl‐2, have reported therapeutic value to treat periodontitis by reducing inflammation and bone‐resorptive cell activity.

Balicatib (formerly AAE581, Novartis) is a CTSK inhibitor with high selectivity properties targeting cathepsin B, L, and S. Balicatib was tested in ovariectomized monkeys over 1 year of treatment. ¹⁵² The anti‐osteoclastic activity was reflected by an increase in the expression of bone markers and bone mineral density in the spine, femur, and hips. In a phase I clinical trial, a 25 mg dose was well tolerated in ovariectomized monkeys inducing significant CTSK repression and new bone formation. ¹⁵³ In postmenopausal women with osteopenia/osteoporosis, a clinical trial was discontinued at phase II due to the cutaneous lesions, such as morphea‐like skin or skin rashes and adverse pulmonary events. ¹⁵⁴ To date, Balicatib treatment has not been reported in periodontitis models.

Odanacatib (formerly MK‐0822, Merck & Co.) is a CTSK inhibitor with high selectivity for cathepsin K. ¹⁵⁵ Odanacatib was tested in ovariectomized monkeys with successful outcomes as observed by significant bone formation without any negative impact on bone turnover. ¹⁵⁶ Odanacatib reached phase III clinical trials in a randomized, double‐blind study with 16 713 osteoporotic postmenopausal women using a 50 mg dose once weekly for 3 years. ¹⁵⁶ Although long‐term treatment with Odanacatib during phase III of the clinical trial increased bone mineral density, trabecular volumetric, and strength of spine and hip compared with placebo, it was discontinued due to an increase in cerebrovascular accidents and adverse cardiovascular events. ONJ, adverse respiratory infections, or systemic sclerosis were not noted. ¹⁵⁷ , ¹⁵⁸ , ¹⁵⁹ Wild‐type female BALB/cj mice challenged with P. gingivalis to induce periodontitis for 56 days and treated with Odanacatib showed a decrease in the numbers of OC, Mφ, and T cells. ¹⁵¹ , ¹⁵² , ¹⁶⁰ Thus, Odanacatib exhibits immunomodulatory properties in conjunction with its anti‐resorptive activity. Indeed, a significant inhibition of downstream Toll‐like receptor (TLR) signaling pathways was observed in gingival epithelial cells indicating that Odanacatib regulates the innate immune response in periodontitis. ¹⁶⁰

Other recent experimental treatments inhibiting CSTK expression include injections of small interfering RNA (siRNA), adeno‐associated virus (AAV), CTSK‐specific inhibitors, BML‐244, or CsinCPI‐2 highlight the therapeutic potential of CTSK inhibitors to treat periodontitis. ¹⁶¹ , ¹⁶² , ¹⁶³ Chen et al., ¹⁶⁴ showed that AAV expressing small interfering RNA (siRNA) (AVV‐sh‐CTSK) silenced CTSK expression in a mice model of periodontitis induced by oral gavage with Pg. Local Injections of AVV‐sh‐CTSK into the palatal gingiva for 7 days showed a significant reduction in gingival inflammation and less alveolar bone resorption. In a double mice model of rheumatoid arthritis (RA) induced by collagen and Pg infection‐induced periodontitis, respectively, injections of AAV containing siRNA‐CTSK in the knee joint and inflamed gingiva resulted in a significant reduction of inflammation in the synovium and alveolar bone loss, respectively. Another study conducted by Pan et al. ¹⁶⁰ using a combinatorial periodontitis and RA model treated with the CTSK inhibitor, BML‐224 showed similar outcomes including anti‐inflammatory responses and reduced alveolar bone loss. Da Ponte Leguizamón et al. ¹⁶¹ demonstrated in vivo therapeutic effects of the phytocystatin CsinCPI‐2, derived from Citrus sinensis. In a murine LIP model, systemic treatment of CsinCPI‐2 resulted in a reduced inflammatory response and less bone resorption due to inhibition of OC differentiation and activation as reflected by low RANKL‐induced TRAP+ cells. ¹⁶¹

A key limitation of CTSK inhibitors is that expression of CTSK is not restricted to bone‐forming cells. Thus, the use of these CTSK inhibitors has been associated with a poor diagnosis of metabolic and immunoregulatory processes. ¹⁵⁰ , ¹⁵¹ , ¹⁵² Importantly, the dual function of CTSK inhibition, anti‐inflammatory and anti‐resorptive, suggests a promising therapeutic strategy. While CTSK inhibitors show promise in treating osteoporosis and other bone diseases by inhibiting bone resorption, their development has been challenged by safety concerns. Further research is needed to overcome these challenges and to develop safe and effective CTSK inhibitors. ¹⁶²

4.2.5. Specialized pro‐resolving mediators

Specialized pro‐resolving mediators (SPM) are endogenous immunoregulatory lipid molecules that play crucial roles in the resolution of periodontal inflammation. ¹⁶³ , ¹⁶⁴ , ¹⁶⁵ , ¹⁶⁶ , ¹⁶⁷ , ¹⁶⁸ , ¹⁶⁹ , ¹⁷⁰ , ¹⁷¹ , ¹⁷² , ¹⁷³ , ¹⁷⁴ SPM are oxylipids mediators, including resolvins, maresins, lipoxins, and protectins, derived from omega‐3 polyunsaturated fatty acid. ¹⁶³ , ¹⁶⁴ , ¹⁶⁵ Experimental studies with SPM have demonstrated enhanced gingival tissue repair and clearance of oral pathogens (Figure 6). This included robust innate and adaptive immune responses by promoting monocyte infiltration, activation of Mφ, inhibition of neutrophil infiltration, and cytokine production driving the resolution of inflammation. ¹⁶³ , ¹⁶⁴ , ¹⁶⁵ , ¹⁶⁶ Indeed, E‐series resolvins (RvE1) interact with BLT1 expressed on neutrophils and chemR23 expressed on Mφ, monocytes, and DC inhibiting their infiltration and cytokine production. ¹⁶⁶ , ¹⁶⁷ The main function of SPM is to restore tissue homeostasis and recent animal studies have shown its potential therapeutic value in periodontitis treatment. For instance, LIP transgenic mice overexpressing human chemR23 (Gao et al., 2013), LIP male Wistar rats (Lee al., 2016), and studies with LIP male rabbits followed by Pg infection (Hasturk et al., 2006; Hasturk et al., 2007) treated with local RvE1 resulted in reduced inflammatory bone resorption associated with a reduced OC activity. ¹⁶⁸ , ¹⁶⁹ , ¹⁷⁰ , ¹⁷¹ Hasturk et al. (2021) tested the efficacy of Lipoxin A4 (LXA4), an SPM that acts via GPC‐receptor, and observed a significant reduction in inflammation in patients with gingival inflammation by modulating inflammatory immune pathways. The phase 1 RCT indicated that after 28 days of treatment with an oral rinse containing a LXA4 mimetic, methyl ester‐benzo‐lipoxin A4 (BLXA4), the level of gingival inflammation was substantially reduced along with a reduction of periodontal pocket thereby suggesting LXA4 as a potential novel therapeutic treatment for periodontitis. ¹⁷² Mizraji et al. ¹⁷³ tested the efficacy of RvD2 in BALB/c mice in a Pg‐induced experimental periodontitis model for 8 weeks and observed significantly reduced inflammation and alveolar bone loss in mice with experimental periodontitis. Qualitative assessment of the above‐mentioned animal studies ¹⁶⁸ , ¹⁶⁹ , ¹⁷⁰ , ¹⁷¹ , ¹⁷² , ¹⁷³ was performed by a systematic review using the ARRIVE guidelines (arriveguidlines.org) ¹⁷⁴ and noted that Resolvins demonstrate efficacy in the treatment of periodontitis by inhibiting the destructive inflammatory process and loss of alveolar bone in laboratory‐induced periodontitis under controlled experimental conditions. ¹⁷⁴

FIGURE 6.

Immunomodulatory therapy adjuncts to scaling root and planing. Non‐surgical immunomodulatory therapies: (A) Specialized pro‐resolving mediators (SPMs) and (B) Biological therapies targeting IL‐6, TNFα improves alveolar bone homeostasis by decreasing inflammation, regulating immune responses, and reducing OC activity.

4.2.6. Biological therapies

Inhibitors of pro‐inflammatory mediators TNFα, IL‐6, IL‐1β regulating inflammatory responses have been tested in experimental models of periodontitis ¹⁷⁵ , ¹⁷⁶ , ¹⁷⁷ , ¹⁷⁸ , ¹⁷⁹ (Figure 6). A recent study analyzed the anti‐inflammatory effects of tocilizumab, a monoclonal antibody targeting IL‐6, in a mice model of LIP. Results demonstrated a significant reduction in bone resorption, gain of tooth attachment, and reduced inflammatory responses. ¹⁷⁶ Multiple studies have demonstrated that TNFα, a key signaling modulator of OC activation in periodontal pathology, is an excellent therapeutic candidate. ¹⁷⁷ , ¹⁷⁸ , ¹⁷⁹ Indeed, two studies by the same group evaluated the effects of blocking TNFα activation by systemic administration of Etanercept in mice with concomitant diabetes mellitus (DM) and periodontitis. ¹⁷⁷ , ¹⁷⁸ After 5 weeks of treatment, results indicated that anti‐TNFα treatment improved the metabolic process in mice with DM and reduced alveolar bone resorption. Another feasibility study in a rat model of Pg LPS‐induced periodontitis, treated with AAV expressing TNF receptor immunoglobulin Fc (TNFR:Fc), reported reduced pro‐inflammatory responses and OC activity. ¹⁷⁸ Similar results were observed in another study using pentoxifylline as a TNFα blocker in a mice model with concomitant arthritis‐associated periodontitis. ¹⁷⁹ Treatment with this drug demonstrated reduced joint inflammation, reduced pro‐inflammatory responses, decreased infiltration of neutrophils, and diminished OC activity. Since periodontitis is a complex chronic inflammatory disease often associated with other chronic comorbidities, a combinatorial use of inhibitors targeting different inflammatory cytokines may yield better clinical outcomes, specifically in the later (moderate–severe) stages of the disease. Finally, it is important to evaluate systemic side effects or significant adverse events associated with inhibitors of pro‐inflammatory mediators in mice models of periodontitis. However, new approaches based on site‐restricted biologics, which do not affect immune activity in other non‐target tissues/organs, are highly desirable.

4.2.7. Non‐coding RNAs

Dysregulation of non‐coding RNAs targeting key osteolytic genes may contribute to the impairment of alveolar bone homeostasis and the progression of periodontitis. Multiple studies have shown the potential therapeutic benefits of targeting microRNAs (miRNA) in in vitro and in vivo models of periodontitis. ¹⁸⁰ , ¹⁸¹ , ¹⁸² Epigenetic regulation by miRNA can modulate target gene expression through post‐transcriptional mechanisms. ¹⁸¹ , ¹⁸² , ¹⁸³ , ¹⁸⁴ Several miRNAs have been reported to exhibit immunomodulatory functions in the pathobiology of periodontitis and its progression, along with a promising value as diagnostic markers ¹⁸¹ , ¹⁸² , ¹⁸³ , ¹⁸⁴ , ¹⁸⁵ , ¹⁸⁶ , ¹⁸⁷ , ¹⁸⁸ , ¹⁸⁹ , ¹⁹⁰ , ¹⁹¹ , ¹⁹² , ¹⁹³ , ¹⁹⁴ , ¹⁹⁵ ; Figure 7. Multiple reports have demonstrated a pathophysiologic role of miR‐146 in periodontitis, where levels are significantly increased compared to healthy gingiva. ¹⁸⁴ , ¹⁸⁵ , ¹⁸⁶ , ¹⁸⁷ Mechanistically, overexpression of miR‐146 is correlated with a significant decrease of pro‐inflammatory cytokines, IL‐1β, IL‐6, IL‐8, and TNFα by targeting NF‐κB. ¹⁸⁴ , ¹⁸⁵ , ¹⁸⁶ , ¹⁸⁷ Reduction in the levels of pro‐inflammatory cytokines attenuates the differentiation of bone‐resorbing cells. ¹⁸⁶ Thus, miR‐146 acts as a negative feedback regulator of periodontal inflammation and may be of significant therapeutic value to be further tested in future clinical trials. ¹⁸⁷ Similarly, miR‐21 and miR‐200c have been shown to exert anti‐inflammatory and anti‐osteoclastic functions by suppressing NF‐κB signaling and RANKL/OPG. ¹⁸⁸ , ¹⁸⁹ , ¹⁹⁰ Conversely, miR‐223, a hematopoietic cell‐specific miRNA, has been reported to play a role in alveolar bone loss in periodontitis. ¹⁹¹ High levels of miR‐223 promoted differentiation and recruitment of neutrophils and osteoclastogenesis while inhibiting OB differentiation by targeting nuclear factor 1‐A (NF1‐A). ¹⁹¹

FIGURE 7.

Therapeutic targeting of non‐coding RNAs in periodontitis. miR‐146 and miR‐21 have an anti‐inflammatory role in mice models of periodontitis. Both target NFκB, reducing pro‐inflammatory cytokine expression and preventing OC differentiation and activation. miR‐200c exerts an anti‐inflammatory role in a rat model of periodontitis by targeting IL‐6, IL‐8, and Interferon‐1 related development regulator (IFRD1) and by reducing the RANKL/OPG ratio. miR‐223 has an inflammatory role in periodontitis. High levels of pro‐inflammatory miR‐223 impair bone turnover by inhibiting bone‐forming cells and promoting OC differentiation and activity. miR‐26a‐5p has an anti‐inflammatory role in human‐inflamed gingiva. High levels of miR‐26a‐5p are associated with resolution of inflammation by targeting PLCβ1 which is associated with the production of pro‐inflammatory cytokines. Mice model of LIP induces the overexpression of Malat‐1 and downregulation of miR‐30 expression by Malat‐1 sequestration. MALAT‐1 is upregulated while miR‐30b, an anti‐inflammatory miRNA, is downregulated causing induced expression of pro‐inflammatory macrophages, impaired coordinated innate immune responses, and unsuccessful resolution of inflammation.

Expression of miR‐142‐3p is downregulated in inflamed gingiva. ¹⁹² , ¹⁹³ , ¹⁹⁴ , ¹⁹⁵ Valverde et al., ¹⁹⁴ demonstrated that miR‐142‐3p targets multiple genes (including Vinculin, Dab2, and Skap2) that regulate cytoskeletal rearrangement and cell movement. In vivo, delivery of miR‐142‐3p in a murine LIP model showed reduced pro‐inflammatory cytokine (IL‐6 and TNFα) levels, neutrophil infiltration, and Th17 polarization [Valverde et al., Unpublished results]. In subjects with periodontitis, miR‐26a‐5p is downregulated in inflamed gingiva, while its cognate mRNA target PLCβ1 expression was upregulated compared to healthy control subjects. After non‐surgical therapy (SRP), expression of miR‐26a‐5p and PLCβ1 returned to levels observed in healthy subjects and correlated with improved clinical parameters suggesting their diagnostic and prognostic value. miR‐26a‐5p targets pathways related to host defense and wound healing, which are critical in periodontal pathogenesis and its resolution. ¹⁹⁵

Besides miRNAs, long noncoding RNAs (lncRNAs), which are defined as regulatory non‐protein coding RNAs longer than 200 nucleotides, have recently gained attention in periodontal pathobiology. ¹⁹⁶ , ¹⁹⁷ , ¹⁹⁸ , ¹⁹⁹ , ²⁰⁰ , ²⁰¹ , ²⁰² , ²⁰³ , ²⁰⁴ LncRNAs regulate transcriptional, post‐transcriptional, translational, and epigenetic changes by physically interacting with DNA, RNA, miRNA, or proteins. ¹⁹⁶ , ¹⁹⁷ , ¹⁹⁸ , ¹⁹⁹ , ²⁰⁰ , ²⁰¹ , ²⁰² , ²⁰³ , ²⁰⁴ Several studies have indicated a pathological role of lncRNA in chronic inflammatory diseases including periodontitis. ¹⁹⁶ , ¹⁹⁷ , ¹⁹⁸ , ¹⁹⁹ , ²⁰⁰ , ²⁰¹ , ²⁰² , ²⁰³ , ²⁰⁴ For instance, Li et al., (2022) demonstrated the anti‐inflammatory role of lncRNA Nron in experimental periodontitis. Localized delivery of Nron in OC‐specific Nron KO mice inhibited alveolar bone resorption by promoting nuclear transport of NF‐kB repressing factor (NKRF), which significantly repressed Nfatc1 and induced resolution of inflammation. ¹⁹⁹ Zhu et al. ²⁰⁰ reported that lncRNA APDC, an ortholog of human lncRNA ANRIL, plays an anti‐inflammatory role in periodontitis progression as observed by exacerbated bone loss and overt pro‐inflammatory cytokine expression in KO mice subjected to experimental periodontitis.

Studies from our laboratory have demonstrated the role of lncRNAs MALAT‐1, LIN01010 LRRC75A‐As1, and GAPLINC in periodontitis ²⁰¹ , ²⁰² , ²⁰³ , ²⁰⁴ ; Figure 7. In both periodontitis subjects and in vivo periodontitis murine model, MALAT1 was upregulated, while miR‐30b was downregulated in inflamed gingiva. MALAT1 sequesters miR‐30b by direct interaction to promote the inflammatory M1 Mφ phenotype as observed by higher M1 marker expression. Furthermore, MALAT1 knockdown attenuates bacterial phagocytosis, antigen processing, and inflammatory cytokine secretion. ²⁰² Similarly, Valverde et al. ²⁰³ demonstrated a novel function of myeloid cell‐specific lncRNAs, LRRC75A‐As1 and GAPLINC. Both lncRNAs were observed to be upregulated in inflamed gingiva of periodontitis subjects. RNAi of LRRC75A‐As1 and GAPLINC resulted in a significant reduction of M1 Mφ expression markers without an impact on M2 markers. Thus, LINC01010 s can regulate key innate immune activities and polarization of Mφ, which are critical players in periodontitis progression and its resolution. ²⁰⁴

Together these findings indicate an important role of miRNAs and lncRNA‐miRNA regulatory networks in inflammatory pathways involved in periodontitis and could serve as novel therapeutic targets in periodontitis treatment or diagnosis. Future studies focusing on pre‐clinical models and clinical trials testing the validity of noncoding RNAs as a therapeutic option to treat or diagnose periodontitis are needed.

4.2.8. Antimicrobial photodynamic therapy (aPDT)

Antimicrobial photodynamic therapy (aPDT) is another alternative therapeutic approach based on the principle that visible light activates a non‐toxic molecule called photosensitizer, resulting in the generation of reactive oxygen species that non‐specifically kills bacteria via an oxidative burst. ²⁰⁵ , ²⁰⁶ , ²⁰⁷ , ²⁰⁸ , ²⁰⁹ , ²¹⁰ , ²¹¹ , ²¹² , ²¹³ , ²¹⁴ However, studies have shown that aPDT is less aggressive in removing the commensal biofilms compared to regular systemic antibiotic therapy. ²⁰⁵ , ²⁰⁶ , ²⁰⁷ , ²⁰⁸ , ²⁰⁹ , ²¹⁰ , ²¹¹ , ²¹² , ²¹³ , ²¹⁴ Multiple split‐mouth RCTs have reported that the use of aPDT as an adjunct to SRP, or when aPDT is applied multiple times in periodontitis patients, improves periodontal diagnostic indicators including a substantial decrease in BOP due to a reduction in periodontopathogens burden and associated inflammatory mediators (TNFα and IL1β) thus promoting restoration of periodontal homeostasis. ²⁰⁵ , ²⁰⁶ , ²⁰⁷ , ²⁰⁸ , ²⁰⁹ , ²¹⁰ , ²¹¹ , ²¹² , ²¹³ , ²¹⁴

4.3. Emerging treatment modalities for periodontitis

Alternative therapies using the above‐described anti‐resorptive and immunomodulatory drugs require tailored approaches to treat periodontitis. Novel therapeutic adjuncts to SRP include probiotics, statins/fibrates, vitamins, and omega‐3 fatty acids. ¹⁰⁶ , ²¹⁵ , ²¹⁶ , ²¹⁷ , ²¹⁸ , ²¹⁹ , ²²⁰ , ²²¹ , ²²² , ²²³ , ²²⁴ , ²²⁵ , ²²⁶ , ²²⁷ , ²²⁸ , ²²⁹ , ²³⁰ , ²³¹ , ²³² , ²³³ , ²³⁴ , ²³⁵ , ²³⁶ , ²³⁷ , ²³⁸ , ²³⁹

As adjuncts to SRP, probiotics are new therapies targeting periodontopathogens aimed at the restoration of the healthy oral microbiome and thus a healthy periodontium. ¹⁰⁶ , ²¹⁵ , ²¹⁶ , ²¹⁷ , ²¹⁸ , ²¹⁹ , ²²⁰ The World Health Organization has defined probiotics as “living microorganisms that, when administered in appropriate amounts, confer health benefits to the host”. ²¹⁵ Lactobacillus and Bifidobacterium are the most common probiotics used in periodontitis treatment. ²¹⁸ , ²¹⁹ Probiotics regulate the host's inflammatory response by secreting their own antimicrobial peptides or anti‐inflammatory molecules. In a recent systematic review, 10 RCTs assessed the impact of probiotics on clinical, microbial, and immunological outcomes. ²¹⁷ , ²¹⁸ , ²¹⁹ , ²²⁰ , ²²¹ As an adjunct to SRP, significant clinical benefits with greater PD reductions and CAL were noted after 12 months.

Studies indicate that periodontitis patients are more likely to have unfavorable serum lipid profiles and hence higher levels of inflammation, compared to periodontally healthy individuals. ²²² , ²²³ , ²²⁴ The most commonly prescribed oral lipid‐lowering agents are statins and fibrates. Statins are used in patients with hypercholesterolemia while fibrates are useful for decreasing serum triglyceride levels. ²²² , ²²³ , ²²⁴ , ²²⁵ Statins are inhibitors of HMG‐CoA reductase with anti‐inflammatory, anti‐coagulant, and antioxidant properties. ²²¹ The anti‐inflammatory properties of statins have stimulated interest in the treatment of periodontitis. ²²² Clinical investigations have reported that subgingival application of statin gels (atorvastatin, simvastatin, or rosuvastatin) as adjuncts to SRP significantly reduced levels of pro‐inflammatory mediators. Hence, an increase in anti‐inflammatory marker expression and a reduction in immune cells infiltration and bone resorption were noted. ²²² Further, a retrospective cohort study of over 50 000 patients reported a 25.7% reduction of chronic periodontitis risk for >3 years in patients prescribed with a combinatorial treatment of statins and fibrates. ²²⁰

The role of nutrition in host‐modulation to prevent oral diseases and/or as an adjunct to periodontal therapy has gained attention. Recent studies have described the current understanding of the impact of impact of the diet supplemented with vitamins and other micro/macronutrients on preventing and treating periodontitis. ²²⁶ For instance, dietary nitrate and omega‐3 fatty acids have been proposed to suppress periodontal inflammation by different mechanisms. A recent RCT reported that subjects with generalized stage III or IV periodontitis receiving high doses of omega‐3 polyunsaturated fatty acids for 6 months resulted in an improved resolution of inflammation as noted by decreased PD and bleeding on probing (BOP). ²²⁷ , ²²⁸ , ²²⁹ , ²³⁰ , ²³¹ However, the long‐term benefits of periodontal health remain unclear. In experimental periodontitis, animals fed with a dietary inorganic nitrate exhibited higher levels of NO, an osteoprotective and immunosuppressive free radical. This finding correlated with reduced pro‐inflammatory cytokine IL‐1β levels, endothelial inflammatory pathways, and tissue oxidative stress. Of note, higher anti‐inflammatory cytokine IL‐10 levels were also reported. ²³²

Resolvins are specialized SPM derived from omega‐3 fatty acids with anti‐inflammatory properties that suppress neutrophil infiltration and promote anti‐inflammatory cytokine production. ¹⁶³ , ¹⁶⁴ , ¹⁶⁵ , ¹⁶⁶ , ¹⁶⁷ , ¹⁶⁸ , ¹⁶⁹ , ¹⁷⁰ , ¹⁷¹ , ¹⁷² , ¹⁷³ , ¹⁷⁴ , ²²⁷ , ²²⁸ , ²²⁹ , ²³⁰ , ²³¹ Two independent meta‐analyses of different clinical studies reported that omega‐3 fatty acid administration as an adjunct to SRP reduced PD and improved CAL in periodontitis subjects compared to placebo + SRP treatment. ²²⁷ , ²²⁸ , ²²⁹ , ²³⁰ Further, a recent RCT concluded that the intake of omega‐3 fatty acids as an adjunct to SRP reduced periodontitis progression by reducing periodontal pocket depths and increasing alveolar bone attachment. ²²⁵ Additional clinical studies are needed to confirm the functional efficacy of omega‐3 fatty acids as a therapeutic adjunct for the treatment of periodontitis.

Emerging evidence of the importance of nutritional modulation of periodontal inflammation was reported by the European Federation of Periodontology's European Workshop on Periodontology. ²³¹ Vitamins that confer anti‐inflammatory and anti‐oxidative effects have been harnessed as a novel therapeutic approach to manage periodontitis. ²²⁷ , ²²⁸ , ²²⁹ , ²³⁰ , ²³¹ In brief, a mice model of LIP treated with vitamin C reported inhibition of inflammation and reduced bone resorption associated with less OC differentiation and activation. ²³² Vitamin B was administered daily in a rat model of LIP for 11 days resulting in bone tissue repair due to an increase in OB activity and reduced OC differentiation and activity. ²³³ Vitamin D exert immuno‐modulatory properties inhibiting the production of pro‐inflammatory cytokines and reducing the overactivation of immune cells such as CD4+, Th1, Th2, and Th17. ²³⁴ In addition, there is credible evidence that supports the role of Vitamin K, in promoting OB differentiation and reducing OB apoptosis thereby playing a key role in bone homeostasis. ²³⁵ , ²³⁶ , ²³⁷ , ²³⁸ , ²³⁹ Koshihara et al. ²³⁷ demonstrated the role of Vitamin K1 (phylloquinone) and K2 (menaquinone) in mesenchymal stem cells differentiation to OB by upregulating bone marker genes such as BMP‐2, and OPG and concomitant inhibition of RANKL or NF‐kB activation in OB. Yamaguchi and Weitzmann ²³⁸ analyzed the efficacy of Vitamin K1 and K2 in the treatment of osteoporosis in ovariectomized mice and concluded that Vitamin K2 had a greater effect compared to Vitamin K1 in preventing bone density loss in mice fed with a high‐fat diet compared to a normal diet. However, in a rat model of LIP, single treatments with either vitamin D3, vitamin K2 or a combination of both after SRP did not show any reduction in periodontitis‐associated pro‐inflammatory markers and OC activity‐associated bone resorption. ²³⁹ Together, these findings show that Vitamin K1 and K2 play a differential roles in alveolar bone homeostasis; albeit the results were not consistent and further studies are needed.

In summary, additional pre‐clinical testing and clinical trials using novel, non‐invasive therapeutic approaches are urgently needed. It will be important to take into consideration dosage, standardization, and patient compliance, among others.

5. CONCLUSIONS

Numerous molecular and cellular dynamics affect alveolar bone metabolism in periodontal health and disease. This comprehensive narrative review has detailed the current knowledge of impaired OB‐OC axis in periodontitis and the role of immune cells in controlling bone homeostasis and remodeling. The functional diversity of these factors may influence bone homeostasis during different stages of periodontitis as well as treatment outcomes. Therefore, a broader understanding of the cell‐specific dysregulated genes controlling bone remodeling beyond OB‐OC interactions will shed light on the underlying mechanisms and facilitate the development of novel therapeutic approaches. Strategies aimed at preventing the initiation of periodontitis or ameliorating the pathophysiologic events at the earlier stages of disease progression will minimize the need for surgical intervention. As stated earlier, adjunctive use of antibiotics after SRP has shown improved clinical outcomes but concerns regarding antibiotic resistance motivate the search for new, alternate drugs to treat periodontitis. A diverse spectrum of anti‐resorptive (bisphosphonates, OPG‐Fc, RANKL inhibitors, and strontium ranelate) and immunomodulatory agents (CTSK inhibitor, specialized pro‐resolving mediators, biological therapies, and noncoding RNAs) have shown promising outcomes in preventing or restoring the bone homeostasis in experimental periodontitis. The use of these candidate targets should be considered with caution only after comprehensive assessment in preclinical and Phase 1 clinical trials. For instance, the risk to ONJ development after bisphosphonate administration or DRONJ in the case of OPG‐Fc and RANKL inhibitors limits their clinical use. Clinical trials testing the efficacy of CTSK inhibitors (Balicatib and Odanacatib) and other experimental treatments (injections of small interfering RNA (siRNA), adeno‐associated virus (AAV), CTSK‐specific inhibitor, BML‐244, or CsinCPI‐2) have yielded promising results by reshaping bone metabolism or tissue immunity in periodontitis. However, adverse side effects (cerebrovascular accidents and cardiovascular events) have been reported.

Therapeutics targeting periodontitis, a host immune response‐associated disease, should be aimed at mitigating endogenous host mediators of inflammation and its resolution. Treatment with SPMs and biological therapies have shown promising outcomes by specifically targeting inflammatory genes associated with bone metabolism. Additionally, specific noncoding RNAs have been identified with significant roles in regulating immunity and osteoclastic activity. Further studies and clinical trials are needed to validate their use as therapeutic agents. Other alternative emerging treatment options such as probiotics, statins, vitamins, and omega‐3 fatty acids have gained attention due to their anti‐inflammatory and anti‐resorptive properties with limited or no side effects. However, the effective doses of these natural antimicrobial compounds need to be determined and their antimicrobial mechanisms should be further delineated before their use as therapeutic adjuncts. Novel microbiome‐based approaches have demonstrated exciting outcomes in treating experimental periodontitis; however, further research is needed to improve their efficacy and to understand their long‐term effects. Interaction of candidate drug targets with modifiable factors including smoking, nutrition, and oral hygiene should also be investigated to assess their therapeutic efficacy.

Current treatment strategies remain moderately effective in delaying disease progression and regenerating the periodontium and are expensive. There is a need for the development of more cost‐effective and innovative therapeutic approaches that can predict the onset of disease and arrest its progression. Specifically, personalized treatment plans based on precision medicine tools will become mainstream. To monitor the progression of periodontitis and post‐treatment outcomes, identification, and validation of specific and sensitive biomarkers will provide a better assessment of periodontal health that may help clinicians personalize treatment strategies for each patient. While the significance of personalized medicine based on prognostic biomarkers of periodontitis is immense, it remains largely understudied. Changes in cellular and molecular mediators precede the clinical manifestation of periodontitis and its resolution. Therefore, novel approaches aimed at harnessing the potential of tissue‐relevant biological changes will help us understand the disease and advance therapeutic intervention. This can be aided by comprehensive multi‐omic approaches to comprehensively screen host and pathogen‐derived molecules to develop unique markers of disease severity and response to periodontal therapies. Large, multicenter‐randomized controlled trials involving different genders, races, and age groups should be undertaken to evaluate the clinical value of predictive markers of periodontitis to personally prevent or treat the disease.

FUNDING INFORMATION

NIDCR/NIH; contract graft numbers: R01DE027980 (ARN), R56DE033249 (ARN), DE021052 (AG), DE031737 (AG), and DE021052 (SN); NEI/NIH contract grant number R01EY033622 (ARN); The Brodie Endowment Fund (AG).

CONFLICT OF INTEREST STATEMENT

The authors have no conflicts of interest.

Valverde A, George A, Nares S, Naqvi AR. Emerging therapeutic strategies targeting bone signaling pathways in periodontitis. J Periodont Res. 2025;60:101‐120. doi: 10.1111/jre.13326

DATA AVAILABILITY STATEMENT

No original data were used in this manuscript.