Maintaining homeostatic control of periodontal bone tissue

Alveolar bone is a unique osseous tissue due to the integration of the teeth and the proximity of dental plaque biofilms. Periodontal health and homeostasis are mediated by a balanced host immune response to polymicrobial oral biofilms. Shifts in the composition and quantity of microbes within dental plaque biofilms can drive a local proinflammatory immune response state in the epithelial and gingival barrier connective tissues. Infiltrating proinflammatory immune cells within the inflamed connective tissue secrete local factors that induce paracrine signaling to subjacent bone cells. Sustained chronic inflammation disrupts “coupled” osteoclast-osteoblast actions, which ultimately result in alveolar bone destruction.

This chapter will provide an overview of alveolar bone physiology and will highlight why the oral microbiota is a critical regulator of alveolar bone remodeling. The ecology of subgingival plaque biofilms will be reviewed considering our understanding that periodontitis is a polymicrobial disruption of host homeostasis. The pathogenesis of periodontal bone loss will be explained from both a historical and current perspective, providing the opportunity to revisit the role of fibrosis in alveolar bone destruction. The molecular basis of host-microbe interactions will be discussed in the context of the commensal oral flora in periodontal health and pathogenic shifts in the oral microbiota during periodontal disease states. The role of periodontal innate and adaptive immune cell interactions with bone cells will be reviewed based on our current understanding of osteoimmunological mechanisms influencing alveolar bone remodeling. Lastly, probiotic/prebiotic therapeutic interventions in the oral microbiota will be evaluated as a potential therapy to support alveolar bone homeostasis/ prevent periodontal bone loss.

1 |. ALVEOLAR BONE ANATOMY/PHYSIOLOGY

The alveolar process (commonly referred to as alveolar bone) is dependent on the development, eruption, and maintenance of the teeth.^1,2 The primary functions of the alveolar bone are to protect the roots of the teeth and support masticatory function. Like nonoral skeletal tissues, the alveolar bone functions as a source of hematopoietic and mesenchymal stem cells and is a reservoir for calcium, phosphorus, and magnesium. Alveolar bone is responsive to calciotropic hormones, such as parathyroid hormone and calcitonin, which regulate serum calcium homeostasis. Alveolar bone metabolism is also influenced by endocrine signaling effects mediated by changes in sex hormones and circulating inflammatory factors.

Alveolar bone is composed of alveolar bone proper, supporting trabecular bone, and supporting cortical bone (ie, lingual and buccal cortical plates) (Figure 1). The alveolar bone proper is a 0.1–0.4 mm thick layer of bone that lines the alveolus (tooth socket) and supports the attachment of periodontal ligament Sharpey’s fibers.^3–5 The outer surface of the alveolar bone proper is surrounded by supporting trabecular and cortical bone. The lingual/buccal cortical plates merge with the alveolar bone proper at the alveolar bone crest.^4,6,7 Depending on the morphology of the roots and the buccal-lingual dimensions of the alveolar process, trabecular bone can be interposed between the alveolar bone proper and lingual/buccal cortical plates.^4,6,7 The trabecular number, thickness, and organization of the alveolar bone are highly variable and do not appear to be influenced by age.^5,8

FIGURE 1.

Murine alveolar bone anatomy. Twelve-week-old male C57BL6 mouse: serial frontal sections. A, Through the mesial root of the mandibular first molar. B, Through the furcation of the mandibular first molar. P, periosteum; E, endosteum; B.Ct.B, buccal cortical bone; L.Ct.B, lingual cortical bone; Ma, marrow; Tb.B, trabecular bone

Alveolar bone surfaces are lined by membranes (ie, periodontal ligament, periosteum, endosteum) that have a dense blood supply and serve as a robust source of progenitor cells. The periodontal ligament lines the inner surface of the alveolar bone proper; periosteum lines the outer surface of the lingual/buccal cortical plates; and endosteum lines the inner (endocortical) surface of the lingual/buccal cortical plates and interposed trabeculae. Recent seminal reports have applied cell lineage tracing techniques to discern the source of mesenchymal progenitor cells where bone-forming osteoblast cells originate. Orthopedic investigations of nonoral skeletal sites delineated that osteoblasts originate from mesenchymal progenitors derived from the bone marrow, endosteum, and periosteum.^9–11 Orthopedic osseous wound healing studies further determined that the endosteum and periosteum are the primary sources of osteoblasts during osseous regeneration.^9–11 Having direct implications for alveolar bone remodeling and regeneration, a timely report utilized cell lineage tracing and mineral double-labeling approaches to compare the periodontal ligament, periosteum, and endosteum as sources of osteogenic progenitors.¹² This seminal study showed that bone-forming osteoblastic cells robustly originate from mesenchymal progenitors derived from the periodontal ligament, periosteum, and endosteum. Highlighting the importance of the periodontal ligament as a source of osteoblasts within the alveolar bone, progenitor cells originating from the periodontal ligament displayed a two to threefold higher mineral deposition rate than progenitor cells originating from the periosteum and endosteum.¹²

The alveolar bone medullary space, which is located between the endocortical surfaces of the cortical plates and around the interposed trabecular bone, is interspersed with hematopoietic marrow, adipocytes, and blood vessels.^5,8 The adult bone marrow houses hematopoietic stem cells, which are self-renewing and multipotent progenitor cells that sustain erythropoiesis, myelopoiesis, and lymphopoiesis.^13,14 Stem cell niches are tissue microenvironments that support the maintenance of stem cells and regulate their functions through the production of local factors.^13,14 Current research supports the notion that hematopoietic stem cell niches in bone marrow are endosteal and/or perivascular in nature,^13–16 and there is evidence that stromal-osteoblastic cells and osteoclastic cells produce local factors that influence the maintenance and modulate the function of hematopoietic stem cells.^13–16 These hematopoietic stem cells and downstream progenitor cell populations play important roles in supporting tissue homeostasis and regeneration, both in health and disease.^15,16 Future research is necessary to determine whether the hematopoietic stem cell niche housed within alveolar bone is similar to nonoral skeletal sites. Furthermore, periodontal disease has unknown effects on local hematopoietic stem cell niches.

2 |. BONE CELLS: OSTEOBLASTS, OSTEOCYTES, OSTEOCLASTS

Osteoblasts are bone-forming cells that are derived from the mesenchymal lineage (Figure 2). Osteoblast lineage cells include osteoblast precursors, osteoblasts, bone lining cells, and osteocytes.^17–19 The transcription factors runt domain–containing transcription factor, osterix, and activating transcription factor 4 regulate osteoblast differentiation.^17–19 Runt domain–containing transcription factor is required for the commitment of mesenchymal progenitors to the osteoblast lineage. Runt domain–containing transcription factor and downstream osterix are necessary for osteoblast differentiation and function. Activating transcription factor 4, which is independent of runt domain–containing transcription factor and osterix, is required for osteoblast differentiation and function. WNT, bone morphogenetic protein, fibroblast growth factor, and insulin-like growth factor signaling support osteoblast differentiation and function, whereas Notch signaling inhibits osteoblast differentiation.^17–19 Osteoblasts secrete an extracellular matrix primarily composed of type I collagen fibers and noncollagenous proteins.^20,21 The newly formed bone matrix is initially unmineralized, which is commonly referred to as osteoid. Secreted noncollagenous proteins, such as alkaline phosphatase, osteocalcin, and bone sialoprotein, support mineralization of the bone matrix.^20,21 The mineralized inorganic phase of the bone matrix is hydroxyapatite, Ca₁₀(PO₄)₆(OH)₂.^20,21 Osteoblasts eventually undergo apoptosis, become quiescent bone lining cells, or develop into osteocytes.

FIGURE 2.

Bone cells: osteoclasts, osteoblasts, and osteocytes. Twelve-week-old male C57BL6 mouse trabecular bone. Tartrate-resistant acid phosphatase–positive osteoclasts (red arrows) resorbing old bone, cuboidal bone forming osteoblasts (green arrows), and bone-embedded osteocytes (black arrows)

Osteocytes are terminally differentiated osteoblast lineage cells that are entombed within the mineralized bone matrix (Figure 2).^20,22 While osteoblasts make up roughly 4%−6% of bone cells, osteocytes astonishingly account for more than 90% of bone cells.^20,22 Osteocytes embedded within the bone matrix form dendritic processes. The osteocyte cell body is encased in an osseous space called the lacuna, and the dendritic processes travel through the bone matrix in small canals known as canaliculi. Osteocyte dendritic processes facilitate communication with other osteocytes, cells lining the bone surface, vascular spaces, and the bone marrow environment.^22–24 Osteocytes play important roles in sensing mechanical loading forces on bone²⁵ and are responsive to circulating endocrine signaling mediators.²⁶ Osteocytes express biologic factors that critically regulate osteoblast activity, including dickkopf WNT signaling pathway inhibitor 1 and sclerostin. Dickkopf WNT signaling pathway inhibitor 1 is expressed broadly across cell types, whereas sclerostin is primarily synthesized by osteocytes. Both dickkopf WNT signaling pathway inhibitor 1 and sclerostin are negative regulators of the Wnt/β-catenin pathway in osteoblasts, which suppresses osteoblast-mediated bone formation.^22–24

Osteoclasts are multinucleated bone resorbing cells that are derived from monocyte-macrophage precursor cells (Figure 2). Macrophage colony-stimulating factor^27,28 and receptor activator of nuclear factor-kappa B ligand (RANKL)^29–33 are signaling factors that are critical and necessary for osteoclastogenesis. The receptor activator of nuclear factor kappa B (RANK) is expressed on osteoclast precursors and osteoclasts. Macrophage colony-stimulating factor signaling at its cognate receptor, c-Fms, induces the expression of RANK³⁴ and supports the proliferation and differentiation of osteoclast precursor cells.³⁵ RANKL binding at its receptor on osteoclast precursors induces the expression of the transcription factor, NFATc1, which leads to osteoclast differentiation.^36–38 RANKL:RANK binding on osteoclast cells mediates the expression of effector genes that drive osteoclast maturation, function, and survival.^36–38 Dendritic cell–specific transmembrane protein^39,40 and osteoclast stimulatory transmembrane protein⁴¹ are RANKL-induced fusion proteins critical for osteoclast maturation. RANKL-induced expression of the β3 integrin subunit is necessary for αVβ3-mediated binding to the bone matrix.^36–38 Osteoclasts bound to the bone matrix form a sealing zone, where they secrete hydrogen ions and lytic enzymes that resorb the underlying osseous tissue. The secretion of the osteoclast proteolytic enzymes, tartrate-resistant acid phosphatase and the collagenase cathepsin K, are dependent on NFATc1-mediated RANKL signaling.^36–38 RANKL-mediated osteoclastogenesis is critically regulated by its physiologic decoy receptor osteoprotegrin.^42–45 Osteoprotegrin binding of RANKL inhibits RANKL signaling at the RANK receptor, which downregulates osteoclastogenesis. Appreciating that the RANKL:osteoprotegrin ratio determines the availability of free RANKL in the local environment, the RANKL:osteoprotegrin axis must be evaluated when assessing the potential of RANKL to drive osteoclastogenesis.

3 |. RANKL:OSTEOPROTEGRIN AXIS

Clinical and preclinical studies have discerned that dental plaque biofilms upregulate RANKL, downregulate osteoprotegrin, and increase the RANKL:osteoprotegrin ratio.^46–48 Exogenous osteoprotegrin administration in rodent experimental periodontal disease models suppressed osteoclastogenesis and blunted periodontal bone loss,^49,50 which demonstrates that the RANKL:osteoprotegrin axis critically regulates periodontitis-driven alveolar bone loss. The molecular underpinnings defining the cellular sources of RANKL and osteoprotegrin have been recently elucidated.

Diverse cells synthesize RANKL and osteoprotegrin under physiologic conditions, which supports basal osteoclastogenesis and homeostatic bone remodeling. Osteoblasts were initially perceived to be the primary source of RANKL^30,51 and osteoprotegrin.⁵² A study in transgenic mice with inducible ablation of mature osteoblastic cells challenged the notion that osteoblast-derived RANKL regulates basal osteoclastogenesis.⁵³ Osteoblast-ablated transgenic mice had impaired bone formation but lacked alterations in osteoclast-mediated bone resorption.⁵³ Timely studies utilizing mesenchymal stem cell and osteocyte-specific conditional RANKL knockout models later discerned that RANKL derived from osteocytes, not osteoblasts, critically supports basal osteoclastogenesis and physiologic bone remodeling.^54–56 T and B-cell–specific conditional RANKL knockout models did not demonstrate differences in bone mass, which implies that lymphocyte-derived RANKL does not regulate physiologic bone remodeling.⁵⁷ Timely investigations in lymphocyte-specific knockout mice have revealed that B cells and T cells play important roles in the production of osteoprotegrin under physiologic conditions.^58,59 B cell knockout mice have elevated bone resorption and reduced bone mass, which is secondary to deficient osteoprotegrin synthesis in the bone marrow environment.⁵⁸ Furthermore, immunophenotyping in wild-type mice astonishingly revealed that 64% of marrow osteoprotegrin synthesis is attributed to the B cell lineage.⁵⁸ T cells promote skeletal homeostasis through the expression of CD40 ligand, which indirectly regulates B cell osteoprotegrin synthesis. CD40 ligand activation of the CD40 costimulatory receptor induces B cell osteoprotegrin production,^44,58 which supports basal osteoclastogenesis and physiologic bone remodeling.^58,59

Dental plaque biofilms have been reported to upregulate RANKL and downregulate osteoprotegrin, resulting in an enhanced RANKL:osteoprotegrin ratio that drives osteoclastogenesis and catabolic alveolar bone loss.^46,47 Whereas both supragingival and subgingival biofilms have the ability to upregulate the RANKL:osteoprotegrin ratio, subgingival biofilms profoundly skew the RANKL:osteoprotegrin axis.⁴⁸ RANKL derived from diverse cell types critically regulates periodontitis-induced alveolar bone loss. Kawai et al initially reported that B and T lymphocytes are the primary source of RANKL in the bone resorptive lesion of periodontal disease.⁶⁰ The report was based on immunofluorescent confocal microscopy analysis of CD3⁺RANKL⁺ T cells, CD20⁺RANKL⁺ B cells, and CD14⁺RANKL⁺ monocytes in healthy versus diseased clinical gingival tissue isolates. Notably, the study did not assess the impact of periodontitis on RANKL expression by osteoblasts, osteocytes, or periodontal ligament cells. Pacios et al later reported that osteoblast lineage cells play an essential role in periodontal bone loss through the activation of nuclear factor-kappa B.⁶¹ Transgenic mice with osteoblast lineage–specific depletion of nuclear factor-kappa B signaling were orally inoculated with Porphyromonas gingivalis and Fusobacterium nucleatum. Study outcomes revealing that wild-type mice had increased alveolar bone loss, osteoclastogenesis, and RANKL expression led the authors to postulate that osteoblast RANKL contributes to periodontal bone loss.⁶¹ Graves et al employed transgenic mice with osteocyte-specific deletion of RANKL to discern that osteocytes play an important role in periodontitis through expression of RANKL.⁶² Oral inoculation of transgenic mice with P. gingivalis and F. nucleatum showed that osteocyte RANKL critically regulates bacteria-driven osteoclastogenesis and periodontal bone loss. Most recently, Tsukasaki et al applied the murine ligature periodontitis model to transgenic mice with targeted deletion of RANKL in B cells, T cells, osteoblastic cells, and periodontal ligament cells.⁶³ Study outcomes discerned that osteoblast RANKL and periodontal ligament RANKL most substantially contributed to periodontal bone loss. T-cell–derived RANKL induced periodontal bone loss to a lesser extent than osteoblast RANKL and periodontal ligament RANKL, whereas B-cell–derived RANKL had no effect. These studies support the notion that periodontitis-associated biofilms upregulate RANKL across diverse cell types to drive osteoclast-mediated alveolar bone loss. Future investigations are indicated to determine the role of cell-specific osteoprotegrin alterations in periodontitis-driven catabolic effects on alveolar bone.

4 |. BONE REMODELING

Bone remodeling (turnover) is the skeletal renewal process in which myeloid-lineage–derived osteoclastic cells resorb old bone matrix, and mesenchymal-lineage–derived osteoblastic cells subsequently form new bone matrix. Skeletal homeostasis occurs when osteoclast-osteoblast actions are balanced (“coupled”), and there is no net gain/loss of osseous tissue. Skeletal bone loss occurs when the actions of osteoclastic cells exceed those of osteoblastic cells, and there is a net loss of osseous tissue.^64–67 Osteoclast-osteoblast–mediated bone remodeling processes are modulated by mechanical loading forces, paracrine/juxtracrine signaling interactions with nearby bone and immune cells, and endocrine signaling events induced by circulating hormones and immune factors.

Bone remodeling takes place in an asynchronous manner throughout the skeleton and occurs at distinct anatomic sites known as basic multicellular units.⁶⁴ The basic multicellular unit consists of osteoclastic cells and osteoblastic cells that remodel (turnover) the bone through distinct and sequential phases: activation, resorption, reversal, formation, and termination.⁶⁸ The healthy adult skeleton contains an estimated one million active basic multicellular units, which turn over approximately 10% of the entire skeleton per year.^69,70 Trabecular bone is estimated to remodel at an average rate that is seven times more rapid than cortical bone,^69,70 which highlights that trabecular bone may be more susceptible to inflammatory bone loss. Considering that the estimated lifespan of bone-resorbing osteoclasts is 2 weeks and bone-forming osteoblasts is 3 months in the basic multicellular unit,^69,70 alterations in osteoblast activity can substantially impact skeletal homeostasis.

Saffar and coworkers^71,72 performed seminal research in the golden hamster, which delineated that periodontal disease causes alveolar bone loss through the disruption (“uncoupling”) of balanced osteoclast-osteoblast–mediated bone remodeling. These reports showed that dietary plaque-induced periodontal inflammation critically altered the actions of osteoclasts and osteoblasts within the basic multicellular unit.^71,72 Experimental periodontitis-induced alveolar bone destruction was driven by the following changes in physiologic bone remodeling: upregulated activation of bone cells initiating small foci of bone remodeling; increased osteoclast-mediated bone resorption; increased reversal (empty resorption lacunae lined by mononuclear cells), and decreased osteoblast-mediated bone formation.^71,72 Importantly, these seminal studies clearly delineated that periodontitis-driven alveolar bone destruction is secondary to enhanced osteoclast-mediated bone resorption and suppressed osteoblast-mediated bone formation.

Early work by Irving et al⁷³ lends support to the view that osteoblasts critically regulate periodontal bone loss. Weanling gnotobiotic rats were mono-infected with gram-positive bacteria: Actinomyces naeslundii, Actinomyces viscosus, or Streptococcus mutans. Osteoblast activity was evaluated via ³H proline labeling, and osteoclast activity was assessed by the acid phosphatase technique. Mono-infection with each of the three gram-positive bacteria induced periodontal disease as evidence by the plaque accumulation, apical migration of the junctional epithelium, and alveolar bone destruction. Notably, the alveolar bone destruction was attributed to cessation of active bone formation, not enhanced osteoclastogenesis. In order to validate that the aforementioned study outcomes were not due to animal species or gnotobiotic status, weanling conventional hamsters were subsequently superinfected with A. naeslundii. Periodontal bone loss occurred more slowly in the hamster model than in the rat model but was defined by severely impaired osteoblastogenesis.⁷³

Irving et al also carried out a follow-up investigation to determine whether gram-negative bacteria cause periodontal bone destruction through actions on osteoblasts or osteoclasts.⁷⁴ Weanling gnotobiotic rats were mono-infected with a gram-negative, anaerobic rod isolated from a case of human periodontitis. Periodontal disease was characterized by minimal plaque formation, apical migration of the junctional epithelium, and alveolar bone destruction. Alveolar bone destruction was attributed to severely suppressed active bone formation and substantially enhanced osteoclastogenesis. Irving et al speculated that the alveolar bone loss that occurred in the absence of gross plaque accumulation was likely attributed to endotoxin.⁷⁴ The work by Irving and coworkers⁷³ importantly reveals that both gram-positive and gram-negative bacterial biofilms have catabolic effects on alveolar bone, which are mediated in part through anti-osteoblastic actions.

Despite knowledge that periodontitis-driven bone loss is in part attributed to suppressed osteoblastogenesis,^71,72 the osteoblastic cell lineage is, for the most part, overlooked by periodontal researchers. However, there is indirect evidence from recent studies that further highlights the importance of the osteoblastic cell lineage in the pathogenesis of periodontal bone loss (Figure 3). Exogenous intermittent administration of the first 34 amino acids of parathyroid hormone is a US Food and Drug Administration–approved anabolic drug for the treatment of osteoporosis. Exogenous intermittent parathyroid hormone 1–34 has anabolic effects on bone through stimulating osteoblast-mediated bone formation.^18,75 Exogenous intermittent parathyroid hormone 1–34 administration in the rat ligature periodontitis model has been shown to prevent/protect against periodontitis-associated alveolar bone loss,^76,77 and clinically has been shown to adjunctively enhance periodontal surgery outcomes.⁷⁸ In January 2019, the Food and Drug Administration recommended approval of another anabolic drug, romosozumab, for the treatment of postmenopausal osteoporosis. Romosozumab is a monoclonal antibody that stimulates bone formation by binding to sclerostin, the osteocyte-derived protein that suppresses osteoblastogenesis.^18,79 Interestingly, sclerostin-antibody administration has been shown to support alveolar bone regeneration in the rat ligature periodontitis.^80,81

FIGURE 3.

Periodontal immune microenvironment. Subgingival plaque biofilm derived microbe-associated molecular patterns stimulate the periodontal immune response. Neutrophils are recruited to the junctional epithelium via chemokine gradients. Innate immune cells, such as macrophages and dendritic cells, secrete matrix metalloproteinases and proinflammatory cytokines—that is, tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6)—which disrupt connective tissue homeostasis. Osteoblasts, osteocytes, periodontal ligament cells, and lymphocytes (ie, T cells, B cells) secrete receptor activator of nuclear factor-kappa B ligand (RANKL) and other proinflammatory factors, which support osteoclastogenesis and inhibit osteoblastogenesis. B cells synthesize antibodies that drive the humoral response. Osteocytes secrete sclerostin to suppress osteoblastogenesis. CEJ, cementoenamel junction; DC, dendritic cells; IFNy, interferon-gamma; Mac, macrophages; MAMPs, microbe-associated molecular patterns; MMP, matrix metalloproteinases; OB, osteoblasts; OC, osteoclasts; OPG, osteoprotegrin; PDL, periodontal ligament; PMN, neutrophils; SOST, sclerostin

5 |. ALVEOLAR BONE: A UNIQUE OSSEOUS TISSUE

Alveolar bone is a unique osseous tissue owing to its integration with the dentition and close proximity to oral biofilms. Bone remodeling in the alveolar bone complex has been reported to take place at a rate that is three to sixfold more robust than at nonoral skeletal sites.^82–84 Mechanical loading studies estimate that functional strains are two to fourfold higher in alveolar bone than at nonoral skeletal sites,^85–88 which implies that occlusal forces transmitted through the fibrous junction of the periodontal ligament contribute to the robust remodeling of alveolar bone. Periodontal research studies revealing that oral biofilms significantly modulate alveolar bone metabolism in periodontal health⁸⁹ and disease^71,72 highlight that oral biofilms critically regulate alveolar bone remodeling.

The host is colonized by diverse microorganisms at mucosal barrier surfaces exposed to the external environment. The collection of microbes colonizing distinct anatomic sites are referred to as microbiota communities.^90–92 The oral cavity is a distinct anatomic barrier environment to colonizing microbes due to the presence of a transmucosal organ, the tooth, which supports vigorous biofilm formation and is integrated with alveolar bone. The spatial relationship of the oral microbiota to alveolar bone is unique, in that no other microbiota community (ie, gut, skin, urogenital, lung) colonizes an external body surface directly integrated with osseous tissue.

The dentogingival junction, which consists of the epithelial and connective tissue attachment to the tooth,^93,94 protects the subjacent alveolar bone from microbes resident in the dental plaque biofilm.^95,96 Whereas epithelial tissues typically act as an impermeable barrier to microbial biofilms colonizing external body surfaces, the junctional epithelial attachment at the tooth surface is highly permeable.^95,97 The periodontal innate immune response defends against microbial invasion of the permeable junctional epithelium via the continuous transit of gingival crevicular fluid^98,99 and leukocytes into the gingival sulcus.^100–103 Notably, polymorphonuclear cells (neutrophils) transiting through the gingival vascular plexus to the junctional epithelium emigrate to the gingival sulcus, and they do not infiltrate or reside in the connective tissue extracellular matrix.^95,96,104 Polymorphonuclear cells emigrating into the gingival sulcus form a barrier wall between the epithelium and plaque biofilm, and thus have been considered the first line of the periodontal immune defense.^97,105,106 Dentogingival biofilms stimulate immune response effects in barrier epithelial and underlying connective tissues, which induce paracrine signaling that influences alveolar bone homeostasis.^{95,96,104,107}

6 |. SUBGINGIVAL BIOFILM ECOSYSTEM: INTER ACTIONS WITH THE HOST

Advances in the understanding of periodontal biofilm ecosystems and the host immune response have led to evolving theories of periodontal disease pathogenesis. Early observations demonstrating that increased bacterial plaque accretions were associated with periodontal disease sites shaped the “nonspecific plaque hypothesis.” This hypothesis is based on the notion that the increased quantity of bacteria, irrespective of the presence of specific bacteria, stimulates periodontal tissue destruction.^108,109 Advances in bacterial culturing techniques and the advent of whole-genome deoxyribonucleic acid probes enabled researchers to identify prominent bacteria in dental plaque biofilms from healthy versus diseased sites. Investigations of subgingival plaque biofilms from active periodontal disease sites led to the “specific plaque hypothesis.” This hypothesis dictates that infection by a specific putative periopathogenic bacterium (eg, P. gingivalis, Aggregatibacter actinomycetemcomitans) or a small cluster of interacting putative periopathogenic bacteria (eg, “red complex”: P. gingivalis, Tannerella forsythia, Treponema denticola) drives periodontitis-associated alveolar bone loss.^109,110

Oral biofilms are dynamic polymicrobial communities that involve interspecies synergies and competition, which have implications for the periodontal immune response and alveolar bone homeostasis.^111–113 Oral inoculation–induced experimental periodontitis investigations have been carried out to discern the role of single bacteria versus a consortium of bacteria in the etiology of periodontal bone loss. Mono versus polymicrobial oral infection models have been employed to evaluate the interactions of red complex (P. gingivalis, T. denticola, T. forsythia) periopathogenic bacteria in periodontitis-driven alveolar bone loss. F. nucleatum was also investigated based on evidence that it serves as a bridge bacterium between early and late-colonizing plaque biofilm species. Kesavalu et al¹¹⁴ subjected rats to P. gingivalis, T. denticola, or T. forsythia monomicrobial oral infections or combined polymicrobial oral infections with or without F. nucleatum. Rats that were infected with the polymicrobial consortium exhibited increased alveolar bone loss compared with rats infected with one of the microbes, which was independent of the presence of F. nucleatum.¹¹⁴ Polak et al orally infected mice with P. gingivalis or F. nucleatum alone or both bacteria together.¹¹⁵ Combined F. nucleatum/P. gingivalis infection relative to monoinfections induced more alveolar bone loss in vivo and increased tumor necrosis factor and interleukin-1beta levels in the subcutaneous chamber model. Orth et al¹¹⁶ orally inoculated mice with P. gingivalis or T. denticola alone or coinfected with P. gingivalis/T. denticola. Four doses of a co-inoculum at 1:1 ratio of P. gingivalis and T. denticola (5 × 10⁸ total bacterial cells) was shown to induce the same level of bone loss as four doses of 1 × 10¹⁰ P. gingivalis cells. Co-inoculum induction of T cell proliferation and interferon-gamma–dominant responses relative to mono-inoculum treatments suggest that P. gingivalis and T. denticola act synergistically to stimulate the host immune response and drive periodontal bone loss. Settem et al subjected mice to monomicrobial infection with F. nucleatum or T. forsythia alone or coinfection with both bacteria.¹¹⁷ Coinfection of F. nucleatum/T. forsythia was reported to be more potent than monomicrobial infections in inducing nuclear factor kappa B activity and proinflammatory cytokine secretion (ie, interleukin-1beta, interleukin-6, tumor necrosis factor) in monocytic cells and primary murine macrophages. In vivo comparison demonstrated that coinfection relative to monomicrobial infections resulted in increased gingival inflammatory infiltrate, enhanced osteoclast cell numbers lining bone, and synergistic effects on alveolar bone loss.¹¹⁷ However, Yamazaki et al utilized periopathogenic bacteria (P. gingivalis, Filifactor alocis, and F. nucleatum) or healthy periodontal bacteria (A. naeslundii, Streptococcus mitis, and Veillonella rogosae) in germ-free mice to find no alveolar bone resorption in either group.¹¹⁸

Periodontitis is currently understood to be a polymicrobial disease that results from complex interactions among the commensal microbiota, host susceptibility, and environmental factors. Periodontal pathogenesis appears to be driven by an excessive proinflammatory immune response to shifts in the normal microbiota, which are exacerbated by disease-associated bacterial species. Investigations in the germ-free versus specific-pathogen-free mouse model have discerned that the oral commensal microbiota induces a low-grade proinflammatory immune response state that drives alveolar bone loss in health.^89,119 We have recently shown that antibiotic perturbation of the commensal microbiota induces a supraphysiologic proinflammatory immune response state that has catabolic effects on the skeleton, both at oral (unpublished data) and nonoral¹²⁰ sites. Hajishengallis et al provided evidence supporting the notion that proinflammatory alveolar bone loss is exacerbated by periopathogenic bacteria–induced shifts in the normal microbiota.¹¹⁹ The seminal murine experimental periodontitis study demonstrated that low-abundance P. gingivalis triggers changes to the amount and composition of the oral commensal microbiota, which drives proinflammatory periodontal bone loss.¹¹⁹

The application of powerful 16S ribosomal RNA gene amplification and sequencing techniques has enabled investigators to characterize the bacterial microbiome (genomic complement) of microbiota communities. The oral microbiota is a diverse microbial community made up of roughly 1000 bacterial species.^121–123 454-Pyrosequencing of 16S ribosomal RNA gene amplicons in clinical subgingival plaque isolates has revealed that the subgingival microbiota consists of roughly 700 bacterial species.¹²⁴ Advanced knowledge about the composition of subgingival bacterial biofilms in health and disease calls into question whether periodontitis is secondary to infection by empirically defined putative periopathogenic bacteria.

Griffen et al reported that subgingival plaque from chronic periodontitis versus healthy patients demonstrated an increased diversity, both in the number of species and the combination of richness and evenness reflected by the Shannon index.¹²⁴ The bacterial species more prominent in health were not lost in disease but constituted a smaller fraction of the total community. The investigation confirmed associations of P. gingivalis, T. forsythia, and T. denticola with disease. However, the study revealed substantial numbers of additional species associated with disease that are not empirically defined putative periopathogenic bacteria. F. alocis, a bacterium not previously associated with periodontitis, was one of the top three associated species.¹²⁴

Abusleme et al found that subgingival plaque from chronic periodontitis versus healthy patients demonstrated a higher bacterial load and richness.¹²⁵ Empirically defined putative periopathogenic bacteria were present at low levels in health, which calls into question whether infection by specific microbes induces the host proinflammatory response that mediates periodontal tissue destruction. Based on findings that a higher community biomass was associated with disease, the supraphysiologic host immune response causing periodontal breakdown may be secondary to an overall greater bacterial challenge. Considering that periodontal disease was characterized by the emergence of newly abundant taxa concurrent with an increase in bacterial biomass,¹²⁵ the report provides evidence supporting the postulate that the pathogenesis of periodontitis is secondary to ecological shifts in the subgingival microbiome.¹²⁶

Our current understanding of periodontitis as a polymicrobial disruption of host homeostasis^104,107 is centered on the bacterial component of the microbiota. However, the oral microbiota is comprised of diverse microbial species, including bacteria, fungi, viruses, archaea, and other eukaryotes.^112,123,127 Recent advances in 18S ribosomal RNA and 28S ribosomal RNA genomic sequencing have begun to define the mycobiome (fungal component of the microbiome), which has led to research discerning the impact of the mycobiota (fungal component of the microbiota) on the host immune system.^128,129 Candida albicans has recently been identified as the single, ubiquitous member of the microbiota that is central for the systemic induction of antifungal T_H17 cell–mediated immunity.^130,131 C. albicans is considered a commensal fungus with pathogenic potential that colonizes the gut, skin, and oral cavity at low levels during health. Bacher et al delineated that C. albicans is responsible for the systemic induction of human antifungal T_H17 responses.¹³⁰ Shao et al demonstrated that intestinal colonization of mice with C. albicans induces systemic expansion of fungal-specific T_H17 CD4⁺ T cells and interleukin-17 responsiveness that protects against C. albicans invasive infection.¹³¹ Notably, C. albicans–induced T_H17/interleukin-17–mediated immunity was shown to confer protection against invasive extracellular pathogens that extend beyond the fungal microorganisms, including Staphylococcus aureus.¹³¹

T_H17 cell–mediated immunity has been shown to be essential for oral mucosal host defense against C. albicans–mediated oral candidiasis¹³² and periopathogenic bacteria–induced periodontal bone loss.¹³³ However, the relationship between interkingdom microbial interactions and oral immunity is poorly understood. C. albicans and F. nucleatum species demonstrate strong coaggreagation,^134,135 which has immunoregulatory effects on macrophage immune defense that appear to favor sustained colonization.¹³⁶ The cooperation of C. albicans and F. nucleatum highlights the potential for interkingdom interactions in plaque biofilms, which may have implications for the pathogenesis of periodontitis-driven alveolar bone loss.

7 |. PATHOGENESIS OF ALVEOLAR BONE DESTRUCTION: RANGE OF EFFECTIVENESS

Subgingival plaque biofilms induce a supraphysiologic proinflammatory immune response state in the gingival connective tissue, leading to paracrine signaling that drives alveolar bone destruction. Page and Schroeder estimated that the “range of effectiveness” of subgingival plaque to induce alveolar bone destruction is about 2.5 mm.¹³⁷ This postulate is supported by Garant and Cho’s theory that dental plaque–induced bone resorption factors in the connective tissue have an “effective radius of action,”¹³⁸ and by Waerhaug’s work showing that the linear distance from the apical extension of the subgingival plaque to the alveolar crest ranged from 0.5 mm to 2.7 mm.¹³⁹ Histologic investigations performed by Lindhe and Ericsson in the beagle dog ligature model¹⁴⁰ and by Rowe and Bradley in clinical autopsy specimens¹⁴¹ have shown that the proximity of the inflammatory infiltrate to the alveolar bone correlates with the number of osteoclasts and resorption lacunae present.

Chronic pathophysiologic stimulation by dental plaque biofilms leads to the excessive production of proinflammatory mediators, which can ultimately have catabolic effects on balanced tissue remodeling processes. Subgingival plaque–induced supraphysiologic immune and inflammatory responses result in pathophysiologic levels of prostaglandins, proteases, matrix metalloproteinases, cytokines, and other host enzymes being released from epithelial cells, fibroblasts, polymorphonuclear leukocytes, monocytes, macrophages, osteoblasts, or other host cells.^142–144 The chronic proinflammatory state dysregulates fibroblast-mediated remodeling of the gingival connective tissue and the periodontal ligament, which leads to reduced collagen content and compromised tissue integrity.^142,143 Periodontal ligament collagen fibers irreversibly detach from the root surface, which results in the junctional epithelium extending apically. This facilitates further apical extension of subgingival biofilms (Figure 4). As the periodontal pocket deepens, the anaerobic microenvironment favors gram-negative periopathogenic bacteria, increasing the site’s risk for disease progression.^137,145

FIGURE 4.

Localized periodontitis at maxillary right first molar. Apical migration of subgingival biofilm–mediated alveolar bone destruction at the distobuccal root

The inflammatory infiltrate spreads deeper within the periodontitis-afflicted connective tissue, driving proinflammatory paracrine signaling effects that dysregulate “coupled” osteoclast-osteoblast–mediated bone remodeling processes. Diverse cells within the local periodontal environment synthesize increased levels of RANKL and lower levels of osteoprotegrin, which skews the RANKL:osteoprotegrin axis to favor osteoclastic bone resorption.^46,47 A multitude of cells in the local microenvironment synthesize proinflammatory cytokines (ie, tumor necrosis factor, interleukin-1beta, interleukin-6, interleukin-17) that further upregulate RANKL production and/or have synergistic effects on RANKL signaling, which exacerbates osteoclast bone resorptive processes (Figure 3).^142–144 Furthermore, interleukin-1beta, interleukin-6, and tumor necrosis factor have potent anti-osteoblastic actions that suppress osteoblast differentiation and function (Figure 3).^18,144 The subgingival plaque–induced proinflammatory host immune response state therefore drives periodontal bone destruction through enhanced osteoclast activity and/or suppressed osteoblastic activity.

8 |. PATHOGENESIS OF ALVEOLAR BONE DESTRUCTION: REVISITING FIBROSIS

Page and Schroeder¹⁴⁶ laid the foundation for our understanding of cellular changes in epithelial and gingival connective tissue during periodontal health and disease. The investigation depended on histopathologic and ultrastructural analysis of periodontal tissues specimens. Despite not having advanced molecular-cellular assays available, the report was written from the perspective that the host immune response to colonizing oral biofilms drives the pathogenesis of periodontitis.¹⁴⁶ The seminal work is still considered to be the most influential manuscript on periodontal disease pathogenesis due to current knowledge that the host immune response to plaque biofilms drives periodontal tissue destruction.

Page and Schroeder proposed that the pathogenesis of periodontitis occurred via a succession of stages (ie, initial lesion, early lesion, established lesion, advanced lesion), which resulted in pathologic changes to the structure and function of the periodontium.¹⁴⁶ We will focus on the advanced lesion, since it is the only stage afflicted by appreciable bone loss. The advanced lesion is characterized by a dense infiltrate of lymphocytes, macrophages, and plasma cells that spreads deep through the gingival connective tissue, progressive loss of collagen subjacent to the pocket epithelium that continues to migrate apically/laterally, and destruction of the periodontal ligament attachment and supporting alveolar bone. Bone destruction initiates along the crest of the interdental septum, which results in fibrotic gingival thickening. Exposed marrow spaces become hypercellular, undergo fibrosis, and transform to dense fibrotic scar tissue.

Page and Schroeder posited that, “Generally, bone destruction begins along the crest of the interdental septum around the communicating blood vessels.¹⁴⁶ As the marrow spaces are opened, both red and white marrow become hypercellular, undergo fibrosis and become transformed into scar like connective tissue.” Weinmann introduced the alveolar bone fibrosis theory, which was realized during a histologic investigation of the effects of chronic periodontitis in human periodontium specimens collected at autopsy.¹⁴⁷ The author explained that the inflammatory infiltrate in the superficial connective tissue leads to resorption of the alveolar bone crest, an inflammatory reaction following along the blood vessels into the bone marrow spaces, and a transformation of the fatty marrow into fibrous marrow.¹⁴⁷ Moskow and Polson performed an extensive histological investigation evaluating the extension of the inflammatory infiltrate in human periodontitis.¹⁴⁸ The study included 350 autopsy and surgically retrieved segments of human jaws. The authors reported that the inflammatory infiltrate extends into the alveolar process and elicits a response. Penetrations of inflammatory cells into the marrow was commonly associated with fibrosis of the fatty marrow. The authors state, “Our observations are in agreement with those of Weinmann (1941), in that a major pathway of the inflammatory infiltrate in periodontitis is through the intra-alveolar vessels.”¹⁴⁸

The clinical studies performed by Weinmann,¹⁴⁷ Page and Schroeder¹⁴⁶, and Moskow and Polson¹⁴⁸ are, astonishingly, the only known published reports that have addressed the alveolar bone fibrosis phenomena. Personal communication with Roy Page provided valuable insight as to why the fibrosis mechanism of periodontal pathogenesis has been overlooked. Experimental periodontitis model limitations are largely responsible. Preclinical models do not recapitulate the long-term chronic proinflammatory immune response state induced by subgingival biofilms in chronic periodontitis. Small rodent animal models typically do not have naturally occurring periodontal disease. Furthermore, the standard experimental approaches employed to induce periodontitis (ie, periopathogen oral inoculation, ligature placement) have limited capacity to alter the indigenous subgingival microbiota and are too short in duration.

Fibrosis is the common pathologic outcome across many chronic inflammatory diseases. Extensive research efforts have begun to define the pathophysiology of fibrotic conditions. Reviewing this knowledge lends strong support to the postulate that chronic periodontitis–induced alveolar bone destruction occurs in part through fibrosis. General fibrosis principles that apply to all fibrotic diseases include the following:

1. Fibrosis is caused by unresolved chronic inflammation that dysregulates physiologic tissue remodeling and leads to aberrant fibrous connective tissue deposition.

2. Fibrosis ultimately results in loss of tissue architecture and progressive loss of organ function.^149–151

In line with the first principle, chronic periodontitis has been shown to dysregulate balanced (“coupled”) osteoclast-osteoblast–mediated alveolar bone remodeling,^71,72 leading to the deposition of fibrous scar-like tissue within the alveolar bone marrow compartment.^146–148 In line with the second principle, chronic periodontitis ultimately results in loss of alveolar bone architecture and progressive loss of periodontal function, which is synonymous with loss of dentoalveolar support that can terminally result in tooth loss.

Cutting-edge microbiome research has recently revealed that distinct microbiota communities critically regulate the pathogenesis of fibrotic conditions afflicting the liver,^152,153 lungs,^154–156 and skin.^157,158 Dysfunction of the gut epithelial barrier leads to increased translocation of gut bacterial by-products/metabolites into the portal venous circulation, which contributes to the development and progression of nonalcoholic fatty liver disease.^152,153 Dysbiosis of the lung microbiota has been shown to exacerbate lung fibrosis via upregulating a profibrotic inflammatory cytokine network.¹⁵⁶ Furthermore, the skin microbiota can have potent proinflammatory immunomodulatory actions that induce fibrous healing (scarring) of skin wounds.¹⁵⁸ Knowledge that nonoral microbiota–host immune response effects contribute to fibrotic conditions further supports the notion that periodontitis-induced alveolar bone destruction occurs in part through fibrosis. Future research is critically needed to discern the role of fibrosis in the pathophysiology of clinical periodontal bone loss.

9 |. OSTEOIMMUNOLOGY: INTERACTION OF IMMUNE CELLS: BONE CELLS

The field of osteoimmunology has demonstrated that the immune cell interactions with bone cells regulate skeletal development and homeostasis, under physiologic and pathophysiologic conditions.^58,159–161 Osteoimmunological studies have led to explorations of dissecting the crosstalk between bone cells and immune cells in the regulation of bone turnover.^58,159–161 The commensal microbiota is a critical regulator of the host immune response and immunological education.¹⁶² Importantly, the commensal microbiota has recently been shown to have immunomodulatory actions that regulate osteoclast-osteoblast–mediated bone remodeling processes.^89,163

Page and Schroeder provided a sound conceptual framework for the understanding of the impact of subgingival biofilms on cellular changes in the periodontium.¹⁴⁶ Whereas Page and Schroeder focused on macrophages, lymphocytes, and plasma cells,¹⁴⁶ the field of periodontal research has carried out elaborate investigations further defining the interactions of subgingival biofilms and specific adaptive and innate immune cells (Figure 5). The focus of this section is centered on the current understanding of oral microbiota-periodontal immune processes regulating alveolar bone homeostasis in health and disease.

FIGURE 5.

Periodontal osteoimmunology. The subgingival plaque biofilm induces a multitude of immune cells to mount the periodontal immune response. There is an increase in neutrophil chemotaxis, as well as differentiation of naive CD4⁺ T cells into several subsets. T_H1 cells secrete interferon-gamma (IFNγ), which can have pro-osteoclastogenic properties. T_H17 cells secrete interleukin-17 (IL-17) and tumor necrosis factor (TNF), which induce osteoblasts to synthesize proinflammatory/pro-osteoclastic factors. T regulatory cells and T_H2 cells secrete interleukin-10 (IL-10) and interleukin-4 (IL-4), which have anti-osteoclastogenic effects. T_H22 cells and T_H9 cells produce interleukin-22 (IL-22) and interleukin-9 (IL-9), factors that currently have unclear roles in periodontal osteoimmunology. B cells, natural killer (NK) cells, and natural killer T (NKT) cells can secrete receptor activator of nuclear factor-kappa B ligand (RANKL), whereas mast cells can produce interleukin-17 (IL-17) to support osteoclastogenesis. Myeloid-lineage cells, such as myeloid-derived suppressor cells (MDSC), macrophages, and dendritic cells (DC), synthesize proinflammatory cytokines—that is, TNF, interleukin-1 (IL-1), interleukin-6 (IL-6)—that support osteoclastogenesis. M-CSF, macrophage colony-stimulating factor; T_REG, T regulatory cells

9.1 |. CD8⁺/CD4⁺/gamma delta T cells

T cells are the critical players in adaptive immunity that regulate the function of antigen-presenting cells and B-cell–mediated humoral immunity.⁶⁶ T cells recognize immunological epitopes through T-cell receptors and through antigens presented on major histocompatibility complex molecules from antigen-presenting cells. Unlike T cells that are specific for self-antigens expressed by thymic epithelial cells, commensal microbiota–specific T cells do not undergo negative selection in the thymus and are present in healthy individuals despite the constant stimulation of their cognate antigens.¹⁶⁴ Furthermore, T cells are essential for productive, long-lasting immune responses and immunological memory.

CD8⁺ T cells function in monitoring shifts in peptide antigens presented on major histocompatibility complex class I molecules, which are expressed on all nucleated cells. CD8⁺ T cells also possess cytotoxic properties, where they can control other immune cells to avoid excessive immune activation and its pathological consequences.¹⁶⁵ CD4⁺ T cells recognize both self and foreign peptides presented by the major histocompatibility complex class II pathway,¹⁶⁶ allowing for a robust and lasting immune response and the establishment of effective immune memory.¹⁶⁷ Gamma delta (γδ) T cells are an unconventional, less prominent T cell population that have important immune surveillance properties at epithelial and mucosal barriers,¹⁶⁸ including the oral cavity.^169,170 There are specific γδ T-cells subsets that are adapted to specific barrier sites. This cell population can be induced by the commensal microbiota and pathogens, influencing such properties as generation, effector functions, proliferation, and maintenance.^168–170 Interestingly, oral mucosal homeostasis is orchestrates by mutual interplay between interleukin-17–producing γδ T cells and the microbiota.^170,171

Under pathologic conditions, studies have clarified how T cells can influence bone remodeling through direct expression of pro-osteoclastic cytokines and indirect signaling to stromal-osteoblastic cells.^30,172,173 Kong et al showed that T cells isolated from global RANKL knockout mice produced less interleukin-2 and interferon-gamma, which was attributed to a developmental defect of these T-cells and not a direct effect of RANKL on mature T cells.³⁰ RANKL expression in T cells is induced by T-cell receptor engagement with an antigen receptor,¹⁷² which is dependent on T-cell receptor activation-induced calcium ion mobilization.¹⁷³ T cells have been shown to critically regulate osteoclastogenesis through the expression of RANKL and other biologic mediators.^66,174

Extensive periodontal research has been performed to discern the role of T cells in periodontitis-driven alveolar bone loss. Investigations from the late 1980s to 1990s often referred to the ratio of CD4:CD8 T cells as means to determine the extent of the immune response in periodontal lesions. Studies have reported a decreased CD4:CD8 ratio in periodontitis lesions compared with healthy sites or peripheral blood,^175–178 although an increased CD4:CD8 ratio¹⁷⁹ or no changes in CD4:CD8 have also been reported.^180,181 Investigations characterizing T lymphocytes present in gingival tissues afflicted by periodontitis determined that T cells are primed to become memory T cells.^182,183 During the progression of periodontal disease, subgingival plaque bacterial antigens are taken up by innate immune cells. These innate immune cells are believed to migrate to the cervical lymph nodes, where they present antigens to prompt the adaptive immune response. This allows for T cell activation leading to memory T cells, which have been found in the peripheral blood of chronic periodontitis patients versus healthy control subjects.¹⁸⁴

Several studies used severe combined immunodeficient mice, which lack both T and B cells, to begin to delineate the role of T cells in periodontitis-induced alveolar bone loss. Teng et al transferred human peripheral blood leukocytes from periodontal disease patients into non-obese diabetic/severe combined immunodeficient mice and then orally challenged with A. actinomycetemcomitans.⁵⁰ Outcomes from the investigation suggest that A. actinomycetemcomitans–driven periodontal bone loss is mediated in part by RANKL expression on T cells.⁵⁰ Baker et al orally infected severe combined immunodeficient mice and immunocompetent control mice with P. gingivalis. Severe combined immunodeficient mice were more resistant to oral infection–induced periodontal bone loss, which implies that T cells and B cells significantly modulate P. gingivalis–driven alveolar bone destruction.¹⁸⁵ Baker et al employed the P. gingivalis oral infection model in mice deficient in CD4⁺ T-cell signaling to major histocompatibility complex class II (A_β-knockout mice) and mice lacking major histocompatibility complex class I signaling to CD8⁺ T cells and NK1⁺ T cells (B₂m-knockout mice).¹⁸⁶ A_β-knockout mice (deficient in major histocompatibility complex class II–responsive CD4⁺ T cells) exhibited less alveolar bone loss than wild-type mice did, whereas B₂m-knockout mice (deficient in major histocompatibility complex class I–responsive CD8⁺ T cells and NK1⁺ T cells) were not protected against P. gingivalis–induced alveolar bone loss.¹⁸⁶ These outcomes support the notion that CD4⁺ T cells are important effectors of alveolar bone loss consequent to P. gingivalis oral infection.¹⁸⁶

Though CD8⁺ T cells and γδ T cells may not be the primary inducer of periodontitis-driven alveolar bone loss, they likely play important roles modulating the host response to subgingival biofilms in periodontal disease. Importantly, CD8⁺ T cells suppress interferon-gamma–producing cells and favor humoral immune responses.¹⁸⁷ In peripheral blood mononuclear cells isolated from chronic periodontitis patients, activated cytotoxic CD8⁺CD28⁺ T cells were significantly elevated in periodontitis patients compared with healthy controls.¹⁸⁸ γδ T cells are present in epithelial and connective tissues in both healthy^189–191 and chronically inflamed gingiva.^189,192 Gemmell and Seymour reported that the proportion of γδ T cells was not significantly altered in gingival tissues’ isolates from patients afflicted by gingivitis and periodontitis.¹⁹³ Tsukasaki et al evaluated changes in γδ T-cell frequency within gingival tissues of mice subjected to the ligature periodontitis model.⁶³ Experimental periodontitis did not alter γδ T-cell frequency within gingival tissues,⁶³ which is in line with previously reported clinical findings.¹⁹³

Effector CD4⁺ T cell subsets, including T_H1, T_H2, T regulatory, T_H17, T_H9, and T_H22, have begun to be dissected to understand their specific roles in osteoimmunology and periodontitis-driven alveolar bone loss.

9.2 |. T_H1/T_H2 cells

T_H1 cells are CD4⁺ T-effector cells that target intracellular pathogens and are specifically defined by secretion of interleukin-2, interferon-gamma, and tumor necrosis factor.^194,195 Interferon-gamma has been shown to have both pro and antiresorptive actions.^196–201 Conversely, T_H2 cells function in extracellular pathogen control and are specifically defined by interleukin-4, interleukin-5, and interleukin-13 cytokine secretion. Furthermore, T_H2 cells potentiate humoral responses, supporting B cell proliferation and differentiation. T_H1 and T_H2 cells are mutually opposing and self-sustained, because interferon-gamma and interleukin-4 antagonize one another.^194,195

T_H1-derived interferon-gamma was initially reported to inhibit osteoclastogenesis, where interferon-gamma receptor–deficient mice had increased osteoclasts and enhanced bone loss.¹⁹⁶ Interferon-gamma also has inhibitory actions on osteoclast precursors¹⁹⁷ and tumor necrosis factor-α–induced bone marrow macrophage-derived osteoclasts.¹⁹⁸ To the contrary, other studies reported that interferon-gamma enhances osteoclastogenesis.^199–201 However, after much debate on whether interferon-gamma promotes or inhibits osteoclastogenesis, Gao et al determined that interferon-gamma dynamically regulates osteoclastogenesis.²⁰² Interferon-gamma directly targets osteoclast precursors to inhibit osteoclast formation, and indirectly promotes osteoclast differentiation and maturation through antigen-dependent T cell activation, which induces T cells to secrete the pro-osteoclastic factors RANKL and tumor necrosis factor.²⁰² Thus, interferon-gamma has direct anti-osteoclastogenic actions and indirect pro-osteoclastogenic actions, which are dependent upon physiologic conditions.

T_H2 cytokines interleukin-4, interleukin-10, and interleukin-13 have been reported to have anti-osteoclastogenic functions.^203–207 In RANKL-induced osteoclastogenesis, interleukin-4 inhibited NFATc1 expression by antagonizing nuclear factor kappa B signaling.²⁰³ Interleukin-10 also has inhibitor functions on NFATc1 expression and its nuclear translocation in osteoclastogenesis.²⁰⁴ Interleukin-4, interleukin-10, and interleukin-13 have been shown to upregulate osteoprotegrin and downregulate RANKL synthesis.^205,206 Interleukin-10 also inhibits osteoclastogenesis by downregulating the production of pro-osteoclastogenic cytokines, such as tumor necrosis factor, interleukin-1beta, and interleukin-6.²⁰⁷

Reports have evaluated T_H1 and T_H2 characteristic cytokine expression in clinical periodontal tissues. Several studies purported more T_H1 involvement over T_H2 in chronic periodontitis.^208,209 Investigations conveyed early onset periodontitis patients had increased interleukin-4 in gingival tissues.^210–212 Others have postulated that T_H2 cells are prominent in early onset and chronic periodontitis,^213,214 and T_H2 cytokines may be an exacerbating factor in progression from gingivitis to chronic periodontitis.²¹¹ Contradictory to these findings, several studies found that T_H1 and T_H2-related cytokines were comparable in vitro²¹⁵ and between diseased and healthy periodontal tissues.²¹⁶

Experimental periodontitis models have been utilized to define the role of T_H1 and T_H2 cells in inflammatory periodontal bone loss. An in vitro study found that P. gingivalis–specific T-cell lines produce both T_H1 and T_H2 cytokines.²¹⁷ Another investigation found that P. gingivalis–specific clone T cells initially primed by cross-reactive F. nucleatum antigens were polarized to the T_H2 subset, whereas T-cells stimulated with P. gingivalis alone maintained the profile of T_H1 subset cells.²¹⁸ Kawai et al aimed to evaluate the role of T cells, specifically using T-cell receptor/CD28–dependent T_H1 or T_H2 clones in rats.²¹⁹ Gingival injection of A. actinomycetemcomitans antigen and lipopolysaccharide induced local bone resorption after the transfer of antigen-specific T-cell receptor/costimulatory B7–dependent T_H1 clone cells but not after transfer of T_H2 clone cells.²¹⁹ Teng et al found that increased interferon-gamma was correlated with A. actinomycetemcomitans–specific RANKL⁺CD4⁺T_H-cell–mediated alveolar bone loss during the progression of periodontal disease.²²⁰ Furthermore, Alayan et al reported that both T_H1-cytokine (interleukin-12p40, interferon-gamma, tumor necrosis factor)–deficient mice and T_H2-cytokine (interleukin-10, interleukin-4)–deficient mice exhibited significantly more alveolar bone loss than wild-type control mice.²²¹ T. forsythia has been shown to drive alveolar bone loss through a T_H2 bias.²²² The aforementioned work demonstrates the complexity of subgingival plaque polymicrobial species in the modulation of T_H1/T_H2-mediated immunity in periodontitis-induced bone loss.

Historically, evaluating the T_H1/T_H2 cellular balance was considered an important outcome when assessing periodontal bone loss. The proposed theory was that T_H1 cells mediate early stable lesions, whereas T_H2 cells attempt to maintain homeostasis. However, as the disease progresses, T_H2 cells become more prominent beyond the established lesion (reviewed by Gemmell and Seymour²²³). This notion is supported by the presence of elevated B cell activation and plasma cells that infiltrate inflamed tissue, where the T_H2-derived cytokine interleukin-4 promotes B cell activation and proliferation. Because T_H1 and T_H2 influences on alveolar bone loss clinically and experimentally are not straightforward, this calls for ongoing studies to define the molecular underpinnings of each cell in periodontal disease progression.

9.3 |. T regulatory cells/T_H17 cells

T regulatory cells maintain immune homeostasis by ameliorating inflammation through cytokine production of interleukin-10, interleukin-12, and transforming growth factor beta. In clinical periodontitis, T regulatory cells have been shown to be present during disease.^224–227 T regulatory cell presence throughout progressive stages of periodontitis may imply a compensatory mechanism aiming to alleviate tissue destruction from the overreaction of the immune system. Yet, T regulatory cells have been shown to have high plasticity, where they can lose immunosuppressive function under chronic periodontal inflammation.^228,229 Thus, it may be proposed that T regulatory cells may be present in early lesions to mitigate the adaptive immune response, but then, as the disease progresses, T regulatory cells are dominated by other proinflammatory cells and factors.

Kobayashi et al employed the murine P. gingivalis oral inoculation model to evaluate the induction of interleukin-10–producing CD4⁺ T cells in chronic periodontitis. The number of FoxP3⁺CD4⁺CD25⁺ T regulatory cells was increased in the experimental group during the late stage of infection, which correlated with elevated interleukin-10–producing CD4⁺ T cells.²³⁰ Garlet et al showed that A. actinomycetemcomitans oral inoculation in mice resulted in elevated CD4⁺CD25⁺ and CD4⁺FoxP3⁺ cells, characterizing the presence of T regulatory cells in the periodontal environment in a late stage after infection. T regulatory cell–associated cytokines, including interleukin-10 and transforming growth factor beta, were synthesized in kinetics that resemble T regulatory cell migration. Treatment with glucocorticoid-inducible tumor necrosis factor receptor (T regulatory cell function inhibitor) exacerbated inflammation and periodontal bone loss, which suggests the presence of T regulatory cells attenuates the severity of periodontitis.²³¹ Furthermore, T regulatory cell migration and recruitment have been shown to be essential for ameliorating experimental periodontitis-induced alveolar bone loss, which may be dependent upon CCR4-CCL22 signaling.^232,233 Other periodontal disease studies have proposed that T regulatory cells may be transdifferentiated into T_H17 cells during intermediate stages of disease.^63,229 Tsukasaki et al reported that murine experimental periodontitis-induced oral infection converted FoxP3⁺ T cells into T_H17 cells. These bone-damaging exFoxP3 T_H17 cells, which initially may suppress local infection and protect against pathogens, also contributed to alveolar bone resorption.⁶³ Clinically, interleukin-17/FoxP3 double-positive cells were detected in periodontal lesions of chronic periodontitis tissues.²²⁹ In contrast, Parachuru et al reported no detection of interleukin-17/FoxP3 double-positive cells in active periodontal disease lesions.²³⁴

T_H17 cells sustain homeostasis at mucosal barrier sites by regulating the microbiota, and they modulate autoimmune conditions, such as multiple sclerosis and rheumatoid arthritis.²³⁵ T_H17 cells are induced from naive T cells via antigen presentation or through stimulation by proinflammatory cytokines, such as interleukin-1beta, interleukin-6, and interleukin-23.²³⁵ To the contrary, interferon-gamma and interleukin-4 can inhibit T_H17 differentiation. T_H17 cells predominantly secrete interleukin-17, as well as interleukin-21, interleukin-22 and tumor necrosis factor.²³⁵ Recent timely reports have highlighted the interplay between T_H17 cells and the oral microbiota, which have been shown to have implications for alveolar bone homeostasis in health and disease.^{63,89,236,237} T_H17 cells are of interest in bone remodeling due to interleukin-17 having potent pro-osteoclastic actions. Sato et al found that interleukin-17 has no effect on osteoclast precursor cell differentiation in the RANKL-macrophage colony–stimulating factor-osteoclast culture system.²³⁸ This implies that interleukin-17 promotes neighboring cells in the microenvironment to drive osteoclastogenesis, which is consistent with a report showing that interleukin-17 promotes osteoclastogenesis through osteoblastic cell–mediated RANKL induction.²³⁹

T_H17 cells, , since their discovery, have been of great interest in periodontal disease and alveolar bone loss.^240,241 Increased T_H17 cells and interleukin-17 expression have been observed clinically and are elevated in isolates from periodontitis versus gingivitis patients.^242–246 Interleukin-17 has also been reported to be increased in gingival isolates from active versus inactive periodontitis lesions.^226,247,248 Interleukin-23, which can promote T_H17 cell differentiation, has been reported to be increased in periodontitis lesions with elevated interleukin-17 expression.^245,247 Dutzan et al designated T_H17 cells as the primary cellular source of interleukin-17 in clinical periodontitis, where there was minimal interleukin-17 contribution from CD8⁺ T cells, γδ T cells, and non-T-cell lineages.²⁴⁹

Interleukin-17 receptor alpha–deficient mice (C57Bl/6 and Balb/c) inoculated with P. gingivalis have significantly more alveolar bone loss due to defects in the chemokine-neutrophil recruitment axis.^133,250 Yu et al found this outcome to be sex dependent, with females having more severe periodontal bone loss, due to defective production of anti–P. gingivalis immunoglobulin G and the chemokines CXCL1 and CXCL2.²⁵⁰ Eskan et al showed that antibody neutralization of interleukin-17A blunted periodontal inflammation and alveolar bone loss in the P. gingivalis oral inoculation model.²⁵¹ In vitro studies have delineated that specific serotypes of A. actinomycetemcomitans²⁵² and P. gingivalis²⁵³ induce characteristic T_H1/T_H17 transcription factors and cytokines. A modified T. forsythia strain, with an altered O-glycan surface composition, has been reported to induce T_H17-linked mobilization of neutrophils to the gingival tissues and suppress P. gingivalis–driven alveolar bone loss in mice.^254,255 These reports highlight the complexity of the subgingival plaque polymicrobial ecosystem in the modulation of the T_H17/interleukin-17 periodontal immune response.

Whereas early investigations evaluated the T_H1/T_H2 balance in periodontal disease, reports have evolved into describing the T_H17/T_H1 paradigm²⁵⁶ and T_H17/T regulatory cell paradigm.²⁵⁷ The T_H17/T_H1 paradigm is centered on discerning which cell is more destructive, whereas the T_H17/T regulatory cell paradigm is focused on T regulatory cell amelioration of inflammation and periodontal bone loss. Monasterio and coworkers recently employed the murine oral inoculation periodontitis model to show that the T_H1/T_H17 immune response and periodontal bone loss is strain dependent.^258,259 Bi et al reported increased T_H1/T_H17 cells in periodontitis-afflicted gingival isolates, whereas T_H2/T regulatory cells were upregulated in healthy gingiva.²⁶⁰ Serotype b of A. actinomycetemcomitans induced elevated T-bet⁺interferon-gamma⁺ and RAR-related orphan receptor gamma t⁺interleukin-17⁺ T cells in periodontal lesions, which were associated with increased RANKL and alveolar bone resorption.²⁵⁸ Capsular-defective mutant strains of P. gingivalis promoted less bone loss and decreased T_H1/T_H17 cells and related cytokines compared with the encapsulated W50 wild-type strain.²⁵⁹ Investigations focusing on the T_H17/T regulatory cell paradigm have reported an upregulated T_H17/T regulatory cell balance in the progression of ligature-induced periodontitis, both in the rat model²⁶¹ and rhesus monkey model.²⁶² Supporting these findings, Wang et al showed that vaccination with P. gingivalis ameliorates murine experimental periodontitis by offsetting the T_H17/T regulatory cell imbalance.²⁶³ Recently, Cafferata et al reported alterations in the T_H17/T regulatory cell axis but found that interleukin-35 treatment inhibited alveolar bone loss by modulating the T_H17/T regulatory cell ratio with increased T regulatory cell–associated cytokines.²⁶⁴ Taken together, the balance of T_H17 cells to T_H1 cells and T regulatory cells may provide insight into defined T_H17 cell parameters that correlate with periodontal disease progression.

9.4 |. T_H9 and T_H22 cells

Most recently, there have been further discoveries into defining additional CD4⁺ T-cell helper subsets, such as T_H9 and T_H22 cells. T_H9 cells have been described in allergy and asthma, where they require transforming growth factor beta and interleukin-4 for their induction.^265,266 T_H9 cells are defined by the intracellular transcription factors GATA3 and interferon regulatory factor 4 and primarily secrete interleukin-9, driving immunoglobulin E/immunoglobulin G production.^265,266 T_H22 cells are involved in skin immunity and homeostasis, where they can primarily secrete interleukin-22 and tumor necrosis factor.^267–269 T_H17 and T_H22 cells both can express the transcription factor RAR-related orphan receptor gamma t, but T_H22 cells are also identified by aryl hydrocarbon receptor expression. Notably, T_H22 cells synthesize interleukin-22, which is significant for host barrier defense against bacterial infections.^267–269 It is currently unclear whether T_H9 and T_H22 cells are critical players in osteoimmunology and alveolar bone homeostasis. Of interest, several clinical research investigations have begun to elucidate the role of T_H9 and T_H22 cells in periodontal disease.^270,271 Diaz-Zuniga et al evaluated the interleukin-9 and interleukin-22 cytokine levels in gingival crevicular fluid samples and biopsies and their association with periodontal disease severity.²⁷⁰ Increased levels of interleukin-22 and aryl hydrocarbon receptor were detected in patients with periodontitis compared with gingivitis and healthy individuals, and a significant correlation was reported between secreted interleukin-22 and clinical attachment level of the sampled periodontal pockets.²⁷⁰ Higher interleukin-9 levels were detected in gingivitis patients than in periodontitis and healthy counterparts.²⁷⁰ Osteoclasts stimulated with tissue homogenates obtained from periodontitis versus healthy patients demonstrated enhanced resorptive activity, which was dependent on the presence of interleukin-22.²⁷⁰ The same research group isolated dendritic cells and naive CD4⁺ T cells from healthy donors and stimulated with different serotypes of A. actinomycetemcomitans or purified lipopolysaccharide.²⁷¹ A. actinomycetemcomitans serotype b increased levels of interleukin-22 and aryl hydrocarbon receptor in T lymphocytes.²⁷¹ Further research is indicated to discern the role of the T_H9 and T_H22 helper T cell subsets in periodontal health and disease.

9.5 |. B cells

B cells derive from the hematopoietic lineage, where they are considered positive immune regulators due to antibody production. B cells are professional antigen-presenting cells as well as producers of cytokines that promote multiple humoral responses. Importantly, stromal-osteoblastic cells have been shown to support the commitment and differentiation of all stages of B cell development.²⁷² Activated B cells express RANKL, which critically regulates skeletal homeostasis and disease.^66,273 Interestingly, B cells can also inhibit osteoclast formation and viability through transforming growth factor beta secretion.²⁷⁴

The seminal report by Page and Schroeder described the plasma cells/B cells as a minor component of the inflammatory infiltrate detected within the early lesion of chronic periodontitis.¹⁴⁶ Disease progression led to an increased presence of these cells in the affected connective tissue. The advanced lesion, which is the only stage afflicted by appreciable bone loss, was dominated by plasma cells/B cells.¹⁴⁶ Subsequent studies have confirmed that plasma cells/B cells are the most prominent cellular constituent of the infiltrate in advanced lesions of chronic periodontitis.^{183,275–278} Interestingly, Jing et al identified CD138⁺CD38⁺ plasma cells as major contributors to the secretion of interleukin-35 and interleukin-37, which are postulated as anti-inflammatory cytokines in chronic periodontitis.²⁷⁹

Experimental animal models challenged by P. gingivalis²⁸⁰ and A. actinomycetemcomitans²⁸¹ oral inoculation found that the serum antibody response preceded alveolar bone loss, and bacterial-responsive B cells were responsible for this bone loss. P. gingivalis–induced experimental periodontitis studies performed in mice deficient in immunoglobulin D (IgD)²⁸² and immunoglobulin M (IgM)^283,284, which have critical defects in B cell maturation, clearly demonstrate that B cells contribute to periodontal bone loss. Baker et al studied the contribution of B cell IgD to alveolar bone loss by carrying out P. gingivalis oral infection in IgD-deficient mice. P. gingivalis–specific antibody was lower and oral colonization was higher in IgD-deficient mice, yet alveolar bone loss was completely absent.²⁸² Diminished proportions of CD4⁺ T cells and CD69⁺ activated B cells in IgD-deficient mice support the notion that IgD mediates alveolar bone resorption through antigen-specific coactivation of B cells and CD4⁺ T cells.²⁸² Oliver-Bell et al reported that IgM-deficient mice were also protected from P. gingivalis–mediated periodontal bone loss, which was attributed to reduced RANKL-expressing B cells.²⁸³ Abe et al demonstrated that tumor necrosis factor ligand superfamily member 13 and B-lymphocyte stimulator are elevated in experimental murine periodontitis and clinical periodontitis.²⁸⁴ Tumor necrosis factor ligand superfamily member 13 and B-lymphocyte stimulator are tumor necrosis factor ligand family cytokines that are important for B cell proliferation, maturation, and survival. The upregulated expression of tumor necrosis factor ligand superfamily member 13 and B-lymphocyte stimulator correlated with increased numbers of B cells/plasma cells in gingival tissue isolates from both mice and humans.²⁸⁴ Ligature-induced periodontitis in IgM-deficient mice resulted in less alveolar bone loss compared with wild-type controls.²⁸⁴ Antibody neutralization of tumor necrosis factor ligand superfamily member 13 or B-lymphocyte stimulator lowered B cell numbers in the gingival tissue and inhibited alveolar bone loss in wild-type mice, which was not observed in IgM-deficient mice.²⁸⁴

Because activated B cells express RANKL,^66,273 studies have attempted to elucidate the role of activated B cells in periodontal disease. Han et al immunized mice with A. actinomycetemcomitans and isolated activated B cells from immunized and nonimmunized mice.²⁸¹ A. actinomycetemcomitans–specific activated B cells secreted greater RANKL levels in vitro than nonimmunized B cells did. A. actinomycetemcomitans–specific activated B-cell culture supernatants induced superior osteoclast outcomes in the RAW264.7 cell osteoclast culture system. Kanzaki et al evaluated RANKL expression on B cells and T cells within gingival tissue isolates from periodontitis patients, finding that RANKL expression was more profound on B cells than on T cells.²⁸⁵ Isolated B cells promoted osteoclast differentiation and function in the RAW264.7 cell osteoclast culture system.²⁸⁵ Demoersman et al showed that peripheral blood–activated B cells from chronic periodontitis patients versus healthy controls expressed more RANKL.²⁸⁶

Memory B cells can drive a proinflammatory infiltrate within active periodontal lesions through cytokine production, such as tumor necrosis factor or interleukin-6, or enhance synthesis of matrix metalloproteinases to influence tissue breakdown.^277,287 Mahanonda et al demonstrated that memory B cells reside in the connective tissue near the junctional epithelium in normal gingiva,²⁸⁷ suggesting that memory B cells may be important in regulating periodontal health and homeostasis. Demoersman et al found memory B cells to be the predominant B cell subset in periodontitis patients compared with healthy controls.²⁸⁶ Experimental models of periodontitis performed by Han and coworkers^288–290 have further defined the role of memory B cells in periodontal bone loss. In one report, rats were primed with systemic lipopolysaccharide injections and then memory B cells were isolated and adoptively transferred into rats that were subjected to lipopolysaccharide systemic challenge compared with vehicle control.²⁸⁸ Memory B cells were reported to enhance alveolar bone loss and osteoclast differentiation and maturation in lipopolysaccharide-challenged rats compared with control.²⁸⁸ In a rat ligature-induced periodontitis model, memory B cell numbers were suppressed but were found to express higher levels of RANKL than control did.²⁹⁰ Han et al further defined memory B cells in a study in which rats were primed with systemic injections of P. gingivalis.²⁸⁹ Memory B cells were then isolated and adoptively transferred into rats that were subjected to the ligature-induced periodontitis. Results from this study indicate that switched memory B cells (CD27⁺CD38⁻IgD⁻) expressed more RANKL, interleukin-6, and interleukin-1beta than naive, antibody-secreting, and unswitched memory B cells (CD27⁺CD38⁻IgD⁺) did.²⁸⁹ Switched memory B cells also induced greater periodontal bone loss and upregulated proinflammatory cytokines, T_H1 cells, and T_H17 cells in the gingiva and cervical lymph nodes.²⁸⁹ Though reports differ in outcomes concerning whether activated B cells or memory B cells express more RANKL, both B cell subsets have been shown to play important roles in regulating periodontal disease–driven alveolar bone loss.

Additional B cell subsets have been identified, including B1, B2, and regulatory B cells (also known as B10 cells). B1 and B2 cells are traditional B cells that can give rise to long-lived plasma cells and memory cells. Regulatory B cells are identified by the expression of interleukin-10.²⁹¹ Ongoing studies have begun to identify these B cells subsets in periodontal health and disease. Donati et al evaluated gingival tissues from chronic periodontitis patients and found B1 and B2 cells constituted a larger proportion of cells than T cells and plasma cells.²⁷⁷ In contrast, Demoersman et al reported that B1 cells are diminished in peripheral blood of periodontitis patients compared with healthy patients.²⁸⁶ Timely reports have characterized regulatory B cells to play a critical role in suppressing inflammatory responses in vitro^292,293 and ameliorating alveolar bone loss in vivo.^293–295 Liu et al performed an in vitro study in which isolated splenic B cells were immunized with P. gingivalis and treated with CD40 ligand and CpG to induce B cell–derived toll-like receptor signaling.²⁹² Immunized B cells secreted elevated interleukin-10 levels, compared with nonimmunized B cells.²⁹² Based on these in vitro findings, further studies characterized regulatory B cells in rodent experimental periodontitis models. Hu et al used the rat ligature-induced periodontal disease model, with or without local CD40 ligand injections into the palatal gingiva.²⁹³ CD40 ligand elevated regulatory B cells, increased gingival Il10, decreased RANKL, and blunted alveolar bone loss.²⁹³ Local injection of CD40 ligand and CpG into the gingiva was studied by Yu et al in a murine ligature model²⁹⁴ and by Wang et al in a P. gingivalis-lipopolysaccharide systemic administration model.²⁹⁶ CD40 ligand and CpG–induced regulatory B cells alleviated periodontal bone loss.^294,296 Zhao et al found that CD40 ligand and CpG induced regulatory B cells ameliorate ligature-induced periodontitis in a toll-like receptor-9–independent manner.²⁹⁵ Similarly, Shi et al adoptively transferred periodontopathogenic-specific regulatory B cells, which alleviated alveolar bone loss, increased interleukin-10 production, and reduced interleukin-17 and T_H17 cells in the murine gingiva.²⁹⁷

Advancing knowledge about B cell subsets and their diverse roles in periodontal health and disease will provide opportunity for the development of therapeutic targets in the B cell lineage for the treatment of periodontitis.

9.6 |. Natural killer cells/natural killer T cells

Natural killer cells are cytolytic effector lymphocytes that are important in viral and bacterial infections and malignancies. Natural killer cells have several activating receptors, such as natural cytotoxicity receptors (NKp30, NKp44, and NKp46), of which only NKp46 has a mouse ortholog (NCR1). Peripheral natural killer cell homeostasis is mediated by interleukin-15, while natural killer cells can produce interferon-gamma as well as proinflammatory and anti-inflammatory cytokines, such as tumor necrosis factor and interleukin-10, respectively.²⁹⁸ Notably, natural killer–mediated interferon-gamma production modulates T cell responses in secondary lymphoid organs as well as impacts T cell and B cell immunity.²⁹⁸ Invariant natural killer T cells are defined by NK1.1 expression, which recognizes glycolipids:CD1d complexes expressed by antigen-presenting cells.²⁹⁹ Natural killer T cells also recognize pathogenic bacterial antigens and self-antigens.²⁹⁹ The difference between these cell types is that natural killer cells are large granular lymphocytes that mature in circulation, whereas natural killer T cells are a type of T cell that maturates in the thymus.^298,299

Under pathologic conditions like rheumatoid arthritis, natural killer cells contribute to osteoclastogenesis by expressing soluble and surface-bound RANKL and macrophage colony-stimulating factor.³⁰⁰ In vitro studies have elucidated natural killer cell interactions with osteoclasts and osteoblasts.^301,302 In the presence of interleukin-15, synovial fluid–derived natural killer cells were co-cultured with autologous monocytes, which differentiated monocytes into osteoclasts.³⁰¹ Takeda et al isolated whole bone marrow, extracted either natural killer cells or T cells, and co-cultured with osteoblasts in the presence or absence of prostaglandin E2 and/or interleukin-15.³⁰² Osteoblast numbers were decreased by dose-dependent interleukin-15 in whole marrow cultures and cultures lacking T cells.³⁰² Elevated osteoblast numbers were observed in co-cultures lacking natural killer cells, which was attributed to interleukin-15 activation of natural killer cell–induced osteoblastic cell apoptosis.³⁰²

There are few studies determining the role of natural killer cells in the initiation and pathogenesis of periodontitis. Tsoumis et al reported a positive correlation between peripheral blood natural killer cell cytotoxicity and clinical periodontitis progression,³⁰³ and Fujita et al performed an immunohistochemical study that demonstrated natural killer cell accumulation in gingival tissue afflicted by periodontal disease.³⁰⁴ Chaushu et al orally inoculated P. gingivalis or F. nucleatum in mice deficient for the natural killer cell receptor NCR1. The NCR1 knockout mice, compared with wild-type mice, exhibited less alveolar bone loss when infected by P. gingivalis and were completely protected against F. nucleatum–induced alveolar loss.³⁰⁵ These studies imply that natural killer cells regulate the pathogenesis of periodontal disease and related alveolar bone loss.

Natural killer T cells have been shown to be more prominent in periodontitis lesions versus gingivitis lesions.^306,307 Yet, Nowak et al reported natural killer T cells were not present in clinical chronic periodontal disease lesions.³⁰⁸ Baker et al orally inoculated P. gingivalis in β₂m-knockout mice that are deficient in major histocompatibility complex class I–responsive CD8⁺ T cells and NK1⁺ T cells.¹⁸⁶ The β₂m-knockout mice had similar alveolar bone loss to wild-type mice, which implies that natural killer T cells do not modulate periodontitis-driven alveolar bone loss.¹⁸⁶ Aoki-Nonaka et al orally inoculated P. gingivalis in CD1d knockout mice, which are deficient in responsive natural killer T cells.³⁰⁹ The CD1d knockout mice exhibited no alveolar bone loss, suggesting that natural killer T cells contribute to periodontal bone loss. The limited studies discerning the role of natural killer cells and natural killer T cells in periodontitis demonstrate the need for ongoing investigations.

9.7 |. Mast cells

Mast cells differentiate from the hematopoietic stem cell lineage and mature in peripheral connective tissues. Mast cells regulate mucosal epithelial barrier homeostasis by inducing both innate and adaptive immune cells through the production of proinflammatory mediators, such as tumor necrosis factor, nitric oxide, and matrix metalloproteinases.³¹⁰ Mucosal tissues, such as the gut³¹¹ and oral cavity,³¹² contain mast cells that are continuously being exposed to foreign pathogens and allergens. Mast cells have been characterized in healthy gingiva,^312,313 but they expand in inflamed tissues.^314,315

Mast cell populations were observed to be increased in chronic periodontitis.^316–318 Timely studies have elucidated the role of mast cells in periodontal disease progression, where the degree of degranulation correlates with disease severity.^319–321 Interestingly, when mast cells were inhibited by lodoxamide ethyl in beagle dogs, alveolar bone loss was ameliorated.³²² In contrast, Gemmell et al reported that tryptase⁺ mast cells were deceased in clinical periodontal lesions, suggesting there is not an increase in mast cell migration into diseased tissue.²¹⁴ More recently, Huang et al reported higher toll-like receptor-4 expression on mast cells within gingival tissues of chronic periodontitis patients, suggesting that mast cell toll-like receptor-4 expression may mediate the disease process.³²³ Interleukin-31³²⁴ and interleukin-33³²⁵ have been investigated in relation to the role of mast cells in experimental periodontal disease models. Tada et al reported that P. gingivalis induces the production of interleukin-31 by human mast cells, which downregulated tight junction molecules to promote gingival epithelial barrier permeability.³²⁴ Koseoglu et al found that interleukin-33, which supports mast cell development, exhibited a positive correlation with the number of mast cells in a rat ligature periodontitis model.³²⁵ Upregulated interleukin-33 expression correlated with increased levels of RANKL and alveolar bone loss.³²⁵

Recognizing that mast cells have been linked with interleukin-17 production, Parachuru et al evaluated healthy, gingivitis, and chronic periodontitis tissues to discern the cellular source of interleukin-17.³²⁶ Interleukin-17⁺–labeled cells were neither CD4⁺ nor CD8⁺, but were tryptase⁺, a selective marker for mast cells.³²⁶ Furthermore, an experimental mouse model of periodontitis presented mast cells as contributors to P. gingivalis–induced alveolar bone loss by increasing tumor necrosis factor and interleukin-6 production.³²⁷ Though mast cells may be important in recognition and clearing of early infections, a constant inflammatory milieu may promote mast cells to contribute and exacerbate later stages of periodontal disease.

9.8 |. Neutrophils

Neutrophils are the first responders in primary host immune defense.³²⁸ When proinflammatory cytokines and chemokines are present, neutrophils utilize adhesion molecules to attach to endothelial cells within the blood vessels of gingival lamina propria.^{95,104,328,329} It has been estimated that about 30,000 neutrophils transit through the gingival tissue per minute,¹⁰³ where this recruitment is necessary for periodontal tissue homeostasis.¹⁰⁴ Individuals afflicted by neutrophil defects have a higher incidence of periodontal disease than healthy individuals do.³³⁰ Clinical neutrophil defects have facilitated characterizing the role of neutrophils and their function in the periodontal innate immune defense.¹⁰⁴ In periodontal disease, neutrophils have enhanced survival, which may contribute to the chronic proinflammatory state that drives periodontal tissue destruction. Though neutrophils have been extensively characterized as an important immune cell in periodontitis (reviewed by Hajishengallis and Korostoff³³¹), this cell population continues to be of interest owing to its close proximity to the oral microbiota and the modulation of other immune cells in periodontal health and disease.

9.9 |. Myeloid-derived suppressor cells

Myeloid-derived suppressor cells are an immature myeloid precursor cell population derived from bone marrow that regulate the innate and adaptive immune responses.^332,333 Whereas myeloid-derived suppressor cells were initially studied in cancer, research advances have elucidated that myeloid-derived suppressor cells regulate immune responses and the pathogenesis of several inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, and periodontal disease.^332–334 Under homeostatic conditions, myeloid-derived suppressor cells comprise about 30% of normal bone marrow cells.³³⁵

Chronic inflammation at peripheral sites drives recruitment of myeloid-derived suppressor cells, which promote ongoing tissue damage through several mechanisms. Primarily, myeloid-derived suppressor cells suppress T cell activation and proliferation,³³⁶ but also modulate the T regulatory cell population through major histocompatibility complex class II³³⁷ and inhibit natural killer cell functions.^338,339 Furthermore, because myeloid-derived suppressor cells are an immature progenitor population, these cells can differentiate into other myeloid/monocyte lineage cells, like macrophages, plasmacytoid dendritic cells, inflammatory monocytes, and granulocytes, which is dependent upon proinflammatory factors in the local microenvironment.^340,341 Interestingly, seminal reports have shown that myeloid-derived suppressor cells can be osteoclast progenitor cells in vivo and can be cultured into osteoclasts in vitro under inflammatory conditions.^342–345 Cytokines such as tumor necrosis factor, interleukin-1beta, CCL3, and CCL4, which have pro-osteoclastic effects,^346–348 support myeloid-derived suppressor cells recruitment to inflammatory sites.^349,350 We recently published an initial report showing that perturbations in the microbiota elevate myeloid-derived suppressor cells in bone marrow.¹²⁰ Antibiotic disruption of the commensal microbiota increased myeloid-derived suppressor cells in the bone marrow environment, which was linked to increased osteoclastogenesis through the major histocompatibility complex class II antigen presentation pathway.¹²⁰

Though myeloid-derived suppressor cells have been well characterized in several chronic inflammatory conditions, little is known about their function in periodontal health and disease. Ezernitchi et al described the role of myeloid-derived suppressor cell–mediated immunosuppression in a murine model challenged by P. gingivalis injection, reporting that upregulated myeloid-derived suppressor cells suppressed T cell intracellular signaling.³⁵¹ Arjunan et al drove monocytes down the myeloid-derived suppressor cell lineage in vitro, through stimulation of P. gingivalis.³⁵² Both acute and chronic P. gingivalis oral infections in mice induced myeloid-derived suppressor cells, which suppressed cytotoxic T cells and upregulated FoxP3⁺ T cells.³⁵² Su et al found that P. gingivalis oral infection expanded three subpopulations of myeloid-derived suppressor cells in mice: CD11b⁺Ly6G⁺⁺Ly6C⁺, CD11b⁺Ly6G⁺Ly6C⁺⁺, and CD11b⁺Ly6G⁺Ly6C⁺.³³⁴ Only the CD11b⁺Ly6G⁺Ly6C⁺⁺-expressing cells suppressed T cell proliferation and were able to differentiate into osteoclasts in vitro.³³⁴ Recently, Cheng et al reported that periodontal inflammation supports the recruitment of distant metastatic cancer cells through myeloid-derived suppressor cells and related chemokines.³⁵³ Currently, there are no known studies attributing myeloid-derived suppressor cells’ immunosuppressive effects directly on alveolar bone homeostasis. The role of myeloid-derived suppressor cells and their immunosuppressive mechanisms influencing alveolar bone remodeling in periodontal health and disease remain to be defined.

9.10 |. Macrophages

Macrophages share a common progenitor cell population with myeloid-derived suppressor cells, monocytes, dendritic cells, and osteoclasts, and they are responsible for mediating inflammation, phagocytic activities, and immune surveillance via antigen presentation. These cells can polarize into proinflammatory-like (M1) or anti-inflammatory-like (M2) cells, dependent upon factors within the microenvironment.³⁵⁴ Defining the role of macrophages in osteoimmunology, bone marrow macrophages were identified in close proximity to osteoblasts, suggesting that they communicate with bone cells and support bone remodeling processes, specifically producing bone morphogenetic proteins to promote osteoblast maturation.^355,356 Furthermore, macrophage depletion models support the notion that macrophages are critical osteoimmune cells that modulate both osteoblast and osteoclast processes.^357,358 Alternatively, activated macrophages have been characterized as regulators of skeletal homeostasis, yet macrophage effects on bone remodeling in the alveolar bone complex are not as well understood.

Periodontal research has determined that macrophages play critical roles in periodontal tissue destruction (reviewed by Sima and Glogauer³⁵⁹). Recent investigations have focused on how macrophage polarization may be a vital target for monitoring disease periodontal progression. Studies imply that M1 macrophages are contributory in osteoclastogenesis with P. gingivalis infection in vitro^360,361 and in vivo.^360,362 M1 macrophages have been shown to induce interferon-gamma or interleukin-6 production and exacerbate alveolar bone loss.^360,362 Huang et al hypothesized that P. gingivalis and A. actinomycetemcomitans would induce M1-type cells, whereas oral commensal bacteria would promote more of an M2-like response.³⁶¹ M1 macrophage output from P. gingivalis challenge increased proinflammatory cytokines with less promotion of T cell recruitment, whereas A. actinomycetemcomitans challenge elevated both the proinflammatory cytokines and T-cell chemokines.³⁶¹ In contrast to these findings, Yamaguchi et al found that M1 macrophages inhibited RANKL-induced osteoclastogenesis, which was interferon-gamma dependent.³⁶³

Macrophage skewing toward an M2 phenotype has also been investigated. Huang et al reported that M2 macrophages were induced by oral commensals, but not P. gingivalis or A. actinomycetemcomitans.³⁶¹ Yu et al found M2 macrophages to be slightly enhanced in comparison with M1 macrophages in vitro and in vivo.³⁶⁰ Zhuang et al showed that induction of M2 macrophages prevented periodontal bone loss, utilizing both the P. gingivalis oral inoculation and ligature-induced experimental periodontitis models.³⁶⁴ Though considerable research efforts have been centered on macrophage polarization/skewing in periodontitis, the function of these cells has not been extensively investigated in periodontal health and homeostasis.

9.11 |. Dendritic cells

Like other professional antigen-presenting cells, such as macrophages and B cells, myeloid lineage–derived dendritic cells are necessary for thymic T cell education for immune tolerance and T cell interactions in lymphoid tissues in health and immune challenges.³⁶⁵ Dendritic cells function in immune surveillance, which is achieved through antigen presentation by major histocompatibility complex class I and class II complexes. These dynamic mechanisms are essential for innate-adaptive immune crosstalk presenting self/nonself-peptides to the adaptive immune system. Dendritic cells are critical in taking in nonself-antigen, migrating to secondary lymphoid tissues to present to T cells, promoting an adaptive immune response.

Dendritic cell subsets can be divided into plasmacytoid dendritic cells, Langerhans cells, and conventional dendritic cells. Plasmacytoid dendritic cells derive from lymphoid progenitors and are proinflammatory in nature due to production and secretion of type I interferons, interleukin-6, tumor necrosis factor, and proinflammatory chemokines.³⁶⁵ Plasmacytoid dendritic cells have also been associated with increased levels of CCL2, CCL5, and CXCL10 that may promote RANKL, thus leading to osteoclastogenesis induction.^366,367 Conventional dendritic cells reside in connective and lymphoid tissues, where they express high levels of CD11c. These cells commonly present antigen and secrete multiple cytokines to stimulate specific T cell subsets, thus skewing T cells subsets for secretion of interferon-gamma, RANKL, and interleukin-17, promoting osteoclastogenesis.³⁶⁸ Immature conventional dendritic cells survey connective tissues for potential foreign threats, but, when activated, these cells will migrate and travel to nearby lymph nodes to present foreign antigens to T cells for an adaptive immune response.³⁶⁹ Langerhans cells are a distinct dendritic cell subset that express the C-type lectin receptor langerin (CD207). These cells reside in the skin epidermis and oral mucosal epithelium, where they function in immune surveillance by promoting either tolerance to self-antigen or an immune response to activate T cells.^370–372 Langerhans cells are derived from pre-dendritic cells and monocytes through a possible transforming growth factor beta 1 signaling mechanism to promote mucosal homeostasis and prevent tissue damage.³⁷³

As previously stated, dendritic cells share a common progenitor with osteoclasts. Therefore, several reports have shown that dendritic cells can develop into osteoclasts.^374,375 Murine bone marrow and splenic CD11c⁺ dendritic cells develop into TRAP⁺ cathepsin K⁺ functional osteoclasts in a RANKL/RANK-dependent manner, both in vitro³⁷⁵ and in vivo.³⁶⁸ These findings were supported by an investigation which showed that immature dendritic cells derived from spleen and marrow developed into osteoclasts in a macrophage colony-stimulating factor/RANKL–induced in vitro system, whereas mature conventional dendritic cells and plasmacytoid dendritic cells did not.³⁷⁴ Importantly, these reports suggest that immature dendritic cells can act as osteoclast precursor cells.

Because of the multifunctionality and common cellular lineage that dendritic cells share with other immune cells, dendritic cells have been investigated in experimental periodontitis models to understand dendritic cell–mediated disease initiation, progression, and support of osteoclastogenesis. A study investigating the role of Langerhans cells in oral mucosal immunity found that in vivo Langerhans cell depletion in P gingivalis-induced murine periodontitis led to increased T cell and B cell infiltration in periodontal tissues and exacerbated alveolar bone loss.³⁷⁶ However, a separate report stated that Langerhans cells were not necessary for P. gingivalis–induced alveolar bone loss.³⁷⁷ Furthermore, Bittner-Eddy et al reported that oral mucosal Langerhans cells promoted T_H17 differentiation, and Langerhans cell ablation resulted in a T_H1-driven response but T regulatory cells were unaffected.³⁷⁷ The authors speculated that Langerhans cell depletion led to suppression of T_H17-mediated immunity, which was compensated by an upregulation of T_H1-mediated immunity.³⁷⁷

Investigations have utilized experimental periodontal disease models to demonstrate that dendritic cells can drive proinflammatory cytokine production.^378,379 Dendritic cell–mediated osteoclastogenesis has been shown to elevate alveolar bone loss, which is attributed to increased RANKL and numbers of osteoclasts in vitro and in vivo.³⁸⁰ Yet, P. gingivalis oral inoculation allowed for dendritic cells to generate T_H2 responses^381,382 and T regulatory cell induction through the secretion of interleukin-10 and transforming growth factor beta.³⁸³ Alnaeeli et al described dendritic cells as an essential immune cell in the pathogenesis of periodontitis.³⁷⁵ CD4⁺ T cells co-cultured with A. actinomycetemcomitans–primed dendritic cells differentiated into functional osteoclasts.³⁷⁵ Furthermore, adoptively transferred CD11c⁺ dendritic cell–derived osteoclasts induced bone loss in vivo.³⁷⁵ In a P. gingivalis–induced periodontitis model, silencing of cathepsin K led to reduced dendritic cell numbers in periodontal lesions, which was correlated with elevated T_H17 cells.³⁷⁹ Xiao et al carried out a dendritic cell–lineage-specific deletion of FOXO1 in a P. gingivalis and F. nucleatum murine oral gavage model.³⁸⁴ FOXO1-deficient dendritic cells demonstrated impaired recruitment to the oral mucosal epithelium.³⁸⁴ FOXO1-deficient dendritic cells secreted less interleukin-12, which was compensated by elevated interleukin-1beta, interleukin-17, and RANKL.³⁸⁴ Future studies are indicated to further elucidate the specific roles dendritic cells play in mediating oral mucosal immunity and periodontal health and disease.

9.12 |. Highlights of osteoimmune interactions in periodontitis

The immune system plays a critical role in the maintenance of alveolar bone in health and disease. Innate immune cells, such as neutrophils, macrophages, and dendritic cells, have been classified as the early line of defense in periodontal disease. Recent research has highlighted the potential role of myeloid-derived suppressor cells, mast cells, and natural killer/natural killer T cells as candidate modulators of periodontitis. Historically, B cells and CD8⁺ and CD4⁺ T cells were identified as critical promoters in the pathogenesis of periodontal disease and alveolar bone destruction. Advances in characterizing B cell and T cell subsets have begun to elucidate the complexity of the adaptive immune response involved in periodontal bone loss. There has been an extensive focus on the T_H17/interleukin-17 immune response in disease progression, and regulatory B cells have been introduced as protective immune mediators. Ongoing advances in the field of osteoimmunology are necessary to clarify the role of distinct immune cells in periodontal health and disease. This importantly will provide opportunities for therapeutic interventions in the treatment of periodontitis and prevention of alveolar bone destruction.

10 |. THERAPEUTIC INTERVENTIONS: PROBIOTICS AND PREBIOTICS

Probiotic/prebiotic therapies are noninvasive interventions intended to alter indigenous microbiota communities to improve health.³⁸⁵ Probiotic/prebiotic interventions in the gut microbiota modulate the host immune response, which has been shown to protect against skeletal deterioration driven by sex-steroid deficiency.^386–388 There has been a recent surge in probiotic/prebiotic research for the prevention and treatment of periodontal disease. The review will focus on prebiotic/probiotic research that has evaluated alveolar bone outcomes (Table 1). Though there have been a multitude of clinical and preclinical studies, alveolar bone outcomes have predominantly been evaluated in experimental animal models.

TABLE 1.

Summary of preclinical studies testing probiotics on alveolar bone

Probiotic strain	Species tested	Model	Probiotic duration	Outcomes	Reference
Lactobacillus rhamnosus GG	BALB/c mice	Porphyromonas gingivalis, Fusobacterium nucleatum oral gavage	44 d	↓ABL ↓Inflammation ↓# OCs	389
Lactobacillus rhamnosus GG	BALB/c mice	Porphyromonas gingivalis, Fusobacterium nucleatum oral gavage	44 d	↓ABL ↓Duodenum inflammation ↓Ileum IL-6	390
Lactobacillus gasseri SBT2055	BALB/c mice	Porphyromonas gingivalis oral gavage (days 21–35)	35 d	↓ABL ↓TNF, IL-6 ↓Gingival detachment	391
Bacillus subtilis	Wistar rats	Ligature (days 30–44)	44 d	↓ABL, RANKL/OPG ratio ↓Eosinophils	392
Bacillus subtilis	Wistar rats	Ligature (days 1–14)	14 d (days 15–29)	↓# OCs ↓ABL, attachment ↓L-1b, ↑IL-10	392
Lactobacillus brevis CD2	C57BL/6 mice	Ligature	5 d	↓ABL ↓TNF, Il-1b, Il-6, Il-17a ↓Anaerobic microbes, ↑aerobic microbes	393
Bifidobacterium animalis lactis HN019	Wistar rats	Ligature	0, 3, and 7 d	↑OPG, β-defensins ↓Il-1b, Nfkb Altered oral flora ↓Tb.Sp	394
Lactobacillus paracasei NTU-101	Wistar rats	Lipopolysaccharide induction	4 wk	↓ABL ↓TNF, Il-1b, Il-17a ↓Oral bacterial numbers	395
Bifidobacterium animalis lactis HN019	Wistar rats	Ligature (days 1–14)	14 d (days 15–29)	↓ABL, attachment loss ↓# OCs, Il-1b, Cinc ↑Il-10, Tgfb ↑Anaerobic/aerobic ratio	396
Streptococcus sanguinis, Streptococcus salivarius, Streptococcus mitis	Beagle dogs	Natural-occurring periodontitis (12 wk)	Weeks 1, 2, and 4	↓ABL	397
Bacillus subtilis	Wistar rats	Ligature (days 30–44)	44 d	↑Attachment loss, ABL ↑Intestinal villous and crypt depth	398
Bacillus subtilis	Wistar rats	Ligature (days 30–44)	44 d	↓ABL, CTX-1; ↑OPG	399
Saccharomyces cerevisiae	Wistar rats	Ligature (7 d)	96 h	↓ABL 15 d post-ligature; ↓OCs, Tnf, Il-1b 30 d post-ligature	400
Weissella cibaria	ICR mice	Ligature (14 d)	14 d	↓ABL ↓Tnf, Il-1b, Il-6, Il-10	401

Note: Abbreviations: ABL, alveolar bone loss; Cinc, cytokine-induced neutrophil chemoattractant; CTX-1, C-terminal telopeptide of type I collagen; IL/Il, interleukin; Nfkb, nuclear factor-kappa B; OC, osteoclast; OPG, osteoprotegrin; RANKL, receptor activator of nuclear factor-kappa B ligand; Tb.Sp, trabecular spacing; Tgfb, transforming growth factor beta; TNF/TNF/Tnf, tumor necrosis factor.

Probiotics are viable microorganisms that provide benefits to the host.³⁸⁵ Lactobacilli and bifidobacteria are the most commonly used probiotic strains.³⁸⁵ Lactobacilli are indigenous bacteria colonizing the oral cavity and digestive tract, having critical roles in the prevention and treatment of gastrointestinal disorders.⁴⁰² Bifidobacteria naturally occur in a range of ecological niches that are either directly or indirectly connected to the gastrointestinal tract, such as the human oral cavity.⁴⁰² Several strains of probiotics have recently been reported to have beneficial outcomes in the prevention and treatment of periodontal disease. Nackaerts et al performed the first known investigation of probiotic effects on alveolar bone homeostasis. A probiotic combination of Streptococcus sanguinis, Streptococcus salivarius, and S. mitis, was administered three times over 12 weeks in dogs, where probiotic administration restricted naturally occurring alveolar bone loss.³⁹⁷ Other investigations have utilized ligature,^{392–394,396,398–401} perio-pathogen inoculation,^389–391 or lipopolysaccharide administration³⁹⁵ to induce experimental periodontitis in rodent models to evaluate probiotic effects on the oral and gut microbiota, periodontal immune response, and alveolar bone homeostasis. Studies varied in methodology, where diverse probiotics were administered for different durations of time, prior to and/or during experimentally induced periodontitis (Table 1).^{389,391–396,398,399,400} Investigations utilizing a ligature model with a Bifidobacterium probiotic have reported decreased alveolar bone loss,^{392,396,398,399} reduced attachment loss,^392,396,398 increased osteoprotegrin levels,^394,399 suppressed proinflammatory cytokine levels,^394,396 and alterations in anaerobic:aerobic oral microbiota composition.^394,396 Maekawa and Hajishengallis employed a ligature-induced murine periodontitis model.³⁹³ Lactobacillus brevis CD2 was applied topically for 5 days after ligature application, which was found to reduce alveolar bone loss, decrease proinflammatory cytokines (tumor necrosis factor, interleukin-1beta, interleukin-6, interleukin-17), and increase aerobic bacteria while suppressing anaerobic bacteria in the oral cavity.³⁹³ Garcia et al used Saccharomyces cerevisiae to treat experimental periodontitis for 96 hours and the results were evaluated 7, 15, and 30 days following treatment.⁴⁰⁰ Probiotic treatment suppressed alveolar bone loss and upregulated interleukin-10 at 15 days, and it reduced osteoclast numbers and proinflammatory cytokine levels (tumor necrosis factor and interleukin-1beta) at 30 days.⁴⁰⁰ Kim et al utilized the probiotic Weissella cibaria and found decreased proinflammatory cytokines tumor necrosis factor, interleukin-1beta, and interleukin-6 in the gingiva and less alveolar bone loss.⁴⁰¹ Two investigations utilized P. gingivalis and/or F. nucleatum to induce periodontitis in Balb/c mice to determine the effects of probiotics on alveolar bone.^389,391 Gatej et al orally administered Lactobacillus rhamnosus GG 3 days prior to P. gingivalis and F. nucleatum infection and the treatment continued for 44 days.³⁸⁹ L. rhamnosus GG was reported to prevent alveolar bone loss, reduce inflammatory score, and lower numbers of osteoclasts.³⁸⁹ Kobayashi et al orally administered Lactobacillus gasseri SBT2055, which was found to reduce alveolar bone loss, clinical attachment loss, and inflammation in P. gingivalis–induced periodontitis.³⁹¹ Liu et al applied the lipopolysaccharide-induced periodontitis model where rats were fed the probiotic Lactobacillus paracasei NTU-101.³⁹⁵ Dietary L. paracasei NTU-101 decreased alveolar bone loss, suppressed gingival Tnf, Il17a, and Il1b, and reduced the oral microbiota bacterial load.³⁹⁵ Interestingly, there was also an increase in anti–reactive oxygen species in the gingiva and serum of probiotic-treated animals compared with control.³⁹⁵ Notably, two studies employed probiotic use for the treatment of periodontitis and found benefits on intestinal outcomes: Messora et al reported greater intestinal villous and crypt depth,³⁹⁸ and Gatej et al found reduced inflammation in the duodenum and ileum with probiotic use.³⁹⁰ Clinical studies have employed probiotics either as a preventative measure to support periodontal health^403–407 or as an intervention for the treatment of chronic periodontitis.^408–412 However, clinical reports have neglected to quantify probiotic effects on alveolar bone outcomes.

Prebiotics are nondigestible food elements that benefit the host by promoting proliferation and/or function of selective gut microbes to have beneficial effects.³⁸⁵ Certain prebiotics can enhance resident commensal gut bacteria, such as lactobacilli and bifidobacteria, while also providing enhanced functions in deterring pathogenic bacteria colonization.⁴¹³ The primary mechanism of action of prebiotics is assumed to be indirect; that is, facilitating the proliferation of beneficial components of the resident microbiota.⁴¹³ Prebiotics have been shown to improve intestinal absorption and balance of calcium and magnesium,^414,415 as well as improve femoral bone mineral density.⁴¹⁶ There has been a recent increase in studies evaluating prebiotics and their effects on periodontal health and disease. An in vitro study that screened for several prebiotic candidates identified N-acetyl-d-mannosamine to promote biofilm formation of S. mitis and S. sanguinis without influencing pathogenic strains.⁴¹⁷ De Oliveira Silva et al utilized beta-glucans as a prebiotic intervention in rat experimental periodontitis.⁴¹⁸ Beta-glucans treatment reduced alveolar bone loss and gingival interleukin-10 expression.⁴¹⁸ Levi et al utilized mannan oligosaccharide as a prebiotic in rat ligature-induced periodontitis.⁴¹⁹ Mannan oligosaccharide administration was carried out for 30 days, followed by 14 days of ligature-induced periodontitis.⁴¹⁹ Mannan oligosaccharide administration ameliorated alveolar bone loss, decreased interleukin-10, interferon-gamma, tumor necrosis factor, and interleukin-1beta, increased transforming growth factor beta, and elevated intestinal villous height and crypt depth.⁴¹⁹

Future randomized, placebo-controlled, double-blind clinical trials are necessary to discern the potential for probiotics/prebiotics interventions in the microbiome to support alveolar bone homeostasis and prevent periodontal bone loss. Recognizing that probiotic/prebiotic manipulation of the gut microbiota has osteoimmunomodulatory effects that protect against nonoral skeletal deterioration,^386–388 there is high potential for alveolar bone applications.

11 |. CONCLUSION

Alveolar bone is a unique osseous tissue owing to its incorporation with the dentition and proximity to oral biofilms. A balanced host immune response to polymicrobial oral biofilms is required for periodontal health and homeostasis. Further, the oral microbiota is a critical regulator of alveolar bone remodeling. However, shifts in the composition and quantity of microbes within dental plaque biofilms can drive a local proinflammatory immune response in oral barrier sites. Infiltrating proinflammatory immune cells within the inflamed connective tissue secrete local factors that induce paracrine signaling to subjacent bone cells. Prolonged chronic inflammation disrupts “coupled” osteoclast-osteoblast actions, which ultimately results in alveolar bone destruction. Though most studies focus on osteoclastogenesis, we highlight the importance of the osteoblastic cell lineage in the pathogenesis of periodontal bone loss, where the osteoblastic cell lineage, for the most part, is overlooked by periodontal researchers. Highlighted in this review is that knowledge that nonoral microbiota-host immune response effects contribute to fibrotic conditions supports the notion that periodontitis-induced alveolar bone destruction occurs in part through fibrosis. Future research is critically needed to discern the role of fibrosis in the pathophysiology of clinical periodontal bone loss. Furthermore, our current understanding of periodontal osteoimmunology influencing alveolar bone remodeling may play a vital role in alveolar osteoimmune mechanisms. However, with critical gaps in knowledge of several immune cells, this provides an opportunity for further development in periodontal bone osteoimmunology. Lastly, probiotic/prebiotic therapeutic interventions in the oral microbiota as a potential therapy may help to support alveolar bone homeostasis/ prevent periodontal bone loss. Future randomized, placebo-controlled, double-blind, clinical trials are needed to elucidate the noninvasive, therapeutic potential for probiotics/prebiotics interventions in the microbiome to promote alveolar bone homeostasis and prevent periodontal bone loss. Recognizing that probiotic/prebiotic shifts of the gut microbiota have osteoimmunomodulatory protective effects, there is high promise for alveolar bone applications.