Dana T. Graves

Molecular Mechanisms Controlling Bone Formation during Fracture Healing and Distraction Osteogenesis

Fracture healing and distraction osteogenesis have important applications in orthopedic, maxillofacial, and periodontal treatment. In this review, the cellular and molecular mechanisms that regulate fracture repair are contrasted with bone regeneration that occurs during distraction osteogenesis. While both processes have many common features, unique differences are observed in the temporal appearance and expression of specific molecular factors that regulate each. The relative importance of inflammatory cytokines in normal and diabetic healing, the transforming growth factor beta superfamily of bone morphogenetic mediators, and the process of angiogenesis are discussed as they relate to bone repair. A complete summary of biological activities and functions of various bioactive factors may be found at COPE (Cytokines & Cells Online Pathfinder Encyclopedia), http://www.copewithcytokines.de/cope.cgi.

Expression of osteoprotegerin, receptor activator of NF-kappaB ligand (osteoprotegerin ligand) and related proinflammatory cytokines during fracture healing.

Fracture healing is a unique biological process regulated by a complex array of signaling molecules and proinflammatory cytokines. Recent evidence for the role of tumor necrosis family members in the coupling of cellular functions during skeletal homeostasis suggests that they also may be involved in the regulation of skeletal repair. The expression of a number of cytokines and receptors that are of functional importance to bone remodeling (osteoprotegerin [OPG], macrophage colony‐stimulating factor [M‐CSF], and osteoprotegerin ligand [receptor activator of NF‐κB ligand (RANKL)]), as well as inflammation (tumor necrosis factor α [TNF‐α] and its receptors, and interleukin‐1α [IL‐1α] and ‐β and their receptors) were analyzed over a 28‐day period after the generation of simple transverse fractures in mouse tibias. OPG was expressed constitutively in unfractured bones and elevated levels of expression were detected throughout the repair process. It showed two distinct peaks of expression: the first occurring within 24 h after fracture and the second at the time of peak cartilage formation on day 7. In contrast, the expression of RANKL was nearly undetectable in unfractured bones but strongly induced throughout the period of fracture healing. The peak in expression of RANKL did not correlate with that of OPG, because maximal levels of expression were seen on day 3 and day 14, when OPG levels were decreasing. M‐CSF expression followed the temporal profile of RANKL but was expressed at relatively high basal levels in unfractured bones. TNF‐α, lymphotoxin‐β (LT‐β), IL‐1α, and IL‐1β showed peaks in expression within the first 24 h after fracture, depressed levels during the period of cartilage formation, and increased levels of expression on day 21 and day 28 when bone remodeling was initiated. Both TNF‐α receptors (p55 and p75) and the IL‐1RII receptor showed identical patterns of expression to their ligands, while the IL‐1R1 was expressed only during the initial period of inflammation on day 1 and day 3 postfracture. Both TNF‐α and IL‐1α expression were localized primarily in macrophages and inflammatory cells during the early periods of inflammation and seen in mesenchymal and osteoblastic cells later during healing. TNF‐α expression also was detected at very high levels in hypertrophic chondrocytes. These data imply that the expression profiles for OPG, RANKL, and M‐CSF are tightly coupled during fracture healing and involved in the regulation of both endochondral resorption and bone remodeling. TNF‐α and IL‐1 are expressed at both very early and late phases in the repair process, which suggests that these cytokines are important in the initiation of the repair process and play important functional roles in intramembraneous bone formation and trabecular bone remodeling.

Cytokines That Promote Periodontal Tissue Destruction

Although periodontal diseases are initiated by bacteria that colonize the tooth surface and gingival sulcus, the host response is believed to play an essential role in the breakdown of connective tissue and bone, key features of the disease process. An intermediate mechanism that lies between bacterial stimulation and tissue destruction is the production of cytokines, which stimulates inflammatory events that activate effector mechanisms. These cytokines can be organized as chemokines, innate immune cytokines, and acquired immune cytokines. Although they were historically identified as leukocyte products, many are also produced by a number of cell types, including keratinocytes, resident mesenchymal cells (such as fibroblasts and osteoblasts) or their precursors, dendritic cells, and endothelial cells. Chemokines are chemotactic cytokines that play an important role in leukocyte recruitment and may directly or indirectly modulate osteoclast formation. This article focuses on aspects of osteoimmunology that affect periodontal diseases by examining the role of cytokines, chemokines, and immune cell mediators. It summarizes some of the key findings that attempt to delineate the mechanisms by which immune factors can lead to the loss of connective tissue attachment and alveolar bone. In addition, a discussion is presented on the importance of clarifying the process of uncoupling, a process whereby insufficient bone formation occurs following resorption, which is likely to contribute to net bone loss in periodontal disease.

Impaired Fracture Healing in the Absence of TNF‐α Signaling: The Role of TNF‐α in Endochondral Cartilage Resorption

TNF‐α is a major inflammatory factor that is induced in response to injury, and it contributes to the normal regulatory processes of bone resorption. The role of TNF‐α during fracture healing was examined in wild‐type and TNF‐α receptor (p55−/−/p75−/−)‐deficient mice. The results show that TNF‐α plays an important regulatory role in postnatal endochondral bone formation.

The Use of Rodent Models to Investigate Host-Bacteria Interactions Related to Periodontal Diseases

Even though animal models have limitations, they are often superior to in vitro or clinical studies in addressing mechanistic questions and serve as an essential link between hypotheses and human patients. Periodontal disease can be viewed as a process that involves four major stages: bacterial colonization, invasion, induction of a destructive host response in connective tissue and a repair process that reduces the extent of tissue breakdown. Animal studies should be evaluated in terms of their capacity to test specific hypotheses rather than their fidelity to all aspects of periodontal disease initiation and progression. Thus, each of the models described below can be adapted to test discrete components of these four major steps, but not all of them. This review describes five different animal models that are appropriate for examining components of host-bacteria interactions that can lead to breakdown of hard and soft connective tissue or conditions that limit its repair as follows: the mouse calvarial model, murine oral gavage models with or without adoptive transfer of human lymphocytes, rat ligature model and rat Aggregatibacter actinomycetemcomitans feeding model.

Diabetes Enhances Periodontal Bone Loss through Enhanced Resorption and Diminished Bone Formation

Using a ligature-induced model in type-2 Zucker diabetic fatty (ZDF) rat and normoglycemic littermates, we investigated whether diabetes primarily affects periodontitis by enhancing bone loss or by limiting osseous repair. Diabetes increased the intensity and duration of the inflammatory infiltrate (P < 0.05). The formation of osteoclasts and percent eroded bone after 7 days of ligature placement was similar, while four days after removal of ligatures, the type 2 diabetic group had significantly higher osteoclast numbers and activity (P < 0.05). The amount of new bone formation following resorption was 2.4- to 2.9-fold higher in normoglycemic vs. diabetic rats (P < 0.05). Diabetes also increased apoptosis and decreased the number of bone-lining cells, osteoblasts, and periodontal ligament fibroblasts (P < 0.05). Thus, diabetes caused a more persistent inflammatory response, greater loss of attachment and more alveolar bone resorption, and impaired new bone formation. The latter may be affected by increased apoptosis of bone-lining and PDL cells.

Cytokine Profiling of Macrophages Exposed to Porphyromonas gingivalis, Its Lipopolysaccharide, or Its FimA Protein

ABSTRACT To characterize the roles of Porphyromonas gingivalis and its components in the disease processes, we investigated the cytokine profile induced by live P. gingivalis, its lipopolysaccharides (LPS), and its major fimbrial protein, fimbrillin (FimA). Using cytokine antibody arrays, we found that P. gingivalis LPS and FimA induced a similar profile of cytokine expression when exposed to mouse peritoneal macrophages but that this profile differed significantly in response to live P. gingivalis. In vitro, mouse peritoneal macrophages were stimulated to produce interleukin-6 (IL-6), granulocyte colony-stimulating factor, and lymphotactin by live P. gingivalis, but not by P. gingivalis LPS or FimA, while RANTES, gamma interferon, IL-17, vascular cell adhesion molecule 1 (VCAM-1), and vascular endothelial growth factor were induced by P. gingivalis LPS or FimA, but not by live P. gingivalis. In vivo, IL-6 mRNA was strongly induced only by live P. gingivalis while monocyte chemoattractant protein 1 mRNA was strongly induced only by P. gingivalis LPS and FimA in mouse calvarial scalp, further confirming the differences of cytokine profile induced in vitro. Cytokine antibody arrays using toll-like receptor 2 (TLR2)- and TLR4-deficient macrophages revealed that most of the cytokines induced by P. gingivalis LPS or FimA signal through TLR2, while most of cytokines induced by live P. gingivalis signal through both TLR2 and TLR4. Interestingly, the activation of TLR2 by live P. gingivalis inhibited the release of RANTES, VCAM-1, and IL-1α from mouse peritoneal macrophages. A tumor necrosis factor alpha enzyme-linked immunosorbent assay further confirmed that P. gingivalis LPS and FimA activate mouse peritoneal macrophages via TLR2. These results indicate that host immune cells sense live P. gingivalis and its components differently, which translates into the expression of different inflammatory cytokine profiles.

Review of osteoimmunology and the host response in endodontic and periodontal lesions

Abstract Both lesions of endodontic origin and periodontal diseases involve the host response to bacteria and the formation of osteolytic lesions. Important for both is the upregulation of inflammatory cytokines that initiate and sustain the inflammatory response. Also important are chemokines that induce recruitment of leukocyte subsets and bone-resorptive factors that are largely produced by recruited inflammatory cells. However, there are differences also. Lesions of endodontic origin pose a particular challenge since that bacteria persist in a protected reservoir that is not readily accessible to the immune defenses. Thus, experiments in which the host response is inhibited in endodontic lesions tend to aggravate the formation of osteolytic lesions. In contrast, bacteria that invade the periodontium appear to be less problematic so that blocking arms of the host response tend to reduce the disease process. Interestingly, both lesions of endodontic origin and periodontitis exhibit inflammation that appears to inhibit bone formation. In periodontitis, the spatial location of the inflammation is likely to be important so that a host response that is restricted to a subepithelial space is associated with gingivitis, while a host response closer to bone is linked to bone resorption and periodontitis. However, the persistence of inflammation is also thought to be important in periodontitis since inflammation present during coupled bone formation may limit the capacity to repair the resorbed bone.

Initial Characterization of Cells Derived from Human Periodontia

The studies presented in this report describe an initial characterization of cell types derived from explants of human periodontia. Cell cultures were established from human periodontal ligament (PL4, PL7), gingival tissue (GF2), and alveolar bone (BP1) by means of explant techniques and monolayer culture. Cells were studied at passage numbers 2-4 and were characterized on the basis of morphological, biochemical, and proliferative parameters. Subconfluent cells did not have distinct morphologies useful in distinguishing them from one another. At confluence, PL4 and BP1 cells formed multilayered cultures of randomly oriented cells, while PL7 and GF2 cells grew in a monolayer of parallel cells. Biochemically, PL4 and BP1 cells exhibited characteristics consistent with an osteoblast-like phenotype. These included a significant increase in PTH-stimulated cyclic AMP and high basal levels of alkaline phosphatase activity, which were decreased on exposure to PTH and increased after stimulation by 1, 25 dihydroxyvitamin D3. In contrast, PL7 and GF2 cells exhibited basal alkaline phosphatase levels that were low, and cyclic AMP levels were not modulated by PTH stimulation. Cell populations PL7 and GF2 did not proliferate in culture medium supplemented with 3% platelet-poor plasma. After the addition of platelet-derived growth factor (PDGF) to this medium, the proliferation of these cell populations was equal to that in media supplemented with 10% fetal bovine serum. In contrast, PL4 and BP1 cells did proliferate in culture medium supplemented with 3% platelet-poor plasma. The addition of PDGF to the medium resulted in only a moderate increase in the proliferation of cell populations PL4 and BP1. These results establish that both osteoblast-like and fibroblast-like cells can be cultured from adult human periodontal ligament and suggest methods for studying the cells in vitro.

Diabetes-enhanced Inflammation and Apoptosis—Impact on Periodontal Pathology

Diabetes, particularly type 2 diabetes, is a looming health issue with many ramifications. Because diabetes alters the cellular microenvironment in many different types of tissues, it causes myriad untoward effects, collectively referred to as ’diabetic complications’. Two cellular processes affected by diabetes are inflammation and apoptosis. This review discusses how diabetes-enhanced inflammation and apoptosis may affect the oral environment. In particular, dysregulation of tumor necrosis factor and the formation of advanced glycation products, both of which occur at higher levels in diabetic humans and animal models, potentiate inflammatory responses and induce apoptosis of matrix-producing cells. The enhanced loss of fibroblasts and osteoblasts through apoptosis in diabetics could contribute to limited repair of injured tissue, particularly when combined with other known deficits in diabetic wound-healing. These findings may shed light on diabetes-enhanced risk of periodontal diseases.