Analysis of Some Existing Animal Models to Support Research on
Osteoimmunology - Cross-talk between the Immune System and Bone
January 25, 2007
Immunology and Immunotherapy Program
Center for Integrative Biology and Infectious Diseases
National Institute of Dental and Craniofacial Research
National Institutes of Health
Executive Summary
The Immunology and Immunotherapy Program, Center for Integrative Biology and Infectious Diseases (CIBID), National Institute of Dental and Craniofacial Research (NIDCR) conducted a programmatic consultation on January 25, 2007 via teleconference in Bethesda, MD, to gain insight into the strengths and limitations of some animal models used in periodontal research. The purpose of this consultation was to make available information on available animal models to support the broader research community that might apply to the NIDCR Funding Opportunity Announcement (FOA) on “Osteoimmunology - Crosstalk between Immune System and Bone.” The invited participants were asked to discuss the strengths and limitations of existing periodontitis animal models and to highlight the applicability of these models to research on osteoimmunology, specifically within the oral and craniofacial mission of NIDCR.
The participants discussed several of the available animal models in great detail and concluded that there is no perfect animal model to study the group of periodontal diseases. Each model has strengths and limitations. The specific model selected and used depends on the research question being asked. Some pertinent biologic questions for selecting a particular animal model include:
1) How do infectious agents and products in the periodontal diseases activate the innate and adaptive immune responses?
2) Are there any unique aspects of the immunological process related to the oral cavity, considering the role of microorganisms and their effect in periodontitis?
3) Are the outcomes in the pathogenesis of infection in the periodontium different from infections of other mucosal or non-mucosal sites?
4) What is it about the periodontal diseases that cause inflammation-induced destructive loss of bone versus gingivitis, which is characterized by inflammation that does not affect bone? Periodontitis is found only in a subset of the population in which this inflammation leads to destruction of the bone. What are the regulatory features of animals or humans associated with the loss of alveolar bone?
5) How do inhibitors of the innate or adaptive immune response that reduce alveolar bone loss impact the oral microenvironment? Can various host factors be addressed in periodontal models?
6) Are there defects or differences in the way individuals are colonized by bacteria to promote a host response that leads to bone loss? Is it possible to effectively control bacterial infection in a way that modifies the pathogenesis of disease?
7) What is the contribution of the uncoupling of bone resorption and formation to the net loss of bone in the periodontium? What aspects of the host response impair bone formation to cause uncoupling and in which models can this be tested?
Background
The periodontal diseases are a group of disorders of the gums, or gingiva, and the tissues around the teeth. Like most broadly descriptive terms, the periodontal diseases vary in severity, from the reversible mild inflammation called gingivitis, to the sometimes irreversible severe chronic periodontitis that significantly erodes bone and other supporting structures of the tooth, leading to the loss of the tooth. Signs and symptoms include gingival and periodontal inflammation, gingival recession, apical migration of the junctional epithelium with pocket formation, and alveolar bone loss.
The hallmark of periodontitis is bone resorption resulting from the host inflammatory response to microbial challenge. Periodontal infection has several different stages. The initial stage is microbial colonization, transition to infection, transition to bone resorption and then to cellular “coupling”.
Periodontitis can be considered a failure in “coupling” rather than simply a process of bone resorption. As a generalization, the field of periodontal research may not have fully appreciated that bone resorption in a healthy individual is usually coupled with an equivalent amount of bone formation so that the failure to form bone after bacteria-induced resorption is the most striking feature of progressive loss of alveolar bone. Thus it is important to consider the impact on bone formation as well as bone resorption.
The “chronic” nature of some manifestations of periodontitis is not well understood. It has been suggested that human periodontitis occurs in brief episodes of tissue loss followed by long periods of remission termed the “random burst” model or alternatively, there is a chronic slow relatively continuous loss of alveolar bone. To this day neither model has been confirmed. Although little is known about the episodic “bursts”, it is possible that they may occur over periods as brief as days or weeks. Similarly the length of time of bone resorption in the “chronic” model is unknown.
Studying periodontal diseases is extremely difficult. Host immune responses to the complex flora are complicated and have the potential to limit as well as to aggravate the disease. Furthermore, periodontitis can only be studied retrospectively in man, since reliable clinical markers for ongoing tissue destruction (disease activity) are not yet available. Therefore, an animal model in which selected microbiological and immunological parameters can be studied is desirable. Numerous laboratory animal models exist that have been used to study periodontal diseases. (Weinberg et al, J Clin Periodontol 1999). However, cause and effect relationships between specific bacteria and pathologic processes are difficult to establish in non-gnotobiotic conditions. For example, in P. gingivalis (Pg) models the normal murine flora may play an important role in periodontal bone loss, i.e. Pg may indirectly cause bone loss by affecting the murine biofilm. Similarly, A. actinomycetemcomitans Aa models in mice are limited in studying colonization and infectivity by findings that the mouse is not a normal host for Aa.
Murine Models
The rodent model is a commonly used animal model in experimental periodontitis. Some obvious advantages are (1) Rodents are cheaper and easier to handle than some of the larger animal models; (2) There is a tremendous variation in genetically characterized strains with varying alteration in gene related to bone biology and host responses. Also efficient methods for creating specific “knock-out” or “knock-in” models to test human genes of interest are available; (3) Availability of excellent annotated microarray technology and reagents to examine gene expression and bone; (4) The short life span of mice has enabled studies for aging for systemic disease that has provided some guidance to potential mechanisms of human aging.
1) Murine Calvarial Model or the Scalp Model: This model was developed by Dr. Brendan Boyce (Boyce et al, Endocrinology, 1989) to study the effect of cytokines on bone resorption. The model was later adapted to study the effect of bacteria on bone resorption (Zubery et al, Infect Immun 1998) and host-bacteria interactions involving the innate and adaptive immune responses in connective tissue in vivo (Graves et al, J Dent Res, 2001, Leone et al, Infect Immun, 2006).
Strengths
This is an excellent model for studying host-bacterial interactions in the connective tissue setting. The stimulus or bacterial inoculum can be well controlled. Tissues samples obtained are relatively uniform, sufficient for high throughput screening and can be easily used for histologic analysis of both soft tissue and bone. Bone can be easily isolated from soft tissue so that the molecular changes associated with bone resorption and formation can be examined separately. Both innate and adaptive immune responses can be examined in this model. This model is useful in studying host-bacteria interactions in connective tissue, bone resorption and coupling. It can also be used to study the host response to purified bacterial components.
Limitations
This model does not contain a “periodontium” so that it is most useful in investigating specific processes rather than representing an overall disease model. Also, while one can control the challenge, there is little evidence that the bacteria are actually replicating, and it remains unknown how long they remain viable. Since the bacteria are actually delivered into soft tissues this model cannot study microbial components that are crucial for colonization, “invasion”, and even those determinants of physiology that are required for continued infection/colonization. It has been adapted to examine the impact of repeated inoculations over a period of time and thus may mimic some aspects of a sustained infection. This model is not useful for studying bacterial colonization or mechanisms of infection.
2) Murine Oral Gavage Model: This model was developed by Dr. Pamela Baker (Baker et al, Oral Micro Immunol 2000; Baker et al, Infect Immun 2000). In this model, mice are given antibiotics for 10 days to deplete their microflora. After this procedure, the mice are infected orally with microorganism(s) to induce bone loss. It takes about six weeks to develop observable bone loss.
Strengths
This is a non-surgical model which is relatively easy to use. It has been shown to work with multiple oral microorganisms such as Pg, P. gulae and T. forsythia. The model reflects various cellular components of human disease in terms of immune parameters (neutrophils being largely protective and T-cells being largely destructive). This model may be useful in studying colonization and infection with the caveat that it is not a monoinfection model unless germ-free mice are inoculated. It is useful in studying host-bacteria interactions and bone resorption. It may also be useful in studying colonization and infectivity although the pathogenic organisms in the mouse biofilm may not be representative of those in a human biofilm even with the addition of an exogenous bacterium such as Pg.
Limitations
This is not a “real” model for the chronic disease in humans as it leads to more acute colonization by the exogenous pathogens and often requires reinfection to maintain the system. This model has been found to be strain- and gender-dependent (Balb/c mice are more susceptible, C57BL/6 are not; female mice are more susceptible than males). It seems that the oral bacterium, A. actinomycetemcomitans (Aa), may cause only transient infection in this model. It is not useful in studying coupling because the specific time periods frame in which bone resorption and formation occur are difficulty to establish.
3) Humanized HuPBL-NOD/SCID mice: This model was developed by Dr. Andy Yen-Tung Teng (Teng et al, J Periodontol Res. 1999; J Clin. Invest. 2000, Infect Immun 2002 & 2005; Teng, Critical Rev of Oral Biol and Med 2003). In this model, non-obese diabetic (NOD)-SCID mice are reconstituted with human peripheral blood leukocytes (HuPBL engraftments from AgP subjects) followed by oral inoculation of Aa which leads to increased expression of the osteoprotegerin ligand (OPGL, or RANK-L), a key mediator of osteoclastogenesis and osteoclast activation. Inhibition of OPGL via antagonistic osteoprotegrin significantly reduces alveolar bone destruction after bacterial infection. The results support the critical role of microorganism-reactive human CD4+ T cells in periodontal pathogenesis. This suggests that a pathogen-associated human immune repertoire can be established in these mice.
Strengths
Very specific immune questions associated with both Th1- and Th2-related cytokine expressions in vivo can be answered using this model. It is useful in studying host-bacteria interactions in the periodontium and bone resorption.
Limitations
Testing the donor HuPBL samples for sufficient engraftment is required before modeling. The human cell engraftment rate may be variable and the procedure can be lengthy (7-8 weeks). The initiation aspects of periodontal infection cannot be investigated with this model.
Modification of Murine Models to Study Disease.
It is well known that diabetic patients experience a higher risk for severe periodontitis than non-diabetic patients; however, the underlying mechanisms remain unclear. Several of the above models have been modified to study processes specifically related to the impact of diabetes on the periodontium. It should be noted that the strengths and limitations for each model still apply to the diabetic variant with the caveat that diabetes also must be monitored.
4) NOD mice (for type-1 diabetes): It is well know that diabetic patients experience a higher risk for severe periodontitis than non-diabetic patients; however, the underlying mechanisms remains unclear. The NOD mouse is a model of human type-1 diabetes. When this mouse is orally infected with Aa, diabetic NOD mice manifest significantly higher alveolar bone loss than non-diabetic and pre-diabetic NOD mice. (Mahamed & Teng, Diabetes, 2005).
Strengths
The major strength of this model is its wide application to study diabetic mechanisms that lead to exacerbated periodontitis in vivo.
Limitations
The Type 1-diabetes needs to be monitored along with the development of periodontitis.
Calvarial model with db/db mice (for type-2 diabetes) – The db/db mouse becomes obese at an early age and on a C57BLK background male mice become diabetic at approximately 8 weeks of age due to insulin resistance.
Ligature induced periodontitis in the Zucker diabetic fatty rat (ZDF) (type 2 diabetes) – The ZDF rat is the rat equivalent of the db/db mouse. These rats develop diabetes at approximately 8 weeks of age secondary to insulin resistance and obesity.
Rat Models
The rat model of oral infection and periodontal disease has many of the same benefits as the murine models. Fundamentally, the rat has gained popularity because of the increased size of the oral cavity and teeth, allowing acquisition of more specimen material, as well as providing some greater ease in quantifying bone changes.
5) Rat Ligature Model: This has been used as a model of experimental periodontitis for over 40 years. (Rovin S et al, J Periodontal Res 1966; Kenworthy R et al, J Clin Periodontol 1981; Weiner GS et al, J Periodontol 1979; Samejima Y et al, J Periodontal Res 1990; Bezerra MM et al, J Periodontol 2000; Liu R et al, J Dent Res. 2006). The placement of a silk or cotton ligature around the tooth facilitates plaque accumulation. In germ free rats placement of the ligatures does not induce significant gingival inflammation or periodontal bone loss. Topical treatment with chlorhexidine reduces bone resorption and antibiotics reduce both loss of attachment and loss of bone, supporting the role of bacteria in initiating destruction in this model. In contrast, increasing Gram-negative bacterial burden enhances osteoclastogenesis and bone resorption and inhibitors of a host inflammatory response decrease periodontal destruction.
Strengths
This model has a full periodontium therefore one can measure events in a “periodontal context”. This model can be adapted to study bone formation and resorption separately, i.e. coupling. The tissue is well adapted for histologic analysis although the area of analysis is restricted to the mesial aspect of interdental bone due to slow but continuous distal drift of the maxillary molars. It can theoretically be adapted to study innate or innate plus acquired immune response through immunization and is well adapted for application of systemic inhibitors. This model is useful for studying host-bacterial interactions in the periodontium, mechanisms of bone resorption and coupling.
Limitations
Isolation of bone is difficult, limiting the use of the model in high- throughput screening of bone. The number of genetically modified rats is limited. The delivery of etiologic agents to initiate the disease process cannot be precisely controlled. While one can control the challenge, the lack of ability to control sampling and difficulty in quantifying the infection has limited this activity in rats. Also, the ability to effectively infect and maintain the infection during an interval to create bone resorption remains a challenge. This also contributes to variability in experimental animals tied to variations in the actual oral infection. MicroCT technology can provide non-terminal studies of bone loss but the expense and thus limited availability of the technology is a weakness. This model has limited usefulness in studying bacterial colonization and natural mechanisms of infectivity since infection is facilitated by the ligature.
6) Oral Infection Model
In this model, normal rats are orally infected on multiple occasions to establish colonization. This has been accomplished with a range of human oral bacteria, principally Pg and Aa. Recently, studies suppressing the natural oral microbiota with antibiotics (similar to Baker et al.) have enabled oral infections with T. denticola, T. forsythia, F. nucleatum and a polymicrobial challenge. Depending upon the specific model and questions being asked, evaluation can take place from 1-3 months after oral infection. (Sasaki et al, J Periodontal Res. 2004; Kawai et al, Oral Microbiol Immunol 2007 In press; Kesavalu et al, Infect. Immun. 2007).
Strengths
This infection model allows study of immune cell responses, local gingival tissue responses, and bone resorption. There is markedly increased periodontal bone resorption in infected rats. Bacterial infection can generally be demonstrated during the protocols using PCR. Markedly elevated antibody (serum IgG, saliva IgA) and T cell proliferation to some of the bacteria have been observed. This model may be adapted to study colonization or infectivity. However, the results need to be interpreted with caution since the impact of introducing a bacterium to the normal flora may not be simple and one cannot assume that the introduced bacterium is responsible for the pathologic changes, i.e. the introduced bacterium could indirectly cause pathogenesis by modifying the biofilm. This model may be used to study host-bacteria interactions in the periodontium and bone resorption. It is unlikely that this model would be useful for studying coupling.
Limitations
This model does not allow isolation of the immune components. There is limited ability to dissect the individual immune cell contributions to periodontal bone resorption compared to the murine models.
7) Immune cell models: Models of T cell and B cell contributions to alveolar bone resorption (ABL) have been developed in the laboratory of Dr. Martin Taubman. (Kawai et al, J Immunol. 2000; Valverde et al, J Bone Miner Res. 2004; Han et al, J Immunol. 2006). Normal inbred rats are given a gingival microinjection of antigen (eg. AaOmp29) +LPS, or whole Aa. In the T cell model, T cells are adoptively transferred after 2-3 days and evaluation of ABL takes place 10 days after cell transfer. Bone resorption can be quantified in the rats after euthanasia as CEJ-alveolar crest distance with the control side dimension subtracted. In the B cell model, normal inbred rats are injected intraperitoneally with antigen (whole Aa bacteria). Antigen binding B-cells are subsequently isolated and adoptively transferred to congenitally athymic nude rats. Periodontal bone resorption is evaluated after 10 days of transfer.
Strengths
Injection of antigen and LPS into gingiva is required for the T-cell-induced periodontal bone resorption. Stimulated transferred T cells express RANKL and can directly stimulate osteoclast differentiation, resulting in periodontal bone resorption. Also, T cell-induced periodontal bone resorption can be blocked by administration of OPG-Fc (RANKL-dependent), CTLA4Ig (APC-dependent), Kaliotoxin and anti-CD40 ligand MAb. The B cell model showed higher level of RANKL expression in Aa-binding B cells (ABB). ABB-transferred rats demonstrated increased periodontal bone resorption (abrogated by OPG-Fc; RANKL dependent) and osteoclast differentiation on the alveolar bone crest. ABB induce osteoclastogenesis in vitro, which can be blocked by OPG-Fc (RANKL dependent). ABB recipients show elevated serum IgG antibody to Aa. Transferred ABB recovered from gingival tissues of recipients, co-cultured with osteoclast precursor cells (RAW 264.7) induce osteoclastogenesis and bone resorption pit formation. The model is useful in studying specific components of the adaptive immune response in connective tissue of the periodontium and bone resorption. Its usefulness is studying coupling has not been established.
Limitations
Neither of these models represents a chronic periodontal infection. Periodontal bone resorption is dependent on the concentration of transferred T cells or B cells used in the study.
8) LPS-induced alveolar bone resorption:
This model has been recently employed in the laboratory of Dr. Keith Kirkwood (Kirkwood et al, J Pharmacol Exp Ther 2007; Rogers et al, J Perio, 2007). They have established a model of aggressive inflammatory alveolar bone loss in rats using LPS derived from a periodontal pathogen Aa delivered orally three times a week. Bone resorption has been quantitivately measured using microCT. The bone loss is observed within 8 weeks.
Strengths
This model is easily reproducible. Controlled delivery of LPS results in readily demonstrable bone loss and exhibits many features of human periodontal pathology e.g. OC-mediated bone resorption and cytokine expression. The model for useful in studying LPS-host interactions in the periodontium and bone resorption. It could presumably be modified to study other bacterial components such as fimbriae.
Limitations
This is not an infectious, chronic model but an inflammatory induced bone loss model. At this point only Aa LPS has been tested in this model. This model is useful in studying LPS-induced bone resorption. It is not known whether this model can be adapted to study coupling.
Non-human Primate Model
This model primarily using ligature induction of periodontitis in the primates (Holt SC et al, Science, 1988; Beem JE et al, Infect Immun, 1991; Zappa UE et al, J Clin Periodontol, 1986. Heijl L et al, J Periodontol, 1976, Ebersole et al., J Periodont Res, 1999, Ebersole et al., Oral Microbiol Immunol, 2000, Persson et al., Oral Microbiol Immunol, 1994, Schou et al., J Periodontol, 1993, Reynolds et al., J Gerontol 2007, Page RC et al, Oral Microbiol Immunol, 2007). The ligature-induced disease model in nonhuman primates is imperfect relative to human disease, but clearly represents the most accurate model of complex microbial biofilm infections, localized host inflammatory/immune responses, and tissue destruction commensurate with periodontitis. This is a reproducible model that has been validated and enables a true causal study of infection, host responses, and disease progression that can be accomplished over a reasonable timeframe. Additionally, it is clear the extent of disease can be manipulated, and the ability to prolong disease progression for longer intervals is available
Strengths
The range of nonhuman primate species allows some selection for optimizing experimental design for different investigations. The size of the animals allows clinical, radiographic, and potentially even imaging measures that are commensurate with humans. The primates provide a more representative heterogeneity of genetic background comparable to humans. This is significant since genetics clearly provide some contribution to regulating host responses and the biology of disease progression, although this clearly represents a multigenic disease. There is great similarity in the commensal microbiota of primates and humans related to health, gingivitis, and periodontitis. This includes changes in the microbial ecology that is associated with the transition from health to disease. Also, molecular probes for most human oral bacteria that have been studied overlap extremely well with similar bacteria (i.e. same species) in primates. Thus, the microbial triggering of disease in primates in the only model system that reflects the complexity of the polymicrobial challenge by commensal bacteria that occurs in humans.
Available data suggest that the cellular infiltrate and biomolecular responses of inflammation, innate immunity, and adaptive immunity are quite similar in humans and primates and likely follow similar temporal kinetics. This likely does not recapitulate in most murine models of oral monoinfections with exogenous bacteria, or related to local gingival injection of inflammatory stimuli.
Due to the substantial genetic relationship, reagents for various systemic and local analytes in humans can be effectively used in the primates. Due to the size and similarities of human and primate oral cavities, the primates provide a superior ability to determine changes in immune and bone parameters that are related to development from young, to adult, to elderly. A large amount of biological specimen material can be collected thus making studies of immune cells in local tissues or documenting systemic responses more feasible. The availability and similarity in microarray platforms for primates allows broad studies of gene regulation in tissues. Studies of exogenous agents or therapeutics must be considered to be more similar with regards to dosages, actions, pharmacokinetics etc. in primates when compared to rodents.
This model enables studies of vaccine approaches to “controlling” the commensal microbiota and ameliorating disease processes. Studies in the nonhuman primates have been developed to evaluate the transmission of periodontal pathogens and sequence of acquisition of the bacteria in the oral microbiota of infants. This system will enable the exploration of the ontogeny of innate and adaptive immune responses in the local environment reacting to the developing commensal microbial ecology in the oral cavity. Finally, the oral cavity of nonhuman primates can be used as a model system to examine aging and/or drug effects on host responses to microbial challenge of the mucosa.
Limitations
The cost of the primates, including per diem, is high and studies in primates require specialized facilities and uniquely trained personnel to be successful, which adds to the cost. Primate studies are limited in the number of animals that can be used, and any experimental design that would require euthanasia is particularly challenging. While the experimental timeframe for oral bone loss studies in primates is not that different from the murine model, the ability to obtain numerous longitudinal samples during the window of the experiment is more limited with primates, when compared with mice/rats. Immunologic studies that require syngeneic host-cell interactions cannot be done in primates.
References
1) Weinberg MA, Bral M. 1999. Laboratory animal models in Periodontology. Journal of Clinical Periodontology 26: 335-40.
2) Boyce BF, Aufdemorte TB, Garrett IR, Yates AJ, Mundy GR. 1989. Effects of interleukin-1 on bone turnover in normal mice. Endocrinology. 125:1142-50.
3) Zubery Y, Dunstan CR, Story BM, Kesavalu L, Ebersole JL, Holt SC, Boyce BF. 1998. Bone resorption caused by three periodontal pathogens in vivo in mice is mediated in part by prostaglandin Infection Immunity 66:4158-62.
4) Graves DT, Oskoui M, Volejnikova S, Naguib G, Cai S, Desta T, Kakouras A, Jiang Y. 2001. Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment, and osteoclast formation in response to P. gingivalis infection. Journal of Dental Research 80:1875-9.
5) Leone CW, Bokhadhoor H, Kuo D, Desta T, Yang J, Siqueira MF, Amar S, Graves DT. 2006. Immunization enhances inflammation and tissue destruction in response to Porphyromonas gingivalis. Infection Immunity 74:2286-92.
6) Baker PJ, Dixon M, Evans RT, Roopenian DC. 2000. Heterogeneity of Porphyromonas gingivalis strains in the induction of alveolar bone loss in mice. Oral Microbiology and Immunology 15:27-32.
7) Baker PJ, Dixon M, Roopenian DC. 2000. Genetic control of susceptibility to Porphyromonas gingivalis-induced alveolar bone loss in mice. Infection Immunity 10:5864-8.
8) Teng Y-T, Nguyen H, Hassanloo A, Ellen RP, Hozumi N. 1999. Periodontal Immune Responses of Human Lymphocytes in Actinobobacillus actinomycetemcomitans-inoculated NOD/SCID Mice Engrafted with Peripheral Blood Leukocytes of Periodontitis Patients. Journal of Periodontal Research 34:54-61.
9) Teng Y-T A, Nguyen H, Gao X, Kao YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM. 2000. Functional Human T-cell Immunity and Osteoprotegerin-Ligand (OPG-L) Control Alveolar Bone Destruction in Periodontal Infection. Journal of Clinical Investigation 106: R59-R67.
10) Teng YT. 2002. Mixed periodontal Th1-Th2 cytokine profile in Actinobacillus actinomycetemcomitans-specific osteoprotegerin ligand (or RANK-L) - mediated alveolar bone destruction in vivo. Infection Immunity 70:5269-5273.
11) Teng Y-T A, Mahamed D, Singh B. 2005. IFN- positively modulates Actinobacillus actinomycetemcomitans-specific CD4+T cells-mediated alveolar bone destruction in vivo. Infection Immunity 73: 3453-61.
12) Teng YT. 2003. The role of acquired immunity and periodontal disease progression. Critical Reviews of Oral Biology and Medicine 14:237-252.
13) Mahamed DA, Marleau A, Alnaeeli M, Singh B, Zhang X, Penninger J M and Teng Y-TA. 2005.G(-) Anaerobes-reactive CD4+T cells Trigger RANKL-mediated Enhanced Alveolar Bone Loss in Diabetic NOD Mice. Diabetes 54: 1477-1486.
14) Rovin S, Costich ER and Gordon HA. 1966. The influence of bacteria and irritation in the initiation of periodontal disease in germfree and conventional rats. Journal of Periodontal Research 1:193-204.
15) Kenworthy R, Baverel M. 1981. Studies of a periodontal tissue lesion in the rat, untreated or treated with chlorhexidine digluconate. Journal of Clinical Periodontology 8:349-58.
16) Weiner GS, DeMarco TJ and Bissada NF. 1979. Long term effect of systemic tetracycline administration on the severity of induced periodontitis in the rat. Journal of Periodontology 50:619-23.
17) Samejima Y, Ebisu S and Okada H. 1990. Effect of infection with Eikenella corrodens on the progression of ligature-induced periodontitis in rats. Journal of Periodontal Research 25:308-15.
18) Bezerra MM, de Lima V, Alencar VB, et al. 2000. Selective cyclooxygenase-2 inhibition prevents alveolar bone loss in experimental periodontitis in rats. Journal of Periodontology 71:1009-14.
19) Liu R, Bal HS, Desta T, Krothapalli N, Alyassi M, Luan Q, Graves DT. 2006. Diabetes enhances periodontal bone loss through enhanced resorption and diminished bone formation. Journal of Dental Research 85:510-514.
20) Sasaki H, Okamatsu Y, Kawai T, Kent R, Taubman M, Stashenko P. 2004. The interleukin-10 knockout mouse is highly susceptible to Porphyromonas gingivalis-induced alveolar bone loss. Journal of Periodontal Research 39:432-41.
21) Kawai T, Paster BJ, Komatsuzawa H, Ernst CWO, Goncalves RB, Sasaki H, Ouhara K, Stashenko PP, Sugai M, Taubman MA. 2007. Cross reactive immune response to oral commensal bacteria results in an induction of receptor activator of nuclear factor-B ligand (RANKL)-dependent periodontal bone resorption in a mouse model. Oral Microbiology and Immunology. In press.
22) Kesavalu L, Sathishkumar S, Bakthavatchalu V, Matthews C, Dawson D, Steffen M, Ebersole JL. 2007. Rat model of polymicrobial infection, immunity, and alveolar bone resorption in periodontal disease. Infection Immunity 75:1704-1712.
23) Kawai T, Eisen-Lev R, Seki M, Eastcott JW, Wilson ME, Taubman MA. 2000. Requirement of B7 costimulation for Th1-mediated inflammatory bone resorption in experimental periodontal disease. Journal of Immunology 164:2102-9.
24) Valverde P, Kawai T, Taubman MA. 2004. Selective blockade of voltage-gated potassium channels reduces inflammatory bone resorption in experimental periodontal disease. Journal of Bone Mineral Research 19:155-64.
25) Han X, Kawai T, Eastcott JW, Taubman MA. 2006. Bacterial-responsive B lymphocytes induce periodontal bone resorption. Journal of Immunology 176:625-31.
26) Kirkwood KL, Li F, Rogers JE, Otremba J, Coatney DD, Kreider JM, D'Silva NJ, Chakravarty S, Dugar S, Higgins LS, Protter AA, Medicherla S. 2007. A p38alpha selective mitogen-activated protein kinase inhibitor prevents periodontal bone loss. J Pharmacology Experimental Therapeutics 320:56-63.
27) Rogers JE, Li F, Coatney DD, Rossa C, Bronson P, Krieder JM, Giannobile WV, Kirkwood KL. 2007. Actinobacillus actinomycetemcomitans Lipopolysaccharide-Mediated Experimental Bone Loss Model for Aggressive Periodontitis. Journal of Periodontology 78:550-558.
28) Holt SC, Ebersole J, Felton J, Brunsvold M, Kornman KS. 1988. Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 239: 55-7.
29) Beem JE, Hurley CG, Magnusson I, McArthur WP, Clark WB. 1991. Subgingival microbiota in squirrel monkeys with naturally occurring periodontal diseases. Infection Immunity 59:4034-41.
30) Zappa UE, Polson AM, Eisenberg AD, Espeland MA. 1986. Microbial populations and active tissue destruction in experimental periodontitis. Journal of Clinical Periodontology 13:117-25.
31) Heijl L, Rifkin BR, Zander HA. 1976. Conversion of chronic gingivitis to periodontitis in squirrel monkeys. Journal of Periodontology 47:710-6.
32) Ebersole JL, Cappelli D, Mott G, Kesavalu L, Holt SC, Singer RE. 1999. Systemic manifestations of periodontitis in the non-human primate. Journal of Periodontology Research 34:358-362.
33) Ebersole JL, Cappelli D, Holt SC, Singer RE, Filloon T. 2000. Gingival crevicular fluid inflammatory mediators and bacteriology of gingivitis in nonhuman primates related to susceptibility to periodontitis. Oral Microbiology and Immunology 15:19-26.
34) Persson GR, Engel LD, Whitney CW, Weinberg A, Moncla BJ, Darveau RP, Houston L, Braham P, Page RC. 1994. Macaca fascicularis as a model in which to assess the safety and efficacy of a vaccine for periodontitis. Oral Microbiology and Immunology 9:104-11.
35) Schou S, Holmstrup P, Kornman KS. 1993. Non-human primates used in studies of periodontal disease pathogenesis: a review of the literature. Journal of Periodontology 64:497-508.
36) Reynolds M, Branch-Mays G, Dawson III D,. Novak KF, Mattison J, Gunsolley J, Ingram D, Lane M, Roth G, and Novak MJ. 2007. Effects of age, gender, and caloric restriction on inflammatory disease in a non-human primate model. J Gerontology Series A, Biological Sciences (submitted)
37) Page RC, Lantz MS, Darveau R, Jeffcoat M, Mancl L, Houston L, Braham P, Persson GR. 2007. Immunization of Macaca fascicularis against experimental periodontitis using a vaccine containing cysteine protease purified from Porphyromonas gingivalis. Oral Microbiology and Immunology 22:162-168.
List of Participants:
1) Keith L. Kirkwood, DDS, PhD
Assistant Professor
Periodontics and Oral Medicine
University of Michigan
Ann Arbor, MI 48109
2) Martin Taubman, DDS, PhD
Head of the Department of Immunology and Senior Member
Department of Immunology
The Forsyth Institute
Professor, Department of Oral and Developmental Biology
Harvard Medical School and Harvard School of Dental Medicine
Boston, MA 02115
3) Sarah L. Gaffen, PhD
Associate Professor,
Department of Oral Biology
School of Dental Medicine
University of Buffalo
Buffalo, NY 14214
4) Andy Y-T Teng, DDS, MS, PhD
Associate Professor,
Division of Periodontology
Department of Microbiology and Immunology
The University of Rochester
Eastman Dental Center
Rochester, NY 14620
5) Dana Graves, DDS, DMSc
Professor,
Division of Oral Biology
Boston University, Boston, MA 02118
6) Jeffrey L. Ebersole, PhD
Associate Dean for Research and Graduate Studies
University of Kentucky,
College of Dentistry
Lexington, KY 40536
7) Roy Page DDS, PhD
Associate Dean,
Research Center in Oral Biology
Professor, Department of Pathology and Department of Periodontics
University of Washington, Seattle WA 98195
8) Dr. Yongwon Choi, PhD
Professor,
Department of Pathology and Laboratory Medicine
University of Pennsylvania, PA 19104
9) Mark Horowitz, PhD
Professor
Department of Orthopaedics and Rehabilitation
Yale University School of Medicine
New Haven, Connecticut CT 06520
10) Steven R. Goldring, MD
Chief Scientific Officer
Hospital for Special Surgery
New York, NY 10021