Soft tissue ossification and condylar cartilage degeneration following TMJ disc perforation in a rabbit pilot study.

OBJECTIVE: There are limited clinical treatments for temporomandibular joint (TMJ) pathologies, including degenerative disease, disc perforation and heterotopic ossification (HO). One barrier hindering the development of new therapies is that animal models recapitulating TMJ diseases are poorly established. The objective of this study was to develop an animal model for TMJ cartilage degeneration and disc pathology, including disc perforation and soft tissue HO. METHODS: New Zealand white rabbits (n = 9 rabbits) underwent unilateral TMJ disc perforation surgery and sham surgery on the contralateral side. A 2.5 mm defect was created using a punch biopsy in rabbit TMJ disc. The TMJ condyles and discs were evaluated macroscopically and histologically after 4, 8 and 12 weeks. Condyles were blindly scored by four independent observers using OARSI recommendations for macroscopic and histopathological scoring of osteoarthritis (OA) in rabbit tissues. RESULTS: Histological evidence of TMJ condylar cartilage degeneration was apparent in experimental condyles following disc perforation relative to sham controls after 4 and 8 weeks, including surface fissures and loss of Safranin O staining. At 12 weeks, OARSI scores indicated experimental condylar cartilage erosion into the subchondral bone. Most strikingly, HO occurred within the TMJ disc upon perforation injury in six rabbits after 8 and 12 weeks. CONCLUSION: We report for the first time a rabbit TMJ injury model that demonstrates condylar cartilage degeneration and disc ossification, which is indispensible for testing the efficacy of potential TMJ therapies.

Notch Regulates Fibrocartilage Stem Cell Fate and Is Upregulated in Inflammatory TMJ Arthritis.

The Notch pathway is critical for the development of the extracellular matrix in cartilage by regulating both anabolic and catabolic cellular activities. Similarly, Notch signaling plays a biphasic role in adult cartilage health and osteoarthritis by maintaining homeostasis and contributing to degeneration, respectively. The temporomandibular joint (TMJ) is the synovial joint of the craniofacial complex and is subject to injury and osteoarthritis. While Notch has been studied in axial skeletal joints, little is known about the role of Notch in TMJ development and disease. We identified fibrocartilage stem cells (FCSCs) localized within the TMJ condyle superficial zone niche that regenerate cartilage and repair joint injury. Here we investigate the role of Notch in regulating TMJ development and FCSC fate. Using a Notch reporter mouse, we discovered FCSCs localized within the TMJ superficial niche exhibit Notch activity during TMJ morphogenesis. We further showed that constitutively activating Notch promotes FCSC differentiation toward both cartilage and bone lineages, but inhibits adipogenesis. Using a TNF-α-induced TMJ inflammatory arthritis mouse model, we found that the expression of Notch receptors and ligands are upregulated and coupled with cells undergoing cartilage to bone transdifferentiation, which may contribute to TMJ pathogenesis. We also discovered that global Notch inhibition reduces osteogenic and chondrogenic differentiation of FCSCs. Together, these findings suggest that Notch is critical for FCSC fate specification and TMJ homeostasis, and reveal that inhibition of the Notch pathway may be a new therapeutic target for treating TMJ osteoarthritis.

Loss of Discoidin Domain Receptor 1 Predisposes Mice to Periodontal Breakdown.

The discoidin domain receptors, DDR1 and DDR2, are nonintegrin collagen receptors and tyrosine kinases. DDRs regulate cell functions, and their extracellular domains affect collagen fibrillogenesis and mineralization. Based on the collagenous nature of dentoalveolar tissues, we hypothesized that DDR1 plays an important role in dentoalveolar development and function. Radiography, micro-computed tomography (micro-CT), histology, histomorphometry, in situ hybridization (ISH), immunohistochemistry (IHC), and transmission electron microscopy (TEM) were used to analyze Ddr1 knockout (Ddr1-/-) mice and wild-type (WT) controls at 1, 2, and 9 mo, and ISH and quantitative polymerase chain reaction (qPCR) were employed to assess Ddr1/DDR1 messenger RNA expression in mouse and human tissues. Radiographic images showed normal molars but abnormal mandibular condyles, as well as alveolar bone loss in Ddr1-/- mice versus WT controls at 9 mo. Histological, histomorphometric, micro-CT, and TEM analyses indicated no differences in enamel or dentin Ddr1-/- versus WT molars. Total volumes (TVs) and bone volumes (BVs) of subchondral and ramus bone of Ddr1-/- versus WT condyles were increased and bone volume fraction (BV/TV) was reduced at 1 and 9 mo. There were no differences in alveolar bone volume at 1 mo, but at 9 mo, severe periodontal defects and significant alveolar bone loss (14%; P < 0.0001) were evident in Ddr1-/- versus WT mandibles. Histology, ISH, and IHC revealed disrupted junctional epithelium, connective tissue destruction, bacterial invasion, increased neutrophil infiltration, upregulation of cytokines including macrophage colony-stimulating factor, and 3-fold increased osteoclast numbers (P < 0.05) in Ddr1-/- versus WT periodontia at 9 mo. In normal mouse tissues, ISH and qPCR revealed Ddr1 expression in basal cell layers of the oral epithelia and in immune cells. We confirmed a similar expression pattern in human oral epithelium by ISH and qPCR. We propose that DDR1 plays an important role in periodontal homeostasis and that absence of DDR1 predisposes mice to periodontal breakdown.

Fibrocartilage Stem Cells Engraft and Self-Organize into Vascularized Bone.

Angiogenesis is a complex, multicellular process that is critical for bone development and generation. Endochondral ossification depends on an avascular cartilage template that completely remodels into vascularized bone and involves a dynamic interplay among chondrocytes, osteoblasts, and endothelial cells. We have discovered fibrocartilage stem cells (FCSCs) derived from the temporomandibular joint (TMJ) mandibular condyle that generates cartilage anlagen, which is subsequently remodeled into vascularized bone using an ectopic transplantation model. Here we explore FCSC and endothelial cell interactions during vascularized bone formation. We found that a single FCSC colony formed transient cartilage and host endothelial cells may participate in bone angiogenesis upon subcutaneous transplantation in a nude mouse. FCSCs produced an abundance of the proangiogenic growth factor vascular endothelial growth factor A and promoted the proliferation of human umbilical vein endothelial cells (HUVECs). Using a fibrinogen gel bead angiogenesis assay experiment, FCSC cell feeder layer induced HUVECs to form significantly shorter and less sprouts than D551 fibroblast controls, suggesting that FCSCs may initially inhibit angiogenesis to allow for avascular cartilage formation. Conversely, direct FCSC-HUVEC contact significantly enhanced the osteogenic differentiation of FCSCs. To corroborate this idea, upon transplantation of FCSCs into a bone defect microenvironment, FCSCs engrafted and regenerated intramembranous bone. Taken together, we demonstrate that the interactions between FCSCs and endothelial cells are essential for FCSC-derived vascularized bone formation. A comprehensive understanding of the environmental cues that regulate FCSC fate decisions may contribute to deciphering the mechanisms underlying the role of FCSCs in regulating bone formation.

Our Essential and Endangered Dentist-Scientist Workforce.

Future advances in dental medicine rely on a robust and stable pipeline of dentist-scientists who are dedicated to research inspired by the patients' condition. The biomedical research community faces external and internal pressures that have been building over years. This is now threatening the current and future status of basic, translational and patient-oriented research by dentist-scientists who study dental, oral and craniofacial diseases, population sciences, and prevention. The dental academic, research and practicing communities can no longer ignore the warning signs of a system that is under considerable stress. Here, the authors report findings of the Physician-Scientist Workforce Working Group, charged by the National Institutes of Health (NIH) Director, to perform quantitative and qualitative analyses on dentist-scientists by addressing the size, composition and activities of the group, relative to other health professions. From 1999 to 2012, trends in the numbers of grant applications and awards to dentist-scientists point to an overall decline. Disturbing are the low numbers of new investigators who apply for Early Career NIH Programs. While more seasoned dentist researchers enjoy greater success, the average age of first-time funded dentists is 52.7 y for females and 54.6 y for males, with a relatively low number of applications submitted and funded. These new data led the panel to stress the need to expand the capacity of the dentist-scientist workforce to leverage technologies and research opportunities that benefit the profession at-large. Suggestions were made to invest in developing clinical research faculty, including those with foreign degrees, through new training mechanisms. The creation of new alliances between national organizations like the American Association for Dental Research, the American Dental Education Association and the American Dental Association will undoubtedly lead to bold and concerted actions that must be pursued with a sense of urgency. A more supportive culture within dental schools and universities for dentist-scientists is needed, as their success is critical to the future career choices of their mentees. Knowledge Transfer Statement: Advances in dental medicine rely on a pipeline of dentist-scientists who are dedicated to research inspired by the patients' condition. Despite the recent advancement in technology and innovation, the dental community can no longer ignore the various pressures that threaten the future of the dentist-scientist profession. Here, the authors report findings of the Physician-Scientist Workforce Working Group of NIH that were published in 2014, and draw attention to the key issues threatening the NIH-funded pool of dentist-scientists.

Physical pain and the goal of aversively stimulated aggression.

Several studies have indicated that anger arousal elicits instigation to inflict injury, but there is good evidence to date that noninsulting aversive events also create a desire to hurt someone. The verbal hostility or physical aggression displayed in previous investigations of the effects of such aversive stimuli might be expressions of an instigation to hit, but not necessarily to hurt, the available target. Two experiments were designed to demonstrate that painful environmental conditions evoke aggressive inclinations directed toward doing harm even when the available target is not responsible for the suffering. In both studies university women kept one hand in a tank of water that was either painfully cold or much warmer while they delivered rewards and punishments to another woman supposedly in the course of supervising her work. Half of the subjects in each condition were informed that their punishments might hurt their partner, whereas the others were told that these punishments probably would be helpful. In the first experiment the two variables interacted to affect the subjects' behavior only during the first work period. Experiment 2 yielded interaction in both periods for the reward measure. In general, the women exposed to the warmer water tended to deliver the greatest number of rewards when they had been told punishment would hurt, whereas those in the cold-water condition were least rewarding if they had been informed punishment would injure their partner. Citing evidence that a lower number of rewards was somewhat punitive, we conclude that the aversive stimulation had evoked an instigation to do harm, and that the information about the possibility of hurting the partner served as a goal cue facilitating the overt expression of the instigation. Factor analyses of the subjects' feelings in the second study suggested that the women's feelings were organized differently the first and second times they had their hand in the water.

Accelerated osteoarthritis in the temporomandibular joint of biglycan/fibromodulin double-deficient mice.

OBJECTIVE: To investigate whether the absence of biglycan and fibromodulin, two proteoglycans expressed in cartilage, bone and tendon, resulted in accelerated osteoarthritis in the temporomandibular joint (TMJ). METHODS: Histological sections of TMJ from 3-, 6-, 9- and 18-month-old wild-type (WT) and biglycan/fibromodulin double-deficient (DKO) mice were compared. Immuno-stainings for biglycan, fibromodulin and proliferating cell nuclear antigen (PCNA) were performed. RESULTS: Biglycan and fibromodulin were highly expressed in the disc and articular cartilage of the TMJ. At 3 months of age, both WT and DKO presented early signs of cartilage degeneration visible as small acellular areas under the articular surfaces and superficial waving. From 6 months of age, DKOs developed accelerated osteoarthritis compared to WT. At 6 months, small vertical clefts in the condylar cartilage and partial disruption of the disk were visible in the DKO. In addition, chondrocytes had lost their regular columnar organization to form clusters. At 9 months, these differences were even more pronounced. At 18 months, extended cartilage erosion was visible in DKOs when by comparison the thickness of the articular cartilage in WT controls was basically intact. PCNA staining was stronger in 3-month-old WT TMJ fibrocartilage than in 3-month-old DKO TMJ fibrocartilage suggesting that chondrocyte proliferation might be impaired in DKOs. CONCLUSION: The biglycan/fibromodulin double knock-out mouse constitutes a useful animal model to decipher the pathobiology of osteoarthritis in the TMJ.