The protective role of CD73 in periodontitis: preventing hyper-inflammatory fibroblasts and driving osteoclast energy metabolism.

Introduction: Periodontitis is an immune-mediated inflammatory disease affecting almost half of the adult population and is the leading cause of tooth loss in the United States. The role of extracellular nucleotide signaling including nucleotide metabolizing enzyme CD73 adds an important layer of interaction of purine mediators capable of orchestrating inflammatory outcomes. CD73 is able to catabolize 5'-adenosine monophosphate into adenosine at the extracellular level, playing a critical role in regulating many processes under physiological and pathological conditions. Here, we explored the role of CD73 in ligature-induced periodontitis in vivo comparing wild-type C57Bl/6J and CD73-deficient mice. Methods: We assessed gingival levels of inflammatory cytokines in vivo and in murine gingival fibroblasts in vitro, as well as bone loss, and RANKL-induced osteoclastogenesis. We have also analyzed CD73 mRNA in samples derived from patients diagnosed with severe periodontitis. Results: Our results in mice show that lack of CD73 resulted in increased inflammatory cytokines and chemokines such as IL-1ß, IL-17, Cxcl1 and Cxcl2 in diseased gingiva relative to the healthy-controls and in comparison with the wild type. CD73-deficient gingival fibroblasts also manifested a defective healing response with higher MMP-13 levels. CD73-deficient animals also showed increased osteoclastogenesis in vitro with increased mitochondrial metabolism typified by excessive activation of oxidative phosphorylation, increased mitochondrial membrane potential and accumulation of hydrogen peroxide. Micro-CT analysis revealed that lack of CD73 resulted in decreased bone mineral density, decreased trabecular bone volume and thickness as well as decreased bone volume in long bones. CD73 deficiency also resulted in increased alveolar bone loss in experimental periodontitis. Correlative studies of gingival samples from severe (Grade C) periodontitis showed decreased levels of CD73 compared to healthy controls, further supporting the relevance of our murine results. Conclusion: In conclusion, CD73 appears to play a protective role in the gingival periodontal tissue and bone homeostasis, regulating hyper-inflammatory state of stromal fibroblasts and osteoclast energy metabolism and being an important candidate for future target therapies to prevent or control immune-mediated inflammatory and osteolytic diseases.

The effect of shoulder prosthesis stem length on failure due to torsional loading. A biomechanical study in composite humeri.

Background: Shoulder arthroplasty is becoming increasingly common. With evolving implant designs, multiple humeral stem options exist for the surgeon to choose from. New stemless and short-stem systems are modular, remove less native bone stock, and better adapt to patient anatomy. It has been suggested that shorter stem implants may be protective against periprosthetic fracture; however, this has not been mechanistically evaluated. Therefore, this study aimed to biomechanically test synthetic humeri with long-stem, short-stem, and stemless arthroplasty components in a torsional manner to evaluate their response to loading and characterize failure. Methods: Twenty-four synthetic humeri were implanted with long stem, short stem, or stemless uncemented prosthesis, 8 in each group. Humeri were mounted in a custom testing jig with a morse taper interfacing with a mechanical testing system. After a 20N axial force, specimens were torsionally loaded to failure at 15 degrees/sec, with 50 Hz collection. Torque vs. rotation curves were generated for each specimen, and stiffness, yield, ultimate strength, and failure load were measured. ANOVA and post hoc pairwise comparisons were used to assess effect of stem type on mechanical test variable. The association of the stem type with fracture type was analyzed by a Fisher's Exact test. Statistical significance was set at P < .05. Results: During torsional loading, long-stem implants were significantly stiffer than short or stemless implants. The angle of implant yielding was similar across stem designs; however, stemless implants had a lower yield torque. This correlated with a decreased yield energy in stemless compared to short stems as well. Maximum torque and failure torque was also significantly higher in short-stem and long-stem implants compared to stemless. Discussion: Periprosthetic fractures in shoulder arthroplasty are a concern in low-energy trauma, and stem design likely plays a significant role in early implant-bone failure. Our results suggest stemless implants under torsional load fail at lower stress and are less stiff than stemmed implants. The failure mechanism of stemless implants through metaphyseal cancellous bone emphasizes the effect bone quality has on implant fixation. There is likely a balance of torsional stability to survive physiologic loads while minimizing diaphyseal stress and risk of diaphyseal periprosthetic fracture. This combined with revision and fixation options represent decisions the surgeon is faced with when performing shoulder arthroplasty.

Metabolic reprogramming through mitochondrial biogenesis drives adenosine anti-inflammatory effects: new mechanism controlling gingival fibroblast hyper-inflammatory state.

Introduction: Fibroblasts are the dominant stromal cells in the gingival lamina propria with a well-established relevance in regulation of inflammation, and in innate immunity. This is exemplified by their hypersecretion of CXCL8, enhancing leukocyte infiltration in chronic and sustained inflammatory conditions. We have previously shown adenosine to be a key metabolic nucleoside that regulates stromal inflammation, but the underlying mechanisms linking adenosine to the metabolic status of fibroblasts and to the resultant inflammatory response are unclear. This study examined, by seahorse real-time cell metabolic analysis, the bioenergetics of the stromal fibroblast response to extracellular adenosine and IL-1ß, focusing on CXCL8 secretion by primary human gingival fibroblasts (HGF). Methods: Markers of the glycolytic pathway and mitochondrial biogenesis were tracked through immunoblot. Further, the influence of adenosine on mitochondrial accumulation was measured by uptake of MitoTracker Red fluorescent probe and assessment of the role of FCCP (a mitochondrial uncoupler) in CXCL8 secretion and mitochondrial accumulation. Results: Our results show that the anti-inflammatory response of HGF to extracellular adenosine, typified by reduced CXCL8 secretion, is mediated by mitochondrial oxidative phosphorylation, reflected in higher oxygen consumption rate (OCR). In the presence of IL-1ß, adenosine-treated cells induced higher ATP production, basal respiration and proton leak compared to IL-1ß without adenosine. Surprisingly, adenosine had no additional effect on the IL-1ß-induced higher glycolysis rate demonstrated by the extracellular acidification rate (ECAR). In addition, the higher OCR in adenosine-stimulated cells was not due to the mitochondrial fuel dependency or capacity, but due to an increase in mitochondrial biogenesis and accumulation in the cells with concomitant decrease in mitophagy-required p-PINK1 marker. We detected the accumulation of functional mitochondria with increased activation of the AMPK/SIRT1/PGC-1α pathway. The adenosine-induced uptake of MitoTracker was abrogated by PGC-1α inhibition with SR-12898. In addition, the adenosine effects on reduced CXCL8 were ablated by treatment with FCCP, a potent uncoupler of mitochondrial oxidative phosphorylation. Conclusion: Our findings reveal a key role for mitochondrial bioenergetics in regulation of CXCL8-mediated inflammation by HGF through the adenosine/AMPK/SIRT1/PGC-1α axis. Therapeutically targeting this pathway in gingival fibroblasts might be a promising future strategy to modulate stromal-mediated sustained hyper-inflammatory responses.

Macromolecular crowding and decellularization method increase the growth factor binding potential of cell-secreted extracellular matrices.

Recombinant growth factors are used in tissue engineering to stimulate cell proliferation, migration, and differentiation. Conventional methods of growth factor delivery for therapeutic applications employ large amounts of these bioactive cues. Effective, localized growth factor release is essential to reduce the required dose and potential deleterious effects. The endogenous extracellular matrix (ECM) sequesters native growth factors through its negatively charged sulfated glycosaminoglycans. Mesenchymal stromal cells secrete an instructive extracellular matrix that can be tuned by varying culture and decellularization methods. In this study, mesenchymal stromal cell-secreted extracellular matrix was modified using λ-carrageenan as a macromolecular crowding (MMC) agent and decellularized with DNase as an alternative to previous decellularized extracellular matrices (dECM) to improve growth factor retention. Macromolecular crowding decellularized extracellular matrix contained 7.7-fold more sulfated glycosaminoglycans and 11.7-fold more total protein than decellularized extracellular matrix, with no significant difference in residual DNA. Endogenous BMP-2 was retained in macromolecular crowding decellularized extracellular matrix, whereas BMP-2 was not detected in other extracellular matrices. When implanted in a murine muscle pouch, we observed increased mineralized tissue formation with BMP-2-adsorbed macromolecular crowding decellularized extracellular matrix in vivo compared to conventional decellularized extracellular matrix. This study demonstrates the importance of decellularization method to retain endogenous sulfated glycosaminoglycans in decellularized extracellular matrix and highlights the utility of macromolecular crowding to upregulate sulfated glycosaminoglycan content. This platform has the potential to aid in the delivery of lower doses of BMP-2 or other heparin-binding growth factors in a tunable manner.

The Association of Surgical Timing and Injury Severity With Systemic Complications in Severely Injured Patients With Pelvic Ring Injuries.

OBJECTIVES: To evaluate the relationship between timing of definitive fixation, injury severity, and the development of systemic complications in severely injured patients with pelvic ring injuries. DESIGN: Retrospective review. SETTINGS: Level 1 trauma center. PATIENTS: One hundred eighteen severely injured [Injury Severity Score (ISS) ≥ 16] adult patients with pelvic ring injuries undergoing definitive fixation, excluding patients treated with external fixation for hemodynamic instability. INTERVENTION: Early fixation (≤36 hours) in 37 patients and delayed fixation (>36 hours) in 81 patients. MAIN OUTCOME MEASUREMENTS: Systemic complications (acute respiratory distress syndrome, pulmonary embolism, deep venous thrombosis, sepsis, multi-organ failure, and death). RESULTS: The delayed fixation group had a higher ISS and had more patients with chest injuries. There was no detectable difference in the number of patients with systemic complications between early versus delayed fixation groups [8 (22%) vs. 29 (35%), P = 0.1]. The only difference detected in specific complications was a higher incidence of pneumonia with delayed fixation [16 (20%) vs. 0 (0%), P = 0.004] with 11 of the 16 cases being associated with chest injury. Univariate analysis showed an association between complication and time to fixation, ISS, Glasgow Coma Scale, pH, base excess, and injuries to the head, chest, and abdomen. On multivariate analysis, only ISS remained significantly associated with the development of complications [Odds ratio 2.6 per 10 point increase, 95% confidence interval (CI), 1.4-4.4]. CONCLUSIONS: These data suggest that the severity of injury is most highly associated with systemic complications after definitive fixation of pelvic ring injuries. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Fixation of Transverse Acetabular Fractures With Precontoured Plates Alone Causes Fracture Malreduction: A Biomechanical Assessment.

BACKGROUND: Precontoured quadrilateral surface buttress (PQSB) plates have grown in popularity for acetabular fracture fixation. However, our experience has pushed us to hypothesize that their use as sole means of fixation may cause fracture malreduction. A biomechanical model was created to investigate this theory. METHODS: A transverse acetabular fracture was created and reduced anatomically in 18 synthetic hemipelvises. The reduced hemipelvises were fixated using 3 different techniques. Group A fixation included anterior and posterior column screws plus a suprapectineal pelvic reconstruction plate; group B models were fixed using a PQSB plate only; and group C models were fixed with an anterior column screw and a PQSB plate. Acetabular tracking points were placed before final fixation and used to quantify any postfixation displacement. One-way analysis of variance and Tukey HSD testing were used to determine the significant difference (P < 0.05). RESULTS: Models in group B had significant fracture displacement after final fixation when compared with group A and group C models. The average amount of displacement at the anterior column and within the acetabulum was 1.37 mm (95% CI, 1.08-1.65) in group B constructs compared with 0.32 mm (95% CI, 0.22-0.42) and 0.26 mm (95% CI, 0.15-0.38) in groups A and C constructs, respectively. There were no significant differences in displacement after final fixation between group A and group C models. CONCLUSIONS: PQSB plates for acetabular fractures cause malreduction when applied in isolation in this biomechanical model. If a PQSB plate is chosen for fixation, we suggest the use of a columnar lag screw at minimum to hold reduction before plate application.

Precontoured Quadrilateral Surface Acetabular Plate Fixation Demonstrates Increased Stability When Compared With Pelvic Reconstruction Plates: A Biomechanical Study.

OBJECTIVE: To compare the stability of 3 fixation strategies for a transverse acetabular fracture: a reconstruction plate with anterior and posterior column screws (group A); an infrapectineal precontoured quadrilateral surface buttress (iPQSB) plate alone (group B); and an anterior column lag-screw and iPQSB plate (group C). METHODS: A transverse acetabular fracture was created in 18 synthetic hemipelvises. Six were fixed by each of the 3 methods described. Specimens underwent cyclic axial compressive loading to 1700N for 42,000 cycles while anterior and posterior column displacements were measured, followed 4800N for 50 cycles. Displacement and stiffness data were analyzed with analysis of variance and Tukey HSD. A Cox proportional hazards regression model was used to determine survival rate. P values < 0.05 were considered significant. RESULTS: Group C had significantly less posterior column displacement (0.16 ± 0.06 mm) compared with group B (0.38 ± 0.37 mm, P < 0.0001) and group A (0.38 ± 0.37 mm, P < 0.0001). In addition, group A had significantly more anterior column displacement (0.28 ± 0.11 mm) than group B (0.22 ± 0.14 mm, P = 0.0310) and group C (0.18 ± 0.09 mm, P = 0.0001). Group C was 10.5% stiffer than group A (P = 0.0037). Group B had a 7.27x greater rate of failure than group C (95% confidence interval, 1.6-33.2). DISCUSSION AND CONCLUSION: Under anatomical loading, iPQSB plates with anterior column lag-screw fixation demonstrate increased stability in a synthetic bone transverse acetabular fracture model. Based on our data, we support additional evaluation of early weight-bearing after transverse acetabular fracture fixation in patients with healthy bone when an anterior column screw-iPQSB plate construct is used.

Activation of S6 signaling is associated with cell survival and multinucleation in hyperplastic skin after epidermal loss of AURORA-A Kinase.

The role of mitosis in the progression of precancerous skin remains poorly understood. To address this question, we deleted the mitotic Kinase Aurora-A (Aur-A) in hyperplastic mutant p53 mouse skin as an experimental tool to study the G2/M transition in precancerous keratinocytes and AUR-A's role in this process. Epidermal Aur-A deletion (Aur-AepiΔ) led to marked keratinocyte enlargement, pleomorphism, multinucleation, and  attenuated induction of cell death. This phenotype was characteristic of slippage after a stalled mitosis. We also observed altered or impaired epidermal differentiation, indicative of a partial skin barrier defect. The upregulation of mTOR/PI3K signaling was implicated as a mechanism by which keratinocytes may evade cell death after AUR-A deficiency. This was evidenced by the ectopic expression of the pathway readout, p-S6, in the basal layer of Aur-AepiΔ skin and its mitotic upregulation in isolated keratinocytes. We further tested whether our findings were extended to skin carcinoma cells. The chemical inhibition of AUR-A led to a similar mitotic delay, polyploidy/multinucleation, and attenuated cell death in skin cancer cell lines. Moreover, inhibition of mTOR/PI3K signaling ameliorated the effects caused by the deficiency of AUR-A activity but was also associated with the persistence of mitotic p-S6 detection in surviving cancer cells. These results show the induction of multinucleation/polyploidy may be a compensatory state in keratinocytes that allows for cellular survival and maintenance of partial barrier function in face of aberrant cell division or differentiation. Moreover, mTOR/PI3K signaling is active in the mitosis of hyperplastic keratinocytes expressing mutant p53 and is further enhanced by stalled mitosis, indicating a potential resistance mechanism to the use of anti-mitotic drugs in the treatment of skin cancers.