Faculty Bibliography

Osteoarthritis (OA) is themost common form of arthritis, affecting nearly 10%of the US population. There is no therapy to prevent the progression of or reverse OA pathology. Endogenous adenosine 2A receptor (A2AR) stimulation is crucial for chondrocyte viability and cartilage homeostasis, as its downstream signalingmediates inflammation.We have published thatmice lacking the A2AR or ecto-5'nucleotidase (CD73) develop spontaneous OA, suggesting that diminished extracellular adenosine levels promote the development of OA. Since human OA chondrocytes have been found to diminish mitochondrial content, we proposed to test the hypothesis that OA pathogenesis deregulates A2AR signaling at least in part by affecting the cell's capacity for ATP production via reduced content or functionality of mitochondria. Primary neonatal WT and A2ARKO chondrocytes were subjected to Seahorse Mito Stress Kit Assays, which revealed reduced oxygen consumption rates (OCR) at baseline and after mitochondrial uncoupling (corresponding to reduced coupling capacity and ATP production). RNA sequencing DESEQ analysis showed increased metalloproteinases expression along with OA-associated pro-inflammatory pathways. We also saw upregulation of genes involved with aging and the production of nitric oxide (NO) and reactive oxygen species (ROS). Histologic staining for 8hydroxyguanosine (8OH-G) residues as a marker for ROS is also markedly increased in A2ARKO mice as early as 8 weeks. A human chondrocyte cell line, T/C28-a2, was used to determine the effect of IL-1beta-induced inflammation and A2AR stimulation in vitro.Mitochondrial health and functionality was assessed by mean pixel intensity (MPI) of a fluorescent probe for monitoring mitochondrial membrane potential, TMRM, and by measuring OCR. IL-1beta (5ng/mL) incubation for 3 hours followed by A2AR ligation (CGS21680 (CGS; 1uM)) increases mitochondrial membrane potential compared to control, IL-1beta alone and CGS alone as measured by TMRM staining. IL-1beta incubation for 4 hours with a last hour of A2AR stimulation increases basal OCR and maximal respiratory rate. IL-1beta+CGS treated cells had significantly increased ATP production than the control, IL-1beta and CGS treated cells as measured by one-way ANOVA. A2AR ligation improves mitochondrial functionality during inflammation and OA progression. Lack of A2AR signaling not only promotes inflammation and catabolism of cartilage matrix, it also damages the cell's mitochondrial metabolic capacity

Background: Wnt/beta-catenin signaling pathway is a key regulator in bone formation and maintaining bone hemostasis. Wnt signaling upon activation leads to stabilization of the transcriptional regulator beta-catenin and its further nuclear localization. Osteoblast differentiation and proliferation are regulated by a number of local and systemic factors, among which adenosine receptors are prominent during bone development. We recently reported cross-talk between Wnt/beta-catenin pathway and A2aR in fibroblasts; here we seek to determine whether there is similar cross-talk between Wnt signaling and the purinergic adenosine A2A receptor in osteoblasts. Since nuclear translocation of beta-catenin protein is a critical intermediate step in the Wnt signaling pathway, we studied the effect of A2aR signaling on nuclear and cytosolic beta-catenin levels in osteoblasts.
Method(s):We used an osteoblast cell line (MC3T3- E1) as well as primary osteoblasts derived frommesenchymal stem cells ofmice. The cells were treated with CGS21680, a selective A2aR agonist, at doses ranging from 0 to 10muM, and for varying incubation periods from 0 to 240 minutes. Using western blot analysis, the levels of total beta- catenin and phosphorylated beta-catenin at Ser552 (active component) were measured. Additionally, we measured beta-catenin levels in the nuclear extracts of the cells, both before and after A2aR activation. We also analyzed nuclear translocation of p-Ser552 beta-catenin using immunofluorescence (IF) staining.
Result(s): We observed a significant increase (p<0.05), in total beta-catenin (169+/-50% control, n=5) and p-Ser552 beta-catenin (253+/-122%, n=5) levels in MC3T3-E1 cells treated with 1 muM CGS21680 compared to control at 15 minutes following A2aR activation. The immunofluorescence staining results revealed enhanced nuclear accumulation of p-Ser552 beta-catenin by approximately 40% among the osteoblastoid cells treated with CGS21680. Similarly, western blot assays showed a significant increase (p<0.05) in the nuclear translocation of phosphorylated beta-catenin at Ser552 following administration of A2aR agonist inMC3T3-E1 cells (153+/-37%, n=4), as well as osteoblasts derived from mesenchymal stem cells (148+/-31%, n=4).
Conclusion(s): These results demonstrate cross-talk between A2aR and Wnt/beta-catenin signaling pathway in osteoblasts. Moreover, our results suggest that A2aR activation can bypass blockade of Wnt/Frizzled interactions at the cell surface and thereby maintain bone homeostasis

Background:We have previously reported that endogenously produced adenosine, interacting with A2AR, is a critical autocrine factor for maintenance of chondrocyte and cartilage homeostasis and intra-articular injections of liposomal preparations of adenosine inhibit progression of OA in a posttraumatic OA (PTOA) model in rats. We therefore determined whether intra-articular injection of a more selective A2AR agonist could also prevent progression and possibly reverse OA in this model and in the obesity related OA model in mice.
Method(s): PTOA was induced in SD rats following rupture of the anterior cruciate ligament (ACL) by application of external force to the knee. Starting 4 weeks after injury, when OA has already progressed, knees were injected with 100ul of saline, empty liposomes (LIPO) or liposomes containing CGS21680 (LIPO-CGS) every 10 days (6 injections) before sacrifice. The cartilage volume in OA and normal knees was measured by microCTafter staining with hexabrix (40%). Chondrocytes were isolated from neonatal mice and cultured, only first passage chondrocytes were studied. For the obesity-OA model, C57Bl6 mice (5-6/group, 12 weeks old) were fed a 60%fat diet (HFF mice). After 3 months, when OAwas present, mice received intrarticular knee injection (10 mul) of LIPO, LIPOCGS or liposomal adenosine (LIPO-Ado) every 10 days for 4 injections before sacrifice.
Result(s): Injection of LIPO-CGS but not saline or LIPO, significantly reduced swelling of affected rat knees (p<0.001). Surprisingly, there was an increase in tibial and femoral cartilage volume in normal knees treated with intra-articular injections of LIPO-CGS but not LIPO or saline (47% increase in tibia and 22% in femur). More importantly, intra-articular injections of LIPO-CGS, but not LIPO or saline, increased tibial and femoral cartilage volume inOAknees, as compared to normal knees and completely abrogated the histologic evidence of OA as well (OARSI score for CGS21680 0.66+/-0.33 vs 4.55+/-0.82 in the vehicle group and 3.90+/-0.89 in the saline group). There wasmarked chondrocyte proliferation in the deep cartilage of knees of rats treated with LIPO-CGS (Ki67 immunofluorescence). Similarly, LIPO-CGS reversed the OA changes in the obesity related OA model. HFF mice had an OARSI score of 5.17+/-1.84. Treatments with LIPO-Ado and lipo-CGS decreased OA severity (OARSI score 1.33+/-0.81 and 1.83+/-0.98, respectively, p<0.001 vs untreated). A2AR stimulation increased TGF-beta immunostaining in LIPO-CGS-injected joints and increased TGF-beta production by cultured neonatal murine chondrocytes with increased SMAD2/3 phosphorylation and diminished RUNX2 expression.
Conclusion(s): These results demonstrate that intra-articular injection of a long-acting A2AR agonist stimulates chondrocyte and cartilage regeneration, likely by a TGF-beta-dependent mechanism. More importantly, these results indicate that treatment with an A2AR agonist can reverse OA in both traumatic and obesity-related OA

Background: Mutations in plakophilin-2 (PKP2) are the most common cause of familial Arrhythmogenic Right Ventricular Cardiomyopathy, a disease characterized by ventricular arrhythmias, sudden death, and progressive fibrofatty cardiomyopathy. The relation between loss of PKP2 expression and structural cardiomyopathy remains under study, though paracrine activation of pro-fibrotic intracellular signaling cascades is a likely event. Previous studies have indicated that ATP release into the intracellular space, and activation of adenosine receptors, can regulate fibrosis in various tissues. However, the role of this mechanism in the heart, and in the specific case of a PKP2-initiated cardiomyopathy, remains unexplored. Objectives: To investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy. Methods: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. A cardiac-specific, tamoxifen-activated PKP2 knock-out murine model (PKP2cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Results: HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. PKP2cKO transcriptomic data revealed overexpression of genes involved in adenosine-receptor cascades. Istradefylline (an adenosine 2A receptor blocker) tempered the progression of fibrosis and mechanical failure observed in PKP2cKO mice. In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects. Conclusion: Paracrine adenosine 2A receptor activation contributes to the progression of fibrosis and impaired cardiac function in animals deficient in PKP2. Given the limitations of the animal model, translation to the case of patients with PKP2 deficiency needs to be done with caution.

STATEMENT OF PURPOSE/OBJECTIVE:Injuries to the extremities often require resection of necrotic hard tissue. For large bone defects, autogenous bone grafting is ideal, but similar to all grafting procedures, is subject to limitations. Synthetic biomaterial driven engineered healing offers an alternative approach. This work focuses on three-dimensional (3D) printing technology of solid-free form fabrication (SFF), more specifically robocasting/direct write. The research hypothesizes that a bioactive calcium-phosphate scaffold may successfully regenerate extensive bony defects in vivo and that newly regenerated bone will demonstrate mechanical properties similar to native bone as healing time elapses. METHODS:) and hardness (H) using nanoindentation. RESULTS:) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three-time points, while hardness (H) values were equivalent to the native radial bone at 24 weeks. The negative control samples showed limited healing at 8 weeks. CONCLUSIONS:Custom engineered β-TCP scaffolds are biocompatible, resorbable, and can directionally regenerate and remodel bone in a segmental long bone defect in a rabbit model. Custom designs and fabrication of β-TCP scaffolds for use in other bone defect models warrant further investigation.

Craniomaxillofacial congenital anomalies comprise approximately one third of all congenital birth defects and include deformities such as alveolar clefts, craniosynostosis, and microtia. Current surgical treatments commonly require the use of autogenous graft material which are difficult to shape, limited in supply, associated with donor site morbidity and cannot grow with a maturing skeleton. Our group has demonstrated that 3D printed bio-ceramic scaffolds can generate vascularized bone within large, critical-sized defects (defects too large to heal spontaneously) of the craniomaxillofacial skeleton. Furthermore, these scaffolds are also able to function as a delivery vehicle for a new osteogenic agent with a well-established safety profile. The same 3D printers and imaging software platforms have been leveraged by our team to create sterilizable patient-specific intraoperative models for craniofacial reconstruction. For microtia repair, the current standard of care surgical guide is a two-dimensional drawing taken from the contralateral ear. Our laboratory has used 3D printers and open source software platforms to design personalized microtia surgical models. In this review, we report on the advancements in tissue engineering principles, digital imaging software platforms and 3D printing that have culminated in the application of this technology to repair large bone defects in skeletally immature transitional models and provide in-house manufactured, sterilizable patient-specific models for craniofacial reconstruction.