Faculty Bibliography

Background/Purpose: Previous in vivo and in vitro animal studies demonstrate that the adenosine A2A receptor (A2AR) agonist dipyridamole (DIPY) stimulates robust osteogenic differentiation and proliferation without adverse effects on craniofacial suture development. However, no studies to date have been performed on human tissue. This study compares the effects of DIPY, BMP-2, and standard osteogenic media on osteogenic differentiation by human mesenchymal stem cells to lay the foundation for translating this bone tissue engineering approach to pediatric craniofacial reconstruction. Methods/Description: Pediatric mesenchymal stem cells were isolated from surplus bone taken from consented patients undergoing craniofacial surgery. Cells were cultured at early passage for 3 weeks in 1 of 7 experimental conditions: control media; osteogenic media (control + 100 muM beta-glycerophosphate, 0.1 muM dexamethasone and 100 mg/ mL L-ascorbic acid); osteogenic media + 200 ng/mL BMP-2; osteogenic media + 10, 100, 1000, or 10 000 muM DIPY. All experiments were performed in biological triplicates. Samples were analyzed using Alkaline phosphatase (ALP) assay at 6 hours, 24 hours, 48 hours, and 7 days as a marker of early osteogenic differentiation. At the end of the 3-week differentiation period, cells underwent immunocytochemistry to verify phalloidin, osteocalcin, and collagen I expression. Alizarin red staining was used to detect mineralization. Statistical analysis used 1-way ANOVA with Tukeys post hoc correction and multiple t test comparison of means.
Result(s): In all osteogenic conditions, relative peak ALP activity occurred at 48 hours. One thousand micrometer DIPY showed significantly increased peak ALP activity compared to BMP-2 (3.6 +/- 0.1 fold increase vs 3.1 +/- 0.1; P = .006). There was no significant difference between 1000 muM DIPY and osteogenic media (4.1 +/- 0.1; P = .36). At 3 weeks, immunocytochemistry revealed differentiation in all osteogenic conditions compared to control. One thousand micrometer DIPY cells showed greater evidence of mature osteogenic differentiation including cuboidal cell morphology and deposition of collagen I in an extracellular fibrillar network pattern compared to both control osteogenic media and BMP-2. Alizarin red quantification demonstrated significantly increased extracellular matrix mineralization at 100 muM(2.4+/-0.4; P = .002), 1000 muM (4.3+/-0.6; P = .001), and 10 000 muM (5.1 +/- 0.2; P < .0001) DIPY compared to nonosteogenic control medium (1.0 +/- 0.1). Matrix mineralization was not significantly different between BMP-2 (2.4 +/- 0.2) and 1000 muM DIPY (P = .08). ImageJ analysis revealed increased proportion of osteocalcin expressing cells (40.0% +/- 2.8%) in stem cells treated with 1000 muM of dipyridamole compared to control (1.0% +/- 0.6%), osteogenic (5.8% +/- 1.0%), and BMP-2 (16.9% +/- 2.2%; P < .0001).
Conclusion(s): Dipyridamole promotes early osteogenic differentiation and maturation of human bone-derived mesenchymal stem cells. These data suggest that dipyridamole may be an effective tissue engineering strategy for pediatric craniofacial reconstruction

Background/Purpose: Alveolar cleft surgery is the most common bone reconstruction performed in patients with a cleft. Osteogenic agents such as BMP-2 have been used to restore the bony cleft without the morbidity of bone graft, but concerns remain regarding premature fusion of sutures, exuberant bone formation, and malignant degeneration. We have recently demonstrated that dipyridamole-coated, 3D printed bio-ceramic (3DPBC) scaffolds generate comparable bone amounts to BMP2 and significantly greater bone compared to negative controls in short-term growing animal model studies. No detrimental effects to growth sutures were noted in any animals. This study investigates the long-term osteogenic properties, degradation kinetics, and effects on facial growth of these tissue engineering constructs in growing animal models. Methods/Description: Twenty-two 1-month-old (immature) New Zealand white rabbits underwent creation of unilateral 3.5 x 3.5 mm alveolar defects. Each alveolar defect was repaired with either 3DPBC scaffolds coated with 1000 muM dipyridamole (n = 14) or with autogenous bone graft from the radius (n = 8). Six rabbits from the 3DPBC scaffold group were sacrificed at 8 weeks. The remaining rabbits (n = 8 each group) were euthanized following completion of craniofacial growth (6 months). Bone regeneration, scaffold degradation, and maxillary suture patency were calculated using CT images reconstructed and analyzed in Amira software. Facial symmetry was evaluated using dense-surface 3D modeling and validated with bilateral cephalometric measurements of maxillary projection. Bone growth and suture patency were qualitatively evaluated through histologic analysis.
Result(s): After 6 months, animals with defects repaired with 3DPBC scaffolds regenerated an average of 52.9% +/- 3.3% bone (mean +/- SEM), compared to 40.7%+/-4.0% in defects repaired with bone graft (P = .02). This is compared to unoperated alveolus occupied by 39.3% +/- 1.6% bone. Scaffolds showed significant degradation at 6 months (6.7% +/- 1.6%) compared to at 8 weeks (27.1% +/- 1.9%; P >= .001). Morphometric analysis using dense surface modeling showed similar symmetry indices of 55.0 +/- 3.3 for scaffold animals and 61.7% +/- 1.6% for bone graft animals (P = .10). Comparative measurements of operated and unoperated sides showed no significant differences in asymmetry between scaffold and bone graft animals (P = .86). Histologic analysis of scaffold samples revealed vascularized, organized bone within scaffold interstices without evidence of ectopic bone, excess inflammatory cells, or suture fusion.
Conclusion(s): In a growing animal model, dipyridamole-coated 3DPBC scaffolds can regenerate bone comparable to autogenous bone graft by radiographic and histologic analysis. Over 6 months, scaffolds show significant, favorable degradation and do not result in premature suture fusion or disruption of facial growth compared to bone graft. These results support long-term safety and efficacy of this tissue engineering strategy in the repair of alveolar cleft defects

Background/Purpose: Our tissue engineering laboratory has previously demonstrated that dipyridamole-coated, 3D printed bioceramic (3DPBC) scaffolds comprised of B-tricalcium phosphate generate significantly more bone compared to negative controls in short-term growing animal model studies. No detrimental effects to the cranial suture were observed in any experimental animals. The longterm osteogenic efficacy and safety of our 3DPBC scaffold for tissue engineering in growing calvaria was assessed by describing bone regeneration compared to autogenous bone graft, scaffold degradation kinetics, and the effects of the construct on cranial growth over time. Methods/Description: Twenty-two 1-month-old (immature) New Zealand white rabbits underwent unilateral 11-mm craniotomy within 2 mm of the coronal and sagittal sutures. Rabbits' calvarial defects were repaired by 1 of 2 interventions: 3DPBC scaffolds coated with 1000 mM dipyridamole (n = 14) or autogenous calvarial bone graft (n = 8). Six rabbits from the 3DPBC scaffold group were sacrificed at 8 weeks. The remaining rabbits (n = 8 each group) were observed until craniofacial growth was completed (6 months) and then euthanized. Bone regeneration, scaffold degradation, and cranial suture patency were analyzed in Amira software using reconstructed microcomputed tomography (muCT) images. Cranial growth was assessed by comparing bilateral cephalometric measurements based on muCT images. Bone growth and suture patency were qualitatively evaluated through histologic analysis.
Result(s): After 6 months of healing, animals with defects repaired with 3DPBC scaffolds regenerated an average of 53.9% +/- 3.6% (mean +/- SEM) bone, compared to 53.5% +/- 3.6% in defects repaired with bone graft (P = .95). Unoperated calvarial bone porosity was 49.4%+/-2.0%. Scaffolds showed significant degradation at 6 months (15.1% +/-0.7%) compared to 8 weeks (23.2% +/- 0.9%; P<=.001). Comparative measurements of operated and unoperated sides showed no significant differences in asymmetry between scaffold and bone graft animals (P > .24). Analysis of histologic sections revealed well-vascularized, organized bone formation within scaffold interstices with no evidence of ectopic bone formation, excess inflammatory cells, or suture fusion.
Conclusion(s): Dipyridamole-coated 3D-printed bioceramic scaffolds bone regeneration is comparable to autogenous bone graft without showing signs of adverse events such as premature cranial suture fusion, or detrimental effects to facial growth. The scaffold demonstrates favorable absorption kinetics, highlighting the potential for this technology in pediatric bone tissue engineering

Mini-sabbaticals are formal short-term training and educational experiences away from an investigator's home research unit. These may include rotations with other research units and externships at government research or regulatory agencies, industry and non-profit programs, and training and/or intensive educational programs. The National Institutes of Health have been encouraging training institutions to consider offering mini-sabbaticals, but given the newness of the concept, limited data are available to guide the implementation of mini-sabbatical programs. In this paper, we review the history of sabbaticals and mini-sabbaticals, report the results of surveys we performed to ascertain the use of mini-sabbaticals at Clinical and Translational Science Award hubs, and consider best practice recommendations for institutions seeking to establish formal mini-sabbatical programs.

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. The aim of this study was to investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy.
Methods and Results: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. A cardiac-specific, tamoxifenactivated PKP2 knock-out murine model (PKP2-cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Transcriptomic data of PKP2-cKO mice revealed overexpression of genes involved in adenosine-receptor cascades. Treatment with Istradefylline (an adenosine 2A receptorblocker) tempered the progression of fibrosis and mechanical failure observed in PKP2-cKO mice (see Fig. B,C). In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects.
Conclusion(s): 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. (Figure Presented)

There are over 2 million long bone defects treated in the USA annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is standard of care in simple defects, a customized scaffold for large defects in unlimited quantities is not available. Recently, a three-dimensionally (3D) printed bioactive ceramic (3DPBC) scaffold has been successfully utilized in the of repair critical sized long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with Dipyridamole, an adenosine A2A receptor (A_2A R) indirect agonist, because of its known effect to enhance bone formation. Critical-sized full thickness segmental defects (~11mm x full thickness) defects were created in the radial diaphysis in New Zealand White rabbits (n=24). A customized 3DPBC scaffold composed of β-tricalcium phosphate was placed into the defect site. Groups included scaffolds that were collagen-coated (COLL), or immersed in 10μM, 100μM, or 1000μM Dipyridamole solution. Animals were euthanized 8 weeks post-operatively and the radii/ulna-scaffold complex retrieved, en bloc, for micro-CT, histological and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). MicroCT reconstructions illustrated bone in-growth throughout the scaffold, with an increase in bone volume dependent on the Dipyridamole dosage. Histological evaluation did not indicate any adverse immune response while revealing progressive remodeling of bone. These customized biologic 3DPBC scaffolds have the potential of repairing and regenerating bone. This article is protected by copyright. All rights reserved.

BACKGROUND:Alveolar clefts are traditionally treated with secondary bone grafting, but this is associated with morbidity and graft resorption. Although recombinant human bone morphogenetic protein-2 (rhBMP-2) is under investigation for alveolar cleft repair, safety concerns remain. Dipyridamole is an adenosine receptor indirect agonist with known osteogenic potential. This study compared dipyridamole to rhBMP-2 at alveolar cleft defects delivered using bioceramic scaffolds. METHODS:Skeletally immature New Zealand White rabbits underwent unilateral, 3.5 × 3.5-mm alveolar resection adjacent to the growing suture. Five served as negative controls. The remaining defects were reconstructed with three-dimensionally printed bioceramic scaffolds coated with 1000 μm of dipyridamole (n = 6), 10,000 μm of dipyridamole (n = 7), or 0.2 mg/ml of rhBMP-2 (n = 5). At 8 weeks, new bone was quantified. Nondecalcified histologic evaluation was performed, and new bone was evaluated mechanically. Statistical analysis was performed using a generalized linear mixed model and the Wilcoxon rank sum test. RESULTS:Negative controls did not heal, whereas new bone formation bridged all three-dimensionally printed bioceramic treatment groups. The 1000-μm dipyridamole scaffolds regenerated 28.03 ± 7.38 percent, 10,000-μm dipyridamole scaffolds regenerated 36.18 ± 6.83 percent (1000 μm versus 10,000 μm dipyridamole; p = 0.104), and rhBMP-2-coated scaffolds regenerated 37.17 ± 16.69 percent bone (p = 0.124 versus 1000 μm dipyridamole, and p = 0.938 versus 10,000 μm dipyridamole). On histology/electron microscopy, no changes in suture biology were evident for dipyridamole, whereas rhBMP-2 demonstrated early signs of suture fusion. Healing was highly cellular and vascularized across all groups. No statistical differences in mechanical properties were observed between either dipyridamole or rhBMP-2 compared with native bone. CONCLUSION/CONCLUSIONS:Dipyridamole generates new bone without osteolysis and early suture fusion associated with rhBMP-2 in skeletally immature bone defects.