Browsing by Author "Parajuli, Ashutosh"

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    Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes
    (PLOS (Public Library of Science), 2015-05-26) Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.; Padma P. Srinivasan, Ashutosh Parajuli, Christopher Price, Liyun Wang, Randall L. Duncan, Catherine B. Kirn-Safran; Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.
    Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3- E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degeneration
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    Mechanical adaptation of diabetic and perlecan deficient skeletons
    (University of Delaware, 2020) Parajuli, Ashutosh
    Bone, a “smart material”, keeps adapting its structure and mass under mechanical cues to meet its load-bearing function in the body. This amazing capability arises from its living cellular residents, whose sensitivity to mechanical and chemical signals, along with cell-cell communication over time and space, result in an adaptive mechanism, encoded in genes and yet modulated by environments, which works efficiently to ensure the proper maintenance of healthy adult skeleton. However, this robust adaptive process could be impaired by diseases. The overall objective of my thesis was to investigate how diabetes (a metabolic disease condition) and perlecan deficiency (a rare genetic condition, Schwartz-Jampel Syndrome) alter the adaptive response of skeleton to mechanical loading or disuse. The clinical motivation of this line of research is to maximize the anabolic potentials of mechanical stimulation in promoting bone health, a strategy complementary to pharmaceutical interventions. ☐ Given our lab’s interest on osteocytes, the primary mechanosensing cells in bone and the master orchestrator of bone remodeling, I focused my investigations on how hyperglycemia (a complication of uncontrolled diabetes) affects the sensitivity of osteocytes and bone tissue to mechanical loading. I further investigated the effects of deficiency of perlecan, a critical component of osteocyte mechanosensing pericellular complex, on bone cells and bone tissue responses to the removal of loading and subsequent reloading. My results demonstrated the robustness of bone formation driven by mechanical loading in mice with normal or mild diabetes (40% elevated blood glucose), while severe diabetes (>200% elevation) completely abolished bone formation and inhibited osteocyte’s intracellular calcium and other responses to fluid flow. With decreased perlecan content in bone, a severe osteoporotic skeletal phenotype was seen in the trabecular bone compartments of the perlecan deficient mouse mutants. This phenotype was present for both axial bone (vertebrate) and long bones (femur and tibia) and progressively worsen with age, primary due to the upregulation of osteoclastogenesis. Also, for the first time, we found that the perlecan deficient mice, although lost bone similarly as wild type under hindlimb suspension, could partially recover from the bone loss after re-ambulation. ☐ Although my results provide experimental evidence that metabolic and matrix perturbations like diabetes and Schwartz-Jampel Syndrome negatively affect bone, bone cells, and bone adaptation process, the robustness of the bone adaptation process is also evident in the load-induced bone formation found in mildly diabetic mice and the surprising recovery from bone loss after re-loading of perlecan deficient mice. Despite the experiments were performed in mice, these findings could provide insights on managing bone health in patients suffering diabetes (e.g., getting the blood sugar level in control) or Schwartz-Jampel Syndrome (e.g., engaging moderate exercise). Overall, my thesis supports that exercise is a potent anabolic stimulus to bone. Unlocking or restoring its power by regulating the underlying mechanisms could be an effective way to promote bone health.