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Anatomy, Cell Biology & Physiology Department Theses and Dissertations
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Item Approaches to Improve the Structure and Function of the Skeleton in Chronic Kidney Disease(2022-03) Swallow, Elizabeth Anne; Allen, Matthew R.; McNulty, Margaret A.; Moe, Sharon M.; Wallace, Joseph M.Chronic kidney disease (CKD) currently affects ~37 million Americans and causes substantially increased risk of skeletal fracture and fracture-related mortality. Current methods to treat CKD-related bone loss remain alarmingly ineffective. Skeletal fragility in CKD is predominately driven by deteriorations in cortical bone, highlighted by significant cortical porosity development. It is hypothesized that cortical porosity is largely driven by chronically high levels of parathyroid hormone (PTH), which alters the balance of bone remodeling in favor of rampant osteoclast activity and bone resorption. Restricting cortical bone deterioration and the development of cortical pores is likely essential to improve CKD patients’ bone health and reduce their fracture risk. The goal of this series of studies was to answer the following key questions: (1) to what degree do bisphosphonates, an approved pharmacological agent used in metabolic bone disease, accumulate in the skeleton of animals with CKD; (2) can smaller and more frequent doses of bisphosphonates alter skeletal accumulation and improve cortical architecture and the mechanical integrity of bone; (3) can non-bisphosphonate pharmacological interventions more specifically affect cortical bone deterioration. Utilizing epi-fluorescence and two-photon microscopy, our results show that bisphosphonates accumulate more in rats with renal impairment and fractionating bisphosphonates lowered skeletal accumulation irrespective of disease state. Further, studies in both rat and mouse models of CKD demonstrated different bisphosphonate treatments alone do not recover declines in cortical microarchitecture or mechanical properties in CKD. These findings demonstrate that a single intervention is not sufficient in managing CKD-induced bone alterations. Utilizing individual pore tracking analysis, we demonstrated cortical pores can be modulated with therapeutic interventions and can infill, despite the presence of CKD. Potent suppression of PTH led to significant pore infilling while more subtle reductions in PTH, via a calcimimetic, had less striking effects on bone. Calcimimetics mitigated cortical microarchitecture deterioration and reduced the rate of cortical pore expansion. Overall, these findings highlight the importance of PTH management for treating cortical deterioration in CKD. Although bisphosphonates can be utilized in ways that reduce skeletal accumulation, they appear to need co-therapies to reduce skeletal fragility associated with CKD.Item Assessing and Modifying Bone Quality in Chronic Kidney Disease(2015) Newman, Christopher L.; Allen, Matthew R.Chronic kidney disease (CKD) results in an increased fracture risk, partially due to elevations in parathyroid hormone (PTH) that lead to substantial bone loss. On its own, bone loss does not explain bone fragility in CKD, suggesting that changes in skeletal tissue (bone quality) may also be present. Understanding the factors that lead to fracture in these patients will have a substantial impact on patient care and could lead to a better understanding of how to reduce their fracture risk. Due to their suppression of PTH, calcitriol and its analogues are the current standard of care for bone disease in CKD. Yet, surprisingly little is known of their effects on bone. Agents effective in treating osteoporosis are not recommended in advanced CKD due to the lack of data regarding their efficacy and safety in these patients. The goals of the current study were to determine (1) the impact of CKD on bone quality, (2) the ability of calcitriol to improve skeletal parameters, and (3) the efficacy of various pharmacological interventions (calcium, bisphosphonates, anti-sclerostin antibody, and raloxifene) on bone mass, quality, and mechanical properties in CKD bone disease. Using a slowly progressive rat model of CKD, renal and mineral metabolism, bone morphology, bone quality, and bone mechanics (at several length scales) were assessed. Primarily due to elevated PTH, mechanical testing and tissue-level assessments revealed compromised bone quantity (high cortical porosity and low trabecular volume) and quality (high collagen cross-linking and low matrix bound water). Despite clinically relevant reductions in PTH, calcitriol treatment had no positive impact on skeletal properties. Most agents were only effective when PTH levels were normal. Raloxifene, however, led to greater whole bone and material toughness (the ability of bone to tolerate existing damage) despite modest PTH suppression. While the examination of bone quality in CKD is still in its infancy, these results indicate that enhancing bone quality with raloxifene may be an effective means to compensate for bone loss in CKD.Item Bone Perfusion Alterations in Chronic Kidney Disease(2019-05) Aref, Mohammad W.; Allen, Matthew R.; Organ, Jason M.; Tune, Jonathan D.; Wallace, Joseph M.; Moe, Sharon M.Patients with chronic kidney disease (CKD) are at an alarming risk of fracture and cardiovascular disease-associated mortality. There is a critical need to better understand the underlying mechanism driving altered cardiovascular and skeletal homeostasis, as well as any connection between the two. CKD has been shown to have negative effects on many vascular properties including endorgan perfusion. Surprisingly, exploration of skeletal perfusion and vasculature has not been undertaken in CKD. Alterations in bone perfusion are linked to dysregulation of bone remodeling and mass in multiple conditions. An understanding of the detrimental impact of CKD on bone perfusion is a crucial step in understanding bone disease in these patients. The goal of this series of studies was to test the global hypothesis that skeletal perfusion is altered in CKD and that alterations can be modulated through treatments that affect metabolic dysfunction. These studies utilized a rat model of CKD to conduct metabolic assessments, bone perfusion measurements, bone imaging studies, and isolated vessel reactivity experiments. Our results showed that animals with CKD had higher levels of parathyroid hormone (PTH), leading to substantial bone resorption. Bone perfusion measurements showed CKD-induced elevations in cortical bone perfusion with levels progressing alongside CKD severity. Conversely we show that marrow perfusion was lower in advanced CKD. PTH suppression therapy in animals with CKD resulted in the normalization of cortical bone perfusion and cortical bone mass, but did not normalize marrow bone perfusion. These results show a clear association between bone deterioration and altered bone perfusion in CKD. While the relationship of altered bone perfusion and bone deterioration in CKD necessitates further work, these results indicate that determining the mechanisms of bone perfusion alterations and whether they are drivers, propagators, or consequences of skeletal deterioration in CKD could help untangle a key player in CKD-induced bone alterations.Item CaMKK2 Signaling in Metabolism and Skeletal Disease: A New Axis with Therapeutic Potential(2022-07) Williams, Justin N.; Sankar, Uma; Evans-Molina, Carmella; Bonewald, Lynda; Burr, David; Allen, MatthewType 2 diabetes mellitus (T2DM) is a growing problem globally and is associated with increased fracture risk and delayed bone healing. Novel approaches are needed in the treatment of T2DM and the resulting diabetic osteopathy. Recent studies highlight the role of bone as an endocrine organ producing factors that communicate with distant tissues to modulate systemic glucose metabolism. Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is a potent regulator of whole-body energy metabolism, inflammation, bone remodeling and fracture healing. Genetic ablation of CaMKK2 protects from diet-induced obesity, insulin resistance and inflammation, while enhancing pancreatic β cell survival and insulin secretion. Deletion or inhibition of CaMKK2 promotes bone accrual by stimulating osteoblast-mediated bone formation and suppressing osteoclast-mediated bone resorption; however, its specific role in osteocytes, the master regulator of bone remodeling remains unknown. Here we demonstrate that conditional deletion of CaMKK2 from osteocytes enhances bone mass in 3-month-old female, but not male mice, due to suppression of osteoclasts. Conditioned media experiments and proteomics analysis revealed that female osteocytes lacking CaMKK2 suppressed osteoclast formation and function through enhanced secretion of calpastatin, a potent inhibitor of calpains, which are calciumdependent cysteine proteases that support osteoclasts. Further, to determine if CaMKK2- deficient osteocytes regulate whole-body glucose homeostasis, we placed these mice on a high-fat diet (HFD) for a period of 16 weeks. Although the diet did not significantly impact bone mass or strength, we found that conditional deletion of CaMKK2 in osteocytes enhanced bone microarchitecture in 6-month-old male and female mice. We also observed that conditional deletion of CaMKK2 from osteocytes protected male and female mice from HFD-induced obesity and insulin insensitivity. Taken together, these findings highlight CaMKK2 as a potent regulator of osteocyte-mediated modulation of bone remodeling and whole-body energy metabolism.Item Contribution of rankl regulation to bone resorption induced by PTH receptor activation in osteocytes(2012-10-19) Ben-awadh, Abdullah Nasser; Bellido, Teresita M.; Plotkin, Lilian I.; Allen, Matthew R.PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton.Item Disabling the Transcription Factor Nmp4 from Osteogenic Precursors Enhances the Skeleton's Response to the Osteoporosis Drug Parathyroid Hormone(2022-08) Atkinson, Emily Grace; Bidwell, Joseph P.; Robling, Alexander G.; Plotkin, Lilian I.; Wallace, Joseph; Organ, Jason M.; Evans-Molina, CarmellaActivation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis. Yet, FDA-approved osteoanabolics, e.g., parathyroid hormone (PTH) have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice, exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues, therefore, to determine whether the suppression of PTHinduced bone formation is cell autonomous, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4fl/fl) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre+ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre+ targeting mature osteoblasts and (iii) Dmp1Cre+ to disable Nmp4 in transitional osteocytes. Virgin female Cre+ and Cre- mice (10wks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1–34 at 30μg/kg, or vehicle for 4wks or 7wks. The skeletal response was assessed using dual-energy X-ray absorptiometry, microcomputed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4fl/fl;Prx1Cre+ mice recapitulated the global Nmp4-/- skeletal phenotype in the femur, i.e., an enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared to their Nmp4fl/fl;Prx1Cre- controls. This was not observed in the spine, where Prx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4fl/fl;BglapCre+) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4fl/fl;Dmp1Cre+) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4-/- MSPCs drive the enhanced response to PTH therapy, and Nmp4 has stage-specific effects on osteoanabolism.Item Dissecting the cellular and molecular mechanisms mediating neurofibromatosis type 1 related bone defects(2013-06) Rhodes, Steven David; Yang, Feng-Chun; Clapp, D. Wade; Robling, Alexander G.; Bidwell, Joseph P.Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.Item "Do I really have to complete another evaluation?" exploring relationships among physicians' evaluative load, evaluative strain, and the quality of clinical clerkship evaluations(2017-06) Traser, Courtney Jo; Brokaw, James J.Background. Despite widespread criticism of physician-performed evaluations of medical students’ clinical skills, clinical clerkship evaluations (CCEs) remain the foremost means by which to assess trainees’ clinical prowess. Efforts undertaken to improve the quality of feedback students receive have ostensibly led to higher assessment demands on physician faculty; the consequences of which remain unknown. Accordingly, this study investigated the extent to which physicians’ evaluative responsibilities influenced the quality of CCEs and qualitatively explored physicians’ perceptions of these evaluations. Methods. A questionnaire was delivered to physicians (n = 93) at Indiana University School of Medicine to gauge their perceived evaluative responsibilities. Evaluation records of each participant were obtained and were used to calculate one’s measurable quantity of CCEs, the timeliness of CCE submissions, and the quality of the Likert-scale and written feedback data included in each evaluation. A path analysis estimated the extent to which one’s evaluative responsibilities affected the timeliness of CCE submissions and CCE quality. Semi-structured interviews with a subset of participants (n = 8) gathered perceptions of the evaluations and the evaluative process. Results. One’s measurable quantity of evaluations did not influence one’s perceptions of the evaluative task, but did directly influence the quality of the Likert-scale items. Moreover, one’s perceptions of the evaluative task directly influenced the timeliness of CCE submissions and indirectly influenced the quality of the closed-ended CCE items. Tardiness in the submission of CCEs had a positive effect on the amount of score differentiation among the Likert-scale data. Neither evaluative responsibilities nor the timeliness of CCE submissions influenced the quality of written feedback. Qualitative analysis revealed mixed opinions on the utility of CCEs and highlighted the temporal burden and practical limitations of completing CCEs. Conclusions. These findings suggest physicians’ perceptions of CCEs are independent of their assigned evaluative quantity, yet influence both the timeliness of evaluation submissions and evaluative quality. Further elucidation of the mechanisms underlying the positive influence of evaluation quantity and timely CCE submissions on CCE quality are needed to fully rationalize these findings and improve the evaluative process. Continued research is needed to pinpoint which factors influence the quality of written feedback.Item Does time matter? : a search for meaningful medical school faculty cohorts(2014-12) Guillot III, Gerard Majella; Palmer, Megan M.; Dankoski, Mary E.; Nelson Laird, Thomas F.; Seifert, Mark F.; Shew, Ronald L.Background. Traditionally, departmental appointment type (basic science or clinical) and/or degree earned (PhD, MD, or MD-PhD) have served as proxies for how we conceptualize clinical and basic science faculty. However, the landscape in which faculty work has considerably changed and now challenges the meaning of these cohorts. Within this context I introduce a behavior-based role variable that is defined by how faculty spend their time in four academic activities: teaching, research, patient care, and administrative duties. Methods. Two approaches to role were compared to department type and degree earned in terms of their effects on how faculty report their perceptions and experiences of faculty vitality and its related constructs. One approach included the percent of time faculty spent engaged in each of the four academic activities. The second approach included role groups described by a time allocation rubric. This study included faculty from four U.S. medical schools (N = 1,497) and data from the 2011 Indiana University School of Medicine Faculty Vitality Survey. Observed variable path analysis evaluated models that included traditional demographic variables, the role variable, and faculty vitality constructs (e.g., productivity, professional engagement, and career satisfaction). Results. Role group effects on faculty vitality constructs were much stronger than those of percent time variables, suggesting that patterns of how faculty distribute their time are more important than exactly how much time they allocate to single activities. Role group effects were generally similar to, and sometimes stronger than, those of department type and degree earned. Further, the number of activities that faculty participate in is as important a predictor of how faculty experience vitality constructs as their role groups. Conclusions. How faculty spend their time is a valuable and significant addition to vitality models and offers several advantages over traditional cohort variables. Insights into faculty behavior can also show how institutional missions are (or are not) being served. These data can inform hiring practices, development of academic tracks, and faculty development interventions. As institutions continue to unbundle faculty roles and faculty become increasingly differentiated, the role variable can offer a simple way to study faculty, especially across multiple institutions.Item The Effects of Instrument-Assisted Cross Fiber Massage on Ligament Healing(2010-05) Loghmani, Mary T.; Warden, Stuart J.; Burr, David B.; Robling, Alex G.; Seifert, Mark Frederick; Turner, Charles H.Ligament injury is one of the most prevalent musculoskeletal disorders that may lead to disability or disease, such as osteoarthritis. Conservative interventions which accelerate or augment ligament healing are needed to enhance therapeutic outcomes. The purpose of this research agenda was to investigate the tissue level effects of a type of manual therapy, cross fiber massage (CFM), in particular instrument-assisted CFM (IACFM), on ligament healing. Bilateral knee medial collateral ligament (MCL) injuries were created using an established rodent model where one MCL received IACFM treatment and the other untreated MCL served as a within subjects control. The short and long term effects of IACFM on the biomechanical and histological properties of repairing ligaments were investigated. Tensile mechanical testing was performed to determine ligament mechanical properties. Ligament histology was examined under light microscopy and scanning electron microscopy. IACFM was found to accelerate early ligament healing (4 weeks post-injury), possibly via favorable effects on collagen formation and organization, but minimal improvement was demonstrated in later healing (12 weeks post-injury). Regional blood flow and angiogenesis were investigated as possible mechanisms underlying the accelerated healing found in IACFM-treated ligaments. Laser Doppler perfusion imaging was used to investigate vascular function. Micro-computed tomography was used to determine vascular structural parameters. Compared to untreated contralateral injured controls, IACFM-treated injured knees demonstrated a delayed increase in blood flow and altered microvascular structure, possibly suggesting angiogenesis. Mechanotransduction is discussed as a mechanism for the beneficial effects of CFM in that application of a mechanical force was found to enhance biomechanical and histological properties as well as vascular function and structure acutely in healing ligaments. Although this thesis focused on IACFM treatment of injured knee ligaments, it is plausible for concepts to apply to other manual modalities that offer conservative alternatives to invasive procedures or pharmaceuticals in the treatment of soft tissue injuries.