Jeff D. Thostenson

Endogenous glucocorticoids decrease skeletal angiogenesis, vascularity, hydration, and strength in aged mice

Aging or glucocorticoid excess decrease bone strength more than bone mass in humans and mice, but an explanation for this mismatch remains elusive. We report that aging in C57BL/6 mice was associated with an increase in adrenal production of glucocorticoids as well as bone expression of 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1, the enzyme that activates glucocorticoids. Aging also decreased the volume of the bone vasculature and solute transport from the peripheral circulation to the lacunar‐canalicular system. The same changes were reproduced by pharmacologic hyperglucocorticoidism. Furthermore, mice in which osteoblasts and osteocytes were shielded from glucocorticoids via cell‐specific transgenic expression of 11β‐HSD type 2, the enzyme that inactivates glucocorticoids, were protected from the adverse effects of aging on osteoblast and osteocyte apoptosis, bone formation rate and microarchitecture, crystallinity, vasculature volume, interstitial fluid, and strength. In addition, glucocorticoids suppressed angiogenesis in fetal metatarsals and hypoxia inducible factor‐1α transcription and vascular endothelial growth factor production in osteoblasts and osteocytes. These results, together with the evidence that dehydration of bone decreases strength, reveal that endogenous glucocorticoids increase skeletal fragility in old age as a result of cell autonomous effects on osteoblasts and osteocytes leading to interconnected decrements in bone angiogenesis, vasculature volume, and osteocyte‐lacunar‐canalicular fluid.

Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo.

Connexin (Cx)43 is required for inhibition of osteocyte and osteoblast apoptosis by bisphosphonates in vitro. Herein, we evaluated its requirement for the in vivo actions of bisphosphonates using mice in which Cx43 was deleted specifically from osteocytes and osteoblasts (Cx43ΔOb−Ot/− mice). Effective removal of Cx43 was confirmed by the presence of the deleted form of the gene and by reduced mRNA and protein expression in osteoblastic cells and bones obtained from Cx43ΔOb−Ot/− mice. The amino‐bisphosphonate alendronate (2.3 μmol/kg/d) was injected daily into 5‐mo‐old female mice (n = 6–11) for 31 days, starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/d). Cx43ΔOb−Ot/− mice and their littermates (Cx43fl/−, Cx43ΔOb−Ot/+, and Cx43fl/+) gained bone with similar kinetics and exhibited identical bone mass from 2 to 4.5 mo of age, indicating that Cx43 deletion from osteocytes and mature osteoblasts does not impair bone acquisition. In addition, prednisolone induced a similar increase in osteocyte and osteoblast apoptosis in Cx43ΔOb−Ot/− or in control Cx43fl/− littermates. However, whereas alendronate prevented prednisolone‐induced apoptosis in control Cx43fl/− mice, it was ineffective in Cx43ΔOb−Ot/− mice. In contrast, alendronate inhibited glucocorticoid‐induced bone loss in both type of animals, suggesting that inhibition of resorption is the predominant effect of alendronate against the early phase of glucocorticoid‐induced bone loss. Taken together with earlier in vitro evidence, these findings show that Cx43 is required for the anti‐apoptotic effect of bisphosphonates on osteocytes and osteoblasts.

Receptor Activator of Nuclear Factor κB Ligand (RANKL) Protein Expression by B Lymphocytes Contributes to Ovariectomy-induced Bone Loss

Background: The contribution of B lymphocytes to the bone loss caused by estrogen deficiency is unclear. Results: Deletion of the cytokine receptor activator of NFκB ligand from B lymphocytes, but not T lymphocytes, blunted bone loss in ovariectomized mice. Conclusion: Cytokine production by B lymphocytes contributes to ovariectomy-induced bone loss. Significance: This mechanism may be relevant to the mechanisms responsible for postmenopausal osteoporosis. Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.

Comparison of cannabis and tobacco withdrawal: Severity and contribution to relapse

This naturalistic telephone survey study compared perceptions of withdrawal severity in 67 daily cannabis users and 54 daily tobacco cigarette smokers who made quit attempts during the prior 30 days. A Withdrawal Symptom Checklist assessed the severity of abstinence symptoms and a Likert scale assessed perceived relations between abstinence symptoms and relapse. A composite Withdrawal Discomfort Score did not differ significantly between groups (M = 13.0 for cannabis, vs. M = 13.2 for tobacco). Individual symptom severity ratings were also of similar magnitude, except craving and sweating were slightly higher for tobacco. Both groups reported that withdrawal contributed substantially to relapse, and the strength of these ratings was similar across groups. The diverse convenience sample examined in this study adds external validity and generalizability to prior studies that included only users not planning to quit or excluded many common types of cannabis users. The comparable withdrawal experience from these heterogeneous cannabis and tobacco users supports previous findings from controlled laboratory studies and indicates that real-world, frequent cannabis users perceive that withdrawal symptoms negatively affect their desire and ability to quit.

Osteocyte-derived RANKL is a critical mediator of the increased bone resorption caused by dietary calcium deficiency

Parathyroid hormone (PTH) excess stimulates bone resorption. This effect is associated with increased expression of the osteoclastogenic cytokine receptor activator of nuclear factor κB ligand (RANKL) in bone. However, several different cell types, including bone marrow stromal cells, osteocytes, and T lymphocytes, express both RANKL and the PTH receptor and it is unclear whether RANKL expression by any of these cell types is required for PTH-induced bone loss. Here we have used mice lacking the RANKL gene in osteocytes to determine whether RANKL produced by this cell type is required for the bone loss caused by secondary hyperparathyroidism induced by dietary calcium deficiency in adult mice. Thirty days of dietary calcium deficiency caused bone loss in control mice, but this effect was blunted in mice lacking RANKL in osteocytes. The increase in RANKL expression in bone and the increase in osteoclast number caused by dietary calcium deficiency were also blunted in mice lacking RANKL in osteocytes. These results demonstrate that RANKL produced by osteocytes contributes to the increased bone resorption and the bone loss caused by secondary hyperparathyroidism, strengthening the evidence that osteocytes are an important target cell for hormonal control of bone remodeling.

An initial trial of a computerized behavioral intervention for cannabis use disorder

The most potent outcomes for cannabis use disorders have been observed with a combination of three evidence-based interventions, motivational enhancement therapy (MET), cognitive-behavioral therapy (CBT), and abstinence-based contingency-management (CM). Access to this intervention remains limited because of cost and service availability issues. This report describes the initial stages of a project designed to develop and test a computer-assisted version of MET/CBT/CM that could address many of the current barriers to its dissemination. A nonrandomized, 12-week comparison study assigned 38 adults seeking treatment for a cannabis use disorder to either therapist-delivered (n=22) or computer-delivered (n=16) MET/CBT/CM. Attendance, retention, and cannabis use outcomes did not differ significantly between groups, and there were no indications of superior outcomes favoring therapist delivery. Participants provided positive ratings of the computer-delivered sessions. These preliminary findings suggest that computer-assisted delivery of MET/CBT/CM is acceptable to outpatients and does not adversely impact compliance or outcomes achieved during treatment with MET/CBT/CM for cannabis use disorders. Assessment of post-treatment outcomes and replication in randomized trials are needed to determine reliability and longer term effects. As observed in a growing number of studies, computerized therapies have the potential to increase access to, reduce costs, and enhance fidelity of providing evidence-based treatments without sacrificing and possibly enhancing effectiveness.

The Effects of Aging and Sex Steroid Deficiency on the Murine Skeleton Are Independent and Mechanistically Distinct

Old age and sex steroid deficiency are the two most critical factors for the development of osteoporosis. It remains unknown, however, whether the molecular culprits of the two conditions are similar or distinct. We show herein that at 19.5 months of age—a time by which the age‐dependent decline of cortical and cancellous bone mass and cortical porosity were fully manifested in C57BL/6J mice—these animals remained functionally estrogen sufficient. Transgenic mice with conditional expression of mitochondria‐targeted catalase—a potent H2O2 inactivating enzyme—in cells of the myeloid lineage (mitoCAT;LysM‐Cre mice) were protected from the loss of cortical, but not cancellous, bone caused by gonadectomy in either sex. Consistent with these findings, in vitro studies with ERα‐deficient Prx1+ cells and gonadectomized young adult mice showed that in both sexes decreased ERα signaling in Prx1+ cells leads to an increase in SDF1, a.k.a. CXCL12, an osteoclastogenic cytokine whose effects were abrogated in macrophages from mitoCAT;LysM‐Cre mice. In contrast to sex steroid deficiency, the adverse effects of aging on either cortical or cancellous bone were unaffected in mitoCAT;LysM‐Cre mice. On the other hand, attenuation of H2O2 generation in cells of the mesenchymal lineage targeted by Prx1‐Cre partially prevented the loss of cortical bone caused by old age. Our results suggest the effects of sex steroid deficiency and aging on the murine skeleton are independent and result from distinct mechanisms. In the former, the prevailing mechanism of the cortical bone loss in both sexes is increased osteoclastogenesis caused by estrogen deficiency; this is likely driven, at least in part, by mesenchymal/stromal cell–derived SDF1. Decreased osteoblastogenesis, owing in part to increased H2O2, combined with increased osteoclastogenesis caused by aging mechanisms independent of estrogen deficiency, are the prevailing mechanisms of the loss of cortical bone with old age.

Cortical bone loss caused by glucocorticoid excess requires RANKL production by osteocytes and is associated with reduced OPG expression in mice.

Glucocorticoid excess is a major cause of low bone mass and fractures. Glucocorticoid administration decreases cortical thickness and increases cortical porosity in mice, and these changes are associated with increased osteoclast number at the endocortical surface. Receptor activator of NF-κB ligand (RANKL) produced by osteocytes is required for osteoclast formation in cancellous bone as well as the increase in cortical bone resorption caused by mechanical unloading or dietary calcium deficiency. However, whether osteocyte-derived RANKL also participates in the increase in bone resorption caused by glucocorticoid excess is unknown. To address this question, we examined the effects of prednisolone on cortical bone of mice lacking RANKL production in osteocytes. Prednisolone administration increased osteoclast number at the endocortical surface, increased cortical porosity, and reduced cortical thickness in control mice, but none of these effects occurred in mice lacking RANKL in osteocytes. Prednisolone administration did not alter RANKL mRNA abundance but did reduce osteoprotegerin (OPG) mRNA abundance in osteocyte-enriched cortical bone. Similarly, dexamethasone suppressed OPG but did not increase RANKL production in cortical bone organ cultures and primary osteoblasts. These results demonstrate that RANKL produced by osteocytes is required for the cortical bone loss caused by glucocorticoid excess but suggest that the changes in endocortical resorption are driven by reduced OPG rather than elevated RANKL expression.

Relationship of bicipital groove rotation with humeral head retroversion: a three-dimensional computed tomographic analysis.

BACKGROUND Bicipital groove location has been used as a reference for humeral stem orientation in total shoulder arthroplasty to recreate humeral head retroversion. However, anatomic variability has rendered its use for prosthetic orientation problematic in cases of comminuted proximal fractures. We hypothesized that variability in groove rotation is directly related to variability in humeral head retroversion and that by defining the degree of groove rotation, humeral head retroversion can be predicted. METHODS Computed tomographic scans (1-mm sections) were obtained along the entire lengths of thirty-four cadaveric humeri, and three-dimensional models were created by using computer-assisted design software. Humeral head retroversion was determined in reference to the transepicondylar axis. The bicipital groove was mapped from proximal to distal, and the rotation of the groove in relationship to the transepicondylar axis was tracked over the entire length of the groove. The overall groove rotation and the rotation of its proximal, intermediate, and distal 15-mm segments were determined. RESULTS The average humeral head retroversion was 21°, and the average angles of groove rotation in relation to the transepicondylar axis for the overall groove and the proximal, intermediate, and distal segments were 65°, 60°, 63°, and 71° of internal rotation relative to the transepicondylar axis, respectively. Pearson correlation coefficients between bicipital groove rotation and humeral head retroversion were ≥0.78 for all segments. CONCLUSIONS A previously unknown direct correlation between bicipital groove rotation and humeral head retroversion was found to exist. The ability to predict humeral head retroversion when mapping only the distal third of the groove has potentially important clinical implications because the distal third is often the only portion of the groove remaining in patients with a comminuted proximal humeral fracture. This is particularly relevant with computer-navigated surgery.

Suppression of autophagy in osteocytes does not modify the adverse effects of glucocorticoids on cortical bone.

Glucocorticoid excess decreases bone mass and strength in part by acting directly on osteoblasts and osteocytes, but the mechanisms remain unclear. Macroautophagy (herein referred to as autophagy) is a lysosome-based recycling pathway that promotes the turnover of intracellular components and can promote cell function and survival under stressful conditions. Recent studies have shown that glucocorticoids stimulate autophagy in osteocytes, suggesting that autophagy may oppose the negative actions of glucocorticoids on this cell type. To address this possibility, we compared the impact of prednisolone administration on the skeletons of adult mice in which autophagy was suppressed in osteocytes, via deletion of Atg7 with a Dmp1-Cre transgene, to their control littermates. In control mice, prednisolone increased autophagic flux in osteocyte-enriched bone as measured by LC3 conversion, but this change did not occur in the mice lacking Atg7 in osteocytes. Nonetheless, prednisolone reduced femoral cortical thickness, increased cortical porosity, and reduced bone strength to similar extents in mice with and without autophagy in osteocytes. Prednisolone also suppressed osteoblast number and bone formation in the cancellous bone of control mice. As shown previously, Atg7 deletion in osteocytes reduced osteoblast number and bone formation in cancellous bone, but these parameters were not further reduced by prednisolone administration. In cortical bone, prednisolone elevated osteoclast number to a similar extent in both genotypes. Taken together, these results demonstrate that although glucocorticoids stimulate autophagy in osteocytes, suppression of autophagy in this cell type does not worsen the negative impact of glucocorticoids on the skeleton.