Adult Bone Research Articles

Treosulfan-Based Reduced-Toxicity Conditioning for Elderly Patients with High-Risk Myelodysplastic and Myeloproliferative Disease for Allogeneic Hematopoietic Stem Cell Transplantation

Introduction Allogeneic hematopoietic stem cell transplantation (alloHCT) is the only potential curative treatment for patients (pts) with high-risk myeloid malignancies. Despite advances in the supportive care over the past decades, alloHCT is still associated with significant toxicity, especially in the elderly population. The choice of an adequate conditioning regimen has become essential in order to prevent treatment-related morbidity and mortality. Since myeloablative conditioning (MAC) is mostly restricted to the younger population due to the high toxicity, reduced-intensity conditioning (RIC) provides an alternative solution. However, especially for pts with high-risk disease, a potential increase in the incidence of relapse must be taken into account. The advent of reduced-toxicity conditioning regimens (RTC), mostly based on treosulfan in combination with fludarabine, offers a balance between a favorable toxicity profile and a strong anti-neoplastic effect. Considering the increasing rates of alloHCT among older pts in the past decades, finding the optimal conditioning regimen is still an unmet need for this collective. The objective of this study is to analyze the outcome of pts aged ≥60 years who underwent alloHCT for myelodysplastic or myeloproliferative disease and received a treosulfan-based conditioning. In order to maximize the anti-leukemic effect of the conditioning in this population we chose the myeloablative dose of treosulfan of 14g/m2 daily. Materials and methods We retrospectively analyzed the outcome of adult pts ≥60 years with myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) or myelofibrosis (MF) who underwent their first allo-HCT from a matched or mismatched donor over a period of 10 years at the adult Bone Marrow Transplantation Unit of our institution. The conditioning consisted of a combination of treosulfan 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -2, alone or combined with 2 Gy TBI (day 0) for MDS and treosulfan, 14 g/m2/day on days -6 to -4, fludarabine, 30 mg/m2/day on days -6 to -3 for CMML and MF. Endpoints of this analysis were engraftment rate (together with time to engraftment), relapse-free survival (RFS), overall survival (OS), and non-relapse mortality (NRM). Continuous variables were illustrated as median values. NRM, RFS and OS for the whole cohort were analyzed with the Kaplan-Meier method and presented as survival functions. Results Between 01/2012 and 12/2022 91 consecutive elderly pts were eligible for this analysis. Patient and transplant characteristics are listed in Table 1. The median follow-up after allo-HCT was 3.12 years (4 months-10.5 years). Median age was 65 years, 76% (n = 69) of patients had an EBMT (European Society for Blood and Marrow Transplantation) risk score of ≥ 4. In 20 of 91 pts (22%) a 9/10 matched unrelated donor was selected, due to the lack of a more suitable donor. The standard graft versus host disease (GVHD) prophylaxis was a combination of calcineurin inhibitors (CNI) +/- mycophenolate mofetil and Anti-T-lymphocyte globulin (ATG) (n= 85, 94%). Neutrophil engraftment was achieved in 98% (n= 89) of pts. The median time to engraftment was 13 days (12, 15). Fourteen of 91 pts (15%) developed severe (grade III-IV) acute GVHD, and 44% (n= 40) had chronic GVHD of any grade. Considering early toxicity, 2 patients of 91 (2%) died before day +30 after alloHCT due to transplant-related toxicity, overall 6 of 91 (7%) in the first 100 days after alloHCT. The 2-year OS was 55% (95% CI, 44.7-.66.2) (Figure 1) and RFS was 48.5% (95% CI, 38.9-60.5). The 2-year NRM was 37% (95% CI, 33.5-39.5). No significant differences have been observed in the outcome of the different disease entities. Main causes of death were infections (n= 15, 32%), GVHD (n=13, 27.7%) and disease relapse (n= 13, 27.7%). Conclusions Our retrospective analysis suggests that a treosulfan-based RTC with myeloablative potential in an elderly and frail population of pts with high-risk myeloid diseases is feasible, with an excellent engraftment rate and a limited early toxicity, and promotes disease control. We report a 2-year NRM of 37%, which was partially due to severe GvHD in a cohort of pts with a relatively high-proportion of mismatched donors (20%). The role of post-transplant cyclophosphamide as backbone of the GvHD prophylaxis of RTC regimes should be further evaluated, in prospective trials.

Deciphering the Boundaries of KBTBD4-CoREST1 Axis Modulation to Maximally Expand Human HSCs

Background: The UM171 technology has led to major progress in the expansion of cord blood units (CBUs) with low cellularity (Fares et al., Science 2014). This breakthrough has notably increased availability of better HLA-matched CBUs and improvements in clinical outcome (Cohen et al., Lancet Hematol 2020 and Blood Advances 2023, PMID: 37467030; see also Cohen et al. and Milano et al., this meeting). Enhanced HLA matching is a direct consequence of the selection of CBUs which are otherwise too small without expansion. Indeed, with this technology, approximately 50% of units in CB banks are eligible for clinical use, versus 5% without expansion. As recently reported, UM171 promotes the activation of the novel CUL3-KBTBD4 E3 ligase complex leading to the ubiquitylation and degradation of its natural substrate, CoREST1, a repressor complex that includes RCOR1, LSD1, HDAC1/2. This complex is upregulated in human HSC upon their ex vivo culture where it compromises their potential in the absence of UM171 (Chagraoui et al., Cell Stem Cell, 2021). Aims and Methods: The goal of this research is to understand the boundaries of KBTBD4-CoREST1 axis modulation that determine the optimal expansion of human HSCs in vitro and in vivo. More precisely, we monitored the relationship between CoREST1 levels in primary CD34+CD45RA-CD90+ CB cells and determined the impact of maximal CoREST1 degradation on the yield of expanded HSC using a series of functional in vitro and in vivo approaches along with transcriptomic data. Results: Using flow cytometry, we noticed a gradual degradation of RCOR1 with increasing concentrations of UM171 with a plateau at 125nM suggesting that maximal RCOR1 degradation was reached at this dose which represented a 4-fold increase above the concentration of UM171 used in current clinical manufacturing. Of interest, at high concentration of UM171 (i.e.,125nM), total nucleated cell expansion was reduced by approximately 30% while that of primitive CD34+CD45RA-CD90+EPCR+ HSC was maximal and significantly above that measured at 35nM suggesting a preferential impact of CoREST1 degradation on the more primitive cell compartment along with improvement in graft quality. CITE-Seq experiments confirmed the preferential expansion of primitive HSPC including HSCs and LMPPs with a reduction of more mature cellular subsets when cells were exposed to 125nM of UM171 in comparison to 35nM. The in vivo functionality of the 7-day expanded grafts (35nM vs 125nM UM171) were compared by transplanting the outcome of 100 Day 0 CD34+ CB cells in NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) mice, a cell dose representing less than 1 competitive repopulation unit (CRU) of unexpanded cells in this model. Using this low cell dose (limit dilution), we observed that the 125nM expanded graft provided 10-times greater engraftment than observed in mice transplanted with cells exposed to 35nM UM171 with values reaching a median of 5±5% vs. 0.4±0.13%(n=10 mice per condition, p=0.01; Mann Whitney test) of human engraftment at 12 weeks post-transplantation, respectively. Conclusion: Maximal stimulation of CUL3-KBTBD4 by higher concentration of UM171 leads to much better expansion of functional human HSC and opens new avenues for optimal expansion of very small cord blood units and possibly adult bone marrow-derived HSCs which require more than minimal manipulation (e.g., gene therapy). We believe this new data represents a novel milestone in HSC expansion with obvious subsequent clinical benefits.

Leptin receptor+ cells promote bone marrow innervation and regeneration by synthesizing nerve growth factor

The bone marrow contains peripheral nerves that promote haematopoietic regeneration after irradiation or chemotherapy (myeloablation), but little is known about how this is regulated. Here we found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR+) stromal cells is required to maintain nerve fibres in adult bone marrow. In nerveless bone marrow, steady-state haematopoiesis was normal but haematopoietic and vascular regeneration were impaired after myeloablation. LepR+ cells, and the adipocytes they gave rise to, increased NGF production after myeloablation, promoting nerve sprouting in the bone marrow and haematopoietic and vascular regeneration. Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signalling in LepR+ cells, and potentially in adipocytes, increasing their production of multiple haematopoietic and vascular regeneration growth factors. Peripheral nerves and LepR+ cells thus promote bone marrow regeneration through a reciprocal relationship in which LepR+ cells sustain nerves by synthesizing NGF and nerves increase regeneration by promoting the production of growth factors by LepR+ cells.

Hindlimb bones texture through postnatal ages of Rhea americana (Aves: Palaeognathae).

The bone texture of Rhea americana was evaluated through the examination of a postnatal ontogenetic series. The hind limb bone surfaces of specimens of one, three and five months old, and adults were compared to characterize each stage according to the morphological features generated by their differential ossification. The results suggest a similar process of tissue maturation concerning neognathous birds, although with some differences. A spongy or striated surface with abundant pores in the femur and longitudinal grooves in the tibiotarsus and tarsometatarsus characterizes chicks. Vascularity decreases, and the bone texture gradually changes acquiring a more homogeneous surface, to eventually reach the smooth appearance of adult bones. The establishment of particular textures corresponding to different bones and ontogenetic stages permits the accurate interpretation of remains in ecological, archaeological and paleontological contexts in which bones are fragmented and/or incomplete.

Association between normal-weight obesity and bone mineral density in older Korean adults: A population-based cross-sectional study

The effects of normal-weight obesity, which is defined as having a high level of body fat despite a normal body mass index, on the health of bones in older adults are poorly understood. This cross-sectional study examined the relationship between normal-weight obesity and bone mineral density in Korean adults aged 50 years or more (2815 men and 2744 women) from the 2008–2011 Korea National Health and Nutrition Examination Surveys. Between the ages of 50 and 69 years, individuals with normal-weight obesity had a higher risk of low bone mineral density (odds ratio = 1.596, 95 % confidence interval = 1.189–2.141, p = 0.002) compared with individuals with normal-weight non-obesity. However, no significant association between normal-weight obesity and bone mineral density was observed among people between the ages of 70 and 89 years. The study findings support the clinical significance of normal-weight obesity as a proxy biomarker to identify in primary care settings people who are at increased risk of developing osteoporosis at an early stage of aging.

A micro-computed tomography study of the sinus tympani variation in humans.

The posterior part of the tympanic cavity comprises a depression called the sinus tympani (ST). The said structure is of outmost importance, e.g. in surgical procedures involving the middle ear, as a pathology (microbial biofilm or cholesteatoma) present in this difficult to access location might hinder its effective treatment. The aim of the study was to evaluate anatomical variants of the ST in human adult petrous bones. For this purpose, three-dimensional (3D) models of the ST were recreated from micro-computed tomography (CT) scans of 44 dry petrous bone samples (19 female, 25 male), applying 3D Slicer, Meshmixer and MeshLab software. Anatomical variants of the ST were classified in terms of both shape and surface configuration. The internal configuration of the ST was classified as heterogeneous - containing small bony trabeculae and crests up to 1.0 mm in size, contrasting to homogeneous ST that characterizes a relatively smooth interior, or mere presence of minor depressions and mild folds. Female STs were more bowl-shaped (57.9%) than saccular (42.1%), and had heterogeneous surface configuration (52.6%) compared to homogeneous (47.4%). On the contrary, male STs were more saccular (52.0%) rather than bowl-shaped (48.0%), and predominantly had a heterogeneous surface (84.0%) over homogeneous (16.0%). A complex combination of ST features comprised of a saccular shape and heterogeneous surface occurred in 52.0% of males and in 15.8% of females (a statistically significant difference; p = 0.0254, Fisher's exact test) seems to be clinically important because of its potential negative implication on health outcomes after surgery in the case of, for example, cholesteatoma, and it may also favour chronic pathological processes.

Disparities Among Adolescent and Young Adult Patients with Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) in the pediatric population has a 5-year overall survival that exceeds 90%, however, the survival rate drops significantly in the adolescent and young adult (AYA) population. Notably, the AYA contribute to 80% of ALL mortality despite representing less than one-fourth of total ALL cases. The challenges related to caring for adolescents and young adults with malignancies have become increasingly well recognized and the impacts of racial, socioeconomic, and psychosocial disparities as potential contributors to AYA ALL survival rates are only recently being characterized. Methods: We conducted a retrospective cohort study using the Nationwide Inpatient Sample (NIS), the largest all-payer database of hospital admissions in the United States, from January 1, 2011 through December 31, 2020. The study sample consisted of AYA patients 15-39 years of age, as defined by the National Cancer Institute. ALL-related hospitalizations were captured using ICD-9 and 10 codes indicative of ALL. We performed temporal trends analysis to assess inpatient death among ALL hospitalizations by race/ethnicity using joinpoint regression. We used adjusted survey logistic regression analyses to assess associations between race/ethnicity and in-hospital mortality among ALL hospitalizations. Results: Our data captured 138,923 ALL-related hospital encounters of AYA patients from 2011 to 2020. In our cohort, 73.1% of ALL hospitalizations were in patients younger than 30, and 26.9% were in patients between the ages of 30 and 39. AYA NH-Black patients with ALL had twice the odds of inpatient mortality as compared to NH-White patients [odds ratio (OR), 95% confidence interval: 1.75 (1.34-2.29)] (Table 1). While the rate of inpatient mortality improved in the overall AYA population over our 10-year study period, strikingly, the rate of inpatient mortality instead increased in NH-Black patients (Figure 1). In contrast, NH-White patients saw the lowest rate of inpatient mortality, both at the start and at the end of our study period (Figure 1). Although the inpatient mortality rate in Hispanic patients improved over time, these patients remained at an increased likelihood of inpatient mortality compared to their NH-White counterparts [OR 1.32 (1.08-1.61)] (Table 1). Conclusion: Survival gains in the AYA population have improved only modestly compared to that of their pediatric and adult counterparts, and here we report that these survival gains are even further stratified and disparate depending on patient race/ethnicity with worse outcomes in NH-Black patients. It is important to better understand the disparities that impact the AYA population, which is particularly vulnerable to social factors impacting their care. Study limitations include lack of data pertaining to treatment received, including pediatric versus adult treatment protocol or bone marrow transplant status. Disparate access to centers of excellence, stem cell transplant and blinatumomab may certainly be contributors to the disparate outcomes described herein.

FLT3-Ligand Alone Enables the Identification and Maintenance in Vitro of Viable Quiescent Human Fetal Liver Hematopoietic Stem Cells with Maintenance of Their Function

Cells with sustained multi-lineage blood cell regenerative potential are referred to as hematopoietic stem cells (HSCs). Current findings indicate that they are responsible for lifelong blood production, are first detected within the first month of development in humans and are largely created prior to birth after which they expand their numbers in response to physiological demands for normal or enhanced blood cell output requirements. Interestingly, a number of the intrinsically determined functional properties of HSCs also change after birth. These include an apparent decline in their self-renewal potential. Thus, for example, in humans as in mice, cells with long-term (>6 month) HSC regenerative properties present in the developing fetal liver (FL) are a source of HSCs that possess a much higher in vivo regenerative capability than those from older donor sources, such as adult bone marrow or even cord blood (CB). Accordingly, it has been of longstanding interest to better understand the molecular regulation of this high regenerative capacity prevalent in fetal HSCs for potential future therapeutic as well as scientific exploitation. Here, we describe the results of experiments designed to answer the hypothesis that human FL HSCs with human-relevant self-renewal properties can be isolated as a quiescent CD49f+ subset of the GPI80+CD90+CD38-CD45RA-CD34+CD45+ population following their incubation in standard serum-free culture medium supplemented with FLT3-ligand (FLT3-L) alone. Initial experiments showed expression of the CD49f integrin on first trimester hFL cells selectively depleted cells able to produce colonies of granulocytes, macrophages or erythroid cells in standard 2-week methylcellulose cultures containing SCF, GM-CSF, IL6, IL3 and EPO. Conversely, expression of the CD49f integrin selectively enriched for cells with 12-week output capabilities in both growth factor (GF)-supplemented stromal co-cultures and in sublethally irradiated, transplanted immunodeficient NOD-Rag1 -/-IL2Rγc -/- W 41/41 (NRG-W) mice. Initial experiments designed to test the effect of multiple GF and small molecule additives on the maintenance over a 7-day period of this in vivo regenerative ability of the input FL HSC confirmed GPI80 expression to be a continuing positive selective phenotype. In addition, the result of transplant experiments showed that a 2-day incubation in FLT3-L alone maintained the 12-week serially transplantable activity of the HSCs (12-weeks/cycle) as fully equivalent to the unmanipulated input cells and significantly superior (P<0.05) to FLT3-L+IL3+SF (3GF) with or without addition of UM171, stemregenin, or eltrombopag. Interestingly, in vitro monitoring of GPI80+ cells showed FLT3-L alone maintained the survival of only 20% of the input GPI80+ cells compared to any of the 3GF-based conditions. In addition, the time to complete a first division and subsequent divisions of the input cells was delayed and prolonged, respectively, in the FLT3-L versus the 3GF conditions. Subsequent 7-day suspension cultures and 6-week GF-supplemented stromal co-cultures experiments have confirmed the existence of a G0 population within the HSC-enriched subset of human FL cells that best maintains their growth potential in FLT3-L alone. Together, these results demonstrate CD49f expression to be a pervasive marker of human HSCs throughout development and reveal the importance of different GF conditions to support the maintenance of viability and retention of self-renewal capability of human fetal HSCs in contrast to those required to activate/support a rapid initiation of cell division. These findings set the stage for future development of strategies to exploit human FL cells therapeutically and may also be critical to designing conditions that will best support the ex vivo maintenance of HSCs at and after birth.

Spatial Transcriptomics Reveals Distinct Hematopoietic Stem Cell Niches in Mouse Fetal Liver

The hematopoietic stem cell (HSC) microenvironment, termed the niche, supports the proliferation, self-renewal, and differentiation abilities of HSCs. The definitive HSCs emerge from the hemogenic endothelium in the aorta-gonad-mesonephros (AGM) region after E10.5, and then migrate to the fetal liver (FL) after E12.5 for expansion. After E18.5, HSCs migrate to the bone marrow where they reside for the postnatal stage and adulthood. Because the fetal liver is thought to be a harbor for the rapid expansion of HSCs, numerous studies have focused on the FL-HSC niche in the search for novel niche factors and niche cells that support HSC expansion. However, to our knowledge thus far, there have been no successes in translating the niche factors to a clinical application for the expansion of HSCs ex vivo. In this study, we use cutting-edge spatial transcriptomics to comprehensively investigate the transcriptomics and interactions between HSCs and the niche cells in the fetal liver. As a result, we have uncovered two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. To understand the spatial distribution and interactions between FL-HSCs and niche cells, we introduced 2 spatial transcriptomic methods, Slide-seq (10uM resolution), and 10x Genomics Visium (55uM resolution), in our study on E14.5 and E16.5 mouse fetal liver. By integrating with single-cell RNA sequencing, we discovered the spatial transcriptomics of HSCs and potential niche cells, including hepatoblasts, endothelial cells, macrophages, megakaryocytes, and mesenchymal stromal cells (MSCs) in E14.5 and E16.5 mouse fetal liver. Interestingly, we found that MSCs and hepatoblasts were characterized by enriched N-cadherin expression. Both slide-seq and 10x Visium showed that the N-cadherin-expressing MSCs are enriched in the portal vessel area. Importantly, the majority of FL-HSCs are in close proximity to N-cadherin-expressing MSCs and endothelial cells, compared with other potential niche cells, indicating a supportive role of N-cadherin-expressing MSCs and endothelial cells in HSC maintenance. Subsequent CellPhoneDB (CPDB) analysis demonstrated that the N-cadherin-expressing MSCs are major niche-signaling senders with an enriched expression of niche factors and stemness pathway-related ligands. This finding is consistent with our previous finding that N-cadherin-expressing MSCs can maintain reserve HSCs in the adult bone marrow. To investigate the potential role of N-cadherin-expressing cells in supporting FL-HSCs, we generated an N-cadCreER;Cxcl12 and an N-cadCreER;Kitl mouse model to conditionally knockout the well-studied niche factors, Cxcl12 and Kitl (Scf), in N-cadherin-expressing cells. Interestingly, deletion of either Cxcl12 or Kitl through N-cad-CreERT induction resulted in the expansion of myeloid-biased FL-HSCs. Slide-seq further showed that depletion of Cxcl12 through N-cad-CreERT induction led to the repositioning of FL-HSCs from the PV region, which is enriched with MSCs, to the hepatic sinusoidal region, which lacks MSCs. These findings suggested the existence of distinct niches in the fetal liver: the PV niche is composed of N-cadherin + MSCs and peri-PV endothelial cells, and it maintains FL-HSCs with multilineage potential. In contrast, the sinusoidal niche preliminary consists of sinusoidal endothelial cells, hepatocytes, and hepatoblasts, which facilitates the proliferation of FL-HSCs but with a bias toward myeloid lineage. Previous studies suggested that the two forms of Kitl, soluble Kitl (sKitl) and membrane-bound Kitl (mKitl), may have different functions in supporting HSCs. Here, we discovered that mKitl is highly enriched in MSCs, whereas hepatoblasts are the major source of the sKitl. This finding suggests that mKitl may serve as the key niche factor that contributes to the homing and maintenance of FL-HSCs. In summary, by using cutting-edge spatial transcriptomics, we uncovered the existence of distinct niches, the portal-vessel (PV) niche and the sinusoidal niche, in mouse fetal liver and the critical role of N-cadherin + MSCs in maintaining FL-HSCs and a role of hepatoblasts in promoting proliferation. The insight learned from this study enhances our understanding of the complex regulation of HSCs and opens new avenues for potential clinical applications.

Characteristic Alterations of Bone and Mineral Metabolism in Children and Adults with Nephropathic Cystinosis

Characteristic Alterations of Bone and Mineral Metabolism in Children and Adults with Nephropathic Cystinosis

Novel role for alpha-2-macroglobulin (A2M) as a disease modifying protein in senile osteoporosis.

Introduction: In the rapidly aging U.S. population, age-induced bone loss (senile osteoporosis) represents a major public health concern that is associated with a significant increased risk for low trauma fragility fractures, which are debilitating to patients, cause significant morbidity and mortality, and are costly to treat and manage. While various treatments exist to slow bone loss in osteoporosis patients, these suffer from poor tolerability and label restrictions that limit their overall effectiveness. Over the past decade, skeletal stem/progenitor cells (SSPCs), which are the main precursor of osteoblasts and adipocytes in adult bone marrow (BM), have emerged as important players in osteoporosis. Methods: Age-induced skeletal pathology was quantified in elderly (24-month-old) vs. mature (3-month-old) mice by micro-CT and changes in SSPC abundance in the BM of these mice was quantified by fluorescence-activated cell sorting (FACS). SSPCs from elderly vs. mature mice were also analyzed by RNA-Seq to identify differentially expressed genes (DEGs), and gain and loss-of-function studies were performed in human BM-derived mesenchymal stromal cells (BM-MSCs) to assess A2M function. Results: Elderly mice were shown to exhibit significant age-induced skeletal pathology, which correlated with a significant increase in SSPC abundance in BM. RNA-seq analysis identified alpha-2-macroglobulin (A2M), a pan-protease inhibitor that also binds inflammatory cytokines, as one of the most downregulated transcripts in SSPCs isolated from the BM of elderly vs. mature mice, and silencing of A2M expression in human BM-MSCs induced their proliferation and skewed their lineage bifurcation toward adipogenesis at the expense of osteogenesis thereby recapitulating critical aspects of age-induced stem cell dysfunction. Conclusion: These findings identify A2M as a novel disease modifying protein in osteoporosis, downregulation of which in bone marrow promotes SSPC dysfunction and imbalances in skeletal homeostasis.

The bone marrow is the primary site of thrombopoiesis

AbstractMegakaryocytes (MKs) generate thousands of platelets over their lifespan. The roles of platelets in infection and inflammation has guided an interest to the study of extramedullary thrombopoiesis and therefore MKs have been increasingly reported within the spleen and lung. However, the relative abundance of MKs in these organs compared to the bone marrow and the scale of their contribution to the platelet pool in a steady state remain controversial. We investigated the relative abundance of MKs in the adult murine bone marrow, spleen, and lung using whole-mount light-sheet and quantitative histological imaging, flow cytometry, intravital imaging, and an assessment of single-cell RNA sequencing (scRNA-seq) repositories. Flow cytometry revealed significantly higher numbers of hematopoietic stem and progenitor cells and MKs in the murine bone marrow than in spleens or perfused lungs. Two-photon intravital and light-sheet microscopy, as well as quantitative histological imaging, confirmed these findings. Moreover, ex vivo cultured MKs from the bone marrow subjected to static or microfluidic platelet production assays had a higher capacity for proplatelet formation than MKs from other organs. Analysis of previously published murine and human scRNA-seq data sets revealed that only a marginal fraction of MK-like cells can be found within the lung and most likely only marginally contribute to platelet production in the steady state.

Effect of premolar extraction and anchorage type for orthodontic space closure on upper airway dimensions and position of hyoid bone in adults: a retrospective cephalometric assessment.

This study aimed to assess the effect of premolar extraction and anchorage type for orthodontic space closure on upper airway dimensions and position of hyoid bone in adults by cephalometric assessment. This retrospective study was conducted on 142 cephalograms of patients who underwent orthodontic treatment with premolar extraction in four groups of (I) 40 class I patients with bimaxillary protrusion and maximum anchorage, (II) 40 class I patients with moderate crowding and anchorage, (III) 40 class II patients with maximum anchorage, and (IV) 22 skeletal class III patients with maximum anchorage. The dimensions of the nasopharynx, velopharynx, oropharynx, and hypopharynx, and hyoid bone position were assessed on pre- and postoperative lateral cephalograms using AudaxCeph v6.1.4.3951 software. Data were analyzed by the Chi-square test, paired t-test, and Pearson's correlation test (alpha = 0.05). A significant reduction in oropharyngeal, velopharyngeal, and hypopharyngeal airway dimensions was noted in groups I, III, and IV (P<0.001), which was correlated with the magnitude of retraction of upper and lower incisors (r=0.6-0.8). In group II, a significant increase was observed in oropharyngeal and velopharyngeal dimensions (P<0.001). A significant increase in nasopharyngeal dimensions occurred in all groups (P<0.001). Also, in groups I and III, the position of hyoid bone changed downwards and backwards, which was correlated with reduction in airway dimensions (r=0.4-0.6). According to the present results, extraction orthodontic treatment affects upper airway dimensions and hyoid bone position. Maximum anchorage decreases airway dimensions while moderate anchorage increases airway dimensions.

Utility of HR-pQCT in detecting training-induced changes in healthy adult bone morphology and microstructure.

Healthy bone adjusts its traits in an exceptionally coordinated, compensatory process. Recent advancements in skeletal imaging via High-Resolution Peripheral Quantitative Computed Tomography (HR-pQCT) allows for the in vivo 3-dimensional and longitudinal quantification of bone density, microarchitecture, geometry, and parameters of mechanical strength in response to varying strain stimuli including those resulting from exercise or military training. Further, the voxel size of 61 microns has the potential to capture subtle changes in human bone in as little as 8 weeks. Given the typical time course of bone remodeling, short-term detection of skeletal changes in bone microstructure and morphology is indicative of adaptive bone formation, the deposition of new bone formation, uncoupled from prior resorption, that can occur at mechanistically advantageous regions. This review aims to synthesize existing training-induced HR-pQCT data in three distinct populations of healthy adults excluding disease states, pharmacological intervention and nutritional supplementation. Those included are: 1) military basic or officer training 2) general population and 3) non-osteoporotic aging. This review aims to further identify similarities and contrasts with prior modalities and cumulatively interpret results within the scope of bone functional adaptation.

Effect of six weeks of resistance training on bone preservation in older adults: a randomized control trial.

It has been established that chronic resistance exercise contributes to positive changes to bone in older adults. This study evaluated the effect of 6weeks of resistance exercise with either elastic bands or dumbbells vs. a control period on bone morphology of older adults. Fifty-seven adults (mean ± SD; age = 66.5 ± 7.09 yrs; height = 165.2 ± 10.6cm; body mass = 74.5 ± 14.6kg) were randomized into three groups (dumbbell, elastic, or control). Participants underwent a total body dual-energy X-ray absorptiometry (DXA) scan for total body and segmental bone mineral content (BMC) and bone mineral density (BMD) before and following 6-week intervention. Age-matched Z-scores for BMD and BMC were recorded. Data were analyzed using two-way repeated measures ANOVAs and 0.05 significance level. BMCarm improved for the dumbbell group (p = 0.016) after the training, with no change in BMD for any group (p > 0.05). Additionally, significant (time x treatment group) interaction (p = 0.024) of age-matched Z-scores indicated an improvement in only the dumbbell group after 6weeks (p = 0.015), with no change in the elastic group despite them having greater Z-scores than the control group. This study is the first to demonstrate acute normative adaptations as dumbbell-based programs may promote positive maintenance of bone metrics over 6weeks, despite the lack of significant change in absolute BMC or BMD. Adults did not lose relative bone mass with acute exercise using dumbbells as the external load applied and this may lead to positive changes following chronic training. There was no bone-related impact from elastic bands, suggesting a weighted load or force produced relative to gravity is beneficial.

SAT262 Bilateral Stress Fractures Of The Femur In A Patient With HIV On Long-term Tenofovir Disoproxil Fumarate And Short-term Alendronate

Abstract Disclosure: R.J. Mahatme: None. A.D. Lindsay: None. F.S. Mirza: None. Background: Tenofovir disoproxil fumarate (TDF) is a reverse transcriptase inhibitor used for HIV treatment, which has been documented to reduce bone mineral density through enhanced osteoclastic activity, reduced osteoblastic activity, disrupted calcium absorption and renal phosphate wasting. Prolonged use of TDF increases the risk of osteoporosis and fractures. Bisphosphonates (BP) are typically used to prevent osteoporotic fractures by inhibiting bone resorption. However, BP use for more than five years, and concomitant suppression of bone remodeling may be associated with an increased risk of subtrochanteric fractures. We report a case of unusual bilateral subtrochanteric stress fractures of the femur in a patient on long term TDF for HIV, who had taken alendronate for a very short duration. Clinical Case: A 56-year-old woman presented to orthopedics in 2017 with complaint of bilateral leg/hip pain associated with weight bearing. Past medical history was significant for HIV diagnosed in 1995, osteopenia diagnosed in 2015 and fibromyalgia. The patient had been taking TDF as part of HIV therapy since 1995 and had been started on alendronate 15 months prior for osteopenia. X-ray of the hips revealed diffuse osteopenia with areas of possible stress reaction/stress fracture at the proximal third of the right femur and in the subtrochanteric left femur. Nuclear medicine bone scan showed faint foci of increased uptake in the lateral subtrochanteric regions of both proximal femora and midshaft of the right femur suggesting insufficiency fractures. The report noted that these areas were present and unchanged compared to a prior bone scan from 2011. In view of significant discomfort and risk of progression to full fracture, she underwent prophylactic intramedullary nailing of the right femur and was referred to endocrine for bone health evaluation. Endocrine evaluation showed vitamin D insufficiency and suboptimal calcium intake which were both corrected. A call to the pharmacy revealed that she had only filled 3 prescriptions of alendronate even though she was initially prescribed alendronate a year and a half prior to her presentation. Alendronate and TDF were discontinued and tenofovir alafenamide (TAF) was added to her HIV regimen due to its less deleterious effects on the skeletal system. Patient did not report significant improvement in her leg pain after the intramedullary nailing and decided to maintain a conservative approach without intervention for the left femur stress fractures. Conclusion: This is an unusual presentation of bilateral stress fractures of the femur likely related to long term TDF therapy in a relatively young postmenopausal female who had taken alendronate for less than a year of cumulative dose. This case highlights the need to use medications that have less harmful effects on the bone in adults with HIV who are also at an increased risk for low bone density and fractures. Presentation: Saturday, June 17, 2023

FRI673 REMS Technology For The Evaluation Of BMD In Hypothyroidism

Abstract Disclosure: F.A. Lombardi: None. P. Pisani*: None. A.P. Quezada*: None. M. Muratore*: None. M. Di Paola*: None. E. Casciaro*: None. F. Conversano*: None. S. Casciaro: None. *Equal contributors in alphabetical order Thyroid hormones play a crucial role in skeletal development, adult bone turnover, and bone maintenance, but they can also have detrimental effects on bone formation when there is thyroid dysfunction. Thyrotoxicosis (hyperthyroidism), including subclinical illness, can affect bone metabolism, resulting in decreased Bone Mineral Density (BMD) and increased fractures risk, and is a significant contributor to secondary osteoporosis, especially in postmenopausal women. On the other hand, little is known about the impact of overt or subclinical hypothyroidism on bone metabolism. However, hypothyroidism lessens bone turnover, reducing both osteoclastic bone resorption and osteoblastic activity. Such alterations in bone metabolism could potentially result in an increase in bone mineralization, although data on BMD and the risk of fracture in hypothyroidism are lacking and conflicting. Radiofrequency Echographic Multi-spectrometry (REMS) is a novel radiation-free technology for assessing BMD and fracture risk. In this context, the goal of this study was to assess BMD using REMS in patients with and without hypothyroidism, in order to look a possible correlation between the disease, BMD loss and fracture risk. Methods: A sample of 51 Caucasian females with hypothyroidism was compared to a reference group of healthy women who were matched for gender, ethnicity, age, and body mass index (BMI). Using REMS technology, the BMD was measurement at the lumbar spine in all participants at the study. To determine whether the two groups’ lumbar spine BMD value varied, a t-test was run. Results: Patients with and without hypothyroidism were matched in order to get similar anthropometric parameters. In patient with and without hypothyroidism mean ± standard deviation (SD) for age and BMI are (64.8 ± 10.2, 65.2 ± 9.8) and (25.1 ± 3.6, 25.0 ± 3.7), respectively. Patients with hypothyroidism were shown to have a significative lumbar spine BMD difference (0.801 g/cm2 ± 0.063 vs 0.831 g/cm2 ± 0.071, p=0.03) respect to the healthy subjects. Conclusions: Although just a small number of hypothyroidism patients are included in this investigation, without any indication of the drug therapy and its effect, it has been shown that, as compared to controls, women with hypothyroidism have a net decline in REMS-measured BMD values. Based on this thought, REMS technology, thanks to its precision and diagnostic accuracy, might improve the efficient care and follow-up programmes of these patients. Presentation: Friday, June 16, 2023

Topography and indexing of the nutrient foramina of the adult long bones

The primary source of blood supply for long bones comes from the nutrient arteries that pass through the nutrient foramen. Understanding the location and structure of these foramens is important in surgical procedures to ensure that the bone’s blood supply is not disrupted. To study the morphology and topography of nutrient foramina and to determine the foraminal index of the upper and lower limb long bones. The long bones of the upper and lower limbs including 47 humeri, 38 radii, 38 ulnae, 53 femurs, 57 tibiae, and 44 fibulae were studied. The number and position of primary nutrient foramina were noted and the foraminal index was calculated. Results showed that 82.9% of the humeri had a single nutrient foramen, and 17.0% had double foramina. For the radii, 97.4% had single nutrient foramen and 2.6% had double foramina. A single nutrient foramen was observed in all the ulnae. In the lower limb bone, the femurs had 41.5% of single nutrient foramen, 56.6% had double foramina and only 1.9% had triple nutrient foramina. The tibiae showed 93.0% of the single nutrient foramen and only 7.0% had double foramen. In fibulae, only one nutrient foramen was seen. The mean foraminal index was 57.26 for the humerus, 34.82 for the radius, and 39.32 for the ulna. Similarly, the mean foraminal index of the femur was 44.73, the tibia was 32.32 and the ulna was 46.18. Understanding the location of foramina is crucial inmaintaining bone vasculature during surgical procedures, which can improve the success rates of bone grafting, fracture fixation, and replacement surgeries involving the shoulder, elbow, and knee joints.

Different C2 screw placement techniques with mobilization of the vertebral artery in high-riding vertebral artery cases: Cadaver dissection.

In neurosurgery, posterior approaches intended at the craniovertebral junction are frequently used. The most popular procedures for treating upper cervical instability are C1 lateral mass, C2 pedicle, and C1-C2 transarticular screw stabilization. Due to their proximity to neural structures and the presence of the high-riding vertebral artery (VA), these techniques are complicated. The risk of VA damage can be decreased by mobilizing the VA. Using cadaveric specimens in this study was aimed to demonstrate C2 pedicle and C1-C2 transarticular screw placement with VA mobilization and a novel C2 inferior corpus screw placement technique. In this study, twelve adult cadaveric specimens and two adult dry cadaveric C2 bones were used with the permission and decision of the University Research Ethics Committee. Colored silicone was injected into the arteries and veins of these twelve cadaveric specimens. Then, muscle dissection was performed stepwise, and the C2 vertebrae of the cadavers were revealed with a surgical microscope. Each specimen and entire stages of the dissections were recorded photographically. After cadaver dissections, screw placement was performed with three different techniques. Finally, radiological imaging was done with fluoroscopy. After dissection, the lateral mass of the C2 vertebra was observed, and lateral to it, the transverse process and foramen were detected with the help of a hook. Next, the posterior wall of the VA groove was removed using a 1 mm thin plate Kerrison rongeur until the VA loop could partially be observed the VA. This enables us to find the top of the loop of the VA and mobilize it inferiorly using a dissector. Following this step, the C1-2 transarticular, C2 pedicle, and the novel C2 inferior corpus screw placement can be performed safely by directly visualizing the artery. Due to the nearby neurologic and vascular structures, placing the C2 pedicle and C1-2 transarticular screw is a challenging procedure, especially in high-riding VA cases. However, it is possible to place the C2 pedicle, C1-2 transarticular, and novel C2 inferior corpus screw after the mobilization of the VA. This study aimed to show all of them together on a cadaver for the first time, to understand the anatomy of the C2 vertebra, and to use screw placement techniques to minimize the risk of complications.

IĸB Protein BCL3 as a Controller of Osteogenesis and Bone Health.

IĸB protein B cell lymphoma 3-encoded protein (BCL3) is a regulator of the NF-κB family of transcription factors. NF-κB signaling fundamentally influences the fate of bone-forming osteoblasts and bone-resorbing osteoclasts, but the role of BCL3 in bone biology has not been investigated. The objective of this study was to evaluate BCL3 in skeletal development, maintenance, and osteoarthritic pathology. To assess the contribution of BCL3 to skeletal homeostasis, neonatal mice (n=6-14) lacking BCL3 (Bcl3-/- ) and wild-type (WT) controls were characterized for bone phenotype and density. To reveal the contribution to bone phenotype by the osteoblast compartment in Bcl3-/- mice, transcriptomic analysis of early osteogenic differentiation and cellular function (n=3-7) were assessed. Osteoclast differentiation and function in Bcl3-/- mice (n=3-5) was assessed. Adult 20-week Bcl3-/- and WT mice bone phenotype, strength, and turnover were assessed. A destabilization of the medial meniscus model of osteoarthritic osteophytogenesis was used to understand adult bone formation in Bcl3-/- mice (n=11-13). Evaluation of Bcl3-/- mice revealed congenitally increased bone density, long bone dwarfism, increased bone biomechanical strength, and altered bone turnover. Molecular and cellular characterization of mesenchymal precursors showed that Bcl3-/- cells displayed an accelerated osteogenic transcriptional profile that led to enhanced differentiation into osteoblasts with increased functional activity, which could be reversed with a mimetic peptide. In a model of osteoarthritis-induced osteophytogenesis, Bcl3-/- mice exhibited decreased pathological osteophyte formation (P < 0.05). Cumulatively, these findings demonstrate that BCL3 controls developmental mineralization to enable appropriate bone formation, whereas in a pathological setting, it contributes to skeletal pathology.