Bone Lining Cells Could Be Sources of Bone Marrow Adipocytes

BackgroundRecently, lineage-tracing studies demonstrated that parathyroid hormone and anti-sclerostin antibody (Scl-Ab) can convert bone lining cells (BLCs) into active osteoblasts. However, BLCs might also be differentiated into other lineages. Here we investigated whether BLCs could differentiate into bone marrow adipocytes (BMAds) and whether Scl-Ab could suppress this process.MethodsDmp1-CreERt2:mTmG mice were injected with 0.5 mg of 4-hydroxytamoxifen once weekly from postnatal week 4 to week 8. The mice were treated with either vehicle or rosiglitazone for 8 weeks (weeks 12–20). Moreover, they were administered either vehicle or Scl-Ab (50 mg/kg) twice weekly for 4 weeks (weeks 16–20, N = 4–6/group). We chased the GFP+ cells from the endosteal surface to the bone marrow (BM) of the femur. Using immunohistochemical staining, the numbers of perilipin+ or GFP+/perilipin double+ cells in the BM were quantified. In addition, serum N-terminal propeptide of type I procollagen (P1NP) levels were measured at each time point, and bone mass was analyzed at 20 weeks using micro-computed tomography.ResultsScl-Ab administration significantly reversed the decreases in bone parameters induced by rosiglitazone. Plump GFP+ cells, presumably active osteoblasts, and extremely flat GFP+ cells, presumably BLCs, were present on the endosteal surface of the femur at 8 and 12 weeks, respectively, in line with prior findings. When we chased the GFP+ cells, rosiglitazone significantly increased the number of GFP/perilipin double+ BMAds compared to the effects of the vehicle (P < 0.001), and overlapping Scl-Ab administration decreased the number of GFP/perilipin double + BMAd compared to rosiglitazone alone (P < 0.001). In addition, we found that osteoblast lineage cells such as BLCs might express PPARγ on immunohistochemical staining. When rosiglitazone was administered to Rip-Cre:mTmG mice, GFP+ cells were not present on the endosteal surface or in the BM of the femur; however, they were present in the pancreas.ConclusionBLCs could be sources of BMAds, and rosiglitazone could stimulate the differentiation of osteoblast lineage cells into BMAds. Suppression of the differentiation of osteoblast lineage cells into BMAds might contribute to anabolic effects resulting from the pharmacologic inhibition of sclerostin.

Finite Element Analysis of Transhumeral and Transtibial Percutaneous Osseointegrated Endoprosthesis Implantation

Cadaveric mechanical testing of a percutaneous osseointegration docking system (PODS) for osseointegration (OI) prosthetic limb attachment revealed that translation of the exact system from the humerus to the tibia may not be suitable. The PODS, designed specifically for the humerus achieved 1.4–4.8 times greater mechanical stability in the humerus than in the tibia despite morphology that indicated translational feasibility. To better understand this discrepancy, finite element analyses (FEAs) modeled the implantation of the PODS into the bones. Models from cadaveric humeri (n = 3) and tibia (n = 3) were constructed from CT scans, and virtual implantation preparation of an array of endoprosthesis sizes that made contact with the endosteal surface but did not penetrate the outer cortex was performed. Final impaction of the endoprosthesis was simulated using a displacement ramp function to press the endoprosthesis model into the bone. Impaction force and maximum first principal (circumferential) stress were recorded to estimate stability and assess fracture risk of the system. We hypothesized that the humerus and tibia would have different optimal PODS sizing criteria that maximized impaction force and minimized first principal stress. The optimal sizing for the humerus corresponded to implantation instructions, whereas for the tibia optimal sizing was three times larger than the guidelines indicated. This FEA examination of impaction force and stress distribution lead us to believe that the same endoprosthesis strategy for the humerus is not suitable for the tibia because of thin medial and lateral cortices that compromise implantation.

Effects of strong bite force on the facial vertical dimension of pembarong performers

Background: A pembarong performer is a reog dancer who bites on a piece of wood inserted into his/her mouth in order to support a 60 kg Barongan or Dadak Merak mask. The teeth supporting this large and heavy mask are directly affected, as the strong bite force exerted during a dance could affect their vertical and sagital facial dimensions. Purpose: This study aimed to examine the influence of the bite force of pembarong performers due to their vertical and sagital facial dimensions. Methods: The study reported here involved fifteen pembarong performers and thirteen individuals with normal occlusion (with specific criteria). The bite force of these subjects was measured with a dental prescale sensor during its centric occlusion. A cephalometric variation measurement was subsequently performed on all subjects with its effects on their vertical and sagital facial dimensions being measured. Results: The bite force value of the pembarong performers was 394.3816 ± 7.68787 Newtons, while the normal occlusion was 371.7784 ± 4.77791 Newtons. There was no correlation between the bite force and the facial sagital dimension of these subjects. However, a significant correlation did exist between bite force and lower facial height/total facial height (LFH/TFH) ratio (p = 0.013). Conversely, no significant correlation between bite force and posterior facial height/total facial height (PFH/TFH) ratio (p = 0.785) was detected. There was an inverse correlation between bite force and LFH/TFH ratio (r = -.464). Conclusion: Bite force is directly related to the decrease in LFH/TFH ratio. Occlusal pressure exerted by the posterior teeth on the alveolar bone may increase bone density at the endosteal surface of cortical bone.

Alternative Methods of Activation of the Stem Cell Niche Reveal a Spatial Localization of Primitive Cells in the Bone Marrow

Abstract 2396 Osteoblasts are key constituents of the murine hematopoietic stem cell (HSC) endosteal niche, evidenced by the fact that increasing their activity leads to an increase in the number of HSCs. However, recent studies have also suggested a role of the bone resorbing osteoclast in the HSC niche. In these studies we wished to examine whether activation of the osteoblast or the osteoclast had differing effects on the primitive hematopoietic cell populations. To achieve this we pharmacologically activated osteoclasts and osteoblasts using receptor activator of nuclear factor-κB ligand (RANK-L) and parathyroid hormone (PTH), respectively. Our in vitro results demonstrated that both PTH and RANK-L treatment of bone marrow stromal cell populations activated osteoblasts and osteoclasts. Yet, RANK-L treatment, which is able to expand the osteoclastic population to a much higher degree, demonstrated enhanced support of primitive hematopoietic cells in cobblestone area forming cell assays. We next examined the effects of in vivo treatment with RANK-L and PTH by performing histological analyses to study the effect of these treatments on bone composition, and examining the effects on different HSC sub-populations. These data suggested that the effects of our treatment regimens were different according to the region of the bone, with RANK-L treatment having no effect or leading to a reduction in trabecular bone in the metaphysis, but an increase in bone remodeling activity at the endosteal surface of the cortical bone in the diaphysis, while PTH treatment increases bone formation and remodeling activity both in the diaphysis and the trabecular bone of the metaphysis. Correlating with this was our observations that LT-HSC frequency was increased following RANK-L and PTH treatment, yet an increased ST-HSC frequency was only observed following PTH treatment. Direct examination of HSCs in specific regions of the bone indicated that RANK-L treatment preferentially increased the LT-HSCs in the diaphysis region of the bone marrow, while PTH treatment specifically increased the ST-HSC population in the metaphysis. These data not only reveal a supportive role for osteoclasts in the HSC niche suggesting that osteoclast interaction with osteoblasts is an essential component for maintaining HSC population in the bone marrow niche, they also suggest a possible structural organization for localization of LT-HSC vs. ST-HSCs in the long bone with LT-HSCs being more localized in the endosteal surface of the diaphysis area of the long bones as oppose to the ST-HSCs having a preference for the metaphysis of these bones. Disclosures: No relevant conflicts of interest to declare.

Microenvironmental Changes In An In Vivo Model of Myeloid Leukemia Negatively Regulate Osteoblastic Cells.

Abstract 1219 Patients with myelogenous leukemias can present with symptoms of bone pain and pathologic fractures, however little is known about the interactions between malignant cells and the bone marrow microenvironment. Additionally leukemia is known to severely interfere with normal hematopoiesis. To further characterize interactions between leukemic cells and the microenvironment, we used a model of blast crisis CML (bcCML) in which immature murine hematopoietic cells are engineered to express the BCR/ABL and Nup98/HoxA9 translocation products. Injection of these cells into naïve mice results in rapid accumulation of leukemic cells in the bone marrow (Dash et al. PNAS, 2002). We investigated the effect of leukemia on the bone marrow microenvironment by first performing immunohistochemical analyses. Leukemic cells were observed to preferentially localize in close contact with bony trabeculae. In addition, leukemic mice also exhibited a dramatic loss of trabecular bone as measured by micro-CT scanning (22.8 ± 1.5% vs 13.6 ± 1.5%, BV/TV n=5 in each group p=0.0048), prompting us to examine bone resorption and the abundance of osteoclasts. Histologic sections from leukemic mice showed an increase in mature osteoclasts (TRAP+, multinucleated cells) at the endosteal surface of the long bones (51 ± 4 OC/section vs 64 ± 3 OC/section p=0.0229). Additionally leukemic mice had a 50% increase in serum C-telopeptide (CTX), a well-established marker of global bone resorption (15.5 ± 0.3 ng/ml vs 21.8 ± 1.0 ng/ml, p=0.0003). Therefore, the presence of leukemic cells appears to strongly stimulate osteoclastogenesis and bone resorption. In addition to increased osteoclasts, a rapid and severe reduction in bone formation was identified in leukemic mice by decreased serum osteocalcin, a well-established marker of bone formation (70.8 ± 6.9 ng/ml vs 39.9 ± 3.2 ng/ml, p=0.0036). To determine effects of leukemia on bone-forming cells, we analyzed osteoblastic cells from the long bones of leukemic animals. Marrow was flushed from the bone and minced bone fragments were digested in collagenase. Cells isolated in this fashion have strong osteoblastic activity and can support hematopoietic stem cells (HSCs) (Chitteti et al, Blood 2010). Leukemic cells were present in the isolated fraction confirming that leukemic cells were closely associated with the bone and not entirely removed when the marrow was flushed. Total cells isolated by collagenase digestion from the long bones of leukemic mice were cultured with leukemic cells and evaluated for osteoblastic colony-forming ability. These cultures demonstrated a reduced ability to form osteoblastic colonies compared to controls (26 ± 2 colonies vs 13 ± 2 colonies p=0.0014). Freshly isolated cells were CD45 depleted to remove leukemic cells and again evaluated for colony-forming ability. CD45 negative cells from leukemic mice also demonstrated reduced ability to form mineralizing osteoblastic colonies in vitro compared to controls when an identical number of cells were cultured (10 ± 2 colonies vs 0 colonies) suggesting that their previous exposure to leukemic cells in vivo was sufficient to decrease their osteoblastic activity. Further in vivo osteoblastic evaluation in leukemic mice showed reduced immunohistochemical staining for the osteoblastic marker osteopontin at the endosteal surface, supporting a leukemic-induced reduction of the mature osteoblastic population. These data demonstrate a severe reduction in both number and function of osteoblastic cells in a leukemic environment. Together these data show severe effects of leukemia on both osteoblastic and osteoclastic cells, which could contribute to the bone-specific problems associated with leukemic disease. Moreover, we propose that osteoblastic defects observed in this model may contribute to the leukemia-induced inhibitory effects on normal hematopoiesis. Studies are ongoing to assess the microenvironmental support for normal hematopoiesis in the leukemic setting and to identify the leukemic signals that modify the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Novel Role for Host-Derived Megakaryocytes In Facilitating Stem Cell Engraftment through Enhancement of Osteoblastic Niche Restoration Following Radioablation

Abstract 558 The osteoblastic niche is a critical site of engraftment following stem cell transplantation. We have previously demonstrated that the osteoblastic niche expands within the first 48 hours following marrow radioablation. This expansion is accompanied by relocalization of megakaryocytes from their homeostatic location within the central marrow space to the endosteal surface at sites of osteoblast expansion, suggesting that these relocalized megakaryocytes may play a key role in reconstitution of the niche. To determine whether megakaryocytes contribute to the radioablation-induced osteoblast expansion and consequently assist in facilitating engraftment, we have examined osteoblast expansion and stem cell engraftment in wildtype (WT) mice, thrombopoietin receptor (c-Mpl) deficient mice (mpl−/−) that have less than 20% of normal megakaryocyte numbers, and in mice treated with an anti-CD41 blocking antibody (MWREG30). BrdU incorporation and TUNEL assays demonstrated that megakaryocyte relocalization following radioablation occurs through active migration of viable megakaryocytes. mpl−/− mice or anti-CD41 treated WT mice developed less than 20% of the megakaryocyte endosteal migration seen in untreated WT mice 48 hours after radioablation (*P<0.001), and anti-CD41 treatment of irradiated mpl−/− mice reduced endosteal megakaryocytes to baseline pre-radiation numbers. Moreover, mpl−/− mice developed less than 50% of the osteoblast expansion seen in WT mice following irradiation; thus, abrogating megakaryocyte activity blocks critical signals required for osteoblast expansion. Bone marrow expression of IGF-1, a recognized osteoblast growth factor, increased 5-fold within 48 hours after radiation relative to pre-radiation levels in WT mice, but this IGF-1 spike is completely blocked by c-Mpl deficiency, suggesting that megakaryocytes may induce osteoblast expansion through a pathway in which c-Mpl signaling leads to IGF-1 expression. To test the functional significance of radiation-induced megakaryocyte migration and osteoblast expansion, we transplanted lethally irradiated WT or mpl−/− mice with or without anti-CD41 treatment using bone marrow from GFP-transgenic mice. Initial bone marrow engraftment of WT GFP+ donor cells within 24h of transplantation was significantly reduced (*P<0.01 for all groups) in anti-CD41 treated WT (51% of engraftment seen in untreated wildtype recipients), untreated mpl−/− (45%), and anti-CD41 treated mpl−/− (35%) recipients. Expansion of engrafted WT GFP+ donor cells at 3, 5 and 7 days post-transplant was also significantly reduced in untreated mpl−/− recipients (36%, 53%, 63% of untreated WT recipients at 3, 5, and 7 days, respectively, *P<0.05) and anti-CD41 treated mpl−/− recipients (25%, 33%, and 30% at 3, 5, and 7 days, respectively, *P<0.05), with prominent deficits specifically in the reconstitution of the B lymphocyte lineage. Bone marrow cellularity remained significantly reduced in anti-CD41 treated WT and untreated or anti-CD41 treated mpl−/− recipients by 35–45% relative to untreated WT recipients at least out to 3 weeks post-transplant (*P<0.01). Using competitive repopulation secondary transplantation assays performed with marrow harvested from primary recipients at 24h after primary transplantation, we showed that progenitor and short term hematopoietic stem cell (HSC) engraftment was significantly decreased in mpl−/− versus WT primary recipients (*P<0.05). Secondary transplant assays performed with marrow harvested from primary recipients 3 weeks after initial transplantation demonstrated that engraftment and expansion of long term-HSC (*P <0.05) and B cell reconstitution (*P<0.005) are significantly impaired in anti-CD41 treated mpl−/− versus untreated WT recipients. Taken together, our findings demonstrate that host megakaryocytes migrate to the endosteal surface following marrow radioablation where, through the enhancement of osteoblast niche expansion and potential osteoblast-independent effects, they play a pivotal role in facilitating efficient HSC engraftment following transplantation. Further understanding of these stem cell niche restoration pathways may reveal novel therapeutic targets to improve engraftment efficiency in the clinical setting. Disclosures: No relevant conflicts of interest to declare.

IGF1-Mediated Osteoblastic Niche Expansion After Marrow Ablation Enhances Long-Term Hematopoietic Stem Cell Engraftment and Hematopoietic Reconstitution After Bone Marrow Transplantation

Abstract 557 The capacity for hematopoietic stem cells (HSCs) to engraft in bone marrow (BM) and reconstitute long-term multilineage hematopoiesis after transplantation depends on their interaction with a specific microenvironment, the stem cell niche. We previously demonstrated a profound reorganization of the marrow endosteal microenvironment after lethal total body irradiation (TBI) of mice that leads to a rapid restoration of the osteoblastic HSC niche, followed by a transient, reversible expansion of this niche. Here, we show that the niche restoration and expansion is driven by a small number of highly proliferative osteoprogenitors via an IGF1 dependent mechanism, that directly facilities long-term hematopoietic stem cell homing and engraftment. At homeostasis, BM osteoblasts (OB) are organized as a single layer of flattened cells at the endosteal surface. Within 3 h of TBI administered to C57BL6 mice, few OB were identified at the endosteal surface. However, by 6 h post-TBI, a new single layer of OB was evident, followed by a marked increase in OB proliferation resulting in multiple layers of cells at the endosteum. The OB proliferation showed half maximal expansion at 24 h post-TBI and maximal expansion at 48 h, at which time the expanded OB constituted significantly more cell tiers than in unirradiated controls (p<0.005). BrdU incorporation/Ki67 expression dual marker studies, conducted over 48 h post-TBI, revealed clusters of highly proliferative cells that expand to repopulate the endosteum and to drive the osteoblastic niche expansion, suggesting a pool of radioresistant osteoprogenitors as the source of the niche restoration and expansion. Real-time qPCR of BM cells harvested after TBI showed an increased gene expression of Sdf1, Pdgfβ, TGFβ, Fgf2, and notably, Igf1, that peaks at 48 h. Using an ELISA, we, similarly, found an increase in protein expression, relative to unirradiated controls, that also peaks at 48 h (SDF1, 2.3-fold increase, p<0.005; PDGFβ, 6.1-fold, p<0.005; TGFβ, 4.4-fold, p<0.005; FGF2, 3.2-fold, p=0.05; IGF1, 5.3-fold, p<0.005). We propose that this extensive niche remodeling, closely correlated with increased expression of highly relevant cytokines, serves to optimize the osteoblastic niche for homing and engraftment of HSCs. To test the hypothesis, we used competitive repopulation secondary transplantation assays to assess the capacity of the osteoblastic niche to foster HSC engraftment. Primary recipients were transplanted with GFP-transgenic BM (3×106 cells/mouse) at 0.5 h, 24 h, or 48 h (maximal niche expansion) after TBI. At 24 h after transplantation, BM cells (representing 12.5% of the total marrow population) harvested from the primary recipients were transplanted into lethally irradiated secondary recipients with 2×105 competitor (unlabeled) BM cells. Secondary recipients, receiving marrow from primary recipients transplanted at 48 h post TBI, showed greater engraftment at 3 (p<0.05), 6 (p<0.001), and 18 (p<0.05) weeks after BMT compared with the 0.5 h and 24 h groups, indicating that 48 h of niche expansion facilitates homing/early engraftment of short- and long-term hematopoietic repopulating cells. To further define the importance of niche expansion and to determine the contribution of IGF1 signaling, we performed a competitive repopulation secondary transplantation assay with and without picropodophyllin (PPP), a specific IGF1 receptor tyrosine kinase inhibitor. Treated mice were either sacrificed for evaluation of niche expansion or transplanted with GFP-transgenic BM cells at 48 h post TBI. The short half-life of PPP (3 h) indicates that the drug will be eliminated prior to transplantation, 36 h after the last PPP dose. Histological analysis of irradiated, PPP-treated mice revealed that the osteoblastic niche was restored but did not expand to multiple layers at 48 h. Secondary recipients, receiving marrow from PPP-treated primary recipients, showed no evidence of short-term or long-term hematopoiesis in contrast to untreated controls (p<0.05). These data indicate that IGF1 mediated effects on the BM stem cell niche are essential for niche expansion and for engraftment of transplanted HSCs. Collectively, our data indicate that IGF1 mediated osteoblastic niche expansion is a pivotal component of HSC homing and engraftment. Targeting niche expansion may enhance HSC engraftment after clinical BMT, especially if HSC dose is limiting. Disclosures: No relevant conflicts of interest to declare.

Mechanism of Calcium Sensing Receptor Mediated Hematopoietic Stem Cell Lodgment In the Adult Bone Marrow Niche

Abstract 399 The ability of hematopoietic stem cells (HSCs) to maintain an undifferentiated state and undergo self-renewal is partly regulated by external signals originating from the stem cell niche. One receptor expressed on HSCs that is known to be involved in HSC niche biology is the calcium-sensing receptor (CaR). Our previous study using HSCs obtained from the fetal liver of mice deficient in CaR has shown the crucial role of CaR in HSC lodgment and engraftment in the bone marrow (BM), where CaR-/- HSCs lose their ability to lodge in the endosteal surface of the bone, leading to defective engraftment. To further investigate the mechanism of CaR-mediated lodgment of HSCs, we used a pharmacological approach to activate the receptor and assess the in vitro and in vivo effects. Cinacalcet treatment, which acts as a positive allosteric modulator of CaR to increase the sensitivity of the receptor to activation by extracellular Ca2+, leads to a 3-fold increase in primitive hematopoietic cell activity in vitro as assessed by the cobblestone forming cell assay. The increase in activity in vitro does not appear to be an effect of alterations in cell proliferation, cell survival or the expression of cell adhesion molecules such as VLA-4 or L-selectin. Rather, with CaR stimulation, long-term HSCs have an increased adhesion capability to collagen I, a major ECM molecule present predominantly in the BM endosteal region. We also observed that activation of the CaR following Cinacalcet treatment significantly enhances HSC homing to the BM, lodgment at the endosteal surface and in vivo engraftment capabilities as assessed by a competitive repopulation assay. This enhancement of in vivo activity correlates with increased CXCR4 signaling and migration towards an SDF-1alpha stimulus. Signaling through this receptor is known to be important in cell migration, proliferation, survival, and retention of HSCs in the BM following transplantation. Analysis of the CaR on human cells has demonstrated that there is a distinct population of CaR+ cells present in the CD34+ cell population. The frequency of this population varies between approximately 2% on mobilized peripheral blood CD34+ cells and 6% on BM CD34+ cells. Further analysis is being performed to define the identity of this subpopulation of CaR+CD34+ cells. These mechanisms by which the CaR dictates preferential localization of HSCs in the BM endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate. These studies also have implications in the area of clinical stem cell transplantation, where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. Disclosures: No relevant conflicts of interest to declare.