Endosteal Bone Surfaces Research Articles

Can cartilage loss be detected in knee osteoarthritis (OA) patients with 3–6 months' observation using advanced image analysis of 3 T MRI?

Prior investigations of magnetic resonance imaging (MRI) biomarkers of cartilage loss in knee osteoarthritis (OA) suggest that trials of interventions which affect this biomarker with adequate statistical power would require large clinical studies of 1-2 years duration. We hypothesized that smaller, shorter duration, "Proof of Concept" (PoC) studies might be achievable by: (1) selecting a population at high risk of rapid medial tibio-femoral (TF) progression, in conjunction with; (2) high-field MRI (3T), and; (3) using advanced image analysis. The primary outcome was the cartilage thickness in the central medial femur. Multi-centre, non-randomized, observational cohort study at four sites in the US. Eligible participants were females with knee pain, a body mass index (BMI)> or =25 kg/m(2), symptomatic radiographic evidence of medial TF OA, and varus mal-alignment. The 29 participants had a mean age of 62 years, mean BMI of 36 kg/m(2), with eight index knees graded as Kellgren-Lawrence (K&L)=2 and 21 as K&L=3. Eligible participants had four MRI scans of one knee: two MRIs (1 week apart) were acquired as a baseline with follow-up MRI at 3 and 6 months. A trained operator, blind to time-point but not subject, manually segmented the cartilage from the Dual Echo Steady State water excitation MR images. Anatomically corresponding regions of interest were identified on each image by using a three-dimensional statistical shape model of the endosteal bone surface, and the cartilage thickness (with areas denuded of cartilage included as having zero thickness - ThCtAB) within each region was calculated. The percentage change from baseline at 3 and 6 months was assessed using a log-scale analysis of variance (ANOVA) model including baseline as a covariate. The primary outcome was the change in cartilage thickness within the aspect of central medial femoral condyle exposed within the meniscal window (w) during articulation, neglecting cartilage edges [nuclear (n)] (nwcMF x ThCtAB), with changes in other regions considered as secondary endpoints. Anatomical mal-alignment ranged from -1.9 degrees to 6.3 degrees , with mean 0.9 degrees . With one exception, no changes in ThCtAB were detected at the 5% level for any of the regions of interest on the TF joint at 3 or 6 months of follow-up. The change in the primary variable (nwcMF x ThCtAB) from (mean) baseline at 3 months from the log-scale ANOVA model was -2.1% [95% confidence interval (CI) (-4.4%, +0.2%)]. The change over 6 months was 0.0% [95% CI (-2.7%, +2.8%)]. The 95% CI for the change from baseline did not include zero for the cartilage thickness within the meniscal window of the lateral tibia (wLT x ThCtAB) at 6 month follow-up (-1.5%, 95% CI [-2.9, -0.2]), but was not significant at the 5% level after correction for multiple comparisons. The small inconsistent compartment changes, and the relatively high variabilities in cartilage thickness changes seen over time in this study, provide no additional confidence for a 3- or 6-month PoC study using a patient population selected on the basis of risk for rapid progression with the MRI acquisition and analyses employed.

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation

AbstractAdequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-β and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Catabolic Properties of Microdissected Human Endosteal Bone Lining Cells

Bone lining cells cover > 80% of endosteal surfaces of human cancellous bone. Current research assigns to them a dual role: (1) as a biological membrane regulating exchange of substrates between the bone fluid compartment and the extracellular fluid of bone marrow and (2) as a signaling link between the osteocytic network as mechanical receptor and the osteoclastic cell pool for local induction of bone resorption. Furthermore, a catabolic role has been considered. We therefore examined the presence of matrix-metalloproteinases (MMPs) and their physiological tissue inhibitors (TIMPs) as putative proteolytic elements. Firstly, human cancellous bone from 60 patients was examined by immunofluorescence with antibodies against MMPs and TIMPs. Secondly, we applied laser-assisted microdissection (LMD) to isolate bone lining cells from frozen sections of human trabecular bone. mRNA analysis was performed using a single-cell PCR protocol. Three laser microdissection systems were tested: the new generation of Leica LMD and P.A.L.M. laser pressure catapulting (LPC) were compared to P.A.L.M. laser microdissection and micromanipulation (LMM). In a few pooled cell profiles, mRNA of MMP13, MMP14, TIMP1, and CBFA-1 was clearly detected. By immunofluorescence MMP13 and -14 as well as TIMP1 and -2 were strongly present in lining cells, while MMP2, TIMP3, and TIMP4 showed weak or negative signals. Although the functional impact of these enzymatic components remains open, there is additional evidence for a catabolic function of lining cells. The new diode-laser microdissection with LMD and LPC proved to be especially suitable to gain new insights into the properties of bone lining cells.

Solid Tumors Target the HSC Niche to Establish Metastatic Footholds in the Marrow

Metastasis is the most deadly aspect of many solid tissue cancers. The majority of metastases utilize pathways established to regulate hematopoietic stem cell (HSC) homing to gain access to the marrow. Whether solid tumors target the niche to establish footholds in the marrow is unknown. Using prostate caner (PCa) as a model, we determined if PCa and HSC compete for the HSC niche. To determine if disseminated PCa competes for the niche and prevents HSC engraftment, sham-operated or tumor-implanted NOD/SCID mice (Ly5.1) were transplanted with marrow cells from Ly5.2 donor animals. To preserve the niche, the transplanted animals received no preconditioning. At 16 weeks, greater HSC engraftment was observed in sham-operated group than in the tumor-bearing group as reflected by mature HSC progeny in the peripheral blood (0.7 ± 0.4% vs. 0.2 ± 0.2%, p = 0.022). Multiphoton microscopy revealed that PCa and SLAM receptor isolated HSCs co-localize at the endosteal bone surfaces in vivo and in vitro, further suggesting that PCa compete for the niche. Since osteoblasts (OBs) are vital components of the niche, it was determined if alterations in OB numbers also regulates metastasis. Here, vertebrae from control or conditional osteoblast knockout (Col2.3Δ-TK) mice were transplanted into SCID animals. Following treatment with ganciclovir which ablated the osteoblasts, metastasis to these tissues was evaluated. Fewer disseminated PCa were recovered from the Col2.3Δ-TK transplants than vehicle treated transplants (41.6 ± 23.7% vs. 100.0 ± 69.9%, p = 0.009). If metastases target the niche, then we should be able to mobilize metastases using HSC mobilizing regimes. Here, PCa tumors were established and removed. Later G-CSF was administered, and PCa numbers were determined in the blood. More PCa cells were seen in the blood after G-CSF treatments vs. the vehicle group (616.9 ± 463.0 cells vs. 143.7 ± 159.1 cells, p = 0.025). Part of the mechanism responsible for the mobilization of PCa was an increase in the production of MMP-2 and MMP-9 in the marrow. Finally, we evaluated if soluble factors produced by PCa alter HSCs activities. Animals were treated with either control or PCa conditioned medium for 3 days, and then HSC were harvested. Alterations of many mRNA transcripts involved in HSC self renewal were observed in animals exposed to the PCa conditioned medium. Our findings indicate that PCa and HSCs compete for the osteoblastic HSC niche, and that PCa utilize the same mechanisms as HSCs to gain access and egress from the niche. Taken together, these data provide the first evidence that the HSC niche serves as pre-metastatic niche for solid tumors and plays a central role in bone metastases.

Bone Marrow Endothelial Cell Support of Primitive Hematopoietic Cells

Hematopoietic stem cells (HSCs) in the bone marrow (BM) reside in specialized microenvironments known as the stem cell niche. Many reports have found the HSCs to be resident next to the endosteal surface of bone where cells of the osteoblastic lineage are a key component of the so-called endosteal niche. However, HSCs have also been found to reside adjacent to sinusoidal blood vessels. These observations have led to the proposal that HSCs in the adult BM may also reside in a vascular niche. However, the functional role of the vascular niche in hematopoiesis remains to be determined. We wished to evaluate the role that BM endothelial cells (BMECs) play in HSC physiology. To examine this we cultured BMEC-enriched cells in vitro, identified by expression of CD31, Tie-2, VE-cadherin and LDL uptake. We compared these cells to spleen derived ECs and BM stromal cells (BMSCs) in their ability to support primitive hematopoietic cells for extended periods in in vitro culture. We found that BMECs were superior in their ability to support the cobblestone area forming cell activity of VEGF-R1+ HSCs than spleen ECs or BMSCs. We also found that the number of cobblestone area cells was markedly reduced when VEGF-R1− HSCs were cultured on any of the supportive cell layers, however this may be due to an intrinsic difference between these cells as a much higher proportion of VEGF-R1+ HSCs were found to be in the G0 phase of the cell cycle than VEGF-R1− cells. To evaluate the supportive role of BMECs, spleen ECs or BMSCs on hematopoietic progenitor cells (HPC) we cultured purified primitive cells on these supportive layers and the total number of colony-forming unit-culture (CFU-C) cells were examined after 4-days or 7-days co-culture with the feeder cells. The results showed that BMECs or spleen ECs can promote the generation of CFU-C from VEGFR1+ HSCs or VEGFR1− HSCs, yet the tot al number of CFU-C produced from VEGFR1+ HSCs was greater than that from VEGFR1- HSCs. However, both of these cell types were able to support the generation of CFU-Cs to a greater degree than BMSCs. To examine the mechanism of enhanced support of VEGF-R1+ HSCs by the BMECs, we performed real-time PCR analysis for the expression of the VEGF-R1 ligands. Both BMECs and spleen ECs were found to express VEGF-A and –B to similar levels, however the expression of placental growth factor was higher in the BMECs. Whether the increased expression of this factor plays a functional role in the support of the HSCs in currently being evaluated. Our findings suggest that the ECs from BM or spleen can promote the proliferation of hematopoietic progenitor cells, while BMECs can maintain the long-term culture of VEGFR1+ HSCs in vitro. The functional relevance of this in vivo is currently being investigated.

Role of NF-κB in hematopoietic niche function of osteoblasts after radiation injury

Hematopoietic tissue is very sensitive to ionizing radiation (IR). In adult mammalian bone marrow, hematopoietic stem and progenitor cells (HSPC) reside next to the endosteal bone surface, which is lined primarily by osteoblastic cells. In the present study, we proposed to investigate the mechanisms by which osteoblasts in the hematopoietic niche regulate survival, proliferation, and differentiation of HSPC after radiation injury. Human primary CD34+ HSPC were cultured with human fetal osteoblast (hFOB) cell line cells or conditioned medium (CM) from hFOB cells with or without irradiation. Survival, apoptosis, and cell cycle were analyzed using clonogenic and flow cytometric assays. Cytokine and chemokine expression were measured by cytokine array and enzyme-linked immunosorbent assay. Their regulatory activities were assessed by quantitative real-time polymerase chain reaction, small interfering (si)RNA transfection, immunoblotting, and transbinding assays. Survival of gamma-irradiated CD34+ HSPC was significantly enhanced by coculture with hFOB cells or by CM from hFOB cells. There were six factors in hFOB cell lysates and five factors released into hFOB CM enhanced by IR. IR induced phosphorylation of p53, c-Jun, and p38 and downstream p21 expression, as well as cell cycle arrest and apoptosis in hFOB cells. However, IR also induced phosphorylation of nuclear factor (NF)-kappaBp65 (ser536) and NF-kappaB activation in hFOB cells. Inhibition of NF-kappaB expression with siRNA upregulated p21, inhibited release of cytokines and chemokines, and induced hFOB and CD34+ cell apoptosis. NF-kappaB is a radiation-induced prosurvival factor in human osteoblastic cells. NF-kappaB gene knockdown abrogated the hematopoietic niche function of hFOB cells in supporting survival of CD34+ cells after IR.

High-speed evaluation of track-structure Monte Carlo electron transport simulations

There are many instances where Monte Carlo simulation using the track-structure method for electron transport is necessary for the accurate analytical computation and estimation of dose and other tally data. Because of the large electron interaction cross-sections and highly anisotropic scattering behavior, the track-structure method requires an enormous amount of computation time. For microdosimetry, radiation biology and other applications involving small site and tally sizes, low electron energies or high-Z/low-Z material interfaces where the track-structure method is preferred, a computational device called a field-programmable gate array (FPGA) is capable of executing track-structure Monte Carlo electron-transport simulations as fast as or faster than a standard computer can complete an identical simulation using the condensed history (CH) technique. In this paper, data from FPGA-based track-structure electron-transport computations are presented for five test cases, from simple slab-style geometries to radiation biology applications involving electrons incident on endosteal bone surface cells. For the most complex test case presented, an FPGA is capable of evaluating track-structure electron-transport problems more than 500 times faster than a standard computer can perform the same track-structure simulation and with comparable accuracy.

Solitary central osteoma of the jaws: a diagnostic dilemma

Osteomas are relatively rare benign osteogenic neoplasms, characterized by the proliferation of compact and/or cancellous bone. Osteomas can develop as peripheral (periosteal) masses attached to the cortical plates or as central lesions arising from endosteal bone surfaces. Although multiple osteomas of the jaws are a hallmark of Gardner syndrome (familial adenomatous polyposis), nonsyndromic cases are typically solitary. A search of the literature identified 91 well documented cases of solitary osteoma of the jaws published since 1955: 93.4% were peripheral and only 6.6% central, a ratio of 14:1 in favor of peripheral osteomas. Solitary central osteomas of the jaws seem to be very rare, with only 6 such cases reported since 1955. Adding the 4 new cases described here brings the total to 10 central osteomas, 30% occurring in the maxilla and 70% in the mandible. Whereas peripheral osteomas are fairly easy to diagnose, central osteomas pose a more challenging diagnostic problem and need to be differentiated from other similar lesions of the jaws, such as central ossifying fibroma, condensing osteitis, idiopathic osteosclerosis, osteoblastoma, cementoblastoma, and complex odontoma. With such a low number of cases of central osteoma reported, the objectives of the present report are to describe 4 new cases, analyze the clinical and radiographic characteristics, and discuss the features which distinguish these lesions from other similar lesions of the jaws.

New experience regarding various forms of osteoclasis (author's transl)

The authors supplement their 1975 paper on the latest views on osteoclasis with further research on the subject. The complexity of the osteoclastic process is demonstrated, with the participation at times of the nuclei of various bone cells as well as cell organelles, an important role also being played by cell extinction. This complexity of bone resorption relates not only to the endosteal and perosteal bone surfaces, but also appears in osteoclastically active osteocytes. The giant multinuclear cell represents here an absorbent factor, surrounding incompletely demolished bone matrix and cell debris. Submicroscopic histochemical research completes the picture.

Proteasome Inhibitors Stimulate Both Bone Formation and Hair Growth by Similar Mechanisms

We propose that the remodeling process that occurs in localized areas on endosteal bone surfaces and in Haversian canals shares many features in common with the mammalian hair cycle. In both, there are phases of resorption or regeneration, a transition phase, and then a phase of growth, termed anagen in the hair follicle, and formation in the bone remodeling cycle. Furthermore, we suggest that these processes both use the same molecular mechanisms, and specifically the Hedgehog-BMP-Wnt signal transduction cascades. We have found that proteasome inhibitors, which enhance bone formation by effects on these cascades, also stimulate anagen induction and hair growth in the murine and human hair follicle, and propose they do so by effects on similar or identical molecular targets.

Assessing compliance, acceptance, and tolerability of teriparatide in patients with osteoporosis who fractured while on antiresorptive treatment or were intolerant to previous antiresorptive treatment: An 18-month, multicenter, open-label, prospective study

Background: Teriparatide (parathyroid hormone [1-34] [ribosomal DNA origin]) stimulates new bone formation on trabecular and cortical (periosteal and/or endosteal) bone surfaces by preferential stimulation of osteoblastic activity over osteoclastic activity. It has been found to significantly reduce vertebral fractures by 65%, and nonvertebral fragility fractures by 53% in treatment-naive postmenopausal women who have previously suffered a vertebral fracture. Objective: This study examined the compliance, acceptance, and adherance of SC teriparatide 20 pg QD. Methods: In this 18-month, multicenter, openlabel, prospective study, women with postmenopausal osteoporosis, and men >30 years of age with either idiopathic or hypogonadal osteoporosis (with low bone mass [T-score of -1 or worse] and ≥1 fragility fracture), who had experienced a treatment-related adverse event (AE) or an inadequate response while receiving antiresorptive treatment, and who were willing to receive open-label teriparatide for ≥18 months were eligible. Compliance was defined as self-reported use of ≥80% of study medication. Acceptance of the injection pen was determined by scores obtained from questionnaires and rating scales measuring patients' perception. Patients self-reported on injection discomfort, ease of use, and the overall injection administration. Acceptance was assessed at baseline, and 3, 6, and 18 months. AEs were recorded at each clinical visit from the patients' self-reports. At the 3-month visit, a serum calcium level was drawn ≥16 hours after the previous teriparatide dose. Results: In this study, 116 patients—102 women with postmenopausal osteoporosis and 14 men (12 with idiopathic osteoporosis and 2 with hypogonadal osteoporosis)—were assessed for inclusion in the study. The mean (SD) age was 68.8 (11.1) years (range, 40-89 years) and the mean (SD) weight was 60.5 (11.7) kg (range, 37-87 kg). Seventy-three percent of the patients in this study had baseline spinal T-scores ≤-2.5, and 72% had fractured during treatment with an osteoporosis medication. Reported compliance was 89% at 6 months and 82% at 18 months. At baseline, 42% of patients were concerned about injection discomfort, and 43% were somewhat concerned with daily injections, while 7% were quite concerned. At 6 months, most patients reported much less concern (49%) or no concern (42%). Patient perceptions associated with learning how to use the pen injection, attaching the needle, holding the pen, and injecting the dose, improved during the first 6 months of the study. The most commonly reported AEs were dizziness, 12 (10.3%); nausea, 12 (10.3%); back pain, 9 (7.8%); and muscle cramps, 9 (7.8%). No AEs were believed to be associated with the use of the pen injection or teriparatide. Five patients had mildly elevated serum calcium concentrations (maximum value 2.8 mmol/L) at 3 months. However, all were normal on repeat testing ~4 weeks thereafter. Conclusions: This study found that teriparatide pen injection was well accepted in these patients, and acceptance rates improved during the first 6 months of treatment and, thereafter, improved slightly for ~18 months. Reported compliance remained high throughout the study (82%-89%). Teriparatide pen injection was a viable treatment in these osteopenic or osteoporotic patients with fragility fractures.

Bone health status in relation to body adiposity in obese children and youth enrolled in a hospital-based weight management program

Understanding of the pathogenesis of bone fragility in men requires knowledge of its structural basis. There is no evidence that gender differences in fracture rates are explained by gender differences in bone mineral content (BMC) or areal bone mineral density (BMD). This is an untested assumption. The BMD measurement integrates the modeling and remodeling that occurs on the periosteal and endosteal surfaces of bone during growth and aging. The size, shape, and architecture of the bone so formed determine its breaking strength. None of these three-dimensional structural components is “seen” by the dual photons of the densitometer.Men and women attain a similar peak vertebral height during growth. Vertebral width is greater in men, conferring higher BMC and areal BMD, but trabecular number and thickness (trabecular volumetric BMD) is no greater in men than women. Blacks have shorter vertebra than whites, and vertebral width is similar. Trabecular thickness is greater in blacks than whites. Thus, at peak, gender differences in vertebral strength are likely to be size, not BMD, dependent. Racial differences in vertebral strength are likely to be BMD, not size, dependent.Greater periosteal expansion during growth in males than females, and blacks than whites, establishes the gender and racial differences in peak bone size. Men have wider long bones than women. Blacks have wider long bones than whites. The proximity of the endocortical surface to the periosteal surface determines peak cortical width, which is similar in men, women, blacks, and whites. It is the greater distance of the cortical mineral mass from the neutral axis of a long bone in males than in females, in blacks than in whites, and in men with, than men without, fractures, that partly accounts for the greater bone strength in the first mentioned in each group. Thus, at peak, racial and gender differences in long bone strength are likely to be size, not BMD dependent.Trabecular bone loss is similar in men and women. Loss of connectivity is greater in women. Endocortical resorption is greater in women than men, but men lose less cortical width because subperiosteal apposition during aging is greater in men than in women offsetting endocortical resorption. Men with spine fractures have smaller vertebrae because vertebral width is less. Men with hip fractures have smaller femoral neck width. In both types of fractures, there is less bone in the smaller bone-reduced volumetric BMD. The relative contributions of reduced accrual during growth, excessive bone loss during aging, or both to the deficit in volumetric BMD are undefined. No antifracture efficacy trials have been done in men.Reasonable approaches to treatment include the use of testosterone in hypogonadal men, and vitamin D if vitamin D deficiency is present. Calcium supplements may slow endocortical bone loss. Bisphosphonates may increase BMD.

449. Sca-1+ Cell-Based Gene Therapy with a Modified Fibroblast Growth Factor 2 (FGF2) Increased Endosteal/Trabecular Bone Formation in Mice

A major problem in bone-wasting diseases such as osteoporosis is the loss of endosteal bone that results in reduced bone strength and increased fracture risk. Recent advances in gene therapy raise the possibility that ex vivo gene therapy with a bone-forming gene may be used to enhance endosteal bone formation. However, a successful strategy requires that the transduced cells remain at the endosteal bone sites. Sca-1+ cells are a desirable cell vehicle because of their ability to home and engraft into the bone marrow cavity. This study assessed the feasibility of an ex vivo Sca-1+ cell-based systemic FGF2 gene therapy to promote endosteal bone formation. To maximize our chances of demonstrating a biological effect, the human FGF2 transgene was modified to increase protein secretion and stability by adding the BMP2/4 hybrid signal sequence and mutating two key cysteines in the FGF2 molecule. Retro-orbital injection of Sca-1+ cells transduced with the MLV-FGF2 vector into sub-lethally irradiated stem cell deficient (W41/W41) mice resulted in long-term engraftment, marked elevation in serum FGF2, and dose-related endosteal bone formation. Ten weeks post transplantation, serum FGF2 levels in mice transplanted with MLV-GFP-transduced Sca-1+ cells (controls) averaged 36|[plusmn]|6 pg/mL, whereas the serum FGF2 level in mice transplanted with FGF2-expressing cells was significantly increased but highly variable (2502|[plusmn]|2459 pg/mL, p 4,000 pg/mL), endosteal bone formation was so robust that the marrow space was completely filled with bony tissues. There was insufficient calcium available for mineralization of the newly formed bone, leading to hypocalcemia, secondary hyperparathyroidism, and osteomalacia. However, these undesirable side effects appeared to be dose-related. Thus, it may be possible to minimize adverse effects while maximizing efficacy by optimizing transgene expression. In conclusion, these data demonstrate the massive power of our approach to target and deliver a systemic gene therapy to the endosteal bone surface, and provide compelling test-of-principle evidence for the feasibility of a Sca-1+ cell-based ex vivo systemic FGF2 gene therapy to promote endosteal bone formation.

Pelvic Insufficiency Fracture Related to Autosomal Dominant Polycystic Kidney Disease

We report the case of a patient with autosomal dominant polycystic kidney disease (ADPKD) and an insufficiency-type fracture of the pelvis. A 60-year-old Japanese woman was admitted because of pain in the right ischium and pubis that began suddenly with no precipitating cause. Computed tomography showed the bony pelvis to be compressed by enlarged dependent kidneys and an enlarged liver. We relieved compression on the pelvic bones by means of transarterial embolization (TAE) to the kidneys and liver after initiation of hemodialysis therapy. The fracture healed gradually after TAE, and the patient could walk 4 months later. In an iliac bone specimen obtained before TAE, cancellous bone was intact, but periosteal and endosteal surfaces of cortical bone showed marked resorption and were irregular. Normally, many ligaments are connected tightly to the periosteal surface, supporting the cortical bone. However, because of extensive surface resorption associated with pressure from enlarged kidneys, connections between ligaments and the periosteal surface presumably became fragile, promoting an insufficiency fracture from unapparent external forces. Thus, ADPKD is a potential cause of insufficiency fracture owing to abnormalities of cortical bone.

Heterogeneity of engrafted bone-lining cells after systemic and local transplantation

AbstractThe outcome of various osteoprogenitor-cell transplantation protocols was assessed using Col1a1-GFP reporter transgenic mice. The model requires the recipient mice to undergo lethal total body irradiation (TBI) followed by rescue with whole bone marrow. When the mice are rescued with total bone marrow from a Col1a1-GFP transgenic mouse, green fluorescence protein (GFP)-positive donor cells can be observed on most endosteal and trabecular bone surfaces. Although the cells express an osteoblast-restricted GFP, they fail to progress to osteocytes, do not form a mineralized matrix, and do not generate bone nodules in vitro. However when calvarial progenitor cells derived from the same transgenic mice are injected into the bone marrow space, osteogenesis by the donor cells is observed. Using different GFP colors that distinguish the donor and recipient osteoblasts, commingling of the 2 cells types is observed along the mineralizing osteoblast surface as well as within the osteocyte population of the endosteal bone. Despite the ability of the injected progenitor cells to produce bone within the injected bone, they lack the ability to form mineralized bone nodules when explanted to primary osteoblast culture. These reagents and imaging protocols will be useful in evaluating other cells having a better progenitor potential than calvarial-derived stromal cells.

Gene-Expression Profiling of CD34+ Hematopoietic Cells Expanded in a Collagen I Matrix

CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.

Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells

AbstractAlthough recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized critical role for Opn in regulation of the physical location and proliferation of HSCs. Within marrow, Opn expression is restricted to the endosteal bone surface and contributes to HSC transmarrow migration toward the endosteal region, as demonstrated by the markedly aberrant distribution of HSCs in Opn–/– mice after transplantation. Primitive hematopoietic cells demonstrate specific adhesion to Opn in vitro via β1 integrin. Furthermore, exogenous Opn potently suppresses the proliferation of primitive HPCs in vitro, the physiologic relevance of which is demonstrated by the markedly enhanced cycling of HSC in Opn–/– mice. These data therefore provide strong evidence that Opn is an important component of the HSC niche which participates in HSC location and as a physiologic-negative regulator of HSC proliferation.

Deficiency of the G-protein α-Subunit Gsα in Osteoblasts Leads to Differential Effects on Trabecular and Cortical Bone

The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.

Beta-Arrestin2 regulates the differential response of cortical and trabecular bone to intermittent PTH in female mice.

Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female beta-arrestin2(-/-) mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in beta-arrestin2(-/-) and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces. The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity in these compartments may depend on some yet unidentified mechanism(s) of regulation. The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic molecule beta-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo. We used pDXA, muCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 mug/kg/day) in adult female mice null for beta-arrestin2 (beta-arr2(-/-)) and wildtype (WT) littermates (7-11/group). beta-arr2(-/-) mice had significantly lower total body BMD, trabecular bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased midfemoral medullary area. In beta-arr2(-/-) mice, PTH not only improved total body BMD, trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in beta-arr2(-/-) compared with WT. Osteocalcin levels were significantly lower in beta-arr2(-/-) mice, but increased dose-dependently with PTH in both beta-arr2(-/-) and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 mug/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in beta-arr2(-/-). In summary, beta-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.