Early B Cell Factor 1 (EBF1) Regulates Glomerular Development by Controlling Mesangial Maturation and Consequently COX-2 Expression.

BACKGROUND: We recently showed the transcription factor Early B cell factor 1 (EBF1) is essential for the last stages of metanephric development, and that mice globally deficient in EBF1 display impaired maturation of peripheral glomeruli. EBF1 is present within multiple glomerular cell types, including the glomerular mesangium and podocytes. METHODS: To identify which cell type is driving the glomerular developmental defects in the global EBF1 knockout mice, we deleted EBF1 from the mesangium/pericytes (Foxd1-cre) or podocytes (Podocin-cre) in mice. RESULTS: Deletion of EBF1 from Foxd1 lineage cells resulted in hypoplastic kidneys, poorly differentiated peripheral glomeruli, and decreased proximal tubular mass in the outer cortex. Renal insufficiency was apparent at P21 when proteinuria presents, fibrosis of both the glomeruli and interstitium rapidly progresses, microthrombi appear, and hematuria develops. Approximately half of the Foxd1+, Ebf1fl/fl mice die before they are 3 months old. Mice with podocyte-targeted deletion of EBF1 exhibited no developmental abnormalities. Mice with Ebf1 deficiency in Foxd1 lineage cells shared characteristics with Ptgs2/COX-2-insufficient models, and mechanistic investigation revealed impaired calcineurin/NFATc1 activation and decreased COX-2 expression. Deletion of COX-2 from the interstitial/mesangial lineage displayed a less severe phenotype than EBF1 deficiency in mice. Overexpressing COX-2 in the EBF1-deficient mice, however, partially restored glomerular development. CONCLUSIONS: The results suggest that EBF1 regulates metanephric development at the last stages of glomerular maturation through its actions in the stromal progenitor (Foxd1+) lineage where it mediates proper regulation of calcineurin/NFAT signaling and COX-2 expression.

Determination of mesenchymal stem cell fate by pigment epithelium-derived factor (PEDF) results in increased adiposity and reduced bone mineral content.

Pigment epithelium-derived factor (PEDF), the protein product of the SERPINF1 gene, has been linked to distinct diseases involving adipose or bone tissue, the metabolic syndrome, and osteogenesis imperfecta (OI) type VI. Since mesenchymal stem cell (MSC) differentiation into adipocytes vs. osteoblasts can be regulated by specific factors, PEDF-directed dependency of murine and human MSCs was assessed. PEDF inhibited adipogenesis and promoted osteoblast differentiation of murine MSCs, osteoblast precursors, and human MSCs. Blockade of adipogenesis by PEDF suppressed peroxisome proliferator-activated receptor-γ (PPARγ), adiponectin, and other adipocyte markers by nearly 90% compared with control-treated cells (P<0.001). Differentiation to osteoblasts by PEDF resulted in a common pathway that involved PPARγ suppression (P<0.01). Canonical Wnt-ß-catenin signaling results in a MSC differentiation pattern analogous to that seen with PEDF. Thus, adding PEDF enhanced Wnt-ß-catenin signal transduction in human MSCs, demonstrating a novel Wnt agonist function. In PEDF knockout (KO) mice, total body adiposity was increased by >50% compared with controls, illustrating its systemic role as a negative regulator of adipogenesis. Bones from KO mice demonstrated a reduction in mineral content recapitulating the OI type VI phenotype. These results demonstrate that the human diseases associated with PEDF reflect its ability to modulate MSC differentiation.

Selective deletion of the membrane-bound colony stimulating factor 1 isoform leads to high bone mass but does not protect against estrogen-deficiency bone loss.

To better define the biologic function of membrane-bound CSF1 (mCSF1) in vivo, we have generated mCSF1 knockout (k/o) mice. Spinal bone density (BMD) was 15.9% higher in k/o mice compared to wild-type (wt) controls (P < 0.01) and total BMD was increased by 6.8% (P < 0.05). A higher mean femur BMD was also observed but did not reach statistical significance (6.9% P = NS). The osteoclastogenic potential of bone marrow isolated from mCSF1 k/o mice was reduced compared to wt marrow. There were no defects in osteoblast number or function suggesting that the basis for the high bone mass phenotype was reduced resorption. In addition to a skeletal phenotype, k/o mice had significantly elevated serum triglyceride levels (123 ± 7 vs. 88 ± 3.2 mg/dl; k/o vs. wt, P < 0.001), while serum cholesterol levels were similar (122 ± 6 vs. 116 ± 6 mg/dl; k/o vs. wt, P = NS). One month after surgery, 5-month-old k/o and wt female mice experienced the same degree of bone loss following ovariectomy (OVX). OVX induced a significant fourfold increase in the expression of the soluble CSF1 isoform (sCSF1) in the bones of wt mice while expression of mCSF1 was unchanged. These findings indicate that mCSF1 is essential for normal bone remodeling since, in its absence, BMD is increased. Membrane-bound CSF1 does not appear to be required for estrogen-deficiency bone loss while in contrast; our data suggest that sCSF1 could play a key role in this pathologic process. The reasons why mCSF1 k/o mice have hypertriglyceridemia are currently under study.

Osteoclasts lacking Rac2 have defective chemotaxis and resorptive activity.

The role of the small Rho GTPase Rac2 in mature osteoclasts has not been extensively studied. Rac2(-/-) mice are of normal size and have normal tooth eruption. However, femoral cortical thickness was significantly greater in Rac2(-/-) compared to wild-type mice, while percent cortical porosity was lower. As assessed by histomorphometry, trabecular bone mass was significantly higher in male Rac2(-/-) than wild-type animals, although trabecular bone mass was similar when data from male and female animals were combined. There were no significant differences in the number of osteoblasts per bone surface; however, the number of osteoclasts per total bone area tended to be higher in Rac2(-/-) mice and was significantly higher in male Rac2(-/-) mice. In the aggregate, these data suggested a defect in osteoclast function and, consistent with that, rates of bone resorption were significantly reduced in Rac2(-/-) osteoclasts. In addition, Rac2(-/-) osteoclasts had a significantly delayed spreading response to treatment with CSF1 for 15 min. Phalloidin staining showed areas of abnormal actin accumulation and impaired actin ring formation in Rac2(-/-) osteoclasts. Finally, Rac2(-/-) osteoclasts showed a marked defect in chemotaxis toward a point source of CSF1, with a dramatic reduction in migratory rate. Together, these findings indicate an important role for Rac2 in mature osteoclasts.

Targeted overexpression of Dkk1 in osteoblasts reduces bone mass but does not impair the anabolic response to intermittent PTH treatment in mice.

Parathyroid hormone (PTH) is a potent anabolic agent, but the cellular mechanisms by which it increases bone mass are not fully understood. Dickkopf 1 (Dkk1) is an endogenous inhibitor of Wnt signaling and suppresses bone formation in vivo. We sought to determine if Dkk1 and anabolic PTH treatment interact in regulating bone mass. PTH treatment of primary murine osteoblasts for 24 h reduced Dkk1 expression by 90% as quantified by real-time PCR, whereas PTH treatment in vivo reduced Dkk1 expression by 30% when given as a single daily subcutaneous dose. To directly determine whether Dkk1 modulates the anabolic response of PTH in vivo, we engineered transgenic (TG) mice expressing murine Dkk1 under the control of the 2.3-kb rat collagen alpha-1 promoter. TG mice had significantly reduced bone mass, which was accompanied by reduced histomorphometric parameters of bone formation (reduced OV/TV, ObS/OS, and NOb/TAR). Treatment of TG mice and wild-type (WT) littermates with 95 ng/g body weight of human (1-34) PTH daily for 34 days resulted in comparable increases in bone mass at all skeletal sites. Histomorphometric analyses indicated that PTH treatment increased the numbers of both osteoblasts and osteoclasts in WT mice but only increased the numbers of osteoblasts in TG mice. We conclude that overexpression of Dkk1 does not attenuate the anabolic response to PTH in vivo.

The PHEX transgene corrects mineralization defects in 9-month-old hypophosphatemic mice.

Hypophosphatemia is an X-linked dominant disorder resulting from a mutation in the PHEX gene. While osteoblast-specific expression of the PHEX transgene has been reported to decrease the phosphate wasting associated with the disease in male hypophosphatemic (HYP) mice, there are reports that the mineralization defect is only partially corrected in young animals. To test the hypothesis that osteoblast-specific expression of the PHEX gene for a longer time would correct the mineralization defect, this study examined the bones of 9-month-old male and female HYP mice and their wild-type controls with or without expression of the transgene under a collagen type I promoter. Serum phosphate levels, alkaline phosphatase activity, and FGF23 levels were also measured. Mineral analyses based on wide-angle X-ray diffraction, Fourier transform-infrared (FT-IR) spectroscopy, and FT-IR imaging confirmed the decreased mineral content and increased mineral crystal size in male HYP humerii compared to wild-type males and females with or without the transgene and in female HYP mice with or without the transgene. There was a significant increase in mineral content and a decrease in crystallinity in the HYP males' bones with the transgene, compared to those without. Of interest, expression of the transgene in wild-type animals significantly increased the mineral content in both males and females without having a detectable effect on crystallinity or carbonate content. In contrast to the bones, based on micro-computed tomography and FT-IR imaging, at 9 months there were no significant differences between the HYP and the WT teeth, precluding analysis of the effect of the transgene.

The Effects of Fixation and Dehydration on the Histological Quality of Undecalcified Murine Bone Specimens Embedded in Methylmethacrylate.

Histological evaluation is a complex, multistep process culminating in tissue staining. All of the steps leading up to the staining affect the final quality, but too often the effects of these preparations are not given enough consideration. Fixatives in particular usually are chosen not for efficacy but for convenience and availability. This study attempts to create guidelines for selecting fixatives for bone tissue histological evaluation. We compared two of the most widely used fixatives, ethanol and formalin, in their use on mouse tibias embedded in methylmethacrylate and subsequently stained with toluidine blue, safranin O, or Von Kossa. Our results show that ethanol fixation (70%) and subsequent processing in methylmethacrylate gives better staining results for bone cell related elements than fixing in 10% neutral buffered formalin with the same processing and embedding techniques. Further we demonstrated than an additional acetone dehydration and clearing step allowed for even better visualization in bone specimens fixed with 70% ethanol. However, the additional acetone step did not enhance visualization in bone specimens fixed with 10% neutral buffered formalin. Finally, marrow elements were more easily visualized when fixed with formalin as opposed to ethanol.

The anabolic response to parathyroid hormone is augmented in Rac2 knockout mice.

PTH is the only currently available anabolic therapy for osteoporosis. In clinical practice, the skeletal response to PTH varies and because therapy is limited to 2 yr, approaches to maximize the therapeutic response are desirable. Rac2 is a small GTPase that is expressed only in hematopoietic tissue. Rac2(-/-) mice have a slight increase in bone mass and osteoclasts isolated from these animals have reduced basal resorptive activity and reduced chemotaxis. To evaluate the anabolic response to PTH in Rac2(-/-) mice, we treated 18 Rac2(-/-) and 17 control, age-matched wild-type animals once daily for 28 d with 80 ng/g body weight of h(1-34)PTH. Treatment resulted in significantly greater increments in spinal, femur, and total bone density in the Rac2(-/-) as compared with wild-type animals. Microcomputed tomography analysis demonstrated greater increases in trabecular thickness and cortical thickness in the knockout mice. Interestingly, histomorphometric analysis showed an equivalent increase in osteoblast and osteoclast number in response to PTH treatment in both groups of animals. However, as judged by changes in serum markers, the resorptive response to PTH was impaired. Thus, telopeptide of type 1 collagen was 15.9+/-6.9 ng/ml after PTH treatment in the knockout animals and 26.8+/-11.1 ng/ml in the PTH-treated wild-type group. In contrast, serum aminoterminal propeptide of type 1 collagen and osteocalcin were equivalent in both groups. We conclude that, in the genetic absence of Rac2, the anabolic response to PTH is increased. This appears to be due to attenuated resorptive activity of osteoclasts.

The Effects of Storage Conditions on the Preservation of Enzymatic Activity in Bone.

Alkaline phosphatase and acid phosphatase are two major enzymatic measures of osteoblastic and osteoclastic activity, respectively. As a result, the preservation of the enzymes in bone specimens to near in vivo accuracy is essential. Despite standardization of the staining process, several factors related to the storage of blocks and slides before sectioning and staining impact the level of enzymes detected in the tissue. Block condition (intact, faced, or unstained) as well as environment (temperature and length of time in storage) affect alkaline phosphatase preservation while the acid phosphatase enzyme remains unaffected. We conclude that to optimally preserve alkaline phosphatase enzyme, methacrylate-embedded undecalcified murine bones should be stored as intact blocks. After sectioning, the faced blocks should be stored at 4°C for optimal enzyme staining of future sections. Furthermore, it is best to stain sections immediately after sectioning.

Glucose-dependent insulinotropic peptide-overexpressing transgenic mice have increased bone mass.

Glucose-dependent insulinotropic peptide (GIP) is an intestinally secreted hormone the release of which is stimulated by nutrient ingestion. We previously reported that GIP receptors are present in osteoblastic cells and that GIP increases collagen type I synthesis and alkaline phosphatase activity in isolated osteoblasts. We have also shown that osteoclasts express GIP receptors and that GIP inhibits osteoclastic activity and differentiation. In addition, using GIP receptor knockout mice we demonstrated that absence of GIP receptor signaling resulted in a low bone mass phenotype. To further define GIP's role as an anabolic hormone in vivo, we utilized a genetically altered mouse model, a transgenic mouse overexpressing GIP under the control of the metallothionein promoter (Tg+). Tg+ mice had significantly higher mean GIP levels even in the absence of added dietary zinc. Tg+ animals also had a significant increase in markers of bone formation and a decrease in markers of bone resorption. Consistent with these biochemical data, GIP transgenic mice had a significant increase in bone mass as measured by densitometry and histomorphometry. These data support the conclusion that GIP inhibits bone resorption and stimulates bone formation and that excess signaling through the GIP receptor results in gain of bone mass. In view of GIP's role in nutrient absorption, our data suggest that this hormone may serve an important role in linking nutrient ingestion to bone formation.

Selective deletion of the soluble Colony-Stimulating Factor 1 isoform in vivo prevents estrogen-deficiency bone loss in mice.

Neutralizing CSF1 in vivo completely prevents ovariectomy (OVX)-induced bone loss in mice. There are two isoforms of CSF1, soluble (sCSF1), and membrane-bound (mCSF1), but their individual biological functions are unclear. It had been previously reported that mCSF1 knockout (K/O) and wild type (Wt) female mice experience the same degree of bone loss following OVX. In Wt mice the expression of sCSF1 was elevated fourfold in skeletal tissue following OVX while expression of mCSF1 was unchanged. To examine the role of sCSF1 in OVX-induced bone loss, mice were engineered in which sCSF1 was not expressed but expression of mCSF1 was unaffected (sCSF1 K/O). Isoform-specific reverse transcription PCR confirmed the absence of transcripts for sCSF1 in bone tissue isolated from these animals and no circulating CSF1 was detected by ELISA. Surprisingly, there were no significant differences in bone mineral density (BMD) between sCSF1 K/O mice and Wt controls as assessed by dual-energy X-ray absorptiometry and micro-CT. However, one month after OVX, femoral, spinal and total BMD had declined by 11.2%, 8.9%, and 8.7% respectively in OVX-Wt animals as compared to Sham-OVX. In contrast OVX sCSF1 K/O mice showed changes of +0.1%, -2.4%, and +2.3% at the same 3 sites compared to Sham-OVX sCSF1 K/O mice. These data indicate important non-redundant functions for the two isoforms of CSF1 and suggest that sCSF1, but not mCSF1, plays a key role in estrogen-deficiency bone loss.

Effect of daily parathyroid hormone (1-34) on lumbar fusion in a rat model.

BACKGROUND CONTEXT: Despite numerous studies evaluating the anabolic effects of intermittent administration of parathyroid hormone (PTH) on bone, there are no published studies examining its effect on spinal fusion outcomes. PURPOSE: To determine the effect of daily injection of human recombinant PTH(1-34) on posterolateral lumbar fusions in a rat model. STUDY DESIGN: Prospective, case-controlled, preclinical animal study. OUTCOME MEASURES: Manual palpation and serum osteocalcin. METHODS: Single-level, intertransverse process spinal fusions were performed with iliac crest autograft in 56 Sprague-Dawley rats. Animals received daily injections of placebo or PTH(1-34). At 6 weeks, fusion masses were assessed by manual palpation. Serum osteocalcin levels were assessed in a subset of the animals. RESULTS: Manual palpation revealed the control group to have a fusion rate of 37% (10/27) and the PTH(1-34)-treated group to have a fusion rate of 52% (15/29). Mean serum osteocalcin levels were 59.8 and 88.6 ng/L for the control and PTH(1-34) groups, respectively. CONCLUSIONS: There was a trend towards greater fusion rate in the PTH(1-34) group as compared with the placebo group. Further, PTH(1-34) administration was associated with a significant increase in osteocalcin levels. Certainly, further investigations are warranted, as an injectable agent capable of increasing fusion rates would be of great clinical value.

Deletion of Rac in Mature Osteoclasts Causes Osteopetrosis, an Age-Dependent Change in Osteoclast Number, and a Reduced Number of Osteoblasts In Vivo.

Rac1 and Rac2 are thought to have important roles in osteoclasts. Therefore, mice with deletion of both Rac1 and Rac2 in mature osteoclasts (DKO) were generated by crossing Rac1(flox/flox) mice with mice expressing Cre in the cathepsin K locus and then mating these animals with Rac2(-/-) mice. DKO mice had markedly impaired tooth eruption. Bone mineral density (BMD) was increased 21% to 33% in 4- to 6-week-old DKO mice at all sites when measured by dual-energy X-ray absorptiometry (DXA) and serum cross-linked C-telopeptide (CTx) was reduced by 52%. The amount of metaphyseal trabecular bone was markedly increased in DKO mice, but the cortices were very thin. Spinal trabecular bone mass was increased. Histomorphometry revealed significant reductions in both osteoclast and osteoblast number and function in 4- to 6-week-old DKO animals. In 14- to 16-week-old animals, osteoclast number was increased, although bone density was further increased. DKO osteoclasts had severely impaired actin ring formation, an impaired ability to generate acid, and reduced resorptive activity in vitro. In addition, their life span ex vivo was reduced. DKO osteoblasts expressed normal differentiation markers except for the expression of osterix, which was reduced. The DKO osteoblasts mineralized normally in vitro, indicating that the in vivo defect in osteoblast function was not cell autonomous. Confocal imaging demonstrated focal disruption of the osteocytic dendritic network in DKO cortical bone. Despite these changes, DKO animals had a normal response to treatment with once-daily parathyroid hormone (PTH). We conclude that Rac1 and Rac2 have critical roles in skeletal metabolism.

The effect of aging on the skeletal response to intermittent treatment with parathyroid hormone.

Little is known about the modifying effects of age on the skeletal response to intermittent treatment with PTH. We therefore compared the response of 63 aged (18 month old) and 61 young-adult (3 month old) C57BL/6 mice to 4 wk of daily sc injections of either vehicle or h(1-34)PTH at a dose of 95 ng/g body weight. The increase in total body bone mineral density (BMD), compared with vehicle-treated animals, was similar in aged and young-adult mice (+5.6 vs. +6.3%). Aged animals demonstrated a greater increase in spinal BMD than their younger counterparts (+12.0 vs. +5.1%, P = 0.01; absolute increment: 57 x 10(-4) vs. 28 x 10(-4) g/cm(2)). Microcomputed tomography analyses in a subset of the vertebrae showed a trend toward higher L5 trabecular bone volume fraction in the PTH-treated aged animals (+40.2 vs. +19.6%). Vertebral histomorphometry demonstrated a greater PTH-induced increase in osteoblast number in aged vs. young-adult animals (694 vs. 396 cells/mm(2)). In contrast, in the femur the PTH-induced increase in BMD tended to be greater in the young-adult than the aged animals, although this did not reach statistical significance (8.1 vs. 4.2%). The numbers of osteoblast progenitors and mineralizing colonies in cultured marrow were unaffected by PTH treatment in either group. We conclude that aging differentially impacts the regional skeletal response to PTH such that the increase in BMD in the spine is augmented, whereas that in the femur is unaffected. Effects on osteoblast progenitor recruitment do not seem to be the basis for these changes.

Megakaryocyte-osteoblast interaction revealed in mice deficient in transcription factors GATA-1 and NF-E2.

UNLABELLED: Mice deficient in GATA-1 or NF-E2 have a 200-300% increase in bone volume and formation parameters. Osteoblasts and osteoclasts generated in vitro from mutant and control animals were similar in number and function. Osteoblast proliferation increased up to 6-fold when cultured with megakaryocytes. A megakaryocyte-osteoblast interaction plays a role in the increased bone formation in these mice. INTRODUCTION: GATA-1 and NF-E2 are transcription factors required for the differentiation of megakaryocytes. Mice deficient in these factors have phenotypes characterized by markedly increased numbers of immature megakaryocytes, a concomitant drastic reduction of platelets, and a striking increased bone mass. The similar bone phenotype in both animal models led us to explore the interaction between osteoblasts and megakaryocytes. MATERIALS AND METHODS: Histomorphometry, microCT, and serum and urine biochemistries were used to assess the bone phenotype in these mice. Wildtype and mutant osteoblasts were examined for differences in proliferation, alkaline phosphatase activity, and osteocalcin secretion. In vitro osteoclast numbers and resorption were measured. Because mutant osteoblasts and osteoclasts were similar to control cells, and because of the similar bone phenotype, we explored the interaction between cells of the osteoblast lineage and megakaryocytes. RESULTS: A marked 2- to 3-fold increase in trabecular bone volume and bone formation indices were observed in these mice. A 20- to 150-fold increase in trabecular bone volume was measured for the entire femoral medullary canal. The increased bone mass phenotype in these animals was not caused by osteoclast defects, because osteoclast number and function were not compromised in vitro or in vivo. In contrast, in vivo osteoblast number and bone formation parameters were significantly elevated. When wildtype or mutant osteoblasts were cultured with megakaryocytes from GATA-1- or NF-E2-deficient mice, osteoblast proliferation increased over 3- to 6-fold by a mechanism that required cell-to-cell contact. CONCLUSIONS: These observations show an interaction between megakaryocytes and osteoblasts, which results in osteoblast proliferation and increased bone mass, and may represent heretofore unrecognized anabolic pathways in bone.

The cell surface form of colony-stimulating factor-1 is biologically active in bone in vivo.

The specific biological function of the cell surface or membrane-bound isoform of colony-stimulating factor-1 (mCSF-1) is not well understood. To help define the role of this isoform in bone, we developed a transgenic mouse in which targeted expression of human mCSF-1 in osteoblasts was achieved under the control of the 2.4-kb rat collagen type I alpha promoter. Bone density, determined by peripheral quantitative computed tomography, was reduced 7% in mCSF-1 transgenic compared with that in wild-type mice. Histomorphometric analyses indicated that the number of osteoclasts in bone (NOc/BPm, NOc/TAR, OcS/BS) was significantly increased in transgenic mice (1.7- to 1.8-fold; P < 0.05 to P < 0.01) compared with that in wild-type animals. Interestingly, the osteoblast-restricted isoform transgene corrected the osteopetrosis seen in CSF-1-deficient op/op mice. Skeletal growth and bone density in op/op mice expressing mCSF-1 in osteoblasts were similar to those in wild-type mice and were dramatically different from those in the unmanipulated op/op animals. The op/op mice expressing mCSF-1 in bone had normal incisor and molar tooth eruption, whereas the op/op mice evidenced the expected failure of tooth eruption. These findings directly support the conclusion that mCSF-1 is functionally active in bone in vivo and is probably an important local source of CSF-1.

Posterolateral lumbar fusions in athymic rats: characterization of a model.

BACKGROUND CONTEXT: The athymic rat has been used to study the role of osteoinductive products in spinal fusions. This small animal model has been advocated to minimize potential inflammatory responses to allogeneic or xenogenic proteins. Despite past experience, this model has not yet been well characterized. PURPOSE: To further define and validate a posterolateral lumbar fusion model in the athymic rat. STUDY DESIGN/SETTING: Comparison of fusions after animal survival surgery. PATIENT SAMPLE: Forty athymic and 20 normothymic rats. OUTCOME MEASURES: Manual palpation, radiography and histology at 3 and 6 weeks. METHODS: Single-level intertransverse fusions were performed at the L4-L5 level of 40 athymic rats. Twenty rats were implanted with autograft (athymic/autograft), and 20 had no graft placed (athymic/no graft). An additional 20 autograft fusions were performed on normothymic rats (normothymic/autograft). Half were sacrificed at 3 weeks; half were sacrificed at 6 weeks. RESULTS: At 3 weeks, 0% of the athymic/no graft rats fused, 20% of the athymic/autograft rats fused and 20% of the normothymic/autograft rats fused by manual palpation. At 6 weeks, 0% of the athymic/no graft rats fused, 30% of the athymic/autograft rats fused and 40% of the normothymic/autograft rats fused by manual palpation. Radiographs were of limited utility in determining fusion, and histology results were roughly concordant with those of manual palpation. CONCLUSIONS: This work further characterizes the athymic rat posterolateral lumbar fusion model. The absence of a thymus does not appear to affect autograft fusion rates, and no spontaneous fusions were seen when no graft was placed.

Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.

Adipocytes reside in discrete, well-defined depots throughout the body. In addition to mature adipocytes, white adipose tissue depots are composed of many cell types, including macrophages, endothelial cells, fibroblasts, and stromal cells, which together are referred to as the stromal vascular fraction (SVF). The SVF also contains adipocyte progenitors that give rise to mature adipocytes in those depots. Marrow adipose tissue (MAT) or marrow fat has long been known to be present in bone marrow (BM) but its origin, development, and function remain largely unknown. Clinically, increased MAT is associated with age, metabolic diseases, drug treatment, and marrow recovery in children receiving radiation and chemotherapy. In contrast to the other depots, MAT is unevenly distributed in the BM of long bones. Conventional quantitation relies on sectioning of the bone to overcome issues with distribution but is time-consuming, resource intensive, inconsistent between laboratories and may be unreliable as it may miss changes in MAT volume. Thus, the inability to quantitate MAT in a rapid, systematic, and reproducible manner has hampered a full understanding of its development and function. In this chapter, we describe a new technique that couples histochemical staining of lipid using osmium tetroxide with microcomputerized tomography to visualize and quantitate MAT within the medullary canal in three dimensions. Imaging of osmium staining provides a high-resolution map of existing and developing MAT in the BM. Because this method is simple, reproducible, and quantitative, we expect it will become a useful tool for the precise characterization of MAT.

LIM kinase 1 deficient mice have reduced bone mass.

The cytoskeleton determines cell shape and is involved in cell motility. It also plays a role in differentiation and in modulating specialized cellular functions. LIM kinase 1 (LIMK1) participates in cytoskeletal remodeling by phosphorylating and inactivating the actin-severing protein, cofilin. Severing F-actin to release G-actin monomers is required for actin cytoskeletal remodeling. Although less well established, LIMK1 may also influence the cell cycle and modulate metalloproteinase activity. Since the role of LIMK1 in bone cell biology has not been reported, the skeletal phenotype of LIMK1(-/-) mice was examined. LIMK1(-/-) mice had significantly reduced trabecular bone mass when analyzed by microCT (p<0.01). Histomorphometric analyses demonstrated a 31% reduction in the number of osteoblasts (p=0.0003) and a 23% reduction in osteoid surface (p=0.0005). The number of osteoclasts was no different in control and knock out animals. Consistent with the in vivo findings in osteoblasts, the number of osteoblast colony forming units in LIMK1(-/-) bone marrow was reduced by nearly 50%. Further, osteoblasts isolated from LIMK1(-/-) mice showed significantly reduced rates of mineralization in vitro. Osteoclasts from LIMK1(-/-) mice evidenced more rapid cytoskeletal remodeling in response to treatment with CSF1. In keeping with this latter finding, basal levels of phospho-cofilin were reduced in LIMK1(-/-) osteoclasts. LIMK1(-/-) osteoclasts also resorbed dentine slices to a greater extent in vitro and were more active in a pit assay. These data support the hypothesis that LIMK1 is required for normal osteoblast differentiation. In addition, its absence leads to increased cytoskeletal remodeling and bone resorption in osteoclasts.

Increased bone volume and correction of HYP mouse hypophosphatemia in the Klotho/HYP mouse.

Inactivating mutations of PHEX cause X-linked hypophosphatemia and result in increased circulating fibroblast growth factor 23 (FGF23). FGF23 action is dependent upon Klotho, which converts FGF receptor 1 into an FGF23-specific receptor. Disruption of Klotho results in a complex bone phenotype and hyperphosphatemia, the converse phenotype of X-linked hypophosphatemia. We examined effects of disrupting both Klotho and PHEX by creating a double-knockout (Klotho/HYP) mouse. The combined disruption corrected the hypophosphatemia in HYP mice, indicating that Klotho is epistatic to PHEX. FGF23 levels remained elevated in all groups except wild-type, indicating that Klotho is necessary for FGF23-dependent phosphaturic activity. 1,25-Dihydroxyvitamin D levels, reduced in HYP mice, were comparably elevated in Klotho and Klotho/HYP mice, demonstrating that Klotho is necessary for FGF23's effect on vitamin D metabolism. Serum PTH levels were reduced in both Klotho and Klotho/HYP mice. Moreover, the Klotho null phenotype persisted in Klotho/HYP, maintaining the runty phenotype and decreased life span of Klotho null mice. Notably, microcomputed tomography analysis demonstrated greater trabecular bone volume fraction in Klotho/HYP mice than that in all other groups (Klotho/HYP, 56.2 +/- 6.3%; Klotho, 32.5 +/- 10.3%; HYP, 8.6 +/- 7.7%; and wild type, 21.4 +/- 3.4%; P < 0.004). Histomorphometric analysis confirmed the markedly increased trabecular bone density in Klotho/HYP mice and the well-established increase in osteoid volume in HYP mice. These observations suggest that with addition of Klotho loss of function, the overabundant osteoid typically produced in HYP mice (but fails to mineralize) is produced and mineralized in the double knockout, resulting in markedly enhanced trabecular bone density.