Adult Rat Bone Research Articles

Hepatocyte Growth Factor Induces Differentiation of Adult Rat Bone Marrow Cells into a Hepatocyte Lineage in Vitro

Bone marrow (BM) cells originally include alpha-fetoprotein (AFP)- and c-Met [a receptor for hepatocyte growth factor (HGF)]-expressing cells. In vitro treatment of BM cells with HGF induced albumin-expressing hepatocyte-like cells. Furthermore, those hepatocyte-like cells expressed cytokeratins 8 and 18, which are typically expressed in normal adult hepatocytes. These findings demonstrate that BM cells include AFP-expressing hepatic progenitor cells that can be differentiated into hepatocytes by HGF in culture, indicating that such cultures are useful resources for cell transplantation therapy for liver diseases.

Adult rat and human bone marrow stromal cells differentiate into neurons

Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple treatment protocol induced the stromal cells to exhibit a neuronal phenotype, expressing neuron-specific enolase, NeuN, neurofilament-M, and tau. With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF-M. The refractile cell bodies extended long processes terminating in typical growth cones and filopodia. The differentiating cells expressed nestin, characteristic of neuronal precursor stem cells, at 5 hr, but the trait was undetectable at 6 days. In contrast, expression of trkA, the nerve growth factor receptor, persisted from 5 hr through 6 days. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Human marrow stromal cells subjected to this protocol also differentiated into neurons. Consequently, adult marrow stromal cells can be induced to overcome their mesenchymal commitment and may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurologic diseases. J. Neurosci. Res. 61:364–370, 2000. © 2000 Wiley-Liss, Inc.

Skeletal changes in rats bearing mammosomatotrophic pituitary tumors: a model of acromegaly with gonadal dysfunction

Growth hormone (GH) exerts potent effects on bone metabolism, resulting in an increased bone formation in animals and humans. Acromegaly has been associated with increased bone turnover, whereas the net effect of the increased bone metabolism has been obscured because patients with acromegaly are often associated with hypogonadism. We investigated changes in cortical and cancellous bone in adult rats implanted mammosomatotrophic pituitary tumor cells (GH 3) as a model of acromegaly with gonadal dysfunction. Acromegaly model rats were prepared by implanting GH 3 cells into female Wistar-Furth rats at 17 weeks of age. At 28 weeks of age, GH 3-bearing rats (GH rats) showed very high serum GH levels and a moderate increase in serum prolactin levels, resulting in low circulating estradiol levels. The GH rats showed significant increases in body weight and in length and volume of both the femur and vertebral body. Bone mineral content values of either the midfemur or the whole lumbar body were significantly greater in the GH rats compared with littermate controls, while the areal bone mineral density values of the respective bones were not different between the two groups. The parameters of mechanical strength of the femur were significantly larger in the GH rats than in controls, whereas those of the lumbar vertebral body cylinder specimen were not different between the two groups. Respective normalized mechanical parameters of the femur and the vertebral body were the same in the GH rats as in controls. In the midfemur, the GH rats showed a significant increase in the total cross-sectional area without influencing the bone marrow area, resulting in an increase in the cortical bone area and the moment of inertia compared with controls. The indices of periosteal bone formation in the midfemur were greater in the GH rats compared with controls, but the endocortical bone formation and resorption were not different between the two groups. In the vertebral body cancellous bone, the GH rats had an increase in bone turnover rate, whereas the structural parameters were not different between the two groups. These results from GH 3-bearing rats demonstrate that an excess of GH increases cortical bone mass in rats accompanied with estrogen deficiency, while no large effect on vertebral body cancellous bone mass is seen.

Titanium containing amorphous hydrogenated carbon films (a-C : H/Ti): surface analysis and evaluation of cellular reactions using bone marrow cell cultures in vitro

Amorphous hydrogenated carbon (a-C : H) coatings, also called diamond-like carbon (DLC), have many properties required for a protective coating material in biomedical applications. The purpose of this study is to evaluate a new surface coating for bone-related implants by combining the hardness and inertness of a-C : H films with the biological acceptance of titanium. For this purpose, different amounts of titanium were incorporated into a-C : H films by a combined radio frequency (rf) and magnetron sputtering set-up. The X-ray photoelectron spectroscopy (XPS) of air-exposed a-C : H/titanium (a-C : H/Ti) films revealed that the films were composed of TiO 2 and TiC embedded in and connected to an a-C : H matrix. Cell culture tests using primary adult rat bone marrow cell cultures (BMC) were performed to determine effects on cell number and on osteoblast and osteoclast differentiation. By adding titanium to the carbon matrix, cellular reactions such as increased proliferation and reduced osteoclast-like cell activity could be obtained, while these reactions were not seen on pure a-C : H films and on glass control samples. In summary, a-C : H/Ti could be a valuable coating for bone implants, by supporting bone cell proliferation while reducing osteoclast-like cell activation.

Adult rat and human bone marrow stromal cells differentiate into neurons.

Bone marrow stromal cells exhibit multiple traits of a stem cell population. They can be greatly expanded in vitro and induced to differentiate into multiple mesenchymal cell types. However, differentiation to non-mesenchymal fates has not been demonstrated. Here, adult rat stromal cells were expanded as undifferentiated cells in culture for more than 20 passages, indicating their proliferative capacity. A simple treatment protocol induced the stromal cells to exhibit a neuronal phenotype, expressing neuron-specific enolase, NeuN, neurofilament-M, and tau. With an optimal differentiation protocol, almost 80% of the cells expressed NSE and NF-M. The refractile cell bodies extended long processes terminating in typical growth cones and filopodia. The differentiating cells expressed nestin, characteristic of neuronal precursor stem cells, at 5 hr, but the trait was undetectable at 6 days. In contrast, expression of trkA, the nerve growth factor receptor, persisted from 5 hr through 6 days. Clonal cell lines, established from single cells, proliferated, yielding both undifferentiated and neuronal cells. Human marrow stromal cells subjected to this protocol also differentiated into neurons. Consequently, adult marrow stromal cells can be induced to overcome their mesenchymal commitment and may constitute an abundant and accessible cellular reservoir for the treatment of a variety of neurologic diseases.

Long‐Term Effects of Nicotine on Bone and Calciotropic Hormones in Adult Female Rats

This study determined the effects of nicotine on serum concentrations of several calciotropic hormones, and bone formation and resorption end-points in 7 month old, adult female rats. Animals were administered either saline (n= 9/group), low dose nicotine at 3.0 mg/kg/day (n=10/group) or high dose nicotine at 4.5 mg/kg/day (n=11/group) by subcutaneous osmotic minipumps. At the end of a three months treatment period, serum concentrations of calcium, phosphorus, parathyroid hormone, calcitonin, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were determined. Femora, tibiae, and lumbar vertebrae (3-5) were collected and bone parameters evaluated included mineral density and content (femora and vertebrae), strength (femora and vertebrae) and histomorphometry (tibiae). Animals given nicotine had significantly lower levels of 25-hydroxyvitamin D than controls [20.8+/-1.4 ng/ml for the low dose group and 20.7+/-1.0 ng/ ml for the high dose group versus 27.6+/-1.3 ng/ml for the control group (mean+/-S.E.M.), P<0.01]. The high dose nicotine group had smaller vertebral areas (5.4+/-0.2 mm2 versus 6.2+/-0.2 mm2, P<0.05) and a lower bone mineral content than the controls (0.024+/-0.001 g versus 0.030+/-0.001 g, P<0.05). Tibial endocortical mineral apposition rate was also significantly lower in the high dose nicotine group than in the control group (1.06+/-0.13 microm/day versus 1.42+/-0.08 microm/day. P<0.05). No significant treatment differences were detected in bone density, cancellous bone histomorphometry, or bone strength. Results from the present study suggest that nicotine administration may adversely affect bone formation and decrease body storage of vitamin D.

Progesterone- and dexamethasone-dependent osteoprogenitors in bone cell populations derived from rat vertebrae are different and distinct.

Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require dexamethasone (Dex) or progesterone (Prog) to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We now show that the Prog-dependent population cannot be detected in male rats after sexual maturation, but is present in prepubertal rats of both sexes and can be induced in adult male-derived populations by culturing the explants in medium containing 17beta-estradiol (10(-9)-10(-8) M). This suggested that the Prog- and Dex-dependent osteoprogenitors in adult female-derived populations were probably distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate are dependent on the presence of estrogen. We then proceeded to prove this by using replica plating. When one of the paired colonies duplicated was cultured in medium containing Dex (10(-8) M) and the other in medium containing Prog (10(-5) M), 5.0% of duplicates formed bone in Prog only, 11.1% formed bone in Dex only, and 3.4% formed bone in both Prog and Dex. In all cases the size of the bone-forming colonies in Dex-treated cultures was larger than that in Prog-treated cultures, indicating that the effects of Dex on osteoprogenitor proliferation are greater than those of Prog. The results demonstrate the existence of three classes ofosteoprogenitors in adult female rat-derived bone cell populations: a class responding to Dex only, a class responding to Prog only, and a class responding to both Dex and Prog. The results also indicate that the effects of Prog are not mediated by Prog binding to the glucocorticoid receptor and imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog.

Retinoic acid effects on an SV‐40 large T antigen immortalized adult rat bone cell line

Retinoic acid effects on an SV‐40 large T antigen immortalized adult rat bone cell line

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line.

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.

Osteoprogenitor cell frequency in rat bone marrow stromal populations: Role for heterotypic cell-cell interactions in osteoblast differentiation

Glucocorticoids, notably dexamethasone (Dex), have been reported to be a requirement for osteoprogenitor cell differentiation in young adult rat bone marrow stromal cell populations. We have reinvestigated the requirement for Dex and analyzed the frequency of osteoprogenitor cells present. Stromal cells were grown as primary or first subcultures in the presence or absence of Dex and their expression of osteogenic markers (alkaline phosphatase activity, hormone responsiveness, and matrix molecules, including type I collagen, osteopontin, bone sialoprotein, and osteocalcin), as well as their functional capacity to differentiate to form a mineralized bone nodule, were assessed. Dex increased, but was not an absolute requirement for, the expression of osteogenic markers. Bone nodule formation was plating cell density dependent and occurred under all combinations of treatment with or without Dex but was maximal when Dex was present in both the primary and secondary cultures. Dex increased CFU-F by approximately 2-fold, but increased CFU-O (osteoprogenitor cells; bone nodule forming cells) by 5- to 50-fold depending on the cell density and duration of treatment. Neither CFU-F nor CFU-O expression followed a linear relationship in limiting dilution analysis until very high cell densities were reached, suggesting cooperativity of cell types within the population and a multitarget phenomenon leading to osteoprogenitor differentiation. When a large number of nonadherent bone marrow cells or their conditioned medium was added to the stromal cells, osteoprogenitors comprised approximately 1/100 of plated adherent cells and their expression followed a linear, single-hit relationship. By contrast, rat skin fibroblasts or their conditioned medium totally inhibited bone nodule formation. These data support the hypothesis that in marrow stroma, as in other bone cell populations such as those from calvaria, there are at least two classes of osteoprogenitor cells: those differentiating in the absence of added glucocorticoid and those requiring glucocorticoid to differentiate, that more than one cell type is limiting for stromal osteoprogenitor differentiation suggesting a role for heterotypic cell-cell interactions in osteogenesis in this tissue, and that Dex may be acting directly and/or indirectly through accessory cells in the bone marrow to alter osteoprogenitor cell expression.

Nitric Oxide Synthase Expression in Bone Cells

We have localized the expression of the three main nitric oxide synthases (eNOS, bNOS, and iNOS) in bone cells of rats and humans using immunohistochemistry. The predominant isoform expressed in normal adult bone was the constitutive isoform, eNOS, mainly in cells of osteoblastic lineage. In adult bone, the osteoblast lineage cells exhibiting eNOS expression were flat bone lining cells and osteocytes, but cuboidal osteoblasts were consistently negative. Expression for bNOS was not detected in any bone cells. iNOS expression was not detected in any cells of osteoblastic lineage in normal adult rat or human bone, but was observed in cuboidal osteoblasts of adult rats with experimental colitis, in which the suppression in bone formation may be cytokine mediated. Osteoclasts in normal rat tissue showed expression for both eNOS and iNOS, but these were patchy. As for cells of the osteoblast lineage, osteoclasts were negative for bNOS. Thus, our findings support evidence, from in vitro studies and from animal experiments, that nitric oxide may play an important role in the physiology of bone.

Short-term effects of nicotine on bone and calciotropic hormones in adult female rats.

This study assessed the effects of two months nicotine treatment on bone formation and resorption end-points in adult, female rats. In addition, the concentrations of calciotropic hormones which included parathyroid hormone, calcitonin, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in rats (7 months old) were determined. All animals received either saline (n = 7/group), nicotine (3.0 mg/kg/day) (n = 7/group) or nicotine (4.5 mg/kg/day) (n = 7/group) via subcutaneous implantation of osmotic minipumps containing either saline or nicotine for a period of two months. Serum, right tibia, left femur and lumbar vertebra (3-5) were collected for determination of hormonal concentrations as well as various parameters, including histomorphometry, bone mineral density, bone mineral content and vertebral strength. Although nicotine-treated rats showed a lower level of 25-hydroxyvitamin D [54.4 +/- 3.1 ng/ml for the 3.0 mg/kg/day and 55.8 +/- 2.8 for the 4.5 mg/kg/day group] (mean +/- S.E.M.) as compared to controls (74.8 +/- 2.8 ng/ml) (P < 0.01, Newman-Keuls test), no significant difference could be detected for the levels of the remaining hormones. Similarly, no statistical differences were detected on histomorphometric end-points, bone mineral density, bone mineral content and vertebral strength of rats. We conclude that, in spite of lowering serum 25-hydroxyvitamin D by about 30%, nicotine administration of two months duration does not alter bone mass, strength or formation and resorption end-points.

Fluoride Potentiates the Osteogenic Effects of IGF-I in Aged Ovariectomized Rats

The molecular mechanisms whereby fluoride stimulates osteogenic cell proliferation are not clearly established. However, fluoride has been shown to enhance the protein tyrosine phosphorylation of various constituents of intracellular signaling cascades in osteoblastic cells following stimulation of growth factor receptors such as the insulin-like growth factor-I (IGF-I) receptor. Such in vitro findings provided the rationale for testing whether the administration of fluoride could enhance IGF-I effects on bone mass in vivo. Adult ovariectomized osteopenic rats were treated with sodium fluoride at a dose of 6 mg/kg per day in drinking water for 8 weeks in association with IGF-I either at a dose of 2 mg/kg per day, which is capable of increasing bone mass, or at a lower dose without detectable skeletal effects. Bone mineral density (BMD) and content (BMC) were evaluated by dual-energy X-ray absorptiometry at the levels of the lumbar spine and proximal, midshaft, and total tibia before and after 8 weeks of treatment. During this period, fluoride alone did not significantly influence BMD/BMC at any skeletal site. However, it potentiated the effect of the higher dose of IGF-I on bone mass at the level of the proximal tibia. When administered in combination with the lower dose of IGF-I, which per se did not modify bone mass, it appeared to sensitize tibial bone to the effects of IGF-I. These changes were associated with a concomitant increase in osteocalcin, taken as a reflection of bone formation. These results indicate that fluoride could potentiate the osteogenic effects of IGF-I on bone in adult ovariectomized rats.

Derivation of hepato-pancreatic intermediate progenitor cells from a clonal mesenchymal stem cell line of rat bone marrow origin

We have recently established a clonal mesenchymal stem cell line (rBM25/S3) from adult rat bone marrow. The cells have practically unlimited proliferation capacity (over 300 PDL), maintaining multipotency for differentiation. In the present study, we examined the potential for rBM25/S3 cells to differentiate into insulin-secreting cells. When cultured in the presence of HGF and FGF-4 on Matrigel, rBM25/S3 cells expressed genes specific to pancreatic beta-cells as well as those specific to hepatocytes. They still maintained proliferation capacity with a doubling time of approximately 30 h. These hepato-pancreatic intermediate progenitor cells, but not the original undifferentiated rBM25/S3 cells, were induced by the overexpression of PDX-1 to produce significant amounts of insulin in a manner responding to glucose concentration in medium. The present culture system indicates a direction for further studies aimed at the realization of cell transplantation therapy for type I diabetes mellitus.

Progesterone-mediated stimulation of osteoprogenitor proliferation and differentiation in cell populations derived from adult or fetal rat bone tissue depends on the serum component of the culture media.

We have shown previously that progesterone (Prog) and dexamethasone (Dex) stimulate osteoprogenitor proliferation and differentiation in cell populations derived from adult rat vertebrae and in primary cultures of fetal rat calvariae. In these two in vitro systems, osteoprogenitors can be identified by the appearance of colonies of differentiated osteoblasts producing bone (bone nodule formation). Culture conditions supporting proliferation and differentiation of osteoprogenitors include a requirement for the presence of serum in the culture media. Our major interest in the present study was to investigate whether Prog- and Dex-mediated osteoprogenitor proliferation and differentiation was observed to the same degree in different lots of fetal bovine serum (FBS). In addition, we wanted to investigate whether osteoprogenitors present in cell populations derived from fetal calvarial bone and those present in populations derived from adult vertebral bone would respond similarly under the different culture conditions. We found that, in populations derived from adult rat vertebrae, the effects of the serum component of the culture medium on the number of bone nodules induced by Prog and on the dose-dependency of the Prog effect were striking: in culture media containing the most effective serum the number of bone nodules was 22-fold higher than that in the least effective serum. In addition, Prog responses were detectable at 10(-5) M only in some sera but were significant at 10(-7) M in others. The effect of Dex in the adult rat vertebrae-derived populations was much less dependent on the serum used: the number of bone nodules in culture media containing the most effective serum was only 1.3 times greater than that in media containing the least effective serum. In cell populations derived from fetal calvariae, the serum dependence of the Prog response was less pronounced: a 4.3-fold increase over control was observed in the most effective serum, and a 2.4-fold increase in the least effective serum. No effects of the serum component of the culture medium on the Dex response were detectable. Thus, Prog-induced bone nodule formation appears to be strongly dependent on the particular type of FBS used for osteoprogenitors present in bone cell populations derived from adult rat vertebrae but much less so in populations obtained from fetal rat calvariae. Preliminary experiments suggest that the estrogen content of the culture media may be one of the determinants regulating Prog responsiveness of the osteoprogenitors. Dex-induced proliferation and differentiation of osteoprogenitors in bone cell populations derived from both adult rat vertebrae and fetal rat calvariae, on the other hand, did not appear to be strongly dependent on factor(s) present in the FBS component of the culture medium.

In vitro and in vivo effects of ipriflavone on bone formation and bone biomechanics.

Ipriflavone (i.p.) positively affects bone density in postmenopausal osteoporosis, primarily by inhibiting bone resorption. Using in vitro models of human osteoblast differentiation, we have observed that i.p. and some of its metabolites stimulate the expression of bone sialoprotein, decorin, and type I collagen, and facilitate the deposition of mineralized matrix. This suggests that i.p. may stimulate bone formation in addition to its antiresorptive activity. To assess whether these effects translate into an improved bone "quality" in vivo, we measured biomechanical properties, mineral composition, and crystallinity of femurs of 12-week-old, male, Sprague-Dawley rats treated with i.p. for 1 month. i.p. significantly decreased vibration damping, an index of strain energy loss. Because vibration damping increases as bone porosity increases, the results indicate that i.p.-treated bones acquired a higher capacity to withstand dynamic stress. In fact, 1.5-fold higher energy was required to fracture femurs of i.p.-treated rats after a single supramaximal impact. i.p. also increased BMD, assessed by both volume displacement and ash analysis, whereas the relative contents of Ca, P, and Mg in the ashes were not affected. Thus, no gross abnormalities in mineral composition of bone occurred after i.p. administration. As a measure of bone crystallinity, X-ray diffraction analysis was performed. The broadening parameter beta 1/2 for the (310) and (002) reflections was not significantly different between i.p.-treated and control animals. Similarly, there were no differences in serum levels of Ca, Mg, alkaline phosphatase, and type I collagen telopeptides between treated and control animals at the end of the study. Therefore, 1-month treatment with i.p. increased bone density and improved the biomechanical properties of adult male rat bones without altering mineral composition or bone crystallinity.

Ipriflavone does not alter bone apatite crystal structure in adult male rats.

We have previously found that a short-term treatment with high doses of ipriflavone increased bone density and improved the biomechanical properties of adult male rat bones, without altering their mineral composition. To determine whether this effect can be associated with alterations of bone crystal structure, we have performed X-ray diffraction analysis of bones obtained from rats treated with ipriflavone at doses that were effective in inducing favorable changes on bone density and biomechanics. Eighteen-week-old male Sprague Dawley rats were treated by oral route with either ipriflavone (200 or 400 mg/kg/day), or its vehicle for 12 weeks. The treatment was well tolerated and body weight increased to the same extent in all animals. As a measure of bone crystallinity, we examined the (310) and (002) reflections of the X-ray diffraction patterns, corresponding to the directions perpendicular and parallel to the c-axis of the crystals, respectively. No major differences were observed between ipriflavone-treated and control animals for the broadening parameter beta(1/2) for (310) and (002) peaks, as well as for lattice parameters. Therefore, a 12-week treatment with ipriflavone at high doses does not induce significant modifications of bone "crystallinity." Thus, the positive effect of ipriflavone on bone mineral density appears to be associated with an increased apatite crystal formation rather than an increase of crystal size. These results provide further evidence for the safety and usefulness of ipriflavone in the treatment of osteoporotic syndromes.

Phenotype and function of hematopoietic‐derived cells in the CNS of SCID mouse‐lewis rat bone marrow chimeras

Severe combined immunodeficient (SCID) mice previously transplanted with Lewis rat hematopoietic cells (SCID mouse-Lewis rat chimeras) developed experimental autoimmune encephalomyelitis (EAE) following injection with myelin basic protein (BP)-specific Lewis rat T lymphocytes. Rat T cells did not cause EAE in non-chimeric SCID mice. Thus, in addition to BP-specific rat T cells, transplanted rat hematopoietic cells were involved in the development of EAE in SCID mice. In order to examine the role of hematopoietic rat cells in the development of EAE, chimeras were constructed in SCID mice by transplanting 40 × 106 T cell-depleted adult Lewis rat bone marrow cells. Single cell suspensions of brain, blood and spleen from chimeric mice were phenotyped by monoclonal antibody staining specific for mouse or rat cellular differentiation markers at 2 week intervals. Brain cells from chimeric mice were also evaluated for the presence of rat antigen-presenting cells (APC). Four and six weeks after hematopoietic cell transfer, mouse brain contained rat cells expressing the phenotypic markers (CD45+, CD11b/c+) of CNS antigen-presenting cells (APC). Six weeks after hematopoietic cell transfer, rat cells populating the CNS of chimeras were shown to function as APC, stimulating BP-specific Lewis rat T lymphocytes in vitro. © 1996 Wiley-Liss, Inc.

Phenotype and function of hematopoietic-derived cells in the CNS of SCID mouse-Lewis rat bone marrow chimeras.

Severe combined immunodeficient (SCID) mice previously transplanted with Lewis rat hematopoietic cells (SCID mouse-Lewis rat chimeras) developed experimental autoimmune encephalomyelitis (EAE) following injection with myelin basic protein (BP)-specific Lewis rat T lymphocytes. Rat T cells did not cause EAE in non-chimeric SCID mice. Thus, in addition to BP-specific rat T cells, transplanted rat hematopoietic cells were involved in the development of EAE in SCID mice. In order to examine the role of hematopoietic rat cells in the development of EAE, chimeras were constructed in SCID mice by transplanting 40 x 10(6) T cell-depleted adult Lewis rat bone marrow cells. Single cell suspensions of brain, blood and spleen from chimeric mice were phenotyped by monoclonal antibody staining specific for mouse or rat cellular differentiation markers at 2 week intervals. Brain cells from chimeric mice were also evaluated for the presence of rat antigen-presenting cells (APC). Four and six weeks after hematopoietic cell transfer, mouse brain contained rat cells expressing the phenotypic markers (CD45+, CD11b/c+) of CNS antigen-presenting cells (APC). Six weeks after hematopoietic cell transfer, rat cells populating the CNS of chimeras were shown to function as APC, stimulating BP-specific Lewis rat T lymphocytes in vitro.

In vitro andin vivo effects of ipriflavone on bone formation and bone biomechanics

Ipriflavone (IP) positively affects bone density in postmenopausal osteoporosis, primarily by inhibiting bone resorption. Using in vitro models of human osteoblast differentiation, we have observed that IP and some of its metabolites stimulate the expression of bone sialoprotein, decorin, and type I collagen, and facilitate the deposition of mineralized matrix. This suggests that IP may stimulate bone formation in addition to its antiresorptive activity. To assess whether these effects translate into an improved bone “quality” in vivo, we measured biomechanical properties, mineral composition, and crystallinity of femurs of 12-week-old, male, Sprague-Dawley rats treated with IP for 1 month. IP significantly decreased vibration damping, an index of strain energy loss. Because vibration damping increases as bone porosity increases, the results indicate that IP-treated bones acquired a higher capacity to withstand dynamic stress. In fact, 1.5-fold higher energy was required to fracture femurs of IP-treated rats after a single supramaximal impact. IP also increased BMD, assessed by both volume displacement and ash analysis, whereas the relative contents of Ca, P, and Mg in the ashes were not affected. Thus, no gross abnormalities in mineral composition of bone occurred after IP administration. As a measure of bone crystallinity, X-ray diffraction analysis was performed. The broadening parameter β1/2 for the (310) and (002) reflections was not significantly different between IP-treated and control animals. Similarly, there were no differences in serum levels of Ca, Mg, alkaline phosphatase, and type I collagen telopeptides between treated and control animals at the end of the study. Therefore, 1-month treatment with IP increased bone density and improved the biomechanical properties of adult male rat bones without altering mineral composition or bone crystallinity.