Calcitriol but no other metabolite of vitamin D is essential for normal bone growth and development in the rat.

To determine the relative importance of different metabolites of vitamin D in bone growth and development, weanling male rat pups suckled by vitamin D-deficient mothers were given either calcitriol (1,25-dihydroxycholecalciferol) by continuous subcutaneous infusion, oral calcidiol (25-hydroxycholecalciferol), or oral 24,24-difluoro-25-hydroxycholecalciferol, a synthetic compound that can undergo 1-hydroxylation but not 24-hydroxylation, as their sole source of vitamin D for 40 d. Pups raised in the same manner, but given no vitamin D, served as controls. The three metabolites compared were given in doses that restored normal plasma calcium levels and normal increments in body weight. After in vivo double tetracycline labeling, bone histomorphometry by standard methods was performed on one femur and one tail vertebra. There were no significant differences between the three metabolite-treated groups in length, periosteal or endosteal diameter, cortical cross-sectional area, cortical porosity, osteoid thickness and volume, appositional rate and bone formation rate in the femur, or in qualitative and quantitative indices of endochondral ossification in the tail vertebra. All three groups differed markedly from the untreated controls with respect to all measurements. Collectively, the data indicate that neither calcidiol nor any 24-hydroxylated metabolite of calcidiol is needed in the rat (other than as a precursor) for longitudinal or transverse bone growth, for normal endochondral ossification, or for normal periosteal and endosteal formation, mineralization, and resorption of bone. Calcitriol was fully active with respect to each of the indices listed when given in a manner resembling its continuous endogenous production by the kidney, suggesting that previous reports of incomplete skeletal response to calcitriol result from its rapid clearance and infrequent oral administration. We demonstrated that calcitriol is the only metabolite that is both necessary and sufficient for normal bone growth and development in the rat, but our data do not indicate the extent to which its beneficial skeletal effects were mediated by direct action on bone, either of calcitriol itself or of some metabolite thereof, or by restoration of normal plasma levels of calcium and phosphate.

Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis. Implications for the microanatomic and cellular mechanisms of bone loss.

We devised a new method for examining the structural changes that occur in trabecular bone in aging and in osteoporosis. With simultaneous measurement of total perimeter and bone area in thin sections, indirect indices of mean trabecular plate thickness (MTPT) and mean trabecular plate density (MTPD) can be derived, such that trabecular bone volume = MTPD X MTPT. MTPD is an index of the probability that a scanning or test line will intersect a structural element of bone, and is the reciprocal of the mean distance between the midpoints of structural elements, multiplied by pi/2. We applied this method to iliac bone samples from 78 normal subjects, 100 patients with vertebral fracture, and 50 patients with hip fracture. The reduction in trabecular bone volume observed in normal subjects with increasing age was mainly due to a reduction in plate density, with no significant decrease in plate thickness. The further reduction in trabecular bone volume observed in patients with osteoporotic vertebral fracture was mainly due to a further reduction in plate density. There was a relatively smaller reduction in plate thickness that was statistically significant in males but not in females. Only in patients with hip fracture did trabecular thinning contribute substantially to the additional loss of trabecular bone in osteoporosis relative to age. These data indicate that age-related bone loss occurs principally by a process that removes entire structural elements of bone; those that remain are more widely separated and some may undergo compensatory thickening, but most slowly become reduced in thickness. We propose that the process of removal is initiated by increased depth of osteoclastic resorption cavities which leads to focal perforation of trabecular plates; this is followed by progressive enlargement of the perforations with conversion of plates to rods. The resulting structural changes are more severe in osteoporotic patients than in normal subjects, but have been completed in most patients before they develop symptoms.

Identification of the calcium-sensing receptor in the developing tooth organ.

Calcium (Ca2+) is a critical component of tooth enamel, dentin, and the surrounding extracellular matrix. Ca2+ also may regulate tooth formation, although the mechanisms for such action are poorly understood. The Ca2+-sensing receptor (CaR) that is expressed in the parathyroid gland, kidney, bone, and cartilage has provided a mechanism by which extracellular Ca2+ can regulate cell function. Because these tissues play an important role in maintaining mineral homeostasis and because Ca2+ is hypothesized to play a crucial role in tooth formation, we determined whether the CaR was present in teeth. In this study, using immunohistochemistry, CaR protein was detected in developing porcine molars localized in the predentin (pD), early secretory-stage ameloblasts, maturation-stage smooth-ended ameloblasts (SA), and certain cells in the stratum intermedium. CaR protein and messenger RNA (mRNA) were detected also in an immortalized ameloblast-like cell line (PABSo-E) using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and Northern analysis. Based on the observation that the CaR is expressed in cultured ameloblasts, we determined whether increments in medium Ca2+ concentration could activate the intracellular Ca2+ signal transduction pathway. In PABSo-E cells, increasing extracellular Ca2+ in the medium from 0 (baseline) to 2.5mM or 5.0 mM resulted in an increase in intracellular Ca2+ above baseline to 534 +/- 69 nM and 838 +/- 86 nM, respectively. Taken together, these results suggest that the CaR is expressed in developing teeth and may provide a mechanism by which these cells can respond to alterations in extracellular Ca2+ to regulate cell function and, ultimately, tooth formation.

Role of vitamin D in neonatal skeletal development in rats.

The role of vitamin D in rat pup growth and skeletal development without the influence of nutritional factors was investigated. Pups from vitamin D-replete and vitamin D-deficient dams receiving identical amounts of milk for 20 days were compared. Body weight gain, femur ash content and histomorphometric analyses of diaphysial and distal femur were determined. Up to 20 days of age, growth and skeletal development of the pups were completely normal in the absence of vitamin D. Skeletal changes found in vitamin D deficiency were not observed, i.e., there was no increased volume of osteoid or lack of bone mineralization as demonstrated by tetracycline labeling and ash content. Only increased cortical porosity was found in vitamin D-deficient pups. Therefore, abnormalities previously attributed to vitamin D deficiency in neonatal rats can be corrected by sufficient milk consumption and are thus not a direct function of vitamin D.

Increased fluoride content in the femur growth plate and cortical bone of uremic rats.

In chronic renal insufficiency (CRI), serum levels of fluoride (F-) are elevated. However, there is limited information about the effects of F- on bone in CRI. In this study, we determined whether F- content in mineralizing tissue (growth plate, cortical bone, and bone marrow of the femur) is affected by uremia. Adult rats were divided into two groups [sham-operated (S) and 5/6 nephrectomized (Nx)]. At sacrifice, the serum creatinine (mg/dl) in the S and 5/6 Nx animals was 0.37+/-0.09 (mean+/-SD) and 1.10+/-0.34 at 4 weeks, and 0.38+/-0.04 and 0.90+/-0.36 at 8 weeks, respectively. The serum calcium, phosphorus, and parathyroid hormone levels were lower and the serum 1, 25-dihydroxyvitamin D levels were higher in S animals than Nx animals at both 4 and 8 weeks. F- urinary excretion (ppm/24 h) was reduced in Nx animals at 4 weeks (34.0+/-19.2) versus S animals (50.7+/-12.9) (P<0.05). F content (ppm) was significantly increased in the growth plate in Nx animals compared with S animals both at 4 weeks (550+/-167 vs. 353+/-63) and at 8 weeks (654+/-135 vs. 396+/-97), respectively (P<0.01). The F- content in cortical bone was similarly increased in Nx animals compared with S animals, but was only statistically increased at 8 weeks. There was no difference in bone marrow F- content between the two groups. In conclusion, this study suggests that in CRI, there is a rapid increase in F- content of the distal femur in the growth plate region, with a subsequent slower increase of F- content in cortical bone.

Expression of the Hox 2.2 homeobox gene in murine embryonic epidermis.

The expression of the Hox 2.2 gene was studied in mouse fetal skin by in situ hybridization with an antisense RNA probe derived from the homeobox region of this gene. In contrast to the expression of Hox 2.2 in spinal cord, which is strongest in 11-day embryos, and is greatly diminished by day 14 and day 17, the signal for Hox 2.2 in skin could be not be detected in 11-day epidermis, was barely detectable on day 14, became strong on day 17, and decreased in new-born animals (day 19). RNase protection assays using Hox 2.2 homeobox-containing and 3' flanking region probes confirmed that the signals detected in 17-day fetal skin by in situ hybridization represent Hox 2.2 transcripts, and that the message is expressed throughout the day 15 to day 18 period during which the epidermis is undergoing terminal differentiation. RNase protection analysis also revealed two alternatively spliced forms of the Hox 2.2 mRNA are present throughout fetal skin development. Northern gel analysis of 17-day fetal skin using a Hox 2.2 homeobox-containing probe at high stringency showed two bands of 1.6 and 1.9 kb, respectively. The 1.9 kb band was greatly enhanced by hybridization at reduced stringency, suggesting the expression of additional homeobox genes with homology to Hox 2.2. These results suggest that the Hox 2.2 homeobox gene plays a role in epidermal development.

Development and characterization of an SV40 immortalized porcine ameloblast-like cell line.

Enamel is secreted as a protein matrix by the ameloblasts. These same cells then control the maturation of the enamel matrix, secreting proteinases that hydrolyze proteins as mineralization progresses, until mature enamel containing less than 1% protein by weight remains. Further understanding of the factors that control ameloblast function and differentiation requires an in vitro cell culture system. In this study, we report immortalization of enamel organ epithelial cells and the selection of a cell line with characteristics of ameloblasts. Porcine enamel organ cells were dissected from unerupted porcine molars, cultured in serum-free medium, and passaged twice. These cells were transfected with an origin-of-replication defective SV40 plasmid by calcium phosphate precipitation, and a cell line with mRNA expression characteristic of ameloblasts was cloned. This cell line (PABSo-E) expressed mRNA for amelogenin, matrix metalloproteinase-20 (enamelysin), and enamel matrix serine proteinase 1 (EMSP1), but not ameloblastin. PABSo-E cells have been passaged more than 55 times, while continuing to maintain characteristics of ameloblasts. These cells will be useful for future studies of ameloblast function.

Identification of the mineralization front: comparison of a modified toluidine blue stain with tetracycline fluorescence.

A modified toluidine blue method for identification of the mineralization front at the zone of demarcation between bone and osteoid in undecalcified, plastic-embedded sections of bone is described. The intensity of staining is increased both by increasing the pH of the solution and by increasing the duration of staining. The method is reproducible, since measurements of the extent of mineralization front on two nonadjacent sections from the same biopsy in 20 cases had a correlation coefficient of 0.98. The identification of the mineralization front by toluidine blue agrees closely with identification by means of in vivo tetracycline uptake, with a correlation coefficient of 0.97 between measurements of its extent using the two methods on adjacent sections from the same biopsy. It is likely that both toluidine blue staining at pH 6.5 and tetracycline uptake depend on some property of the most recently deposited bone mineral. With either method we find low values for the extent of mineralization front as a fraction of osteoid surface in many patients with osteoporosis. The uniformly normal values for this quantity in osteoporosis reported by other investigators may reflect different criteria for distinguishing osteoid-covered from quiescent bone surfaces.

Erythroid-restricted expression of homeobox genes of the human HOX 2 locus.

We have previously reported that certain members of the HOX 1 and HOX 2 clusters of class 1 homeobox-containing genes showed lineage-restricted patterns of expression in a small series of human hematopoietic cell lines. We now report on the expression patterns of the entire HOX 2 cluster, consisting of nine homeobox genes, in a broad survey of leukemic cell lines of different phenotypes. The most striking observation is that all but one of the HOX 2 genes are consistently expressed in cells with erythroid character and/or potential, but, with rare exception, not in cells with myelomonocytic or T- or B-lymphoid phenotype. By contrast, several genes of the HOX 1 and 3 loci are not expressed in erythroid lines. Within erythroid cell lines, many of the HOX 2 genes are expressed as multiple transcripts. Expression of some HOX 2 genes is detectable in normal human marrow. These data show that in human hematopoietic cell lines HOX 2 homeobox gene expression is largely restricted to cells of erythroid phenotype and suggest that these genes play a role in erythropoiesis.

Primary culture and characterization of enamel organ epithelial cells.

The cells of the enamel organ are programmed by signals such as growth factors and extracellular matrix components to differentiate and form dental enamel. To study how the enamel organ epithelial cells control enamel development, we have begun to characterize a primary porcine enamel organ epithelial cell culture system. The unerupted molars of 3 month old pigs were isolated, the cells were digested into a single cell suspension and grown in media either with or without serum. Expression of amelogenin and ameloblastin mRNA was monitored by RT PCR, and protein secretion was identified by immunohistochemistry. Cells grown in MEM formed a mixed cell population of epithelial- and fibroblast-like cells which grew past confluence, formed nodules, mineralized, and expressed low levels of amelogenin and ameloblastin protein. In LHC-9 media, which is selective for epithelial cells, the cells did not grow past confluence but secreted amelogenin and ameloblastin proteins more strongly. Cell viability was maintained in both serum-free and serum-containing media. However, in the serum-free media, cell proliferation proceeded slowly. Although cells grown in MEM mineralized, the mixed cell population may make studies of specific ameloblast-like cells more difficult. However, cells grown in a culture media selective for epithelial cells will require modifications such as cell immortalization to allow long term studies of cell regulation and interaction. In summary, we have established an enamel organ epithelial cell culture system which will enable us to study the role of ameloblasts in enamel matrix formation, ameloblast regulation, as well as cell-matrix interactions. Selection of specific culture conditions will depend on the questions being addressed in individual studies.

Expression of HOX C homeobox genes in lymphoid cells.

The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

Haploidentical bone marrow transplantation for osteopetrosis.

HLA-matched bone marrow transplantation is an effective form of treatment for some patients with malignant osteopetrosis, a defect of osteoclast function. Following transplant, normal osteoclasts differentiate from donor-derived marrow stem cells and can function normally in some of these patients. For patients without an HLA-matched marrow donor, pharmacologic treatments have not yet proved effective. This article demonstrates that normal osteoclast function can be obtained following the transplantation of HLA-nonidentical marrow that has been purged of T lymphocytes in vitro.

Modulation of homeobox gene expression alters the phenotype of human hematopoietic cell lines.

We have previously reported that certain genes of the HOX2 cluster of homeobox genes on human chromosome 17 are specifically expressed in human leukemic cell lines with erythroid potential, suggesting that these genes are involved in hematopoietic differentiation. We now show that the expression of the HOX 2.2 gene decreases during erythropoietin-induced differentiation of the erythroid cell line MB02. In order to study the role of the HOX 2.2 homeobox gene in hematopoiesis, vectors producing sense or antisense transcripts were introduced into K562 and HEL cells, pluripotent lines with erythroid and myeloid features. Overexpression of HOX 2.2 is associated with loss of erythroid features in both lines and an increase in certain myelomonocytic markers in K562 cells. Expression of antisense HOX 2.2 is associated with an increase in erythroid features in HEL cells and a mild decrease in myeloid characteristics in K562 cells. Overexpression of the adjacent HOX 2.1 gene in K562 cells does not produce similar phenotype changes. These data demonstrate that modulation of a specific HOX 2 homeobox gene can change the phenotype of somatic cells and suggest that certain HOX 2 genes play a role in blood cell differentiation.