Simon C. F. Rawlinson; Ian J. McKay; Mandeep Ghuman; Claudia Wellmann; Paul Ryan; Saengsome Prajaneh; Gul Zaman; Francis J. Hughes; Virginia J. Kingsmill

“ Whilst we have provided culture conditions that favour osteoblasts over other cell types, we accept other cell types may be present. However, in the bone organ we know there are more cell populations present within the samples (for instance, osteocytes, adipocytes, endothelial, neuronal-like hematopoietic cells) . Notwithstanding, in either case, cluster analysis segregates skull and limb bone and isolated osteoblasts into separate populations. ”

“ Even in attempts to generate monocultures, cultures of isolated osteoblasts from bone tissues will contain other cell types. The cells used in these experiments are from third collagenase digests of adult bone and may thus contain osteoblasts along with osteocyte, osteoclast, endothelial, neuronal and adipocyte cells. As a precaution, genetic markers indicative of contamination with another tissue type were assessed. Markers selective for osteoclast, adipocyte, neurofilament, monocyte, muscle and endothelial cell were expressed, if at all, at low levels in the cultures. ”

“ The pilot studies used ten male skeletally mature Black CD57 (24–26 grams) mice divided into two groups to test for the feasibility of gene array in bone organs.
Adult bone-derived cell isolation and culture
Five male CD rats (100–110 grams) were rendered unconscious with CO2 and killed by cervical dislocation. Skull and the cortex of ulnar bones were dissected and cleared of attendant soft tissues, epiphyses and marrow. Individual samples were minced and bone cells isolated by enzymatic digestion. Third digest cell populations were used in the experiments. ”

“ Glycosaminoglycan (Gag) concentrations are higher in limb bone. Osteocyte density in the limb bone was decreased in OVX animals, which is consistent with OVX-induced decreases in other non-cranial skeletal sites [26] . However, the osteocyte density in skull bone was unaffected during growth in an ovarian estrogen-depleted animal. This is consistent with differential responses of regional skull and limb osteoblasts and the pattern of bone loss induced by menopausal hormonal changes. However, we can not preclude that any decrease is partly in response to ageing [27] . ”

“ The lateral aspect of the clavicle is prone to osteoporosis and is consistent with other bones containing a neural crest component (skull vault) being resistant to osteoporosis.
In conclusion, this study shows that genes associated with bone mass and mineral density are differentially expressed in functionally distinct skeletal sites. Our observations also demonstrate developmental gene expression-based “positional identity” in the adult skeleton. ”

“ The Affymetrix Mouse Genome 430 2.0 GeneChip and Rat Genome 230 2.0 GeneChip were used to detail basal gene expression profiles in the bone organ samples. Biotinylated targets suitable for hybridization to the GeneChip probe arrays were prepared from the RNA samples as per ‘One-Cycle Target Labeling' protocol (Affymetrix) . ”

“ A positive strain represents stretching, whilst compression is represented by a negative figure. Direct experimental measurements in a human showed that the strain levels in the parietal bone of the skull are 10 fold lower compared with the tibia [4] . ”

“ The effects of osteotrophic agents on alkaline phosphatase (ALP) activity were assessed in both S-Bdc and L-Bdc. Data was normalised to DNA content. Estrogen (10−8 M) decreased ALP activity in S-Bdc, but increased activity in L-Bdc (Figure 7) . The number of mineralized nodule generation in culture was low (data not shown) , and probably reflects the low levels of osteocalcin expression. ”

“ Quantitative assessment by microdensitometry of polysulphated glycosaminoglycan levels in bone matrix stained with Alcian Blue 8GX. (F) Ovariectomy alters osteoblast incorporation into bone matrix as osteocytes in limb bone, but not skull bone, during growth. S-Bdc and L-Bdc phenotype
Adult bone-derived cells in culture were assessed for differences in proliferation rates and cell size. ”

“ The authors have declared that no competing interests exist.Funding: This work was funded by a NIHR Clinician Scientist Fellowship awarded to VJK. MG is a MRC Clinical Research Training Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ”

“ Table 5 presents validation of the gene array techniques with qRT-PCR, and illustrates the close comparison of fold differences between gene array and qRT-PCR in a range of significant and differentially expressed transcription factors, receptors and matrix proteins.
Table 5 "Validation of gene array with qRT-PCR (Skull/Limb ratio) . (10.1371/journal.pone.0008358.t005) "File:Pone.0008358.t005 The difference in the number of differentially expressed genes in bone (1236) and isolated cell (249) cultures may reflect the populations that comprise the two models. ”

“ The reaction conditions were 70°C for 5 mins (denaturation) , 60 mins 40°C for 60 mins (extension) , 70°C for 15 mins (inactivation) , and then stored at −20°C. Each TaqMan assay was run in four replicates for each RNA sample. 50 ng total cDNA (as total input RNA) in a 10 μl final volume was used for each replicate assay. Assays were run with 2xAbsolute qPCR ROX Master Mix (Abgene) on Applied Biosystems 7900 Fast Real-Time PCR System using universal cycling conditions (10 min at 95°C; 15 s at 95°C, 1 min 60°C, 60 cycles) . The assays and samples were analyzed on 384 well plates. ”