Ultrasound mimics the effect of mechanical loading on bone formation in vivo on rat ulnae.

While the effect of ultrasound as an extreme example of low-magnitude high-frequency stimulation has been explored in the response of bone to injury, little is known about its effect on normal bone. This experiment was designed to test the hypothesis that ultrasound exerts a similar influence on bone as mechanical stimulation at a physiological level. Three groups of female Wistar rats were anaesthetised (6 per group). In one group, the left ulna was loaded cyclically in vivo 40 times, repeated on a further 5 occasions on alternate days. In a second group, transcutaneous low-intensity pulsed ultrasound stimulation was applied to the left ulnae for the same duration as the period of loading. In a third group, loading and ultrasound stimulation were applied concurrently. The right ulna served as non-loaded control in each animal. At the end of the experiment after 14 days, both ulnae were removed. Induced bone formation was assessed by measuring the proportion of medial periosteal bone surface with double label (dLS/BS, %) and by calculation of mineral apposition rate (MAR) from the inter-label distance. All three treatments induced a significant periosteal response, increasing dLS/BS values from <10% in control limbs to >80% in treated limbs. Increases in MAR of experimental ulnae versus contralateral control ulnae were 2.9 (+/-0.9), 8.6 (+/-2.4) and 8.7 microm (+/-3.2) for the ultrasound only, ultrasound and load, and load only groups, respectively. The effects of loading plus ultrasound were not significantly different from ultrasound alone. These data suggest that ultrasound is able to induce changes in bone that share at least some features with mechanical loading.

Isolation of C15: a novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow.

Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V(H) and V(L) gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.

Variations in genome-wide gene expression in identical twins - a study of primary osteoblast-like culture from female twins discordant for osteoporosis.

BACKGROUND: Monozygotic twin pairs who are genetically identical would be potentially useful in gene expression study for specific traits as cases and controls, because there would be much less gene expression variation within pairs compared to two unrelated individuals. However the twin pair has to be discordant for the particular trait or phenotype excluding those resulting from known confounders. Such discordant monozygotic twin pairs are rare and very few studies have explored the potential usefulness of this approach. RESULTS: We studied genome-wide gene expression in primary osteoblast-like culture from marrow aspirates obtained from three pairs of monozygotic twins. We used the latest Affymetrix microchip contains probe sets for more than 20,000 genes. Two pairs were discordant for bone mineral density at the hip by more than one standard deviation, and the third pair was unrelated concordant and used as control. Only 1.5% on average of genes showed variation in expression within pairs as compared to 5% between pairs or over 15% from the literature. Importantly we identified several groups of genes showing variations within the discordant pairs and not within the concordant pair such as chondroitin beta 1,4 N-acetylgalactosaminyltransferase, inhibin beta A, interleukin 1 beta and colony stimulating factor 1 macrophage. These genes are known to have potential roles in bone physiology relating to bone density, osteoporosis and osteoarthritis. CONCLUSION: Using the example of osteoblast-like cells in our monozygotic discordant twins for osteoporosis, we identified genes showing differential expression. Although without further experiment, we cannot confirm or conclude these are genes definitely related to bone physiology, we believe we have shown the potential and cost-effectiveness of further gene expression studies in discordant monozygotic twin pairs. A replication study for confirmation is essential.

STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro.

There is widespread interest in the use of bone marrow stromal cells (BMSC) for tissue reconstruction and repair and for gene therapy. BMSC represent the differentiated progeny of CFU-F, which however comprise a developmentally heterogeneous population as is reflected in the cellular heterogeneity of the cell populations to which they give rise. We have compared the efficacy of monoclonal antibodies recognising a series of stromal antigens, viz. STRO-1, HOP-26, CD49a and SB-10/CD166, as tools for the enrichment of CFU-F prior to culture and as developmental markers for culture-expanded BMSC. In freshly isolated bone marrow mononuclear cells (BMMNC), the proportion of antigen-positive cells was 27%, 46%, 5% and 19% for STRO-1, HOP-26, CD49a and CD166, respectively. All CD49a(+) cells co-expressed STRO-1. The degree of CFU-F enrichment obtained with anti-CD49a (approximately 18-fold) by a one-pass immunoselection strategy was significantly greater than that of all other antibodies tested. BMSC expressed higher levels of all antigens investigated (except for HOP-26) compared with BMMNC. Expression of STRO-1 and CD49a remained restricted to a subset of BMSC, whereas all BMSC were SB-10/CD166 positive. Treatment with dexamethasone (10 nM), which promotes the differentiation and further maturation of cells of the osteogenic lineage in this cell culture system, increased the expression of CD49a and HOP-26. The CD49a(+) and HOP-26(+) fractions of BMSC were further subdivided by dual-labelling with anti-STRO-1 and B4-78 (an antibody recognising the B/L/K isoform of the enzyme alkaline phosphatase), respectively. By using a variety of criteria, the HOP-26 antigen was identified as CD63, a member of the tetraspanin family of proteins thought to modulate integrin compartmentalisation and signalling.

TGFbeta1 limits the expansion of the osteoprogenitor fraction in cultures of human bone marrow stromal cells.

Currently, there is considerable interest in the possibility of using cultured human bone marrow stromal cells (BMSCs) for skeletal tissue engineering. However, the factors that regulate their ex vivo expansion and promote their osteogenic maturation remain poorly defined. Using BMSCs obtained from a large cohort of adult donors, the effects of transforming growth factor (TGF)beta1 on these processes have been determined. BMSCs were found to express TGFbeta receptors (TbetaRs) I, II, III (betaglycan) and CD105/endoglin. The expression of TbetaRs I and II, but not TbetaR III or endoglin, was linked to the cells' state of maturation. Treatment with TGFbeta increased the colony-forming efficiency (CFE) of marrow cell suspensions but reduced the median diameter of the colonies that formed and the number of cells harvested at the end of primary culture. Treatment with TGFbeta also resulted in a significant downregulation in the expression of the developmental markers alkaline phosphatase (AP) and STRO-1. The reduction in AP was due to a decrease in the absolute number of cells expressing this enzyme and in the level (sites/cell) at which it was expressed. Overall, the changes in the expression of STRO-1 and AP are consistent with TGFbeta acting to decrease the size of the osteoprogenitor fraction, and hence the potential clinical utility of the cultured cell population.