Grant Jordan

Further Characterization of Cells Expressing STRO‐1 in Cultures of Adult Human Bone Marrow Stromal Cells

Primitive cells of the osteoblast lineage are not well characterized but are known to be present within the STRO‐1+ fraction of adult human bone and marrow. A survey of human osteosarcoma cell lines revealed that STRO‐1 is expressed by MG‐63 but not SaOS‐2. Among murine cell lines tested, expression of STRO‐1 was detected in the bipotential (adipocyte/osteoblast) line BMS‐2 but not the committed osteoblast precursor MC3T3‐E1. A proportion of cultured adult human bone marrow stromal cells (BMSCs) consistently expressed the STRO‐1 antigen. The expression of a range of cell surface antigens was studied in relation to STRO‐1 by flow cytometry and several, including the bone/liver/kidney isoform of alkaline phosphatase (ALP), were found to subtype the STRO‐1+ population of BMSCs. Further, BMSCs dual‐labeled with antibodies recognizing STRO‐1 and ALP could be assigned to one of four fractions: STRO‐1−/ALP−, STRO‐1+/ALP−, STRO‐1+/ALP+, and STRO‐1−/ALP+. Cells from each fraction could be isolated in high purity and, when recultured, remained viable and exhibited a limited degree of phenotypic stability. Using reverse transcriptase‐polymerase chain reaction, cells in the four fractions were found to express different levels of transcripts for the parathyroid hormone receptor (PTHr) and bone sialoprotein (BSP). The expression of transcripts for the nuclear transcription factor core‐binding factor alpha 1/osteoblast‐specific factor‐2 (CBFA1/OSF2) was restricted to those fractions expressing STRO‐1 and/or ALP. Treatment with 10 nM dexamethasone consistently increased the proportion of cells present in those fractions which expressed the highest levels of transcripts for PTHr and BSP (STRO‐1+/ALP+ and STRO‐1−/ALP+) while simultaneously decreasing the proportion present in the STRO‐1+/ALP− fraction. In conclusion, the expression of STRO‐1 in vitro remains a characteristic of less well differentiated cells of the osteoblast lineage; in cultures of BMSCs and in established human osteosarcoma cell lines, there is an inverse association between the expression of STRO‐1 and ALP; dual labeling of BMSCs with monoclonal antibodies recognizing STRO‐1 and ALP permits the identification and isolation of cells of the osteoblast lineage at different stages of differentiation.

Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1–4

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.

Spatial clustering of remodeling osteons in the femoral neck cortex: a cause of weakness in hip fracture?

Intracapsular femoral neck fractures are associated with decreased cortical width and increased proportions of Haversian canals with diameters greater than the normal mean plus 3 SD (i.e., >385 microm). Such canals might be formed if closely associated resorbing osteons merge; a cortical event analogous with the loss of cancellous connectivity. To test this, we investigated the pattern of osteon distribution in the aging femoral neck to determine if remodeling osteons were distributed in anatomical clusters. Femoral neck biopsies from female patients with intracapsular hip fractures (n = 13) were compared with age/gender-matched cadaveric controls (n = 13). Solochrome-stained sections were analyzed for Haversian canal location, canal diameter, and the presence of an osteoid surface. Clustering was investigated using statistical software with a cluster defined as two or more osteoid-bearing osteon centers within 0.75 mm of each other. Clusters occurred more frequently than would be expected by chance (p < 0.001). Fracture cases had more clusters per unit area (3.14 +/- 0.31 clusters/25 mm2 of cortical bone) than controls (1.89 +/- 0.22) (p = 0.002). In fracture cases, the antero-inferior, antero-superior, and infero-anterior regions had more clusters per 25 mm2 than comparable control regions (ant/inf: 4.12 +/- 0.79, 1.70 +/- 0.60,p = 0.025; ant/sup: 5.31 +/- 1.1, 1.80 +/- 0.59,p = 0.013; inf/ant: 3.15 +/- 0.49, 1.27 +/-0.29, p = 0.004). The mean number of clusters per 25 mm2 per region correlated with the mean porosity per region (adjusted r2 = 0.60;p = 0.014), and the total number of giant canals per region correlated with the total number of clusters per region (adjusted r2 = 0.58; p = 0.011). In conclusion, remodeling osteons are clustered or grouped anatomically, and fracture cases have more clusters than controls. Our data suggest that merging of adjacent, clustered osteons during resorption could lead to the rapid development of canals with excessive diameters and focal weakness. Clustering is greatest in those regions that we have previously shown to have the largest relative reductions in bone strength compared with controls and known to be maximally loaded during a sideways fall. This implicates the remodeling process underlying clustering of remodeling osteons in the aetiology of hip fracture.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis)

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Stratification and Monitoring of Juvenile Idiopathic Arthritis Patients by Synovial Proteome Analysis

Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish patients in whom inflammation extends to affect a large number of joints, early in the disease process. SF samples from 22 JIA patients were analyzed: 10 with oligoarticular arthritis, 5 extended oligoarticular and 7 polyarticular disease. SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression further verified by Western immunoblotting and immunohistochemistry. Hierarchical clustering based on the expression levels of a set of 40 proteins segregated the extended oligoarticular from the oligoarticular patients (p < 0.05). Expression patterns of the isolated protein panel have also been observed over time, as disease spreads to multiple joints. The data indicates that synovial fluid proteome profiles could be used to stratify patients based on risk of disease extension. These protein profiles may also assist in monitoring therapeutic responses over time and help predict joint damage.

The Ratio of Osteocytic Incorporation to Bone Matrix Formation in Femoral Neck Cancellous Bone: An Enhanced Osteoblast Work Rate in the Vicinity of Hip Osteoarthritis

Recently it has been shown that an inactivating mutation in the TGFb-SMAD3 signaling pathway, which increases the conversion of osteoblasts to osteocytes, is accompanied by bone loss combined with increased osteocyte density. We hypothesized that increased matrix TGFb, known to occur in osteoarthritis, might cause the reverse of these effects in man. Because coxarthrosis (cOA) is associated with a reduced risk of femoral neck fracture, whole cross-section femoral neck biopsies were obtained from 11 patients with femoral neck fracture, 14 patients with cOA, and 22 age-and sex-matched controls. Lacunar density (Lc.mm2), osteocyte density (Ot.mm2), and cancellous wall width (Cn.W.Wi), were compared between cases of coxarthrosis, femoral neck fracture (FNF) and controls. In cOA, Lc.mm2 was reduced by 24% (P <0.001) while in FNF it was increased by 20% (P <0.001). Cn.W.Wi was increased in cOA by 22% (P <0.05) and in FNF was reduced by 27% (P <0.001). Lc.mm2 was inversely related to percentage cancellous bone area (adj. r2 = 0.373; P <0.01) and wall widths, r2 = 0.382, P <0.001. The reduction in osteocyte lacunar density coupled with increased wall width is consistant with a model of cOA effects on bone in which increased levels of matrix TGFb might prolong the effective lifespan or work rate of the osteoblast and delay its incorporation into the matrix as an osteocyte. One possible approach to strengthening bone in osteoporosis might be to enhance the effective lifespan of the osteoblast by modulating TGFb-related pathway activity in its local environment.

Increased cancellous bone in the femoral neck of patients with coxarthrosis (hip osteoarthritis): a positive remodeling imbalance favoring bone formation

Osteoporosis is caused by an imbalance between bone resorption and formation which results in an absolute reduction in bone mass. In a previous study we highlighted a condition, osteoarthritis of the hip (coxarthrosis, cOA), where an imbalance between resorption and formation provided beneficial effects in the form of an absolute increase in bone mass. We demonstrated that the femoral neck in patients with cOA had increased cancellous bone area, connectivity and trabecular thickness which might contribute to the protection against fracture associated with the condition. The aim of the present study was to analyze forming and resorbing surfaces in coxarthritic cancellous bone to assess whether increased formation or reduced resorption could be responsible for these structural changes. Whole cross-sectional femoral neck biopsies were obtained from 11 patients with cOA and histomorphometric parameters compared with 14 age- and sex-matched cadaveric controls. The ratio of osteoid surface to bone surface was 121% (p<0.001) higher in the cases but there was no significant difference in resorptive surface. The percentage osteoid volume to bone volume (%OV/BV; +270%, p<0.001) and osteoid width (O.Wi; +127%, p<0.001) were also higher in the cases. This study suggests that the increased cancellous bone mass seen in cases of cOA is due to increased bone formation rather than decreased bone resorption. Investigation of the cellular and biochemical basis for these changes might provide new insights into the pathogenesis of osteoarthritis and highlight novel biological mechanisms regulating bone multicellular unit (BMU) balance that could be relevant to developing new interventions against hip and other osteoporotic fractures.