ABSTRACT

The monoclonal antibody STRO-1 identifies clonogenic bone marrow stromal cell progenitors (fibroblast colony-forming units [CFU-F]) in adult human bone marrow. These STRO-1+ CFU-F have previously been shown to give rise to cells with the phenotype of fibroblasts, adipocytes, and smooth muscle cells. In this study, the osteogenic potential of CFU- F derived from the STRO-1+ fraction of adult human bone marrow was determined. CFU-F were isolated from normal bone marrow aspirates by fluorescence activated cell sorting, based on their expression of the STRO-1 antigen. Osteogenic differentiation was assessed by the induction of alkaline phosphatase expression, by the formation of a mineralized matrix (hydroxyapatite), and by the production of the bone- specific protein osteocalcin. STRO-1+ cells were cultured in the presence of dexamethasone (DEX; 10(-8) mol/L), ascorbic acid 2- phosphate (ASC-2P; 100 mumol/L), and inorganic phosphate (PO4i; 2.9 mmol/L). After 2 weeks of culture, greater than 90% of the cells in each CFU-F colony stained positive for alkaline phosphatase using a monoclonal antibody specific for bone and liver alkaline phosphatase. Alkaline phosphatase activity was confirmed by histochemistry. A mineralized matrix developed in the CFU-F cultures, after 4 weeks of culture in the presence of DEX, ASC-2P, and PO4i. Mineralization was confirmed by both light and electron microscopy. The mineral was identified as hydroxyapatite by electron dispersive x-ray microanalysis and by x-ray diffraction analysis. In replicate cultures, osteocalcin release was shown after exposure of the cells to 1,25-dihydroxyvitamin D3 (10(-7) mol/L) both by radioimmunoassay and Northern blot analysis. This work provides direct evidence that adult human bone marrow-derived CFU-F are capable of differentiating into functional osteoblasts and that osteoprogenitors are present in the STRO-1+ population.

To evaluate the temporal features of physiological fluctuation in serum PTH concentration, we sampled peripheral blood at 4-min intervals for 24 h from five normal men (32.8 yr; range, 26-40 yr) and measured serum PTH levels using a two-site immunoradiometric assay with the exquisite sensitivity and specificity for human PTH-(1-84) (intact PTH). The resultant 24-h time series of serum intact PTH levels were assessed by contemporary techniques in chronophysiology for rhythmic and episodic peak detection. Cosinor analysis disclosed a significant circadian rhythm in serum intact PTH concentrations in all five men, with the mean circadian amplitude and acrophase of 7.2 +/- 4.4 ng/L and 2305 +/- 401 h, respectively (mean +/- SD; n = 5). No apparent fixed ultradian periodicity was found by autocorrelation and spectral analyses. Evaluation of episodic intact PTH pulsatility by Cluster analysis revealed 23.0 +/- 4.4 discrete PTH pulses/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 61.6 +/- 11.1 min. The average duration of a serum intact PTH peak was 42.8 +/- 7.3 min, and its mean incremental amplitude was 12.6 +/- 1.3 ng/L, which corresponded to a 31.8 +/- 5.2% increase above the preceding nadir. Discrete PTH peaks were separated by nonpulsatile valleys which lasted for 17.9 +/- 4.4 min. Cross-correlation between the time series of serum intact PTH and whole blood ionized calcium (Ca2+) was at its maximum (-0.5) at concurrent time points in three subjects, while significant positive correlation between serum intact PTH and simultaneous serum inorganic phosphorus concentrations was observed in four of five subjects. There was no apparent correlation between the levels of serum intact PTH and serum magnesium. Our data show that serum levels of intact PTH, the only biologically active form of PTH in the blood, is characterized by a significant circadian periodicity, spontaneous episodic pulsatility with distinct peak properties, and a significant temporal coupling with Ca2+ and inorganic phosphorus concentrations. We conclude that PTH secretion, as judged by the temporal pattern of serum intact PTH levels, is pulsatile in normal men.

Bone loss around replacement prostheses may be related to the activation of mononuclear phagocytes (MNP) by prosthetic wear particles. We investigated how osteoblast-like cells were regulated by human MNP stimulated by particles of prosthetic material. Particles of titanium-6-aluminium-4-vanadium (TiAlV) stimulated MNP to release interleukin (IL)-1β, tumour necrosis factor (TNF)α, IL-6 and prostaglandin E2 (PGE2). All these mediators are implicated in regulating bone metabolism. Particle-activated MNP inhibited bone cell proliferation and stimulated release of IL-6 and PGE2. The number of cells expressing alkaline phosphatase, a marker associated with mature osteo-blastic cells, was reduced. Experiments with blocking antibodies showed that TNFα was responsible for the reduction in proliferation and the numbers of cells expressing alkaline phosphatase. By contrast, IL-1β stimulated cell proliferation and differentiation. Both IL-1β and TNFα stimulated IL-6 and PGE2release from the osteoblast-like cells. Our results suggest that particle-activated mono-nuclear phagocytes can induce a change in the balance between bone formation and resorption by a number of mechanisms.

CD34 and c-KIT positive cells were only observed in the dentin cavity at 7 days. Cbfa-1 and PCNA scores tended to increase at 7 days after the operation.Conclusion: Mesenchymal stem cells and hematopoietic stem cells in the bone marrow are not involved in the regeneration of the periodontium. Cells which migrated from the residual PDL regenerated new alveolar bone at the early stage and the regeneration around the dentin in the cavity was later than in other parts.

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Summary. We investigated the expression of natural killer cell receptors (NKRs) for HLA-C on peripheral blood mononuclear cells (PBMCs) in 23 allogeneic bone marrow transplantation (allo-BMT) patients to analyse the role of NKRs in alloresponse concerning graft-versus-host disease (GVHD). CD158a expression was low and there was little change in the expression after allo-BMT. Also, there was no difference in the proportion of CD158a /CD3 À after allo-BMT. In contrast, the proportion of CD158b /CD3 À cells, mainly NK cells, increased in the early stage (< 2 months) after allo-BMT and then gradually decreased (3´3 6 2´6% before BMT vs. 15´4 6 8´6% in the early stage after BMT, 8´5 6 4´9% during the period 3±6 months after BMT and 7´0 6 3´0% > 6 months after BMT; P < 0´05). However, CD158b expression on CD3 T cells increased 3 months after allo-BMT (1´1 6 1´1% before BMT vs. 5´1 6 7´7% during the period 3±6 months after BMT and 3´0 6 2´4% > 6 months after BMT, P < 0´05). The highest percentages of CD158 expression in patients without chronic GVHD (cGVHD) and those with cGVHD were compared. The percentage of CD158b /CD3 cells and also that of CD158b /CD8 cells were signi®cantly increased in patients with cGVHD compared with those in patients without cGVHD (2´6 6 2´0% vs. 8´0 6 11´2% and 2´3 6 1´5% vs. 8´3 6 11´7% respectively; P < 0´05). The exact clinical relevance of these CD158b-expressing cells is not clear. However, there is an interesting possibility that CD158b-expressing cells play some role in the regulation of GVHD after allo-BMT.

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