Michel F. Baslé

Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces

SummaryWe report the characterization of human osteoblastic cells that were derived from the surface of trabecular bone fragments. After removal of bone marrow cells, the bone lining osteoblastic cells lining the bone surface were obtained by migration and proliferation from the trabecular surface onto a nylon mesh. The isolated population proliferated in culture and exhibited osteoblastic phenotype. Cultured cells show a regular arrangment in vitro and exhibited multiple interconnecting junctions on scanning electron microscopic examination. Immunocytochemical staining showed that the cells produced almost exclusively type I collagen. Bone-surface-derived cells responded to 1–34 human parathyroid hormone by increasing intracellular cyclic AMP. Cell cultures exhibited high alkaline phosphatase activity, which was unaffected by 1,25 (OH)2 vitamin D. Untreated cells produced high levels of osteocalcin, a bone-specific protein, and they responded to 1,25(OH) vitamin D by increasing osteocalcin synthesis in a dose-dependent manner. Although cells cultured for up to 5 mo. still produced osteocalcin, the response to 1,25(OH)2D decreased after multiple passages. This study shows that the bone cell populations isolated from trabecular bone surface are enriched in osteoblast precursors and mature osteoblstic cells.

Osteoclast ultrastructure in Paget’s disease

The ultrastructural study of osteoclasts in biopsies from 12 patients with Paget’s disease reveals several cytological anomalies. In particular, nuclear inclusions, consisting essentially of striated filaments about 150 Å in diameter, often organized in bundles and sometimes in paracrystalline arrays, appear to be specific to the disease. Morphologically, the inclusions are remarkably similar to those observed in various cases of viral attack, and the hypothesis of the action of a possible external agent in Paget’s disease cannot be ruled out.

Osteoclastic resorption of Ca-P biomaterials implanted in rabbit bone.

SummaryThe nature of the multinucleated cells involved in the resorption processes occurring inside macroporous calcium-phosphate biomaterials grafted into rabbit bone was studied using light microscopy, histomorphometric analysis, enzymatic detection of tartrate-resistant acid phosphatase (TRAP) activity, scanning, and electron microscopy. Samples were taken at days 7, 14, and 21 after implantation. As early as day 7, osteogenesis and resorption were observed at the surface of the biomaterials, inside the macropores. Resorption of both newly formed bone and calcium-phosphate biomaterials was associated with two types of multinucleated cells. Giant multinucleated cells were found only at the surface of the biomaterials; they showed a large number of nuclei, were TRAP negative, developed no ruffled border, and contained numerous vacuoles with large accumulation of mineral crystals from the biomaterials. Osteoclasts exhibited TRAP positivity and well-defined ruffled border. They were observed at the surface of both newly formed bone and biomaterials, around the implant, and inside the macropores. In contact with the biomaterials, infoldings of their ruffled border were observed between the mineral crystals, deeply inside the microporosity. The microporosity of the biomaterials (i.e., the noncrystalline spaces inside the biomaterials) increased underneath this type of cell as compared with underneath giant cells or to the depth of the biomaterials. These observations demonstrate that macroporous calcium-phosphate biomaterials implanted in bone elicit osteogenesis and the recruitment of a double multinucleated cell population having resorbing activity: giant multinucleated cells that resorb biomaterials and osteoclasts that resorb newly formed bone and biomaterials.

Comparison of eight histomorphometric methods for measuring trabecular bone architecture by image analysis on histological sections

Osteoporosis is defined as a disease characterized by low bone mass and microarchitectural deterioration of trabecular bone leading to enhanced bone fragility. Various histomorphometric methods have been described to measure bone architecture on histological sections. However, not all of the methods are strictly equivalent and some of them appear able to detect differences earlier in the course of the disease. We have compared 8 histomorphometric methods known to characterize the architecture of trabecular bone in 154 male osteoporotic patients. Measurements were done on transiliac bone biopsies: Trabecular number, thickness, and separation (Tb.N, Tb.Th, Tb.Sp); Trabecular Bone Pattern Factor (TBPf); Euler‐Poincarés number (E); Interconnectivity Index (ICI); strut analysis of the trabecular network with the ratio of nodes/free‐end (N/F); star volume of the bone marrow (V*m.space) and trabeculae (V*Tb) and the Kolmogorov fractal dimension of the trabecular boundaries (D). Relationships between the various architectural parameters were studied by hierarchical cluster analysis. Linear, hyperbolic, and exponential correlations were found between trabecular bone volume (BV/TV) and architectural parameters. Cluster analysis demonstrates the link between these architectural parameters. ICI, E, and TBPf, which reflect the amount of open/closed marrow cavities clustered together and appeared related to Tb.Sp, V*m.space which are indicators of the mean size of marrow cavities. Tb.Th, V*Tb and N/F flocked together as they reflect the trabecular size. Tb.N and D segregated together and seemed to best describe the trabecular network complexity. These histomorphometric techniques are correlated but correlations may be linear or nonlinear. Several histomorphometric techniques need to be used in parallel to appreciate the pathophysiological mechanisms of osteoporotic states. Microsc. Res. Tech. 45:303–312, 1999.

Proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures: Effects of dexamethasone and calcitriol

During bone loss, osteoblast population can be replaced by adipose tissue. This apparent reciprocal relationship between decreased bone density and increased fat formation can be explained by an imbalance in the production of bone‐forming and fat‐forming cells in the marrow cavity. Thus, osteoblast and adipocyte pathways seem more closely and inversely related. In the present study, we investigated the effects of dexamethasone (dex) and calcitriol [1,25(OH)2D3] on proliferation and differentiation of osteoblasts and adipocytes in rat bone marrow stromal cell cultures. Stromal cells were grown in primoculture in presence of dex and subcultivated in presence of dex and/or 1,25(OH)2D3. Total cell proliferation, osteoblast and adipocyte‐cells number, and ‐mRNA specific markers were used to study the effects of hormonal treatment on stromal cells. Total cell proliferation was stimulated by dex and inhibited by 1,25(OH)2D3. Dex increased osteoblast and adipocyte cell population whereas calcitriol decreased bone‐forming cell number and increased fat cell population. The presence of both hormones led to a strong decrease in osteoblastic cells and to a strong increase in adipocytic cell number. Dex induced mRNA osteoblastic markers expression like bone sialoprotein (BSP) and osteocalcin (OC) and an adipocyte marker expression, the fatty acid binding protein aP2. Calcitriol decreased the dex‐induced BSP expression but stimulated slightly OC and aP2 mRNA. The effects of both hormones was to increase strongly OC and aP2 mRNA. These results support that, in rat bone marrow, adipocyte proliferation and differentiation are stimulated by glucocorticoids and calcitriol which act synergically, whereas osteoblastic cell proliferation and differentiation are increased by dex and inhibited by 1,25(OH)2D3. J. Cell. Biochem. 89: 364–372, 2003.

Thiazolidinediones Induce Osteocyte Apoptosis by a G Protein-coupled Receptor 40-dependent Mechanism

Background: Thiazolidinediones (TZDs) mediate osteocyte apoptosis and sclerostin up-regulation by an unknown mechanism. Results: Osteocyte apoptosis is mediated through activation of Erk1/2 and p38, whereas sclerostin up-regulation is through peroxisome proliferator-activated receptor-γ (PPARγ) signaling. Conclusion: TZDs signal not exclusively through PPARγ, as thought, but also via a surface receptor called GPR40. Significance: Learning how TZDs signal in bone cells is crucial to prevent adverse effects associated with the use of these drugs. Thiazolidinediones (TZDs) represent an interesting treatment of type 2 diabetes mellitus. However, adverse effects such as heart problems and bone fractures have already been reported. Previously, we reported that pioglitazone and rosiglitazone induce osteocyte apoptosis and sclerostin up-regulation; however, the molecular mechanisms leading to such effects are unknown. In this study, we found that TZDs rapidly activated Erk1/2 and p38. These activations were mediated through Ras proteins and GPR40, a receptor expressed on the surface of osteocytes. Activation of this pathway led only to osteocyte apoptosis but not sclerostin up-regulation. On the other hand, TZDs were capable of activating peroxisome proliferator-activated receptor-γ, and activation of this signaling pathway led to sclerostin up-regulation but not osteocyte apoptosis. This study demonstrates two distinct signaling pathways activated in osteocytes in response to TZDs that could participate in the observed increase in fractures in TZD-treated patients.

Excessive bone resorption in human plasmacytomas: direct induction by tumour cells in vivo

Summary. We studied the bone resorption (BR) in 82 patients with overt plasmacytomas (PCT) by histomorpho‐metry. All the solitary PCT and 90% of overt PCT in complete remission lacked excessive BR. In contrast, 73% of the 68 patients with active overt PCT had an excessive BR. This excessive BR was highly related to the presence of tumour cells within the bone sample. Indeed, 92% of patients with active PCT and tumour cells within the biopsy had an excessive BR. On the other hand, 70% of patients with active PCT but lacking tumour cells within the biopsy had normal BR.

Free radicals and side products released during methylmethacrylate polymerization are cytotoxic for osteoblastic cells

Polymerization of orthopedic cements makes use of a peroxide initiator which is decomposed by an accelerator to provide free radicals. Free radicals which act on the monomer molecules are also known to induce cell lesions and cell death. We used an in vitro model of cement polymerization to study the effects of free radicals release on osteoblast-like cells. Initiation of methylmethacrylate was done with benzoyl peroxide and acceleration by N,N-dimethylaniline. Bulk polymerization was done in calibrated test tubes which were left aging until use. Polymers (aged from J1 to J31 days after completion of the polymerization process) were sawed to produce slices. Slices were rinsed in distilled water and free radical release was measured by spectrophotometric titration with p-iodonitrotetrazolium. Saos-2 osteoblast-like cells were cultured in parallel on the slices. Cells appeared to be round and were altered when grown on slices prepared freshly after polymerization. Cytomorphometric analysis of the cell shape (surface area and form-factor polyethylene confirmed that they spread and flatten on slices prepared a long time after polymerization. Free radical release from polymethylmethacrylate cements is a long-lasting event that can induce bone cells alterations in their neighborhood. Two cytotoxic mechanisms were evidenced: (a) polymer slices released a stable toxic component which could be removed by extensive washing; (b) they released free radicals which were still detectable several days after the end of polymerization. The titration curve was a negative exponential.

Bone microarchitecture and bone fragility in men: DXA and histomorphometry in humans and in the orchidectomized rat model.

In men, the risk of fragility fractures increases as bone mineral declines but there is an overlap in the bone mineral density (BMD) measurements between patients with and those without fractures. Biomechanical competence of trabecular (Tb) bone depends on the amount of bone and on microarchitecture. We have developed new histomorphometric methods for evaluating microarchitecture on histological sections. These methods were used in the orchidectomized male rat (ORX--a model of hypogonadism-induced osteoporosis) and on transiliac bone biopsies performed in male osteoporotic patients. ORX rats were studied at 2, 4, 8, and 16 weeks post-ORX. Bone mineral content (BMC) was reduced at 16 weeks. Trabecular bone volume (BV/TV) was significantly decreased from the 4th week. Differences in the sensitivity of the methods were found. Fractal dimension was modified as early as 2 weeks and appeared the most potent descriptor of Tb disorganization. The architectural changes in this model mimic those observed in hypogonadic men. We examined the relationships among BMD, micro-architecture, and vertebral fracture in 108 men with lumbar osteopenia (T-score <-2.5). At least one vertebral fracture was observed in 62 patients and none in 46 patients. After adjusting for age, body mass index (BMI), and BMD, there was no significant difference between the two groups in BV/TV, Tb.Th, and Star volume. In contrast, the mean values of ICI and Tb.Sp were significantly higher whereas Tb.N and nodes were lower in patients with vertebral fracture. Logistic regression analysis showed that ICI, strut analysis, and Tb.N were significant predictors of the presence of vertebral fracture: odds ratios for an alteration of I SD ranged from 1.7 for nodes to 3.2 for ICI. These results strongly suggest that bone Tb microarchitecture is a major and independent determinant of vertebral fracture in men with osteoporosis.

Migration of metal and polyethylene particles from articular prostheses may generate lymphadenopathy with histiocytosis.

Wear particles released from hip or knee prostheses are known to be involved in the fibrohistiocytic membrane interposed between bone and implant. During surgical treatment for pelvic carcinoma (5 cases) and for isolated pseudomalignant lymphadenopathy (4 cases) lymph nodes in 9 patients who had had lower limb articular replacement were harvested. Light microscopy and image analysis of the nodes showed florid endosinusal histiocytosis, predominant in the cortical area. Using Oil Red O staining and polarized light, metal particles and polyethylene particles were detected in the large histiocytes. Scanning electron microscopy with electron backscattering allowed us to localize metal particles and perform elemental microanalysis. Iron, cobalt, chromium, nickel, zirconium, and barium, known to be used in prosthetic and cementing materials, were identified as component of these particles. Large amounts of polyethylene particles appeared in all cases while metal particles were found to be abundant in only 2 cases. Thus, migration of polyethylene debris from the prosthetic site seems to be the major factor in development of the histiocytes induced in satellite lymph nodes.