Normal Mature Bone Research Articles

Osseous choristoma: Report of two cases in oral cavity

Osseous choristoma is the formation of normal mature bone at an abnormal position. These are rare benign tumor-like growth usually asymptomatic except growth or swelling as the clinical presentation. In oral cavity, osseous choristoma is rare and commonly found in posterior third of the tongue. Clinically it is misdiagnosed as malignancy because of its hardness. Clinically, it is also mistaken for other oral cavity lesions, especially those of minor salivary gland lesions of oral cavity. Hence, awareness amongst clinicians, general practitioners and oral pathologists about oral osseous choristomas is necessary. Here, we report two cases of osseous choristoma in the oral cavity located at sites other than tongue.

Strontium ranelate stimulates trabecular bone formation in a rat tibial bone defect healing process.

Systemic administration of strontium ranelate (SrRan) accelerates the healing of bone defects; however, controversy about its action on bone formation remains. We hypothesize that SrRan could affect bone formation differently in normal mature bone or in the bone healing process. Proximal tibia bone defects were created in 6-month-old female rats, which orally received SrRan (625mg/kg/day, 5/7days) or vehicle (control groups) for 4, 8, or 12weeks. Bone samples were analyzed by micro-computed tomography and histomorphometry in various regions, i.e., metaphyseal 2nd spongiosa, a region close to the defect, within the healing defect and in cortical defect bridging region. Additionally, we evaluated the quality of the new bone formed by quantitative backscattered electron imaging and by red picosirius histology. Healing of the bone defect was characterized by a rapid onset of bone formation without cartilage formation. Cortical defect bridging was detected earlier compared with healing of trabecular defect. In the healing zone, SrRan stimulated bone formation early and laterly decreased bone resorption improving the healing of the cortical and trabecular compartment without deleterious effects on bone quality. By contrast, in the metaphyseal compartment, SrRan only decreased bone resorption from week 8 without any change in bone formation, leading to little progressive increase of the metaphyseal trabecular bone volume. SrRan affects bone formation differently in normal mature bone or in the bone healing process. Despite this selective action, this led to similar increased bone volume in both compartments without deleterious effects on the newly bone-formed quality.

Differential regulation of the apoptotic machinery during megakaryocyte differentiation and platelet production by inhibitor of apoptosis protein Livin

Livin is a member of the inhibitor of apoptosis proteins (IAP) family of intracellular antiapoptotic proteins that act by binding and inhibiting caspases. Upon strong apoptotic stimuli, it is then specifically cleaved by caspases to produce a truncated protein (tLivin) with a paradoxical proapoptotic activity. Intriguingly, we have detected robust protein levels of Livin in normal mature bone marrow megakaryocyte (MK) and platelets. To evaluate the potential role of Livin in thrombopoiesis, we used the human BCR-ABL+ cell line, LAMA-84, and cord blood CD34+ cells to induce differentiation toward MKs. Upon differentiation, induced by phorbol myristate acetate and concurrent with increase in Livin protein expression, LAMA-84 cells formed functional platelet-like particles. Livin overexpression in CD34+ progenitor cells induced higher endoreplication in the MKs generated. Furthermore, overexpression of Livin increased the ability of both primary MKs and differentiated LAMA-84 cells to produce functional platelets. In the differentiated LAMA-84 cells, we observed accumulation of proapoptotic tLivin concomitant with increased caspase-3 activity. Downregulation of Livin with small interfering RNA in both leukemic and primary MK cells decreased their ability to produce functional platelets. We suggest that Livin has a role in thrombopoiesis by regulating the apoptotic and antiapoptotic balance in MK endoreplication and platelet production.

The Role of the IAP Livin in Megakaryocytes Differentiation and Thrombopoiesis

AbstractAbstract 3297In this study we observe that Livin plays a role in thrombopoiesis. Livin is a member of the Inhibitor of Apoptosis Proteins (IAP) family of intracellular anti-apoptotic proteins that acts by binding and inhibiting caspases. Previously we found that Livin is unique among the IAP members as upon strong apoptotic stimuli, it is specifically cleaved by caspases to produce a truncated protein (tLivin) that has a paradoxical pro-apoptotic activity. Here we have shown that Livin is expressed in normal mature bone marrow MK and in platelets. Objective:The objective of this study was to evaluate the potential role of Livin in thrombopoiesis. Methods:We studied Livin expression in normal BM using immunohistochemistry staining. We studied the ability of primary MK generated from CD34+progenitor cells over-expressing Livin to produce platelets. While, the human cell line, LAMA-84, was induced by a phorbol myristic acid (PMA) to differentiate to megakaryocytes (MK) to evaluate the potential role of Livin in thrombopoiesis. Results and conclusions:Livin over-expression in CD34+ progenitor cells induced differentiation of these cells into MK and increased the ability of these primary MK to produce platelets. LAMA-84, upon differentiation into MK, produces platelets that are functional and capable of aggregating. This thrombopoiesis was accompanied by increased Livin expression and downregulation of other IAPs: XIAP and Survivin. Our results show that Livin plays a dual role in thrombopoiesis, demonstrating both an anti and pro-apoptotic role in cell activity. Disclosures:No relevant conflicts of interest to declare.

Massive Heterotopic Ossification around the Ankle in a Patient with Macrodactyly of the Foot: A Case Report

A 40-year-old man with macrodactyly of the left great toe presented with progressive enlargement of the ipsilateral ankle and increasing pain after minor trauma. Radiographs and computed tomographic scans of the foot and ankle revealed macrodactyly of the great toe and a large heterotopic ossified mass in the anteromedial aspect of the ankle. The large osseous lesion in the ankle was operatively excised. The lesion was easily excised en bloc after releasing the adhesion and histologically diagnosed as normal mature bone with osteoarthritic changes. Massive heterotopic ossification developing around the ankle in a patient with macrodactyly has not been previously reported. The current case was classified as hyperostotic macrodactyly. Operative treatment relieved the pain and improved the range of the motion of the ankle.

The Role of Livin, An Inhibitor of Apoptosis Protein, in Thrombopoiesis.

The exact mechanism of platelet production is poorly understood. A relationship between activation of the apoptotic cell machinery and the formation of pro-platelets has been established. In this study we show that Livin plays a major role in this process. Livin is a member of the Inhibitor of Apoptosis Protein (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin mRNA and protein are expressed in platelets and Livin was also clearly detected by immunohistochemistry staining in normal mature bone marrow megakaryocytes (MK). Overexpression of Livin was also demonstrated in MK of patients with various hematological diseases such as ITP, MDS, Hodgkin's disease, ET and PV. An in vitro model was established to evaluate the potential role of Livin in thrombopoiesis. The human erythroleukemic cell line, LAMA-84, was induced by a phorbol ester (PMA) to differentiate to MK. Differentiation was characterized by upregulation of the MK marker CD41 from 6% to >60% and downregulation of the erythroid cell marker CD71 from 97% to 70%. Increased cell size, ploidy, and DNA synthesis, all markers of MK differentiation, were detected by flow cytometry. Upon differentiation induced by PMA, LAMA-84 cells formed pro-platelets and produced functional platelets capable of aggregation. This thrombopoiesis was accompanied by Livin overexpression. Moreover, overexpression of Livin by transfection decreased the percentage of the undifferentiated CD71+ cells to 53 % compared to 73 % in control cells (p= 0.007) and increased the production of platelets by two folds compared to control cells. Overexpression of Livin reduced caspase 3 activity and inhibited MK apoptosis. Our results show that anti-apoptotic Livin plays a role in thrombopoiesis possibly by protecting the maturing MK from apoptosis, thus affording more time for efficient platelet production by longer living pro-platelets. We are currently using this new cell line model to further characterize the molecular relationship between apoptosis and thrombopoiesis.

Skeletal affinity of Tc(V)-DMS is bone cell mediated and pH dependent.

In spite of recent advances in bone cellular and molecular biology, there is still a poor correlation between these parameters and data obtained from bone scintigraphy. Diphosphonate derivatives radiolabelled with technetium-99m (Tc-BPs) have long been recognised as bone-seeking agents with an affinity for areas of active mineralisation. However, during clinical trials with a pH-sensitive tumour agent, the pentavalent technetium complex of dimercaptosuccinic acid [Tc(V)-DMS] showed a noticeable osteotropic character only in bone pathologies (bone metastases, Paget's diseases) and lacked accumulation in normal mature bone. To decipher the osteotropic character of Tc(V)-DMS, a study at the cellular level was considered necessary. Moreover, to learn more about the role of Tc bone agents, acid-base regulation by bone tissue or cells was studied. First, biological parameters in body fluid were measured under systemic acidosis, induced by glucose administration, in normal and Ehrlich ascites tumour (EAT)-bearing mice. Then, in vivo biodistribution studies using Tc(V)-DMS or a conventional Tc-BP agent were carried out. The effect of glucose-mediated acidification on the skeletal distribution of the Tc agents in the mice provided valuable hints regarding the differential mediation of bone cells in skeletal tissue affinity for the agents. Thereafter, in vitro studies on osteoblast and osteoclast cells were performed and the comparative affinity of Tc(V)-DMS and Tc-BP was screened under diverse acidification conditions. Moreover, studies were also carried out on acid-base parameters related to the cellular uptake mechanism. Very specific pH-sensitive Tc(V)-DMS accumulation only in the osteoclastic system was detected, and use of Tc(V)-DMS in the differential detection of osteoblastic and osteoclastic metastases is discussed.

Use of the Ilizarov external fixator in the treatment of patients with Ollier's disease.

The authors present the results of treatment of 10 patients with Ollier's disease using the Ilizarov technique. The Ilizarov device was used to treat leg length discrepancy and to enhance the conversion of chondroma cartilage into normal mature bone, with no curettage and bone grafting. The mean duration of treatment was 9.4 months. This technique was highly efficient in treating the disease. It led to conversion of the abnormal cartilage into histologically mature bone in all patients. Some complications were seen, such as decreased knee mobility, which required prolonged use of the device. The Ilizarov technique is successful in treating patients with Ollier's disease despite some complications and the difficulty in using the technique.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.

Separation of normal mature bone marrow plasma cells.

A three-phase purification process for the separation of normal bone marrow plasma cells is described. Material was obtained from haematologically normal humans and baboons. Known monoclonal and polyclonal B cell activators were avoided both in vivo and in vitro. In Phase I, bone marrow fragments were separated from cell suspensions by buoyant density methods. Marrow fragments were shown to be richer in plasma cells than the corresponding marrow cell suspensions. Phase II consisted of culturing fragments by a simple suspension method in which a selective affinity of plasma cells and marrow stromal cells resulted in further concentration of plasma cells with discharge of haemopoietic elements. Maximum concentration of plasma cells occurred within 7 d. In phase III, fragments were disaggregated with trypsin, and the marrow stromal cells were removed by their adherence properties. The resulting non-adherent fraction comprised approximately 85% plasma cells. The process allows for direct quantitative evaluation of normal plasma cell physiology in vitro.