Investigating the effects of bone cement, cyanoacrylate glue and marine mussel adhesive protein from Mytilus edulis on human osteoblasts and fibroblasts in vitro

Bone cement is a widely used standard fixation substance in Orthopaedic Surgery. Cyanoacrylate glue is available for wound closure to supplement suturing. The mussel adhesive protein extracted from Mytilus edulis (Cell-Tak©, BD Biosciences, Heidelberg, Germany) is an experimental fixation device used for in vitro purposes of cell adhesion.

The aim of this study is to introduce a cell culture model investigating the effects of commonly applied and experimental glues on human fibroblasts and osteoblasts in vitro. Cells cultured without additives served as a control group. Microscopic examination was performed to evaluate the morphologic changes. An apoptosis test (Apo-Tag©, Chemicon International, Temecula, CA, U.S.A.) was applied to determine the rate of natural cell death at the end of the study.

It could be demonstrated that morphological changes in bone cement are different in fibroblasts and osteoblasts. Osteoblasts seem to grow on bone cement and develop an orderly formation. Fibroblasts grow in a confluent monolayer around bone cement but do not adhere to the cement itself. This is a desirable effect since most Orthopaedic applications aim at osteointegration as opposed to fibrous tissue overgrowth. Apoptosis attributed to bone cement is comparable to the respective natural rate of apoptosis. Cyanoacrylate glue and the mussel adhesive protein lead to an almost complete apoptosis in the investigated cells. Their routine application should be avoided. The developed cell culture model seems appropriate for performing further investigations.     

Influence of disodium ethane-1-hydroxy-1,1-diphosphonate on vitamin D metabolism in rats

Summary The metabolism and the organ distribution of double labelled vitamin D₃ (1,2-³H-4-¹⁴C-cholecalciferol) has been studied in rats in which the bone mineralization and the intestinal calcium absorption have been inhibited by a large pose (10 mg P/kg s.c. for 7–14 days) of disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The most striking difference found was a reduced accumulation of radioactive cholecalciferol and its metabolites in the kidney of EHDP-treated rats. It is unlikely that this effect was due to an unspecific alteration of the functional renal tissue since blood urea, glomerular filtration rate and renal plasm a flow remained unaltered by this dose of EHDP. The EHDP-treated rats were able to form the metabolite eluted with peak IV of the silicic acid chromatographic system, that is 25-hydroxycholecalciferol. In these vitamin D repleted rats fed a high calcium diet, the tritium deficient metabolite eluted with peak V (1,25-dihydroxycholecalciferol) was only found in the intestinal mucosa of both control and EHDP groups three days after the injection of radioactive cholecalciferol, and this in a very small amount. Therefore no definitive conclusion can be drawn as to a possible interference of EHDP treatment on the production of 1,25-dihydroxycholecalciferol. The change in the renal metabolism of vitamin D in rats treated with a rachitogenic dose of EHDP may be caused by the modifications of the calcium metabolism brought about by the diphosphonate. Its relation, if any, with the decreased calcium absorption remains to be established.     

Transmission electron microscopic demonstration of vimentin in rat osteoblast and osteocyte cell bodies and processes using the immunogold technique

Background: The immunogold labeling technique and transmission electron microscopy were used to demonstrate the expression and position of the intermediate filament vimentin in rat osteoblast and osteocyte cell bodies and cell processes. Conventional light and transmission electron microscopic studies of bone cells demonstrated adjacent cell linkage to be mediated by osteoblast and osteocyte processes present within the canalicular system traversing the bone matrix. The cell processes were filled with densely packed filaments, many of which have been shown previously to be actin microfilaments. The appearance, however, of 10 nm diameter filaments in some cell processes and the fact that the intermediate filament vimentin has been defined in many cells of mesenchymal origin raised the possibility that some of these filaments might be vimentin. The ultrastructural colloidal gold immunochemical technique allowed for demonstration in situ of the expression of vimentin filaments plus accurate definition of their position. Methods: The studies were performed in newborn rat femoral and tibial diaphyseal cortical bone and in 1-week-old repair bone from 2.4 mm diameter defects made through the lateral cortex in 6-week-old rat femurs and tibias. The bone tissues for the immunochemical study were fixed in 1% glutaraldehyde, 4% paraformaldehyde, and 0.1 M phosphate buffer (pH 7.4) for 2 days. Decalcification was performed in 6% EDTA for 2–3 days. Infiltration involved use of Lowicryl resin K4M, and the embedding and curing processes were performed in a cryostat with temperatures ?30°C. An antivimentin monoclonal antibody was used for labeling using the postembedding technique. Effective antibody dilutions ranged from 1:10 to 1:200, with the dilutions of 1:25 and 1:100 showing the best combination of filament labeling with the least matrix background. The grids were exposed to 10 nanometer gold colloid conjugated goat anti-mouse IgM for demonstration of binding. Results: Vimentin immunolabeling was defined clearly in relation to filaments within the osteoblast and osteocyte cell body cytoplasm, throughout the entire length of the osteoblast and osteocyte cell processes, and in close relationship to the intercellular gap junctions which were present within the cell processes both close to the cell bodies and within the canaliculi well away from them. Conclusions: Immunogold labeling demonstrates the presence of the intermediate filament vimentin in osteoblast and osteocyte cell bodies and processes of rat bone. Vimentin distribution is not concentrated to specific areas, is present throughout the extent of the bodies and processes, and is seen immediately adjacent to gap junctions. © 1995 Wiley-Liss, Inc.     

Degradable poly(anhydride ester) implants: effects of localized salicylic acid release on bone

Degradable poly(anhydride ester) implants in which the polymer backbone breaks down into salicylic acid (SA) were investigated. In this preliminary work, local release of SA from the poly(anhydride esters), thus classified as ‘active polymers', on healthy bone and tissue was evaluated in vivo using a mouse model. Degradable polyanhydrides that break down into inactive by-products were used as control membranes because of their chemical similarity to the active polymers. Small polymer squares were inserted over the exposed palatal bone adjacent to the maxillary first molars. Active polymer membranes were placed on one side of the mouth, control polymers placed on the contra lateral side. Intraoral clinical examination showed that active polymer sites were less swollen and inflamed than control polymer sites. Histopathological examination at day 1 showed essentially no difference between control and active polymers. After 4 days, active polymer sites showed epithelial proliferation to a greater extent than the polyanhydride controls. After 20 days, active polymer sites showed greater thickness of new palatal bone and no resorptive areas, while control polymer sites showed less bone thickness as well as resorption including lacunae involving cementum and dentine. From these preliminary studies, we conclude that active polymers, namely poly(anhydride esters), stimulated new bone formation.     

Morphomechanics of the humero-ulnar joint: II. Concave incongruity determines the distribution of load and subchondral mineralization

Background: A deeper joint socket (concave incongruity) is found at most angles of flexion of the humero-ulnar joint and maintained over a wide range of physiological loading. It is, however, unclear how far this incongruity affects the distribution of load and subchondral mineralization of this joint as compared with a congruous configuration. Methods: Two nonlinear, axisymmetrical finite element models with two cartilage layers were constructed, one congruous and one incongruous, with a joint space of realistic magnitude. The distribution of subchondral mineralization was determined by computed tomography osteoabsorptiometry in the same six specimens that were investigated in the first part of the study, and compared with the biomechanical data obtained there and the predictions of the models. Results: In the congruous case, the center of the socket is highly loaded, whereas the periphery does not experience mechanical stimulation. A central bone density maximum is predicted. With concave incongruity the position of the contact areas shifts from the joint margin towards the center as the load increases, and the peak stresses are considerably lower. A bicentric ventro-dorsal distribution pattern of subchondral mineralization is predicted, and this is actually found in the six specimens. Conclusions: Concave incongruity is shown to determine load transmission and subchondral mineralization of the humero-ulnar joint. It is suggested that this shape leads to a more even distribution of stress, provides intermittent stimulation of the cartilaginous tissue, and has beneficial effects on the metabolism, nutrition, and lubrication of the articular cartilage during cyclic loading. © 1995 Wiley-Liss, Inc.     

HIGH-GRADE SURFACE OSTEOSARCOMA OF THE LEFT ILIUM A Case Report and Review of the Literature

A rare case of high-grade surface osteosarcoma of the left ilum is reported. Trephine biopsy performed on a 31-year-old woman suffering from a huge tumor of the left buttock revealed high-grade osteosarcoma, and hemipel-vectomy was undertaken. The surgical specimen showed that the tumor was present on the surface of the left ilium. Ten months after the operation, the patient died of brain metastasis. From these results, we considered this case to be one of high-grade surface osteosarcoma. ACTA PATHOL JPN 38: 235 -240, 1988.     

Determination of specific oxygen uptake rates in human hematopoietic cultures and implications for bioreactor design

Oxygen plays an important role in the cultivation of primary cellsex vivo. In this study, we used hermetically sealed tissue culture well inserts equipped with oxygen electrodes to measure the oxygen utilization of cultured human bone marrow mononuclear cells (BM MNCs). The oxygen uptake rate (OUR) of BM MNCs was determined during a 14-day culture in which both adherent and nonadherent cells were present. Early in the culture, the cells exhibited very low OURs. The specific OURs (uptake rate per cell) were at approximately 0.005 μmol/10⁶ cells/hr shortly after the initiation of culture. The OUR then increased as the cultures developed. After about 8 to 10 days of cultivation the specific OURs had increased to 0.038±0.006 and 0.025±0.005 μmol/10⁶ cells/hr for adherent and nonadherent cells, respectively, after which no further increase was observed. Based on these oxygen uptake rate data, a mathematical model of oxygen diffusion was formulated and use to investigate issues associated with hematopoietic bioreactor design, including initial cell density, medium depth, reactor configuration, and oxygen partial pressure.In situ OUR measurements confirmed predicted oxygen limitations based on the mathematical model and the experimentally determined OURs. High-density hematopoietic cultures present design challenges in terms of sufficient and uniform delivery of oxygen to an active hematopoietic culture. These challenges can be met by using parallelplate bioreactors with thin liquid layers.     

Cytological examination and cellular composition of bone marrow in healthy, adult, cynomolgus monkeys (Macaca fascicularis)

The purpose of this study was to evaluate the cellular composition of the bone marrow of cynomolgus monkeys (Macaca fascicularis). Femoral bone marrow smears from 23 healthy, adult animals (11 males and 12 females) were examined. For each animal, three femoral bone marrow smears were prepared immediately after euthanasia and stained with May-Grünwald-Giemsa. On two of the three smears available, and for each of these smears, a 500-cell differential count was performed and the myeloid: erythroid (M:E) ratio established. The M:E ratio for males varied from 0.67∶1.00 to 1.85∶1.00 with a mean of 1.03∶1.00 and for females from 0.67∶1.00 to 1.63∶1.00 with a mean of 1.02∶1.00. The mean percentage of granulocytic, lymphocytic, plasmacytic and erythroid series was 47.60, 5.44, 1.45 and 46.05% for males and 47.28, 5.12, 1.49 and 46.28% for females. No significant differences were noted between males and females. All cell lines were well represented and showed normal maturation in both sexes. Megakaryocytes were adequate in number and morphology in all animals. Cynomolgus monkeys showed a bone marrow composition similar to rhesus monkeys (Macaca mulatta). Cytological examination of bone marrow was found to be a simple and rapid procedure, well suited to the toxicological research environment. It provided excellent information on cell distribution, morphology and maturation of the haematopoietic system.     

Expression of Functional Molecules in Non-Hodgkin's Lymphoma

It is well known that non Hodgkin's lymphoma (NHL) cells express various antigens which are normally involved in a variety of functions. In addition, NHL is diverse in its proliferative capacity. To investigate the relation between these factors and the clinical picture, 45 cases of NHL were studied by immunohistochemistry using snap-frozen materials obtained before therapy. Reactivities with 27 monoclonal antibodies were examined and the results were correlated with clinical findings. The expression of surface μ and CAM-1 in B-NHLs and CD25 in T-NHLs were significantly associated with bone marrow involvement. B-NHLs without expression of CD21(B2) and T-NHLs with CD25 were seen more frequently in cases with a LDH value of over 500 units/ml. The positivity rate of Ki-67 on B-NHLs was correlated with serum LDH value, NHL histologic classification, and overall survival. These data indicate that immunophenotyping and determination of the proliferative capacity of NHL are of value not only for confirmation of the histopathologic classification of the tumor but also for assessment of clinical behavior.