The alkaline phosphatase activity of pre-osseous tibial cartilage of rachitic bone stored in the deep freeze for two weeks at -25 degrees centigrade was only slightly less than that of fresh controls from the same animals. The deep frozen pre-osseous tissue did not calcify in in vitro calcifying media containing either inorganic phosphorus or organic phosphate ester. The fresh controls calcified equally well in both media. In addition, after deep-freeze storage the tissue hydrolysed the organic phosphate to the same degree as did the fresh tissue. Bones heated at 65 degrees centigrade will calcify in vitro after calcium chloride treatment despite the destruction of phosphatase activity. It appears unlikely that a relationship exists between alkaline phosphatase and the minimal system required for calcification of pre-osseous cartilage in vitro. These findings do not exclude the possibility that alkaline phosphatase plays some critical role in vivo

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic

1. The use of a protein-free synthetic medium has provided a new technical approach to the study of fracture healing in vitro. 2. The tibiae of fourteen-day embryonic chicks were cut in half in the middle of the shaft, the fragments were placed in apposition and the explants grown in vitro for up to sixteen days. The process of bone repair was studied by means of histology and biochemical estimations. 3. The rate of growth in length of fractured bones was greater in an atmosphere containing 50 per cent of oxygen than in one with 20 per cent oxygen, thus emphasising the importance of an adequate oxygen supply for the regeneration of osteogenic cells. 4. The effect of varying the concentration of glucose in the medium was investigated. Two milligrams of glucose per millilitre was the most favourable for healing; higher levels caused fibroblastic changes in the cartilage cells and inhibited the proliferation of osteogenic cells at the fracture site. 5. Histological examination showed that many of the phenomena that occur in the repair of fractures in vivo can be reproduced in vitro in synthetic medium. Similar results were obtained whether the fracture was made in whole bones or in isolated shafts from which the cartilaginous ends had been removed; the latter are more favourable for biochemical study

Ischaemia kills osteocytes, but opinions differ as to how long they can survive. These differences are due to the varying methods of inducing ischaemia, and to the different criteria for diagnosing cell death. Using rabbit bone and a technique of in vitro ischaemia at 37 degrees C, we have shown by electron microscopy that, after up to two hours, the changes which occur are probably reversible; after four hours, the cells were irreversibly damaged. This difference could not be detected by light microscopy. After 24 hours of ischaemia, most lacunae were empty or contained only osteocyte debris. We conclude that osteocytes suffer irreversible damage after in vitro ischaemia of about two hours, which is much the same response as that of most other mammalian cells

Our aim was to determine whether in vitro studies would detect differences in the cellular response to wear particles of two titanium alloys commonly used in the manufacture of joint replacement prostheses. Particles were of the order of 1 μm in diameter representative of those found adjacent to failed prostheses. Exposure of human monocytes to titanium 6-aluminium 4- vanadium (TiAlV) at concentrations of 4 x 10. 7. particles/ml produced a mean prostaglandin E. 2. release of 2627.6 pM; this was significantly higher than the 317.4 pM induced by titanium 6-aluminium 7-niobium alloy (TiAlNb) particles (p = 0.006). Commercially-pure titanium particles induced a release of 347.8 pM. In addition, TiAlV stimulated significantly more release of the other cell mediators, interleukin-1, tumour necrosis factor and interleukin-6. At lower concentrations of particles there was less mediator release and less obvious differences between materials. None of the materials caused significant toxicity. The levels of inflammatory mediators released by phagocytic cells in response to wear particles may influence the amount of periprosthetic bone loss. Our findings have shown that in vitro studies can detect differences in cellular response induced by particles of similar titanium alloys in common clinical use, although in vivo studies have shown little difference. While in vitro studies should not be used as the only form of assessment, they must be considered when assessing the relative biocompatibility of different implant materials

Biofilm-associated infections in wounds or on implants are difficult to treat. Eradication of the bacteria is nearly always impossible, despite the use of specific antibiotics. The bactericidal effects of high-energy extracorporeal shock waves on Staphylococcus aureus have been reported, but the effect of low-energy shock waves on staphylococci and staphylococcal biofilms has not been investigated. In this study, biofilms grown on stainless steel washers were examined by electron microscopy. We tested ten experimental groups with Staph. aureus-coated washers and eight groups with Staph. epidermidis. The biofilm-cultured washers were exposed to low-energy shock waves at 0.16 mJ/mm. 2. for 500 impulses. The washers were then treated with cefuroxime, rifampicin and fosfomycin, both alone and in combination. All tests were carried out in triplicate. Viable cells were counted to determine the bactericidal effect. The control groups of Staph. aureus and Staph. epidermidis revealed a cell count of 6 × 10. 8. colony-forming units/ml. Complete eradication was achieved using the combination of antibiotic therapy (single antibiotic in Staph. aureus, a combination in Staph. epidermidis) and shock wave application (p < 0.01). We conclude that shock waves combined with antibiotics could be tested in an in vitro model of infection

Hydroxyapatite-coated standard anatomical and customised femoral stems are designed to transmit load to the metaphyseal part of the proximal femur in order to avoid stress shielding and to reduce resorption of bone. In a randomised in vitro study, we compared the changes in the pattern of cortical strain after the insertion of hydroxyapatite-coated standard anatomical and customised stems in 12 pairs of human cadaver femora. A hip simulator reproduced the physiological loads on the proximal femur in single-leg stance and stair-climbing. The cortical strains were measured before and after the insertion of the stems. Significantly higher strain shielding was seen in Gruen zones 7, 6, 5, 3 and 2 after the insertion of the anatomical stem compared with the customised stem. For the anatomical stem, the hoop strains on the femur also indicated that the load was transferred to the cortical bone at the lower metaphyseal or upper diaphyseal part of the proximal femur. The customised stem induced a strain pattern more similar to that of the intact femur than the standard, anatomical stem

We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with β-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH). 2. D. 3. , a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and mRNA levels in osteoblast-related genes were enhanced. Our results suggest that the haemarthrosis induced by injury to the anterior cruciate ligament contains osteoprogenitor cells and is a potential alternative source for cell-based treatment in such injury

Particulate wear debris can induce the release of bone-resorbing cytokines from cultured macrophages and fibroblasts in vitro, and these mediators are believed to be the cause of the periprosthetic bone resorption which leads to aseptic loosening in vivo. Much less is known about the effects of particulate debris on the growth and metabolism of osteoblastic cells. We exposed two human osteoblast-like cell lines (SaOS-2 and MG-63) to particulate cobalt, chromium and cobalt-chromium alloy at concentrations of 0, 0.01, 0.1 and 1.0 mg/ml. Cobalt was toxic to both cell lines and inhibited the production of type-I collagen, osteocalcin and alkaline phosphatase. Chromium and cobalt-chromium were well tolerated by both cell lines, producing no cytotoxicity and no inhibition of type-I collagen synthesis. At the highest concentration tested (1.0 mg/ml), however, chromium inhibited alkaline phosphatase activity, and both chromium and cobalt-chromium alloy inhibited osteocalcin expression. Our results clearly show that particulate metal debris can modulate the growth and metabolism of osteoblastic cells in vitro. Reduced osteoblastic activity at the bone-implant interface may be an important mechanism by which particulate wear debris influences the pathogenesis of aseptic loosening in vivo

The haematoma occurring at the site of a fracture is known to play an important role in bone healing. We have recently shown the presence of progenitor cells in human fracture haematoma and demonstrated that they have the capacity for multilineage mesenchymal differentiation. There have been many studies which have shown that low-intensity pulsed ultrasound (LIPUS) stimulates the differentiation of a variety of cells, but none has investigated the effects of LIPUS on cells derived from human fracture tissue including human fracture haematoma-derived progenitor cells (HCs). In this in vitro study, we investigated the effects of LIPUS on the osteogenic activity of HCs. Alkaline phosphatase activity, osteocalcin secretion, the expression of osteoblast-related genes and the mineralisation of HCs were shown to be significantly higher when LIPUS had been applied but without a change in the proliferation of the HCs. These findings provide evidence in favour of the use of LIPUS in the treatment of fractures

The diffusion of Fucidin, gentamicin, and clindamycin from acrylic cement was tested in an in vitro system. The activity of Fucidin was very short-lived and only against gram-positive organisms; gentamicin inhibited gram-positive and gram-negative organisms for twenty-two and eleven days respectively; clindamycin had significant action only against gram-positive organisms and retained some activity for fifty-six days. We suggest that the destruction of organisms in the tissues is more likely to be achieved by topical and intravenous administration of antibiotics during the operation than by incorporation of antibiotic in the cement

Aims

This study aimed to determine if macrophages can attach and directly affect the oxide layers of 316L stainless steel, titanium alloy (Ti6Al4V), and cobalt-chromium-molybdenum alloy (CoCrMo) by releasing components of these alloys.

We isolated multilineage mesenchymal progenitor cells from haematomas collected from fracture sites. After the haematoma was manually removed from the fracture site it was cut into strips and cultured. Homogenous fibroblastic adherent cells were obtained. Flow cytometry revealed that the adherent cells were consistently positive for mesenchymal stem-cell-related markers CD29, CD44, CD105 and CD166, and were negative for the haemopoietic markers CD14, CD34, CD45 and CD133 similar to bone-marrow-derived mesenchymal stem cells. In the presence of lineage-specific induction factors the adherent cells could differentiate in vitro into osteogenic, chondrogenic and adipogenic cells. Our results indicate that haematomas found at a fracture site contain multilineage mesenchymal progenitor cells and play an important role in bone healing. Our findings imply that to enhance healing the haematoma should not be removed from the fracture site during osteosynthesis

In vitro studies on isolated bone cells were undertaken to investigate the presence of transplantation (histocompatibility) antigens. Bone cells were cultured with allogeneic lymphocytes and exposed to cytotoxic sera containing antibodies against transplantation antigens, to determine their antigenic profile. Preliminary results suggest that bone cells may not express lymphocyte stimulating antigens in an active form, at least after the isolation procedure performed. On the other hand, bone cells were killed by cytotoxic antibodies in a specific way, providing evidence for the presence of serologically defined (SD) transplantation antigens on the cell surface. Additional studies with absorbed sera suggest "sharing" of histocompatibility antigens between bone cells and lymphocytes. The relevance of the surface antigens of bone cells to clinical fields such as bone allotransplantation, susceptibility to various orthopaedic diseases and skeletal sarcomata is discussed

We have measured the effect of age on the rate of outgrowth of cells from human trabecular bone, using a quantitative dye-binding technique. In cultures supplemented with autologous serum, there were significant negative correlations between the age of the donor and both the proportion of fragments from which outgrowths were seen after 7 days (r = -0.70; p < 0.001) and the total cell number after 14 days (r = -0.78; p < 0.005). The autologous serum supported greater cell proliferation than did fetal calf serum in all subjects regardless of age. Taken with previous observations that the in vitro growth kinetics of passaged human bone cells are independent of age, our results show that the number of proliferative precursor cells on trabecular-bone surfaces is higher in younger subjects. There is a marked decrease in precursor numbers in the second and third decades of life to a level which is maintained into old age

We have investigated whether the particle-stimulated release of inflammatory cytokines from human primary macrophages in vitro was dependent upon the type of bone cement used. Particles of clinically relevant size were produced from Palacos R without radio-opacifier, Palacos R with BaSO. 4. , Palacos R with ZrO. 2. and from CMW3 which contains BaSO. 4. All four preparations produced significantly greater release of tumour necrosis factor alpha, interleukin-6 and interleukin-1 beta than a negative control but there were no significant differences between them. The differences in the ability to stimulate bone resorption and in clinical performance between proprietary bone cements previously recorded are not explained by the release of the cytokines most commonly implicated in osteolysis

We have studied in vitro the effect of a hydroxyapatite (HA) tricalcium phosphate material coated with hepatocyte growth factor (HA-HGF) on cell growth, collagen synthesis and secretion of metalloproteinases (MMPs) by human osteoblasts. Cell proliferation was stimulated when osteoblasts were incubated with untreated HA and was further increased after exposure to HA-HGF. The uptake of [. 3. H]-proline was increased after treatment with HA. When osteoblasts were exposed to HA-HGF, collagen synthesis was increased with respect to HA. The secretion of MMPs in control cells was undetectable, but in HA and HA-HGF cells MMP 2 and MMP 9 were clearly synthesised. Our results suggest that HA can promote osteoblast activity and that HGF can further increase its bioactivity

Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al. 2. O. 3. and ZrO. 2. ) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 μm). Al. 2. O. 3. was compared with ZrO. 2. at 0.6 μm and with HDP at 4.5 μm. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al. 2. O. 3. particles. When comparing Al. 2. O. 3. and ZrO. 2. at 0.6 μm, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-α also increased with the size and concentration of Al. 2. O. 3. particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 μm and 350 particles per cell (p < 0.05). Release of TNF-α was higher with HDP than with Al. 2. O. 3. particles at 4.5 μm. However, we did not detect any significant difference in the release of TNF-α between Al. 2. O. 3. and ZrO. 2. at 0.6 μm (p > 0.05). We saw no evidence of release of interleukin-1α or interleukin-1ß after exposure to ceramic or HDP particles

Particulate prosthetic materials are often studied by adding them to monocytic cells in vitro and measuring the release of cytokines as an indicator of their inflammatory potential. Endotoxin is known to be a contaminant of particle preparations and also stimulates the release of cytokines. It is usual to use a proprietary endotoxin test to avoid erroneous results. Four different formulations of cement were found to be free from endotoxin using standard, gelclot tests but stimulated different levels of release of cytokines from macrophages. These differences were explained when a more sensitive, kinetic endotoxin assay showed that release correlated with minor contamination with endotoxin. In a repeat experiment using cement particles with low or undetectable levels of endotoxin by kinetic assay, differences in the ability of the formulations to stimulate the release of cytokines were not seen. Endotoxin is adsorbed on to the surface of particles and it is this combination which stimulates increased release of cytokines. In both the above methods for determination of endotoxin, the water in which the particles had been soaked was examined rather than the particles directly. Further investigations showed that a kinetic assay directly on a particle suspension is the most sensitive method to measure contamination with endotoxin

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH). 2. vitamin D. 3. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur. Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH). 2. vitamin D. 3. was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE. 2. Exogenous PGE. 2. (10. −8. to 10. −6. M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE. 2. by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening