Alumina particles influence the interactions of cocultured osteoblasts and macrophages.

The purpose of the current study was to evaluate the effects of alumina particles on secretion of several cytokines involved in bone resorption in cocultures of macrophages and osteoblasts. To distinguish the contribution of each individual cell type, we have established a heterologous in vitro system that makes use of mouse J774 cells and primary cultured human osteoblasts. J744 cells decreased the production of TNF-alpha when they were cocultured with osteoblasts. Treatment of J744 cells with alumina particles increased TNF-alpha secretion, but the induction was lower when cells were cocultured with osteoblasts. Secretion of IL-6 by J744 cells was very low, and increased in the presence of osteoblasts. Alumina particles were only able to stimulate the release of IL-6 by J744 cells when cells were cocultured with osteoblasts. On the other hand, incubation of osteoblasts with alumina particles enhanced the release of IL-6 and GM-CSF. Coculturing osteoblasts with J744 cells induced them to release IL-6 and GM-CSF, and treatment with alumina further increased the secretion of both mediators by osteoblasts. According to these in vitro results, it seems rather plausible that alumina particles are able to initiate an inflammatory response in vivo.

Concentration-dependent effects of titanium and aluminium ions released from thermally oxidized Ti6Al4V alloy on human osteoblasts.

Thermal oxidation treatments of Ti6Al4V, at 500 and 700 degrees C, for 1 h result in the formation of an outer "ceramic" layer of rutile, which enhances osteoblast response. In the present study, we have measured in vitro Ti and Al ion release from Ti64 alloy in the as-received state and after thermal oxidation treatments at 500 or 700 degrees C, to culture medium under standard cell-culture conditions. Concentrations of both Ti and Al released from both thermal oxidation treatments were lower than from polished alloy. Al was released from the treated or untreated surfaces in substantially lower extent than Ti. Titanium and aluminium ions affected primary human osteoblast proliferation, metabolic activity, and differentiation in a dose-dependent manner. Treatments with individual Ti or Al metal ions in similar concentration ranges than released from the surfaces did not alter osteoblast response, which also remained unaffected after treatments with combinations of Ti plus Al applied in the proportional relations than detected in ion-release experiments. We then selected higher concentrations of Ti that impaired osteoblast proliferation and differentiation, while the proportional lower concentrations of Al did not alter osteoblast behavior. In spite of its inert character, it was found that Al significantly enhanced the deleterious effect of Ti on osteoblast differentiation. Therefore, thermal oxidation treatments of Ti6Al4V alloy may improve the biocompatibility of the alloy by reducing both Ti and Al release, and thus attenuating ion-mediated interference with osteoblast differentiation.

Osteoblast response to thermally oxidized Ti6Al4V alloy.

We have recently reported that thermal oxidation treatments of Ti6Al4V at 500 degrees and 700 degrees C for 1 h result in the formation of an outer "ceramic" layer of rutile that do not decrease the high in vitro corrosion resistance of the alloy. In the present work, surface roughness was measured and found marginally increased as a consequence of oxidation of the alloy at 700 degrees C, but not at 500 degrees C. We have evaluated the biocompatibility of the oxidized surfaces, by assessing cell adhesion, proliferation, and differentiation of primary cultures of human osteoblastic cells. Compared with polished alloy, both thermal treatments increased osteoblast adhesion measured as cell attachment, beta1 integrin and FAK-Y397 expression, as well as cytoskeletal reorganization. Compared with treatment at 500 degrees C, thermal oxidation at 700 degrees C enhanced cell adhesion. Treatment at 700 degrees C transiently impaired cell proliferation and viability, which were not altered in alloys oxidized at 500 degrees C. Several markers of osteoblastic differentiation such as procollagen I peptide, alkaline phosphatase, osteocalcin, and mineralized nodule formation were found either unaffected or differentially increased by alloys treated either at 500 degrees or 700 degrees C. In addition, thermal oxidation at 700 degrees C also increased osteoprotegerin secretion. Taken together, our results indicate that thermal oxidation treatments at 500 degrees or 700 degrees C for 1 h improve the in vitro biocompatibility of Ti6Al4V.

Abnormal tyrosine phosphorylation linked to a defective interaction between ADP and its receptor on platelets.

ADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein alpha-subunits, and with rabbit antibodies specific for Gialpha1-3, and Galpha12 all showed normal reactivity when tested against the patient's platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and alpha-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.

Effects of polyethylene and alpha-alumina particles on IL-6 expression and secretion in primary cultures of human osteoblastic cells.

The effect of two biomaterials, polyethylene and alpha-alumina, on interleukin-6 (IL-6) secretion and expression has been studied in human osteoblasts in primary culture. Human osteoblastic cells were derived from fresh trabecular bone explants removed during total knee arthroplasty. On reaching confluence, cells were subcultured in 6 well plates; the resulting subcultures were incubated until confluence and polyethylene or alpha-alumina particles were added to some while the rest were left as controls. The IL-6 mRNA levels were assessed by reverse transcription (RT) followed by polymerase chain reaction (PCR). IL-6 secretion was measured in the conditioned medium. The IL-6 expression was higher in the presence of both biomaterials. Maximum expression occurred in response to a dose of 50 mg particles well with both biomaterials and was greater after polyethylene particle addition than after alpha-alumina particle addition at this dose. The maximum IL-6 secretion elicited by alpha-alumina was produced at 10 mg particles well while maximum response with polyethylene required 50 mg well. At a dose of 10 mg/well, alpha-alumina particles induced more secretion than 10 mg of polyethylene particles. Nevertheless, at a dose of 50 mg/well maximum secretion was produced with polyethylene particles. In conclusion and in our experimental conditions, polyethylene as well as alpha-alumina increased both the expression and the secretion of IL-6 in human osteoblastic cells in primary culture and stimulation from polyethylene appears stronger than that from alpha-alumina at the same dose.

In vitro corrosion behaviour and osteoblast response of thermally oxidised Ti6Al4V alloy.

In this work, the influence of thermal oxidation treatments of Ti6Al4V at 500 degrees C and 700 degrees C for 1 h on the in vitro corrosion behaviour and osteoblast response is studied. The potential of these treatments, aimed to improve the wear surface performance as biomaterial, relies in the formation of an outer "ceramic" layer of rutile. The corrosion behaviour was evaluated in simulated human fluids by electrochemical impedance spectroscopy and anodic polarisation tests. The effect of these thermal oxidation treatments on osteoblastic behaviour was studied in primary cultures of human osteoblastic cells. Results show that thermal oxidation treatments do not decrease the high in vitro corrosion resistance of the Ti6Al4V alloy. Osteoblast adhesion studies indicate that thermal oxidation treatments do not impair the material biocompatibility. Moreover, the thermal oxidation at 700 degrees C enhances the in vitro osteoblastic cell attachment compared to the thermal oxidation at 500 degrees C.

Gracilis muscle transplant for correction of traumatic anal incontinence: Report of a case.

We report a case of traumatic anal incontinence successfully treated by the transplant of a gracilis muscle sling, using the technique described by Pickrell et al. in 1952. Although gracilis muscle transplant has been used in the treatment of congenital anal incontinence, its use in traumatic cases has not been widely accepted. Instead, many techniques offering uncertain results have been employed. We believe that Pickrell's technique is a worthwhile procedure in the presence of traumatic anal incontinence, particularly with noniatrogenic large perineal wounds, and that establishment of a temporary colostomy immediately after the injury, together with use of surgical steel sutures and antibiotics, is very helpful in averting posttransplant infection.

Double heterozygosity of the GPIIb gene in a Swiss patient with Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) results from a qualitative or quantitative defect of GPIIb-IIIa complexes (integrin alphaIIbbeta3). the fibrinogen receptor on platelets. This integrin plays a critical role in platelet aggregation. In this report we describe the molecular abnormalities of a patient with clinical and laboratory findings typical of type I Glanzmann's thrombasthenia. SDS-PAGE with Western blotting revealed an absence of GPIIb but small amounts of normally migrating GPIIIa in his platelets. A non-radioactive PCR-SSCP procedure and direct sequence analysis of PCR-amplified DNA fragments showed the patient to be a compound heterozygote for mutations in the GPIIb gene. A single point mutation (G to A) at nucleotide 1064 of the cDNA derived from the mother's allele led to a Glu324 to Lys amino acid substitution in GPIIb. It was responsible for a MscI restriction site in exon 12 of the GPIIb gene. This amino acid substitution changes the electric charge between the second and third Ca++-binding domains of GPIIb. The second mutation was inherited from his father and is in exon 18 of the GPIIb gene. It was a T --> C base transition at position 1787 of GPIIb cDNA and results in a Ile565 to Thr substitution. The two GPIIb mutations identified in this study will provide new information on GPIIb-IIIa structure and biosynthesis.