L. Munuera

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.

Calcium phosphate-based particles influence osteogenic maturation of human mesenchymal stem cells

Biphasic calcium phosphates (BCPs) consist of a mixture of hydroxyapatite and beta-tricalcium phosphate and are recommended as alternatives or additives to autogenous bone for orthopaedic and dental applications. There is clinical evidence showing particle release from bioceramics, which might impair the ability of human mesenchymal stem cells (hMSC) from bone marrow to proliferate or mature into a functional osteoblast phenotype. This study analyses the influence of BCP particles and their precursors, calcium-deficient apatite (CDA) particles, on in vitro hMSC behaviour. Both types of particles were efficiently internalized by hMSC. Cell viability, morphology and actin cytoskeleton reorganization were unaffected by exposure of hMSC to BCP or CDA particles. Direct exposure to BCP particles impaired hMSC osteogenic differentiation and bone matrix mineralization to a lesser extent than CDA, as assayed by evaluation of alkaline phosphatase activity, osteopontin secretion and mineralized nodule formation. The ability of bioceramic particles to affect osteogenic maturation through modification of soluble factors in media was assayed in an in vitro system that avoids direct cell-particle contact. Indirect exposure to CDA particles severely impaired hMSC osteogenic maturation owing to the uptake of Ca2+ from the culture media. Lower textural properties of BCP and the lack of calcium deficiency in its composition prevented Ca2+ uptake, allowing the development of a functional osteoblast phenotype.

Modulation of the cross-talk between macrophages and osteoblasts by titanium-based particles

Titanium (Ti) and its alloys have widespread uses as implant materials for orthopaedic and dental applications. To improve their surface characteristics, modifications that give rise to an outer ceramic layer of rutile have been developed. It is expected that after a long period of service, rutile particles will arise from these modified surfaces. Rutile particles have recently been proposed as reinforcement agents of substrates designed for bone tissue engineering applications. In this study, the ability of Ti and rutile particles to modulate secretion of soluble factors involved in bone turnover has been assayed in an in vitro co-culture system of macrophages and human osteoblasts that allows the exchange of soluble factors between both cell types without direct cell contact. Exposure of co-cultured macrophages to sub-cytotoxic doses of Ti or rutile particles did not modify the osteoblastic expression of surface RANKL or the secretion of OPG into the media. Both IL-6 and PGE2 levels increased to a similar extent after treatment with rutile or Ti particles. M-CSF and GM-CSF levels were lower after treatment with rutile particles than with Ti. Experiments employing neutralising antibodies indicate that exposure of co-cultured macrophages to both Ti-based particles induces the release of M-CSF, GM-CSF, IL-6 and PGE2 through up-regulation of IL-1beta and TNF-alpha. We comparatively examined the response of co-cultured macrophages, osteoblasts or both types of cells after exposure to particles. The results indicate that interactions of osteoblasts with particles can modulate the extent of the response initiated by macrophages. Maximal levels of secretions of all tested factors were reached after exposure of co-cultured cells to Ti particles, which is suggestive of the lower bioreactivity of rutile particles.

Segmental sensory innervation of the anterior cruciate ligament and the patellar tendon of the cat's knee-

We performed a study in cats to describe and quantify the segmental sensory innervation of the anterior cruciate ligament of the knee. We also studied the patellar tendon to show that transport occurs from an extraarticular, dense connective tissue structure and to obtain comparable quantitative information. We injected a tracer (horseradish peroxidase HRP, coupled to wheat germ agglutinin WGA) in the anterior cruciate ligament and observed the reaction product in the articular nerves of the injected knee and in the cell bodies of ipsilateral dorsal root ganglia. In these experiments, we found an average of 26 (13-52) labeled neurons, mostly large, after injecting the anterior cruciate. More than half of the labeled neurons were found in the dorsal root ganglion of L7 (last lumbar segment in the cat). We counted an average of 204 (17-426) labeled neurons, mostly small, after injecting the patellar tendon. More than half of these labeled neurons were found in the L5 spinal ganglion. No product was observed in contralateral spinal ganglia. Surgical ablation of the medial and lateral articular nerves (MAN and LAN) before injecting HRP-WGA in the anterior cruciate ligament, showed that the remaining afferents in the posterior articular nerve (PAN) projected mainly to L7. After excision of PAN, the projection was maintained through MAN and LAN, mostly to L5. Our quantitative data show that the anterior cruciate ligament is poorly innervated, if compared to the patellar tendon. The anterior cruciate segmental sensory innervation is directed to L7 (corresponding to the main ventral root forming the sciatic nerve in the cat), but also to L5 and L6 (main femoral nerve ventral roots). These segmental data indicate that anterior cruciate innervation influences muscle tone regulation, not only of the hamstrings (neuromuscular system of the sciatic nerve), but also of the quadriceps muscle (neuromuscular system of the femoral nerve).

Identification of differentially expressed genes in trabecular bone from the iliac crest of osteoarthritic patients

OBJECTIVEnOsteoarthritis (OA) is clinically characterized by degeneration of the joints and has been traditionally considered a primary disorder of articular cartilage, with secondary changes in the subchondral bone. The increased bone mass and generalized changes in bone quality observed in osteoarthritic patients suggest that OA may be a primary systemic bone disorder with secondary articular cartilage damage. The iliac crest is a skeletal site distant from the affected joint, with a minimal load-bearing function. To provide evidence that OA is a systemic disorder, we searched for differentially expressed genes in the iliac crest bone of patients suffering from hip OA.nnnMATERIAL AND METHODSnGene expression levels between bone samples collected at surgery from the iliac crest of patients undergoing total hip arthroplasty for primary OA and younger donors, who were undergoing spinal arthrodesis, were investigated by means of oligonucleotide microarrays. To verify data detected by microarrays technology, Real Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays were performed with specimens from osteoarthritic patients and donors, as well as from elderly donors who were undergoing arthroplasty for subcapital femoral neck fracture.nnnRESULTSnThe microarray analysis surveyed 8327 genes and identified 83 whose expression levels differed at least 1.5-fold in the OA group (P<0.005). Comparisons between Real Time RT-PCR data from OA and the two donor groups indicated differential expression of genes involved in bone cell functions in the group of OA patients. The genes identified, including CCL2, FOS, PRSS11, DVL2, AKT1, CA2, BMP6, OMD, MMP2, TGFBR3, FLT1, BMP1 and TNFRS11B, have known roles in osteoblast or osteoclast activities.nnnCONCLUSIONSnThe data from this study identify a set of genes, closely related to bone cell functions, in which differential regulation in osteoarthritic bone distant from the diseased subchondral bone might underlie the etiopathogenesis of OA as a generalized bone disease.

Interventions to improve inpatient osteoporosis management following first osteoporotic fracture: the PREVENT project

ObjectivesTo establish a protocol for the treatment of fragility fractures in the hospital setting based on treatment of osteoporosis.Materials and methodsAn intervention protocol was implemented in patients with fragility fractures based on (1) indicating the diagnosis of osteoporotic fracture in the summary of discharge; (2) “lifestyle recommendations”; and (3) therapy for osteoporosis. Thirty-one hospitals were involved and they were informed of the importance of protocol compliance. In the first phase, a retrospective study was conducted to establish the number of low-energy fractures treated and the percentage of them that had complied with the protocol (nxa0=xa0887). Then, prospectively, the same data were collected for the patients managed for 1xa0year (nxa0=xa06,826) in three sections of 4-month intervals.ResultsThe percentage of compliance increased from 8.2 to 57.2% in the first point, from 12.6 to 42.4% in the second, and from 10.3 to 43.2% in the third.ConclusionThe implementation of programs to improve osteoporosis treatment is very useful for ensuring adherence in the management of osteoporosis following admission due to fragility fracture.

Interactions of human bone cells with diamond-like carbon polymer hybrid coatings

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin-TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration.

Effects of polyethylene and α-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.