Gabor Szalay

Biocompatibility of silver nanoparticles and silver ions in primary human mesenchymal stem cells and osteoblasts

The prevention of implant-related infections is an important issue in medical research. The aim is to exploit the strong antimicrobial effect of silver nanoparticles (AgNP) to develop new antibacterial coatings for implants. However, there is still a serious lack of information on the influence of AgNP on bone metabolism. In the present study we have evaluated the influence of AgNP on cell stress, viability, proliferation and differentiation of primary human mesenchymal stem cells (MSC) and osteoblasts (OB). Finally, cellular uptake of the AgNP was examined. After 21 days impairment of cell viability of MSC and OB occurred at a concentration of 10 μg/g of AgNP. Cytotoxicity and inhibition of proliferation was highly time and dose dependent. No influence on cell differentiation, but an increase in cell stress, was observed. Uptake of AgNP into MSC and OB could be confirmed. In summary, these results demonstrate AgNP-mediated cytotoxicity at higher concentrations. Therefore, a therapeutical window for the application of AgNP in medical products might exist. However, the antibacterial benefits and potential health risks of AgNP need to be weighed in further studies.

Effects of gentamicin and gentamicin–RGD coatings on bone ingrowth and biocompatibility of cementless joint prostheses: An experimental study in rabbits

Antimicrobial coatings are of interest as a means to improve infection prophylaxis in cementless joint arthroplasty. However, those coatings must not interfere with the essential bony integration of the implants. Gentamicin-hydroxyapatite (gentamicin-HA) and gentamicin-RGD (arginine-glycine-aspartate)-HA coatings have recently been shown to significantly reduce infection rates in a rabbit infection prophylaxis model. The purpose of the current study was to investigate the in vitro elution kinetics and in vivo effects of gentamicin-HA and gentamicin-RGD-HA coatings on new bone formation, implant integration and biocompatibility in a rabbit model. In vitro elution testing showed that 95% and 99% of the gentamicin was released after 12 and 24 h, respectively. The in vivo study comprised 45 rabbits in total, with six animals for each of the gentamicin-HA, gentamicin-RGD-HA group and control pure HA coating groups for the 4 week time period, and nine animals for each of the three groups for the 12 week observation period. A 2.0 mm steel K-wire with one of the coatings under test was placed in the intramedullary canal of the tibia. After 4 and 12 weeks the tibiae were harvested and three different areas (proximal metaphysis, shaft area, distal metaphysis) were assessed by quantitative and qualitative histology for new bone formation, direct implant-bone contact and the formation of multinucleated giant cells. The results exhibited high standard deviations in all subgroups. There was a trend towards better bone formation and better direct implant contact in the pure HA coating group compared with both gentamicin coatings after 4 and 12 weeks, which was, however, not statistically significant. The number of multinucleated giant cells did not differ significantly between the three groups at both time points. In summary, both gentamicin coatings with 99% release of gentamicin within 24 h revealed good biocompatibility and bony integration, which was not statistically significant different compared with pure HA coating. Limitations of the study are the high standard deviation of the results and the limited number of animals per time point.

Differences of bone healing in metaphyseal defect fractures between osteoporotic and physiological bone in rats

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (μCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology.

A new animal model for implant-related infected non-unions after intramedullary fixation of the tibia in rats with fluorescent in situ hybridization of bacteria in bone infection

There is no adequate animal model to mimic the difficult clinical situation of infected non-union of the tibia after intramedullary stabilization. The purpose was to establish an animal model of implant-related infected non-unions of the tibia in rats. Furthermore, it was evaluated if detection of bacteria by fluorescent in situ hybridisation (FISH) technique is possible in bone infection. 17 rats were used in which osteotomy of the midshaft tibia was performed and stabilized with an intramedullary device. Two groups were tested: group 1: contamination of the osteotomy site with 10(4) colony forming units (CFUs) of Staphylococcus aureus (11 animals), group 2: no bacterial contamination (6 animals). The animals were sacrificed after 42 days and bone healing and infection were assessed clinically, by X-ray, micro-CT, and microbiological methods including FISH technique using EUB and STAPHY probes. Histology and scanning electron microscopy (SEM) for biofilm formation were performed. All animals of the control group showed uneventful bone healing after 6 weeks without any signs of local infections. 10 of 11 (90.9%) animals of group 1 with bacterial contamination exhibited infected non-union formation with positive clinical, radiological and microbiological infection signs of the tibia but without any systemic infection signs. FISH technique was able to identify bacteria in the infected bone. All intramedullary implants from the infected animals showed positive biofilm formation in SEM. This work presents the first animal model for the induction of intramedullary device-related infected non-union in the tibia and detection of bacteria by FISH technique in infected bone.

Biphasic scaffolds for repair of deep osteochondral defects in a sheep model

BACKGROUND To oppose the disadvantages of autologous osteochondral transplantation in the treatment of deep osteochondral defects such as donor site morbidity, size limitation, and insufficient chondral integration, we developed two biphasic scaffolds of either hydroxylapatite/collagen (scaffold A) or allogenous sterilized bone/collagen (scaffold B) and tested their integration in a sheep model. METHODS We collected chondral biopsies from 12 sheep for the isolation of chondroblasts and cultured them for 4 wk. We created defects at the femoral condyle and implanted either scaffold A or B with chondrocytes or cell free. After 6 wk, animals were euthanized, we explanted the condyles, and evaluated them using histological, immunohistochemical, molecular biological, and histomorphometrical methods. RESULTS Specimens with scaffold A showed severe lowering of the surface, and the defect size was larger than for scaffold B. We found more immune-competent cells around scaffold A. Chondrocytes were scarcely detected on the surface of both scaffolds. Histomorphometry of the interface between scaffold and recipient showed no significant difference regarding tissue of chondral, osseous, fibrous or implant origin or tartrate-resistant acid phosphatase-positive cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed significant up-regulation for collagen II and SOX-9 messenger ribonucleic acid expression on the surface of scaffold B compared with scaffold A. CONCLUSIONS Scaffold B proved to be stable and sufficiently integrated in the short term compared with scaffold A. More extensive evaluations with scaffold B appear to be expedient.

The Biocompatibility of Degradable Magnesium Interference Screws: An Experimental Study with Sheep

Screws for ligament reconstruction are nowadays mostly made of poly-L-lactide (PLLA). However, magnesium-based biomaterials are gathering increased interest in this research field because of their good mechanical property and osteoanabolic influence on bone metabolism. The aim of this pilot study was to evaluate the biocompatibility of an interference screw for ligament reconstruction made of magnesium alloy W4 by diecasting and milling and using different PEO-coatings with calcium phosphates. PLLA and titanium screws were used as control samples. The screws were implanted in the femur condyle of the hind leg of a merino sheep. The observation period was six and twelve weeks and one year. Histomorphometric, immunohistochemical, immunofluorescence, and molecular biological evaluation were conducted. Further TEM analysis was done. In all magnesium screws a clinically relevant gas formation in the vicinity of the biomaterial was observed. Except for the PLLA and titanium control samples, no screw was fully integrated in the surrounding bone tissue. Regarding the fabrication process, milling seems to produce less gas liberation and has a better influence on bone metabolism than diecasting. Coating by PEO with calcium phosphates could not reduce the initial gas liberation but rather reduced the bone metabolism in the vicinity of the biomaterial.

Negative Influence of a Long-Term High-Fat Diet on Murine Bone Architecture

A correlation between obesity and bone metabolism is strongly assumed because adipocytes and osteoblasts originate from the same precursor cells and their differentiation is conversely regulated by the same factors. It is controversially discussed if obesity protects bone or leads to loss of bone mass. Thus, the aim of the present study was to investigate the influence of diet-induced mild obesity (11% increased body weight compared to control) on bone microstructure in mice. Four-week-old male C57BL/6J mice received a high-fat diet (HFD, 60% kcal from fat) and were analyzed by means of dual X-ray absorptiometry, histological methods, real-time RT-PCR, and transmission electron microscopy in comparison to control animals (10% kcal from fat). The cancellous bone mass, collagen 1α1 expression, amount of osteoid, and cohesion of cells via cell-to-cell contacts decreased in HFD mice whereas the bone mineral density and the amount of osteoblasts and osteoclasts were not modified. The amount of apoptotic osteocytes was increased in HFD mice in comparison to controls. We conclude that moderately increased body weight does not protect bone architecture from age-dependent degeneration. By contrast, bone microstructure is negatively affected and reduced maintenance of cell-cell contacts may be one of the underlying mechanisms.

Osteoprotegerin: A new biomarker for impaired bone metabolism in complex regional pain syndrome?

Summary We provide evidence that elevated serum osteoprotegerin reflects pathophysiological processes of complex regional pain syndrome. ABSTRACT Osteoprotegerin (OPG) is important for bone remodeling and may contribute to complex regional pain syndrome (CRPS) pathophysiology. We aimed to assess the value of OPG as a biomarker for CRPS and a possible correlation with radiotracer uptake in 3‐phase bone scintigraphy (TPBS). OPG levels were analyzed in 23 CRPS patients (17 women; mean age 50 ± 9.0 years; disease duration: 12 weeks [IQR 8–24]), 10 controls (6 women; mean age 58 ± 9.6 years) and 21 patients after uncomplicated fractures (12 women; mean age: 43 ± 15 years; time after fracture: 15 weeks [IQR: 6–22]). The CRPS and control patients also underwent TPBS. OPG in CRPS patients was significantly increased by comparison with both control groups (P = 0.001; Kruskal‐Wallis test; CRPS patients: 74.1 pg/mL [IQR: 47.1–100.7]; controls: 46.7 pg/mL [IQR: 35.5–55.0]; P = 0.004; fracture patients: 45.9 pg/mL [IQR: 37.5–56.7]; P = 0.001). As a diagnostic test for CRPS, OPG had a sensitivity of 0.74, specificity of 0.80, positive predictive value of 68% and negative predictive value of 84%. Receiver operating characteristic curve analysis showed an area under the curve of 0.80 (CI: 0.68–0.91). For the CRPS‐affected hand, a significant correlation between OPG and TPBS region of interest analysis in phase III was detected (carpal bones; r = 0.391; P = 0.03). The persistent OPG increase in CRPS indicates enhanced osteoblastic activity shown by increased radiotracer uptake in TPBS phase III. A contribution of bone turnover to CRPS pathophysiology is likely. OPG might be useful as a biomarker for CRPS.

Bone Matrix, Cellularity, and Structural Changes in a Rat Model with High-Turnover Osteoporosis Induced by Combined Ovariectomy and a Multiple-Deficient Diet

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.

Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis

AIMS As the stimulation of the α7-nicotinic acetylcholine receptor (nAChR), which is present in the synovium of patients with rheumatoid arthritis (RA), leads to a decrease in pro-inflammatory cytokines, the α7-nAChR is being discussed as a new therapeutic target. On this background we addressed the question whether α7-nAChR mRNA was differentially expressed in RA compared to osteoarthritis (OA) synovial samples and whether other components of the non-neuronal cholinergic system were also present and differentially expressed in the synovium of patients with RA in comparison to OA. MAIN METHODS The expression of nicotinic and muscarinic acetylcholine receptors (mAChRs), choline and acetylcholine transporters, synthesising and degrading enzymes was determined in human samples of synovial tissue from patients with RA and OA using RT-PCR and immunofluorescence labelling. KEY FINDINGS Compared to OA, patients with RA showed increased expression of nAChR subunit β4 while a decline in subunits α2 and α4 as well as in mAChR M1R was observed. For all other nAChR subunits and mAChRs however there was no significant difference between RA and OA patients. With regard to the ACh transporters and enzymes no expressional changes were observed between OA and RA patients, except for the choline acetyltransferase (ChAT) which was only detected in OA but not in RA synovium. SIGNIFICANCE Our results indicate that besides α7-nAChR other components of the non-neuronal cholinergic system are present and differentially expressed in the synovium of RA and OA patients, which makes them interesting alternative targets in the development of new strategies for RA therapy.