Problems

Biofilm infections are increasingly associated with orthopedic implants. Bacteria form biofilms on the surfaces of orthopedic devices. The biofilm is considered to be a common cause of persistent infections at a surgical site. The growth and the maturation of biofilm are enhanced by the flow of broth in culture environment. In order to reduce the incidence of implant-associated infections, we developed a novel coating technology of hydroxyapatite (HA) containing silver (Ag). We previously reported that the Ag-HA coating inhibits biofilm formation under flow condition of Trypto Soy Broth + 0.25% glucose for 7 days. In this study, we evaluated whether the Ag-HA coating continuously inhibits the biofilm formation on its surface under flow condition of fetal bovine serum, which contains many in vivo substrates such as proteins.

The objective of this study is to determine the knee joint forces when rising from a kneeling position. We have developed a new type of knee prosthesis which is capable of attaining Japanese style sitting. To run the simulations and experiments needed to assess the performance of this prosthesis, it is necessary to know what forces act on the knee during deep flexion. Because these data are lacking, we created a 2D mathematical model of the lower leg to help determine knee joint forces during deep flexion. Healthy subjects of ten males (age of 25±4years, height of 170.3±9.1cm, and weight of 67.0±22.2kg) and five females (25±3years, 161±7.1cm, 47.7±6.2kg) participated in the experiment. Ground reaction force and joints angles were measured using a force plate and a motion recording system respectively. The collected data were entered into our mathematical model, and the muscle forces and the knee joint forces were calculated. To verify our model, we first used it to run simulation of middle and high flexions of the knee joint. In vivo data for these actions are available in the literature, and the results from our simulation were in good agreement with these data. We then collected the data and run simulation when rising from a kneeling position under the conditions shown in Fig. 1. They were a) double leg rising (both legs are aligned) without using the arms, b) ditto but using the arms, c) single leg rising (legs are in the front and the rear respectively) without using the arms, and d) ditto but using the arms. We obtained the following results. The statistics of the maximum values on the single knee joint for each condition were; a) F_max=5.1±0.4 [BW: (force on the knee joint)/(body weight)] at knee flexion angle of Q=140±8°, b) F_max=3.2±0.9[BW] at Q=90±10°, c) F_max-d=5.4±0.5[BW] at Q_d=62±20° for the dominant leg and F_max-s=3.0±0.5[BW] at Q_s=138±6° for the supporting leg respectively, and d) F_max-d=3.9±1.5[BW] at Q_d=70±17° for the dominant, and F_max-s=2.1±0.5 [BW] at Q_s=130±11° for the supporting. We may conclude that the single leg rising should be recommended since the maximum knee joint force did not become large as long as the knee was at deep flexion. The values introduced in this study could be used to assess the strength of the knee prosthesis at deep flexion. To obtain more realistic values of the joint forces, it is necessary to determine the ratio of the forces exerted by the mono-articular and the bi-articular joint muscles.

Bacterial infection related to prosthetic replacement is one of the serious types of complications. Recently, there has been a greater interest in antibacterial biomaterials. In order to reduce the incidence of replacement-associated infections, we developed a novel coating technology of Hydroxyapatite (HA) containing silver (Ag). We reported the Ag-HA coating showed high antibacterial activity against E. coli, S. aureus and methicillin-resistant S. aureus (MRSA) under static condition. However, human bodies have a circulating body fluid, which is not a static condition. And the growth and the maturation of biofilm, which is said that a common course of persistent infections at a surgical site, are enhanced by the flow of broth in culture environment. Therefore, we evaluated whether the Ag-HA coating inhibits the biofilm formation on its surface or not by a biofilm-forming test under flow condition in this study.

Ag-HA or HA powder was sprayed onto the commercial pure titanium disks using a flame spraying system. The HA coating disks were used as negative control. The biofilm-forming methicillin sensitive S. aureus (BF-MSSA; Seattle 1945) strain and the BF-MRSA (UOEH6) strain were used. The pre-culture bacterial suspension (about 10⁵ colony forming units; CFU) was inoculated onto the Ag-HA and HA coating disks. After cultivation at 37 °C for 1 h, the disks were rinsed twice with 500 μL sterile PBS (-) to eliminate the non-adherent bacteria. The number of the adherent bacteria on these disks was counted using culture method. After rinsing, the disks were transferred into petri-dish containing Trypto–Soy Broth (TSB) + 0.25% glucose with a stirring bar on the magnetic stirrer and they were cultured at 37°C for 7 days. In the meantime, the stirring bar was spun at 60 rounds per minute. Then, the disks were immersed in a fluorescent reagent to stain the biofilm. Finally, the biofilm on each disk was observed by a fluorescence microscope and the biofilm-covered rate on the surfaces of them was calculated using the NIH image software.

The number of the bacteria on these disks was not so different between Ag-HA and HA coating after rinsing. After biofilm-forming test, the coverage of the biofilm of BF-MSSA was 2.1% and 81.0% on the Ag-HA and HA coatings, respectively. Similarly, in the case of BF-MRSA, it was 7.7% and 72.0% on the Ag-HA and HA coatings, respectively. Though bacteria slightly adhered, biofilm was hardly observed on the Ag-HA coating. The biofilm on the HA coating was extensive and mature. The inhibition effect of biofilm formation on the Ag-HA coating might be ascribed to the antibacterial effect by Ag ions released from the coating. Because Ag ions have a broad spectrum of antibacterial activity against pathogens, including biofilm forming bacteria, they inhibited the biofilm formation on the Ag-HA coating by killing adherent bacteria. Even in a flow condition, it was suggested that the AgHA shows the antibacterial activity, though the conditions in this work are different from those in living body.

The objective of this study is to introduce the forces acting on the knee joint while ascending from kneeling. Our research group has developed a new type of knee prosthesis which is capable of attaining complete deep knee flexion such as a Japanese style sitting, seiza. Yet we could not set up various kinds of simulation or experiment to assess the performance of our prosthesis because the data about joints' forces during the ascent from deep knee flexion are lacking. Considering this circumstance, we created a 2D mathematical model of lower limb and determined knee joint force during ascent from kneeling to apply them for the assessment of our prosthesis.

Ten male and five female healthy subjects participated in the measurement experiment. Although the measurement of subjects' physical parameters was non-invasive and direct, some parameters had to be determined by referring to the literature. The data of ground reaction force and each joint's angle during the motion were collected using a force plate and video recording system respectively. Then the muscle forces and the joints' forces were calculated through our mathematical model. In order to verify the validity of our model approach, we first introduced the data during the activities with small/middle knee flexion such as level walking and rising from a chair; these kinds of data are available in the literature. Then we found our results were in good agreement with the literature data. Next, we introduced the data during the activities with deep knee flexion; double leg ascent [Fig.1 (a)] and single leg ascent [Fig.1 (b)] from kneeling without using the upper limbs.

The statistics of the maximum values on the single knee joint for all the subjects were; during double leg ascent, F_max = 4.6±0.6 (4.3-5.2) [BW: (force on the knee joint)/(body weight)] at knee flexion angle of b =140±8 (134-147)°, during double leg ascent, F_max = 4.9±0.5 (4.0-5.6) [BW] at b = 62±33 (28-110)° for the dominant leg, and F_max = 3.0±0.5 (22.2-3.8) [BW] at b = 138±6 (130-150)° for the supporting leg respectively. We found that the moment arm length, i.e., the location of muscle insertion significantly affected the results, while ascending speeds did not affect the results much. We may conclude that the single leg ascent should be recommended since F_maxdid not become large while deep knee flexion. The values could be used for assessing the strength of our knee prosthesis from the risk analysis view point.

Chondrosarcoma responds poorly to adjuvant therapy and therefore, new targeted therapy is required. Animal models have been utilised to test therapeutic candidates, however clinically relevant, orthotopic models are lacking. The aim of this study was to develop such a model.

In vitro: two human chondrosarcoma cell lines, JJ012 and FS090, were compared with respect to proliferation, colony formation, invasion, MMP-2 and MMP-9 secretion, osteoclastogenesis, endothelial tube stimulation, and expression of the angiogenic factor VEGF, and the anti-angiogenic factor RECK on western blotting. In vivo: 20,000 cells (JJ012 or FS090) were injected either into the intramedullary canal of the mouse tibia (n=5 for each cell line), or into the tibial periosteum (n=5 for each cell line). Animals were measured, and x-rayed weekly. Once euthanised, tibias and lungs were preserved, embedded and sectioned to determine the presence of tumour and lung metastases.

In vitro: compared with FS090, JJ012 demonstrated significantly higher proliferative capacity at both day two and day four (p=0.017, and p=0.01). JJ012 had a significantly greater ability to invade Matrigel with an average number of 812.5 invading cells, versus 140.8 FS090 cells (p=0.0005). JJ012 readily formed colonies in collagen I, while FS090 formed none. JJ012 conditioned medium stimulated endothelial tube formation and osteoclastogenesis with a greater potency than FS090 conditioned medium. In vivo: tumours formed in the intratibial and periosteal groups injected with JJ012, whilst no mice injected with FS090 cells developed discernable tumours on physical inspection, caliper measurement or histological section. Periosteal tumours grew to three times the non-injected limb size by seven weeks, whereas intratibial injected limbs required 10 weeks to achieve the same extent of tumour growth. All JJ012 periosteal tumours resulted in lung micrometastases, while only 2/4 JJ012 intratibial tumours demonstrated metastases. Lung metastases stained positive with Von Kossa and alizarin red stains, indicating a tendency for calcification, which is similar to metastases in the human disease. Sectioned tumour tissue demonstrated features of grade II-III chondrosarcoma. Similarities with the human disease were also noted on the X-ray, including endosteal scalloping, and cortical thickening.

Both intratibial and periosteal JJ012 models replicate the site, morphology, and many behavioural characteristics of human chondrosarcoma. Local tumour invasion of bone and spontaneous lung metastasis offer valuable assessment tools to test the potential of novel agents for future chondrosarcoma therapy.

Introduction and aims

After internal hemipelvectomy for malignant pelvic tumors, pelvic reconstruction is necessary for eventual weight bearing and ambulation. Non-vascularised, fibular grafts (NVFG) offer fast, and stable reconstruction, post- modified Enneking's type I and I/IV resection. This study aimed to evaluate the success of graft union and patient function after NVFG reconstruction.