Summary

Coagulase-negative staphylococci, including S. epidermidis, have emerged as the leading pathogens of hospital-acquired biomaterial-related infections. These infections can be clinically indolent and challenging also for diagnostic imaging. In the current model of catheter-related infections, ⁶⁸Ga-labeled Siglec-9 PET/CT imaging was able to detect peri-implant S. epidermidis bone infections.

Background and aim: Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into osteogenic and chondrogenic pathways. MSCs are among the key repair cells in fracture healing and implant osseointegration. They are also an attractive tool of cell therapy in reconstruction procedures of bone. Minipigs are a large-animal model recommended for preclinical studies of orthopaedic bone implants. Minipigs are claimed to have bone physiology close to humans, but their MSC characteristics are poorly defined. The aim of this study was to isolate and characterize minipig bone marrow and peripheral blood derived MSCs in comparison of human MSCs.

Methods: Five male minipigs (weight 36.2 ± 2.2 kg) were subjected for experimental femoral osteotomy, which was fixed with either compression plate or intra-medullary nailing. Before surgery, bone marrow (BM) sample (2–4 ml) was aspirated from the posterior iliac crest and a peripheral blood (PB) sample (20 ml) was also collected. Mononuclear cells (MNC) were isolated by Ficoll gradient centrifugation. MSCs were cultured and selected by plastic adherence. Cell morphology was evaluated during the whole culture period and proliferation capacity was examined by determining the number of population doublings (PDs) at the end of each passage. Osteoblastic differentiation capacity was investigated by culturing MSCs in the presence of beta-glycerophosphate, dexamethasone and ascorbic acid. The lineage phenotype was studied by alkaline phosphatase and von Kossa staining.

Results: MNC were successfully isolated from all BM and PB samples. Plastic adherent cells obtained fibroblast-like morphology and proliferated over time in culture. The maximum PDs were 3.4 ± 0.7 and 4.3 ± 0.5 for BM- and PB-derived cells, respectively. The maximum PD capacity of PB-derived cells was significantly higher than that of BM-derived cells (p=0.027). However, when cultured in osteoblastic induction medium, only BM-derived cells were capable of differentiating into alkaline phosphatase positive osteoblasts with an occasional presence of von Kossa-stained mineralized bone nodules. The maximum PDs of minipig BM-derived MSCs were similar to those of human BM-MSCs isolated from young adult fracture patients.

Conclusion: We successfully isolated plastic adherent MSCs from minipig bone marrow samples, which proliferated and differentiated into cells of osteoblastic lineage. BM-derived porcine MSCs had similar morphology to human MSCs. There were marked inter-individual variations in the proliferation and differentiation capacity of minipig MSCs, resembling the observations in humans. No circulating MSCs could be detected in minipigs before surgery and this confirmed our previous observation in humans.

Introduction: In postmenopausal female patients with hip osteoarthritis, osteoporosis as well as altered local trabecular bone architecture may lead to a increased migration of uncemented hip prostheses.^1,2 The aim of this study was to determine whether 3D bone architecture and mechanical properties of intertrochanteric cancellous bone in the proximal femur predict RSA migration of uncemented femoral stems.

Materials and Methods: The study population consisted of 61 female patients with primary hip osteoarthritis. Informed consent was obtained prior to any study-related procedures. The Ethical Committee approved the study protocol.

All patients underwent a total hip replacement (ABG II, Stryker) with uncemented proximally hydroxyapatite-coated femoral stem with tantalum RSA markers. Ceramicceramic bearing surfaces were used. A uniplanar type of RSA setup was applied. The RSA examinations were performed postoperatively and at 3, 6, and 12 months.

During surgery, cancellous bone biopsy was taken from the proximal femur from the site of stem implantation. The specimens were scanned with micro-CT. 3D analysis of trabecular bone geometry and BMD was performed using CTAn software. After micro-CT imaging, the trochanteric cancellous bone specimens were subjected to a destructive compression test. Maximum force and stiffness were calculated. Linear regression analysis was applied to study correlations between different parameters investigated.

Results: The patients exhibited major differences in the density and structural quality of intertrochanteric cancellous bone. Significant correlations were found between the measured biomechanical parameters and the structural parameters calculated from micro-CT data.

Unexpectedly, the quality of intertrochanteric cancellous bone did not predict RSA migration of the femoral stems. The femoral stems reached high stability within 3 months and there were no significant differences in the axial and rotational migration of the femoral stems between the patients with normal or poor quality of the intertrochanteric cancellous bone.

Discussion: The 3D bone architecture, mineral density and mechanical properties of the local intertrochanteric cancellous bone do not seem to serve as predictors of femoral stem migration. The observation suggests that the significance of cancellous bone quality for the initial stability of uncemented femoral stems has been over-emphasized.

Introduction: Drug delivery systems (DDSs) using resorbable materials have been developed for local therapy of adult osteomyelitis. An ideal DDS would provide controlled release of antibiotic for an extended period and have an osteoconductive component for spontaneous restoration of bone stock.

Materials and Methods: The developed DDS consisted of three components: poly(DL)-lactide (PDLLA), ciprofloxacin (AB) and bioactive glass (BG) as the osteoconductive component. Based on in vitro studies, the composite provides a long-lasting release (> 3 months) of the ciprofloxacin at therapeutic levels. The localized osteomyelitis model (Stage IIIA) was applied in adult male New Zealand white rabbits (n=30). A cortical bone window was drilled in the proximal tibial metaphysis and filled with bone cement. 0.1 ml of Staphylococcus aureus lxl0⁵ 1/ml was injected into the defect. Infection was allowed to develop for two weeks, when the bone cement was surgically removed (debridement) and osteomyelitis was confirmed by positive bacteriology. In treated experimental animals, antibiotic containing composite (AB-PDLLA-BG) was impacted into the infected medullary space. In untreated infection control group, the infected the medullary space was subjected only to surgical debridement. In sham-treated control group, the infected medullary space was filled with a composite without antibiotic (PDLLA-BG). In the negative control group, the injection of bacterial suspension was replaced by saline injection. The treatment response was evaluated by FDG-PET and pQCT at 3 and 6 weeks. Concentration of ciprofloxacin was also measured from bone tissue. The statistical significance of the differences was calculated using paired t-test and one-way ANOVA with Tukey t-test.

Results: Before infection treatment, 96% of the animals had positive bacterial cultures, while none of the negative control group had positive cultures. At sacrifice, all animals in untreated and sham-treated control groups had culture positive infection, while all bone cultures were negative in treated animals. However, three treated animals had culture positive soft-tissue infection. In untreated infection control group, the FDG uptake was increased many-fold compared with the negative control group both at 3 and 6 weeks. The treatment with AB-PDLLA-BG significantly decreased the FDG uptake and the difference was highly significant (p=0.013) compared the untreated animals. Based on pQCT imaging, the cortical defect healing was faster in treated and negative control animals than in untreated and sham-treated groups. In treated animals, the local therapy resulted in high bone concentration of ciprofloxacin.

Conclusions: The current experiment confirmed by collaborative results of both bacteriologic, FDG-PET and pQCT studies that the local infection therapy by the selected antibiotic composite was successful in bone eradication of Staphylococcus aureus pathogen.

Aims: Bioactive glasses are a family of silica-based synthetic biomaterials, which form chemical bonding with the surrounding bone. The limiting biologic factors of the bonding process are poorly understood. The hypothesis of the current study was that there are species-specific differences in the incorporation of bioactive glasses due to anatomic and physiologic factors. Methods: Conical porous implants made of sintered bioactive glass or titanium microspheres (Ø 250–300 μm) were surgically implanted bilaterally into the cortex of tibias or femurs in sheep, dog and rabbit. Implant incorporation was evaluated by means of push-out testing, pQCT, his-tomorphometry, BEI-SEM, and EDXA. The comparison was made at 12 weeks. A total of 176 implants were analysed. Results: Between the three species, there were significant differences in the extent of new bone ingrowth and in the mechanical strength of implant fixation. The rabbit showed the highest amount of bone ingrowth into both bioactive glass and titanium implants. Also the shear strength of the implants was superior in the rabbit compared with the dog and the sheep. Histological pattern of new bone ingrowth into bioactive glass structures was similar in the dog and in the rabbit. In contrast, the ingrowth of new bone failed into bioactive glass implants in the sheep. Conclusions: Based on these results, the sheep represents a divergent model for bone healing studies of bioactive glass. Long bones of the sheep contain yellow (fat) marrow and we assume that the poor healing response reflects the deficiency of marrow-derived osteoprogenitor cells.

Introduction: Positron emission tomography (PET) using F-18 ßuorodeoxyglucose (FDG) is a promising new imaging modality for bone infections. The method is based on intensive cellular use of glucose during infection. The aim of the current study was to establish the FDG-PET characteristics of normal bone healing and bone infection under standardized conditions. Methods: A modiþed osteomyelitis model of Mader and Fitzgerald was applied in the rabbit (n=12). A metaphyseal defect of the proximal tibia was þlled with bone cement. A predetermined amount (0.1 ml) of Staphylococcus aureus (strain 52/52A/80, 1x10⁵/ml) suspension was injected into the defect. The control animals received an equal saline injection without bacteria. Bone cement was removed from each animal at 2 weeks. During the follow-up, FDG-PET and pQCT were performed at 3 weeks and 6 weeks. Osteomyelitis was conþrmed with bacterial cultures at the time of cement removal and again at sacriþce at 6 weeks. Results: Compared with the contralateral intact tibia, control defects healing without infection showed an increased uptake (p=0.019) at 3 weeks but their FDG-PET tended to normalize within 6 weeks. In the osteomyelitis group, the uptake did not decrease over time and was signiþcantly (p< 0.001) increased both at 3 weeks and at 6 weeks compared with intact bone uptake. The uptake of the infected region was also signiþcantly higher than that of non-infected control defects. Conclusions: Standardized osteomyelitis of the current model was shown to result in an intense continuous FDG-PET activation, which is higher than the transient response of a healing bone defect.

Aims: The present study examined the effect of ade-novirus-mediated recombinant human BMP-2 (RAd-BMP-2) gene therapy combined with bioactive glass (BG) microspheres in promotion of new bone formation. Methods: Harlan Dawley female rats (n=72) underwent unilateral surgery of right or left tibia in a random order. A round cortical window (⁻ 2.8 mm) was drilled into the anteromedial cortex of the proximal tibia. A smaller unicortical hole (⁻ 1.0 mm) was drilled 5 mm distally. Bone marrow was removed and the medullary space between the cortical holes was þlled with BG microspheres. Adenoviral vectors RAdBMP-2 carrying the BMP-2 gene or RAdLacZ harbouring the E. coli LacZ reporter gene were injected locally into the medullary spaces. The control defects were þlled with BG microspheres only. Empty control defects were left to heal without any þlling. The rats were killed 4 days, 2 and 8 weeks after surgery and the tibias were harvested for analyses. At each time point, six animals were used for pQCT, radiography, BEI-SEM and histomorphometric analyses. Results: All BG-þlled defects showed a time-related increase of intramedullary new bone. At 8 weeks, there was signiþcantly more new bone in defects treated with BG and RAdBMP-2 gene than in defects left to heal without þlling (p=0.003) (BG + RAdBMP-2: 25.0 ± 6.0% and empty control defects: 12.3 ± 3.8%). Also defects þlled with BG only showed higher new bone formation than empty control defects, but this was not statistically signiþcant (p=0.10) (BG: 19.9 ± 7.3%). Conclusions: The current study showed that local BMP-2 gene therapy enhances new bone formation on bioactive glass microspheres.

Aims: In a recent study, chemical microroughening of bioactive glass surface was shown to promote attachment of osteoblastic cells and osseointegration of porous bioactive glass implant. The current in vivostudy employed molecular biologic techniques to clarify the osteogenic effects of smooth and microrough glass surfaces. Methods:Using a rat model, a portion of the medullary canal in the proximal tibia was evacuated and filled with microroughened or smooth bioactive glass microspheres. The primary bone healing response and subsequent remodelling were analysed at 1, 2, and 8 weeks, respectively. The expression of various genes for the bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis. Results: The microroughened bioactive glass microspheres were found to induce higher mRNA levels for osteopontin and lower levels for osteonectin at 2 weeks after operation when compared to smooth control micropheres. At 8 weeks, the MMP-9 expression levels were significantly higher with microroughened bioactive glass microspheres. Conclusion: Microroughening of the bioactive glass surface triggered temporal changes in the expression of specific genes.