Establishment of a new methicillin resistant Staphylococcus aureus animal model of osteomyelitis.

PURPOSE: The increase in methicillin-resistant Staphylococcus aureus (MRSA) infections is currently a major health care problem. Vancomycin is still often the first-line anti-microbiological agent for treating such infections; however, a recent decline in efficacy of vancomycin in MRSA infections has raised concerns and accelerated the search for new antibiotics. The aim of this study was to establish a MRSA peri-implant osteomyelitis animal model for future testing of new anti-microbiological agents under typical MRSA infection conditions. METHODS: Eighteen randomised NZW-rabbits underwent a standardised surgical procedure with the insertion of a femoral bone implant. Animals were then divided into group 1 (MRSA inoculation, no antibiotics; M/N), group 2 (MRSA inoculation, Vancomyin; M/V), and group 3 (no MRSA inoculation, no antibiotics; N/N). The primary study outcome parameters were animal leucocyte count, animal weight, and animal body temperature at one, seven, and 42 days after surgery. Additionally, a histo-morphometrical score was established and adjusted to a modified histological Smeltzer score. RESULTS: Macroscopic and histo-morphometrical findings showed a peri-implant osteomyelitis in group 1 with both increased acute and chronic infection parameters in M/N, as compared to M/V and N/N, indicating that vancomycin treatment prevented typical morphological changes of MRSA peri-implant osteomyelitis. Similarly, there was a reduction in animal weight and increase in leucocyte count and body temperature in group 1 (each p < 0.005). Vancomycin treatment again resulted in significantly reduced leucocyte count and body temperature, and increased animal body weight. CONCLUSIONS: Here we have established a peri-implant MRSA osteomyelitis model that successfully combined clinical and laboratory outcome parameters of infection with histo-morphometrical results; this model appears to be valuable for future experimental use and therapeutic monitoring of new anti-microbiological MRSA drugs.

Bone remodelling around the Metha® short stem implant - Clinical and dual-energy x-ray absorptiometry (DXA) results.

OBJECTIVE: Bony ingrowth of short stems is under investigation. METHOD: Over one year bony reactions around the Metha® stem were investigated using the DXA method in a standardized zonal system (19 ROIs). Clinical results were documented using the HHS. RESULTS: HHS significantly improved from 46 points to 97 points. After one year bone marrow density (BMD) increased in the lateral distal and lateral proximal areas as well as in the mid medial area. BMD decreased in the proximal medial and mid lateral areas. CONCLUSION: The concept of metaphyseal ingrowth was confirmed, but distal bony reactions need further investigation.

The influence of surface coatings of dicalcium phosphate (DCPD) and growth and differentiation factor-5 (GDF-5) on the stability of titanium implants in vivo.

Mechanical stability of implants is usually tested by pull out or push out tests which destroy the interface between the implant and bone. Pull out tests do not ideally reflect the clinical situation. In contrast, applying submaximal load leads to more physiologic micro-displacement between implant and bone. The aim of this study was to evaluate a new non-destructive mechanical testing device on different modifications of titanium implants. In 18 rabbits we investigated the influence of a dicalcium phosphate (DCPD) coating, or of a growth and differentiation factor-5 (GDF-5) coating, or a combination of both on the stability of titanium implants. The stability of implant was assessed by a non-destructive micro-measurement. In the same specimens the interface was investigated by micro-CT and histological evaluation. Surface modifications had a positive effect on the implant stability regarding displacement (p=0.001). Mechanical stability correlated with the quality of peri-implant tissue. Micro-displacement correlated negatively with the bone formation around the implants in histomorphometric evaluation (p=0.02). Amount of peri-prosthetic soft tissue showed a positive correlation with micro-displacement (p=0.01). Our findings indicate the positive effect of DCPD and GDF-5 coatings on stability of titanium implants. Results demonstrate the non-destructive testing to be an effective method to evaluate mechanical stability of implants.

[Method of non-destructive mechanical testing of new surface coatings for prostheses]. / Methodik zur zerstörungsfreien Testung von Oberflächenbeschichtungen in der Endoprothetik.

Research into new surface coatings and surface processing methods for prostheses is subject to numerous studies. The aim of this study was to test an innovative biomechanical measuring method for the examination of the ingrowth of bone implants. Using a transcortical model, coated (n=14) or uncoated (n=14) titanic cylinders were implanted into the lateral condyle of 28 New Zealand White Rabbits. After 6 weeks or 6 months the animals were sacrificed and the osseointegration of the implants was evaluated biomechanically and histologically. Up to traction of 50 N the load dependent movement between bone and testing cylinder did not lead to a destruction of the bone-implant-interface. Therefore, biomechanical and histological investigations could be performed in the same specimen. The results of both evaluations showed a significant correlation (correlation coefficient -0.79; p < 0.01) and were absolutely reproducible. With the method of non-destructive mechanical testing, it is possible to halve the number of required animals. Additionally, the results of the biomechanical and histological analysis can be compared and thus serve as an internal control. In summary, the method of non-destructive mechanical testing represents an ideal tool to study new surface coatings and surface processing methods for prostheses.

Vascular endothelial growth factor gene-activated matrix (VEGF165-GAM) enhances osteogenesis and angiogenesis in large segmental bone defects.

UNLABELLED: Healing of fractures is dependent on vascularization of bone, which is in turn promoted by VEGF. It was shown that 0.1 and 1 mg of pVEGF165-GAM led to a significant increase in vascularization and bone regeneration in defects that would otherwise have led to atrophic nonunions. INTRODUCTION: One reason for lack of bone healing in nonunions is the absence of vascularization. In skeletogenesis, which is tightly linked to angiogenesis, vascular endothelial growth factor (VEGF) promotes the vascularization of the growth plate and transformation of cartilage to bone. We postulate that a gene-activated matrix (GAM), created with a plasmid coding for human VEGF165, coated on a collagen sponge could efficiently accelerate bone healing in large segmental defects. MATERIALS AND METHODS: Sixty New Zealand white rabbits received a 15-mm critical size defect on one radius, which was filled with either 0.1 or 1 mg plasmid-DNA as GAM. Radiographs were obtained every 3 weeks. After 6 or 12 weeks, animals were killed. New bone was measured by microCT scans. Vascularity was measured using anti-CD31 staining of endothelial cells in 18 regions of interest per implant. RESULTS: Scaffold and control plasmid showed no defect healing, whereas most of the animals in the VEGF groups showed partial or total bone regeneration. Significantly more bone was found in the VEGF groups, with no significant differences between the 0.1- and 1-mg groups. Immunohistochemical staining of endothelial cells revealed that the VEGF groups showed two to three times the number of vessels and a significantly larger endothelial area after 6 weeks. Twelve weeks after surgery, the amount of vascularization decreased, whereas more new bone was detectable. CONCLUSIONS: The rabbit critical size defect was appropriate in size to produce atrophic nonunions. We showed that angiogenesis and osteogenesis can be promoted by a VEGF165-GAM that is an appropriate tool to induce bone healing in atrophic nonunions.

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.

Comparable short-term results seen with standard and high-flexion knee arthroplasty designs in European patients.

PURPOSE: In a prospective study we evaluated outcomes of total knee arthroplasty (TKA) procedures in 62 patients with a follow up of 12 months to test our hypothesis that high-flexion CR (cruciate retaining) TKA provides a better range of motion and better outcome than standard CR design in Central European patients with their special problems and demands. METHODS: Patients were randomly divided into two groups (high flex vs. standard). The outcome was determined by measuring maximum knee flexion and using the KS-score and the SF-36 score. RESULTS: After 12 months ability to flex the knee significantly improved in both groups to 115° (SD 11) in the high flex group versus 119° (SD 12) in the standard group. There was no difference between the designs regarding maximum flexion (p = 0.78). Overall clinical rating scores significantly improved in both groups, but there was no difference between groups at one year after surgery (p (KSS) = 0.7 and p (SF-36) = 0.63). KS-score values improved from 25 points to 89 points for standard TKAs and from 20 points to 90 points for high flex TKAs. SF-36-score "Physical Functioning" values improved from 33 points to 66 points for standard TKAs and from 27 points to 63 points for high flex TKAs. CONCLUSION: Our results confirm known good results of the procedure and suggest that the benefit of high flex knee designs is similar to standard knee designs one year after index surgery. Further studies are required to evaluate long-term results of both designs.

Implant stability in the treatment of MRSA bone implant infections with linezolid versus vancomycin in a rabbit model.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant healthcare burden. Vancomycin and linezolid exhibit potent clinical and microbiological activity in MRSA infections. Our purpose was to investigate the efficacy of linezolid versus vancomycin in experimental implant infections and the influence on implant stability in a rabbit model. Thirty-six female New Zealand White rabbits received surgical insertion of titanium implants into their distal femurs and were randomly assigned to six groups (A: infected, no treatment; B: infected, vancomycin; C: infected, linezolid; D: no infection, no treatment; E/F: no infection, vancomycin or linezolid, respectively). Antibiotics were administered, and plasma levels determined. Bone-implant specimens were tested for mechanical stability of fixation. Quantitative histomorphometry of bone and soft tissue was performed using computerized image analysis. Plasma levels of linezolid and vancomycin were within the respective therapeutic ranges. Microbiological analysis of specimens from infected rabbits showed MRSA tissue colonization in all untreated animals, in two of six vancomycin-treated animals, and in none of the linezolid-treated animals. Antibiotic treatment improved mechanical stability significantly (p = 0.004) with both vancomycin and linezolid. Mechanical testing correlated with histomorphometry results. A significant negative correlation was found between displacement of the implant and the percentage of calcified tissue around the implant, and a significant positive correlation was found between displacement of the implant and the amount of noncalcified tissue. Our data indicate that both treatment regimens improved implant stability.

Improved osseointegration of PTFEP-coated titanium implants.

BACKGROUND: Clinical success of bone implants is critically related to the interaction between the implant surface and the surrounding tissue. The polymer poly(bis(trifluoroethoxy)phosphazene) (PTFEP) is a promising, highly biocompatible surface coating which also inhibits the adsorption of granulocytes, macrophages, inflammatory cells, bacteria, and platelets. However, there is limited clinical experience of PTFEP as a coating for bone implants. Therefore PTFEP-coated titanium implants in an animal model were examined. MATERIAL/METHODS: PTFEP-coated titanium cylinders were implanted into the lateral femoral condyles of rabbits. Osseointegration was examined six weeks and six months after implantation using a non-destructive mechanical pull-out measurement and a histological analysis. RESULTS: The results indicate improved osseointegration of PTFEP-coated implants. Six weeks after implantation, the PTFEP-coated implants showed a higher stiffness (pull-out length [pol]=7.1+/-2.0 microm) compared with uncoated cylinders (pol=10.2+/-3.4 microm, p<0.05). Six months after implantation, the mechanical properties of both implants had adjusted, and histological analysis revealed an increased bone-implant interface of PTFEP-coated cylinders compared with the first 6 weeks (17.5% vs. 8.2% in controls, p<0.05). CONCLUSIONS: Taken together, the results of this preliminary study indicate promising applications of PTFEP as a coating material for bone implants.

Local application of a collagen type I/hyaluronate matrix and growth and differentiation factor 5 influences the closure of osteochondral defects in a minipig model by enchondral ossification.

This pilot study evaluated the effect of growth and differentiation factor-5 (rhGDF-5) combined with a collagen type I/hyaluronate matrix (c/h) on osteochondral defect repair in a minipig model. Defects created in both medial femoral condyles of 20 minipigs were treated with c/h (n = 10), c/h + rhGDF-5 (n = 10) or were left empty. After 3 and 12 months, five animals of each group were sacrificed. Evaluation included macroscopic and histological scoring and quantitative histomorphometry of synthesized bone. C/h and c/h + rhGDF-5 treatment increased trabecular bone formation in the upper third of the defect compared to empty controls, showing significance for c/h + rhGDF-5 (p = 0.05) but not between c/h and c/h + rhGDF-5 treatment. Cartilage regeneration and macroscopic outcome were not improved by c/h or c/h + rhGDF-5 treatment. Since c/h remnants were seen even one year postoperatively in the defect, possibly inhibiting further bone and cartilage healing, other matrices in combination with rhGDF-5 may provide further improvement in osteochondral defect treatment.

Evaluation of mineralized collagen and alpha-tricalcium phosphate as scaffolds for tissue engineering of bone using human mesenchymal stem cells.

Owing to their plasticity and high proliferation capacity in vitro, mesenchymal stem cells (MSC) isolated from human bone marrow are promising candidates for use in tissue engineering approaches for the repair or replacement of mesenchymal tissues such as bone, cartilage or tendon. In keeping with the tissue engineering concept, these cells are cultivated on three-dimensional (3D) scaffolds to replace 3D tissue defects. Among the scaffolds tested for tissue engineering of bone, those containing phosphorus and calcium, as natural bone does, are the most promising candidates for this purpose. In this study, MSC from five patients were isolated from bone marrow. After in vitro expansion, cells were cultivated and differentiated towards the osteogenic lineage on mineralized collagen sponges and alpha-tricalcium phosphate (alpha-TCP). To analyze how appropriate these scaffolds would be for tissue engineering purposes, we established an in vitro characterization system to describe seeding efficiency, cell distribution and proliferation behavior on each scaffold. Real-time reverse transcriptase polymerase chain reaction quantification of important genes involved in osteogenic differentiation [e.g. bone sialoprotein (BSP), bone morphogenic protein 2, alkaline phosphatase and osteocalcin] was used to monitor the differentiation process of cells seeded on mineralized collagen and alpha-TCP. Using this in vitro characterization, we were able to demonstrate effective 3D growth of MSC on both scaffolds investigated. Improved osteogenic differentiation was observed on the scaffolds as compared to control monolayers. Of the two matrices, mineralized collagen was superior to alpha-TCP with regard to seeding efficacy (98 vs. 67%, p = 0.0003), increase in osteogenic marker genes (BSP expression on day 24, Pcollagen vs. TCP = 0.046) and 3D cell alignment (cell infiltration up to 500 vs. 200 microm). In conclusion, our data suggest that mineralized collagen is a promising candidate for use as a scaffold in tissue engineering of bone.

Immunohistochemical demonstration of -(carboxymethyl)lysine protein adducts in normal and osteoarthritic cartilage.

N(epsilon)-(carboxymethyl)lysine (CML) is an advanced glycation end product formed by non-enzymatic glycation and oxidation of proteins. The distribution pattern of CML-modified proteins in normal and osteoarthritic (OA) cartilage was investigated using specific antibodies. In healthy articular cartilage, immunoreactivity for CML was preferably found in the extracellular matrix (ECM) of the superficial layer. In OA samples, CML immunoreactivity was not restricted to the ECM of the superficial layer. Interestingly, OA chondrocytes showed a remarkable cytoplasmic immunoreactivity for CML. With the help of a western blot analysis CML-modified proteins between 68 and 39 kDa could be demonstrated in OA cartilage samples. These results suggest that the accumulation of CML adducts contributes to the matrix damage in osteoarthritis. Therefore, the inhibition of CML accumulation may represent an effective therapeutic strategy to prevent severe OA cartilage injury.

Effects of local application of growth and differentiation factor-5 (GDF-5) in a full-thickness cartilage defect model.

Our purpose in this study was to investigate the effects of growth and differentiation factor-5 (GDF-5) on cartilage and subchondral bone in a rabbit model harboring an osteochondral defect for a period of 6 months. Absorbable composites were implanted in adult rabbits (18 controls, 18 animals with collagen-I matrix, and 18 animals with matrix plus GDF-5). After 4, 8, or 24 weeks the specimens were studied by histology, microcomputed tomography (microCT) and flow-cytometric analysis (FACS). Implantation of GDF-5 resulted in an improved histological appearance. This was the result of significantly improved defect filling at 4 and 8 weeks. At 24 weeks, however, there was no difference between the groups. The histological examination disclosed a predominance of fibrocartilage, and remodeled subchondral bone was also observed. MicroCT documented normal bone density in all groups, excluding subchondral sclerosis. At 24 weeks, FACS analysis revealed high apoptotic activity in the GDF-5-treated group. As far as we are aware, this is the first report of the effects of GDF-5 in a full-thickness cartilage defect model. We assume that recombinant GDF-5 contained on the carrier is probably able to accelerate the regeneration of the osteochondral defect owing to its availability. However, control of the protein delivery may require further investigation.