Antibacterial activity of bone allografts: comparison of a new vancomycin-tethered allograft with allograft loaded with adsorbed vancomycin.

Bacterial contamination of bone allograft is a significant complication of orthopedic surgery. To address this issue, we have engineered a method for covalently modifying bone allograft tissue with the antibiotic vancomycin. The goal of this investigation was to compare the biocidal properties of this new allograft material with those of vancomycin physisorbed onto graft material. The duration of antibiotic release from the vancomycin-modified allograft matrix was determined, and no elution was observed. In contrast, the adsorbed antibiotic showed a peak elution at 24h that then decreased over several days. We next used an Staphylococcus aureus disk diffusion assay to measure the activity of the eluted vancomycin. Again we found that no active antibiotic was eluted from the covalently modified allograft. Similarly, when the vancomycin-modified allograft morsel was used in the assay, no measurable elution was observed; amounts of antibiotic released from the adsorbed samples inhibited S. aureus growth for 4-7 days. Probably the most telling property of the allograft was that after 2 weeks, the tethered allograft was able to resist bacterial colonization. Unlike the elution system in which vancomycin was depleted over the course of days-weeks, the antibiotic on the allograft was stably bound even after 300 days, while its biocidal activity remained undiminished for 60 days. This finding was in stark contrast to the antibiotic impregnated allograft, which was readily colonized by bacteria. Finally we chose to evaluate three indicators of cell function: expression of a key transcription factor, expression of selected transcripts, and assessment of cell morphology. Since the tethered antibiotic appeared to have little or no effect on any of these activities, it was concluded that the stable, tethered antibiotic prevented bacterial infection while not modifying bone cell function.

Topographic features retained after antibiotic modification of Ti alloy surfaces: retention of topography with attachment of antibiotics.

Periprosthetic infection is increasingly prevalent in orthopaedics with infection rates of 2% to 15% after total hip arthroplasty. To effectively decrease bacterial attachment, colonization, and subsequent development of periprosthetic infection, we previously described a method to covalently bond vancomycin to smooth Ti alloy surfaces. To attach vancomycin, the Ti surface is first passivated to create a fresh oxide layer. Previously, passivation has been achieved with an H2SO4/H2O2 etch that can destroy the topography of the underlying implant. Passivation by hydrothermal aging as well as by H2SO4/H2O2 incubation produced a robust oxide layer, but only hydrothermal aging left the geometry unaltered. These hydrothermally passivated Kirschner wires and smooth or beaded Ti surfaces were chemically coupled with vancomycin. Antibiotic-coupled samples representing all three geometries were uniformly covered with antibiotic, resisted colonization by Staphylococcus aureus for longer than 8 hours, and retained their biocompatibility as assessed by normal attachment and morphology of preosteocytic MLO-A5 cells. Using this technique, we believe it is possible to passivate many complex implant designs/geometries as a first step toward covalent bonding of antibiotics or other bioactive factors.

Controlled release of vancomycin from thin sol-gel films on implant surfaces successfully controls osteomyelitis.

Peri-prosthetic infection remains a serious complication of joint replacement surgery. Herein, we demonstrate that a vancomycin-containing sol-gel film on Ti alloy rods can successfully treat bacterial infections in an animal model. The vancomycin-containing sol-gel films exhibited predictable release kinetics, while significantly inhibiting S. aureus adhesion. When evaluated in a rat osteomyelitis model, microbiological analysis indicated that the vancomycin-containing sol-gel film caused a profound decrease in S. aureus number. Radiologically, while the control side showed extensive bone degradation, including abscesses and an extensive periosteal reaction, rods coated with the vancomycin-containing sol-gel film resulted in minimal signs of infection. MicroCT analysis confirmed the radiological results, while demonstrating that the vancomycin-containing sol-gel film significantly protected dense bone from resorption and minimized remodeling. These results clearly demonstrate that this novel thin sol-gel technology can be used for the targeted delivery of antibiotics for the treatment of periprosthetic as well as other bone infections.

Development of the terminally differentiated state sensitizes epiphyseal chondrocytes to apoptosis through caspase-3 activation.

The maturation of epiphyseal chondrocytes is accompanied by dramatic changes in energy metabolism and shifts in proteins concerned with the induction of apoptosis. We evaluated the role of mitochondria in this process by evaluating the membrane potential (Delta psi m) of chondrocytes of embryonic tibia and the epiphyseal growth plate. We observed that there was a maturation-dependent change in fluorescence, indicating a fall in the Delta psi m. The level of mitochondrial Bcl-2 was decreased during maturation, while in the same time period there was an obvious increase in Bax levels in the mitochondrial fraction of the terminally differentiated chondrocytes. Bcl(xL), another anti-apoptotic protein, was also robustly expressed in the mitochondrial fraction, but its expression was not dependent on the maturation status of the chondrocytes. We found that caspase-3 was present throughout the growth plate and in hypertrophic cells in culture. We blocked caspase-3 activity and found that alkaline phosphatase staining and mineral formation was decreased, and the cells had lost their characteristic shape. Moreover, we noted that the undifferentiated cells were insensitive to elevated concentrations of inorganic phosphate (Pi). It is concluded that during hypertrophy, the change in membrane potential, the increased binding of a pro-apoptotic protein to mitochondria, and the activation of caspase-3 serve to prime cells for apoptosis. Only when the terminally differentiated chondrocytes are challenged with low levels of apoptogens there is activation of apoptosis.

Transforming growth factor-beta1 (TGF-beta1) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression.

Regulated splicing of fibronectin (FN) occurs during the mesenchymal to chondrocyte transition and ultimately results in the relative enrichment of an extra domain B (EDB) exon-containing FN isoform with the suggestion that FN isoforms may play a functional role in chondrogenesis. Promotion of chondrogenesis can also be achieved by treatment with transforming growth factor-beta (TGF-beta), which also regulates FN isoform expression. We have examined the effects of TGF-beta treatment on the assumption of the chondrogenic phenotype in the teratoma-derived cell line ATDC5 and tested whether these effects on chondrogenesis are paralleled by appropriate changes in FN isoform expression. ATDC5 cells were maintained in a pre-chondrogenic state and, in this state, treated with 10 ng/ml TGF-beta. The cells started to elaborate a matrix rich in sulfated proteoglycans, such that within the first 12 days of culture, TGF-beta1 treatment appeared to slightly accelerate early acquisition of an Alcian blue-stained matrix, and caused a dose- and time-dependent decrease in collagen type I expression; changes in collagen type II expression were variable. At later times, cells treated with TGF-beta became indistinguishable from those of the controls. Interestingly, TGF-beta treatment caused a significant dose- and time-dependent decrease in the proportion of FN containing the extra domain A (EDA) and the EDB exons. These data suggest that TGF-beta induces the early stages of chondrogenic maturation in this pre-chondrogenic line and that TGF-beta treatment increases expression of FN isoforms that lack the EDA and EDB exons.

The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin.

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, (beta)ig-h3, is a highly conserved transforming growth factor-beta-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length (beta)ig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on (beta)ig-h3-coated plates and developed actin stress fibers. Purified (beta)ig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the (beta)ig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of (beta)ig-h3 in the ECM.