No abstract

Peri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections. b s t r a c tPeri-prosthetic infections are notoriously difficult to treat as the biomaterial implant is ideal for bacterial adhesion and biofilm formation, resulting in decreased antibiotic sensitivity. Previously, we reported that vancomycin covalently attached to a Ti alloy surface (Vanc-Ti) could prevent bacterial colonization. Herein we examine the effect of this Vanc-Ti surface on Staphylococci epidermidis, a Gram-positive organism prevalent in orthopaedic infections. By direct colony counting and fluorescent visualization of live bacteria, S. epidermidis colonization was significantly inhibited on Vanc-Ti implants. In contrast, the gram-negative organism Escherichia coli readily colonized the Vanc-Ti rod, suggesting retention of antibiotic specificity. By histochemical and SEM analysis, Vanc-Ti prevented S. epidermidis biofilm formation, even in the presence of serum. Furthermore, when challenged multiple times with S. epidermidis, Vanc-Ti rods resisted bacterial colonization. Finally, when S. epidermidis was continuously cultured in the presence of Vanc-Ti, the bacteria maintained a Vanc sensitivity equivalent to the parent strain. These findings indicate that antibiotic derivatization of implants can result in a surface that can resist bacterial colonization. This technology holds great promise for the prevention and treatment of periprosthetic infections.

Previously, we noted that inorganic phosphate (P i ), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000 Bone adapts to mechanical and physiological stress by a unique form of tissue replacement contained within discrete structures defined as basic multicellular units (2). Within each of these units, the actual process of bone removal is carried out by osteoclasts; replacement bone matrix is synthesized and mineralized by cells of stromal origin, osteoblasts. Examination of resorbing sites in developing skeletal tissues indicates that many of the cells are apoptotic (3). Thus, there is evidence of DNA fragmentation in osteoclasts, osteoblasts, and osteocytes (4, 5). In contrast, in mature skeletal tissues, only about 1-2% of all bone cells are dying or dead. In both the developing and mature skeleton, most of the apoptotic cells are confined to bone remodeling sites or locales of high bone turnover (5-8).How osteoclasts communicate with and regulate the life history of other cells of the basic multicellular unit is a topic of intense debate. It is clear that osteoclast differentiation and activation are dependent on paracrine signals received from stromal cells in the multicellular unit (9). Within the past four years, chemical modulators of these processes have been identified, and recent evidence indicates that growth factors provide survival signals that result in bone cell proliferation and depression of the apoptotic process (5, 6, 10). In addition, it has been demonstrated that a number of pharmacological agents can induce osteoblast apoptosis in vitro (5, 10 -12). Surprisingly, however, little is known of events that promote osteoblast death in situ.Although the resorption process may generate agents that stimulate osteoblast proliferation, it is probable that products of the resorbing bone may also stimulate bone cell death. Recent work from this laboratory has clearly demonstrated that one of the ions present in the bone matrix, inorganic phosphate (P i ), induces apoptosis of cultured human osteoblasts and chondrocytes (1, 13). Since Ca 2ϩ as well as P i are released from the bone apatite lattice during the resorption process, the possibility exists that Ca 2ϩ may influence P i -mediated bone cell apoptosis. To test the hypothesis that this ion pair may trigger the death program, we examine the effect of P i and Ca 2ϩ on human osteoblast-like cells. We ask the questions, Can Ca 2ϩ modulate P i -induced cell death, and, if so, is death mediated by apoptosis? Using a cell culture system, we demonstrate that Ca 2ϩ accentuates the apoptogenic effect of P i . In addition, we provide evidence for the involvement of mitochondria and intracellular Ca 2ϩ in the apoptotic pathway activated by the ion pair. MATERIALS AND METHODSCell Culture-Specimens of human bone were obtained during dental surgery performed at the Hospital of the University of Pennsylvania and during spinal surgery performed at the Children's Hospital of Philadelphia...

The goal of this review is to examine the fate of the hypertrophic chondrocyte in the epiphyseal growth plate and consider the impact of the cartilage microenvironment on cell survival and apoptosis. Early investigations pointed to a direct role of the hypertrophic chondrocyte in osteogenesis. The terminally differentiated cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens. We present a new concept: hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis.

Periprosthetic infection is a devastating consequence of implant insertion and can arise from hematogenous sources or surgical contamination. Microbes can preferentially colonize the implant surface and, by forming a biofilm, escape immune surveillance. We hypothesized that if an antibiotic can be tethered to a titanium alloy (Ti) surface, it will inhibit bacterial colonization, prevent biofilm formation, and avert late-stage infection. To test this hypothesis, a Ti rod was covalently derivatized with vancomycin. Reaction efficiencies were evaluated by colorimetric and spectrophotometric measurements. The vancomycin-modified surface was stable in aqueous solutions over extended time periods and maintained antibiotic coverage, even after pressfit insertion into a cadaverous rat femora. When evaluated using fluorescently labeled bacteria, or by direct colony counts, the surface-bound antibiotic prevented bacterial colonization in vitro after: (1) exposure to high levels of S. aureus; (2) extended incubation in physiological buffers; and (3) repeated bacterial challenges. Importantly, whereas the vancomycin-derivitized pins prevented bacterial colonization, S. aureus adhered to control pins, even in the presence of concentrations of vancomycin that exceeded the strain MIC. These results demonstrate that we have effectively engineered a stable, bactericidal Ti surface. This new surface holds great promise in terms of mitigating or preventing periprosthetic infection. ß