In vitro and in vivo efficacies of teicoplanin-loaded calcium sulfate for treatment of chronic methicillin-resistant Staphylococcus aureus osteomyelitis

Abstract

The in vitro and in vivo therapeutic efficacies of teicoplanin-loaded calcium sulfate (TCS; 10% [wt] teicoplanin) were investigated in a rabbit model of chronic methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. The in vitro elution characteristics of teicoplanin from TCS pellets were realized by carrying out an evaluation of the release kinetics, recovery rate, and antibacterial activity of the released teicoplanin. Chronic osteomyelitis was induced by inoculating 10(7) CFU of a MRSA strain into the tibial cavity of rabbits. After 3 weeks, the animals were treated by debridement followed by implantation of TCS pellets in group 1, calcium sulfate (CS) pellets alone in group 2, and intravenous (i.v.) teicoplanin (6 mg/kg of body weight every 12 h for three doses and then every 24 h up to 4 weeks) in group 3. Animals in group 4 were left untreated. After 6 weeks, the efficacy of the osteomyelitis treatment was evaluated by hematological, radiological, microbiological, and histological examinations. In vitro elution studies showed sustained release of teicoplanin at a therapeutic level over a time period of 3 weeks. The released teicoplanin maintained its antibacterial activity. In vivo, the best therapeutic effect was observed in animals treated with TCS pellets, resulting in significantly lower radiological and histological scores, lower positive rates of MRSA culture and bacterial load, and excellent bone regeneration compared with those treated by CS alone or i.v. teicoplanin, without any local or systemic adverse effects. TCS pellets are an effective alternative to i.v. teicoplanin for the treatment of chronic MRSA osteomyelitis, particularly because teicoplanin is delivered locally while the TCS pellets simultaneously promote bone defect repair.

FIG. 1.

Release of teicoplanin from teicoplanin-impregnated calcium sulfate pellets (a) and cumulative percentage (b) as functions of time of immersion in PBS (means ± standard deviations). L, liter; w/w, weight, weight.

FIG. 2.

(a) Changes in body weight of animals at sacrifice compared to preoperative weights (means ± standard deviations) exhibited a significant increase in group 1 and a decrease in groups 2 and 4. (b) Changes in WBC counts of animals showed a significant increase in groups 2 and 4 and no significant changes in groups 1 and 3 compared to preoperative levels (means ± standard deviations). L, liter.

FIG. 3.

Serum concentrations of teicoplanin in groups 1 and 3 (means ± standard deviations).

FIG. 4.

Representative radiographs (1, 2, 3, and 4 correlate to group number). Experimental tibial osteomyelitis occurred 3 weeks after induction of MRSA, as evaluated by bone destruction (white arrow), periosteal new bone formation (black arrow), and sequestral bone formation (white arrowhead) (1a). Bone windows (black arrowhead) and implantation of pellets (star) were observed after debridement (1b and 2a). Healing of osteomyelitis was observed 6 weeks after debridement and implantation of TCS in group 1 (1c). Debridement without implantation of pellets is illustrated for group 3 (3a). Osteomyelitis had deteriorated in various degrees 6 weeks after treatment in groups 2, 3, and 4 (2b, 3b, and 4, respectively).

FIG. 5.

Histological and radiographical scoring showed that significantly lower scores occurred in groups 1 (TCS pellets) and 3 (i.v. teicoplanin) than in control groups, as well as between group 1 and 3 (means ± standard deviations). The filled circle and filled upright triangle indicate significant differences (P < 0.05) compared with preoperative scores and scores in groups 2, 3, and 4. The filled inverted triangle and filled square indicate differences (P < 0.05) compared with groups 2 and 4.

FIG. 6.

Representative photomicrographs of longitudinal sections of tibiae (hematoxylin and eosin stained). (A) Typical signs of osteomyelitis prior to surgery included destruction of bone (white arrow), reactive hyperostosis (white arrowhead), fibrosis (black arrow), and the presence of pus cells in the medullary cavity (black arrowhead) (×40 magnification). (B) Six weeks after debridement and implantation of TCS pellets, a large amount of new bone formation (white arrows) with the degradation of the pellets (black arrows) occurred in group 1, accompanied by mild fibrosis (×40 magnification). (C) A high-magnification (×200) image from group 1 shows the newly formed bone, accompanied by infiltration by macrophages (white arrowheads), indicating the phagocytic process of the host to dissolve the biodegradable CS. (D) Six weeks after implantation of CS pellets alone in group 2, the pellets were mostly resorbed and surrounded by chronic fibrosis with multinucleated granulocytes (black arrow), proliferated foamy histiocytes (white arrow), and no obvious new bone formation (×40 magnification). (E and F) Moderate to severe inflammation remained in group 3 (E) and group 4 (F) 6 weeks after treatment (×40 magnification).

FIG. 7.

Quantitative microbiological data are plotted as the number of CFU per gram of bone sample. The individual data (diamonds), the medians (black bars), and the approximate detection limit (dotted line; 2 × 10³ CFU/g) are indicated. Group 1 (TCS pellets) had a bacterial load at autopsy that was significantly lower than the preoperative level (P < 0.001) and those of group 2 (CS pellets; P < 0.001), group 3 (i.v. teicoplanin; P = 0.010), and group 4 (untreated; P < 0.001). Additionally, group 3 (i.v. teicoplanin) exhibited a significantly lower CFU count than either group 2 (P = 0.033) or group 4 (P = 0.001).