In vivo effect of quaternized chitosan-loaded polymethylmethacrylate bone cement on methicillin-resistant Staphylococcus epidermidis infection of the tibial metaphysis in a rabbit model

Abstract

Infection of open tibial fractures with contamination remains a challenge for orthopedic surgeons. Local use of antibiotic-impregnated polymethylmethacrylate (PMMA) beads and blocks is a widely used procedure to reduce the risk of infection. However, the development of antibiotic-resistant organisms make the management of infection more difficult. Our in vitro study demonstrated that quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan [HACC])-loaded PMMA bone cement exhibits strong antibacterial activity toward antibiotic-resistant bacteria. Therefore, the present study aimed to investigate the in vivo antibacterial activity of quaternized chitosan-loaded PMMA. Twenty-four adult female New Zealand White rabbits were used in this study. The right proximal tibial metaphyseal cavity was prepared, 10(7) CFU of methicillin-resistant Staphylococcus epidermidis was inoculated, and PMMA-only, gentamicin-loaded PMMA (PMMA-G), chitosan-loaded PMMA (PMMA-C), or HACC-loaded PMMA (PMMA-H) bone cement cylinders were inserted. During the follow-up period, the infections were evaluated using X rays on days 21 and 42 and histopathological and microbiological analyses on day 42 after surgery. Radiographic indications of bone infections, including bone lysis, periosteal reactions, cyst formation, and sequestral bone formation, were evident in the PMMA, PMMA-G, and PMMA-C groups but not in the PMMA-H group. The radiographic scores and gross bone pathological and histopathological scores were significantly lower in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.05). Explant cultures also indicated significantly less bacterial growth in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.01). We concluded that PMMA-H bone cement can inhibit the development of bone infections in this animal model inoculated with antibiotic-resistant bacteria, thereby demonstrating its potential application for treatment of local infections in open fractures.

FIG 1

Clinical assessments of animals from various groups. (A) Temperature changes. (B) Weight changes. (C) WBC changes.

FIG 2

Lateral X-ray photographs of the right tibia, 3 and 6 weeks after surgery. Typical signs include osteolytic lesions (asterisks), periosteal new bone formation (arrowheads), and sequestral bone formation (arrows).

FIG 3

Radiographic scores. Score assessments indicated no significant differences among the PMMA, PMMA-G, and PMMA-C groups (P > 0.05). *, P < 0.05, compared with the other groups.

FIG 4

Gross appearance (A) and scores (B) of longitudinal sections of the tibiae. The PMMA, PMMA-G, and PMMA-C groups exhibited signs of purulent infection, while the PMMA-H group presented no obvious signs of infection. *, P < 0.05, compared with the other groups.

FIG 5

Quantification of bacteria obtained from explanted cements and bone tissue on the day of sacrifice. (A) Quantity of CFU per explanted PMMA-based bone cement. *, P < 0.01, compared with other groups; #, P < 0.05, compared with the PMMA or PMMA-C group. (B) Growth of bacterial colonies in bone cements on TSA. (C) Quantity of CFU per gram of bone. *, P < 0.01, compared with the PMMA or PMMA-C group; #, P < 0.05, compared with the PMMA-G group; $, P < 0.05, compared with the PMMA or PMMA-C group.

FIG 6

Representative photomicrographs of longitudinal sections from proximal tibiae, with hematoxylin and eosin staining. Column A, overview of signs of osteomyelitis in the proximal metaphysis. Magnification, ×0. Column B, moderate-to-severe inflammation with massive enlargement and destruction of bone tissue (black arrows) and fibrosis (white arrows) in the PMMA, PMMA-G, and PMMA-C groups and mild inflammation in the PMMA-H group. Magnification, ×40. Column C, moderate-to-severe inflammation with intramedullary abscesses and acute or chronic inflammatory cells (white arrowheads) around necrotic bony trabeculae (black arrowheads) in the PMMA, PMMA-G, and PMMA-C groups and mild inflammation in the PMMA-H group. Magnification, ×200.

FIG 7

Histological scores for each parameter in each group. The histological intraosseous acute inflammation (IAI), intraosseous chronic inflammation (ICI), periosteal inflammation (PI), and bone necrosis (BN) scores and total scores for the PMMA-H group were significantly lower than those observed in the PMMA, PMMA-G, and PMMA-C groups. *, P < 0.05.