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A Bioresorbable Calcium Phosphate Delivery System with Teicoplanin for Treating MRSA Osteomyelitis

Lazarettos, J*; Efstathopoulos, N*; Papagelopoulos, P J; Savvidou, O D; Kanellakopoulou, K; Giamarellou, H; Giamarellos-Bourboulis, E J; Nikolaou, V*; Kapranou, A§; Papalois, A; Papachristou, G*

Clinical Orthopaedics and Related Research: June 2004 - Volume 423 - Issue - p 253-258
doi: 10.1097/01.blo.0000127422.06956.35
SECTION II: ORIGINAL ARTICLES
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To assess the effectiveness of calcium phosphate as a delivery system of teicoplanin, methicillin-resistant Staphylococcus aureus osteomyelitis was induced in 36 rabbits. Osteomyelitis was induced by inoculating 107 cfu of methicillin-resistant Staphylococcus aureus isolate into a 2-mm hole at the upper ⅓ of the femur for 3 weeks, when all animals had reoperations, and calcium phosphate cement with 3% teicoplanin was implanted. Animals were divided into six groups of six animals each, sacrificed at Weeks 1, 2, 3, 4, 5, and 6, respectively, after implantation. One rabbit in each group was used as a control. Substantial clinical improvement of the rabbits was observed after implantation, accompanied with sterile cultures of bone after the second week of treatment. Throughout the same period, 105 to 108 cfu/g of methicillin-resistant Staphylococcus aureus isolate was cultured from the control samples. Bacterial eradication signified a considerable decrease of the total histologic scores of osteomyelitis compared with controls, accompanied with newly growing host bone. The calcium phosphate with teicoplanin delivery system seems promising for treatment of bone infection attributable to methicillin-resistant Staphylococcus aureus. In addition, this mixture allows filling of bone defects by new host bone.

From the *Second Department of Orthopaedics, University of Athens, Athens, Greece; †First Department of Orthopaedics, University of Athens, Athens, Greece, ‡Fourth Department of Internal Medicine, University of Athens, Athens, Greece; §Department of Medicine, “Tzanio” General Hospital, Piraeus, Greece; Experimental Research Unit of Elpen Co., Athens, Greece.

Received: April 28, 2003

Revised: August 25, 2003; February 2, 2004

Accepted: March 8, 2004

Correspondence to: Panayiotis J. Papagelopoulos, MD, DSc, Athens University Medical School, 4 Christovassili Street, 15451, Neo Psychikon Athens, Greece. Phone: 011-30-210-6721355; Fax: 011-30-210-6721355; E-mail: pjp@hol.gr.

The treatment of chronic bone infections is a challenging problem for orthopaedic surgeons and infectious diseases specialists. This treatment commonly requires long periods of hospitalization, repeat surgical interventions, and can result in side effects from the prolonged systemic administration of antibiotics. Because of these complications, the quality of life of these patients is severely compromised. In addition, there are important social and financial consequences.4

Adequate surgical debridement of the infected area and systemic long-term administration of antibiotic chemotherapy is an accepted method for treatment of chronic osteomyelitis.8,31,32 However, the efficacy of local chemotherapy against chronic osteomyelitis has been recognized the last two decades and several methods for locally controlled release of drugs have been developed.5,15,21 Polymethylmethacrylate (PMMA) is a commonly used medium of controlled antibiotic release in clinical use.40,42 Although this material is a well-established controlled release system, it is not compatible with thermosensitive antibiotics and requires a two-stage procedure involving implantation of antibiotic beads and eventual removal for subsequent bone grafting.5,26

Calcium phosphate is a biomaterial with a chemical and crystal structure similar to that of bone.33 This material has been used in clinical practice to repair bone defects. After implantation, calcium phosphate is absorbed gradually and is replaced by newly growing bone tissue.12,37

We investigated the effectiveness of the calcium phosphate cement enriched with 3% teicoplanin in an experimental methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model with special attention given to bacterial eradication.

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MATERIALS AND METHODS

Thirty-six adult male New Zealand rabbits, with an average weight of 3000 g (range, 2750 g–3210 g), were used. The protocol for animal subjects was approved according to the state’s relevant laws and regulations. International directives for handling experimental animals used in medical research were followed.l,9,30

Animals were divided into six groups of six animals each, depending on the time from implantation to sacrifice, with the animals being sacrificed at 1, 2, 3, 4, 5, and 6 weeks, respectively, after implantation. One rabbit in each group was used as a control. Three stages were followed in the experiment design.

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Stage I

Experimental osteomyelitis was induced at the upper ⅓ of the right femur, according to the modified model of experimental osteomyelitis induction described previously.11,28,29,33,34 After general anesthesia was administered, a direct 3-cm skin incision was made at the outer surface of the right hip and the trochanter was exposed. Using a drill, a small 2-mm diameter hole was made at the area between the greater and the lesser trochanter. Thereafter, 100 μL of cultured MRSA (inoculum 108 cfu/mL as suggested in a previous study22) was injected, and a sterilized metal needle (2.5 cm length and 0.7 mm diameter) was inserted to stimulate local inflammation. The hole was sealed with surgical wax. The strain of MRSA used, obtained from a patient with chronic osteomyelitis, had a minimum inhibitory concentration (MIC) for teicoplanin equal to 0.5 μg/mL.

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Stage II

Three weeks after induction of osteomyelitis the needle was removed and was sent for quantitative culture with a small bone fragment. A mixture of calcium phosphate cement with teicoplanin 3% (2400 mg cement powder mixed with 74.5 mg teicoplanin) was injected in the gap.

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Stage III

One group of six animals was sacrificed per week, for 6 consecutive weeks. Small bony fragments were cut by a sterile drill and placed into separate plastic sterile containers for quantitative culture. The animals’ femurs were removed, had radiographic evaluation, and were sent for histopathologic examination.

Segments of bone, drawn at Stages II and III, were washed with sterile NaCl, weighed, and homogenized. A 0.1-mL aliquot was diluted 1:10 in sterile NaCl consecutive times. Another aliquot of 0.1 mL of each dilution was plated on blood agar. Plates were incubated at 35°C and the number of viable colonies was counted in each dilution and multiplied by the appropriate dilution factor. The number of viable cells was expressed as cfu/g; the lowest limit of detection was 10 cfu/g.

The number of viable bacterial cells in bone was represented by its log10 value. Log10 changes between surgery in Stages III and II were expressed by its mean values (± SD). Comparisons between times were done using ANOVA with the Bonferroni correction. Any value of p ≤ 0.05 was considered significant.

For all animals, in each of the above stages, a clinical assessment was done based on the following criteria: (a) malaise; (b) elevated body temperature; (c) decrease of body weight; (d) pain on palpation; and (e) abscess formation. Body temperature was estimated by placement of a thermometer in the rectum (Omron, Germany). Body weight was decreased by a denial of food intake attributable to infection. Animals were weighed weekly to assess if they regained their initial weight before induction of osteomyelitis. Each criterion was scored as 1 point for its presence and 0 for its absence so that a semiquantitative clinical performance score was counted. The total score was expressed by its mean (± SD) for each group of animals at the respective week of sacrifice after implementation of the calcium cement with teicoplanin.

Pathologic findings were evaluated by a semiquantitative total histology score as suggested by Tarbox et al39 with separate grading from 0–4+ (0: absence, 1+: scarce, 2+: mild, 3+: moderate, 4+: intense) for the presence of acute and chronic inflammatory reaction, new vessel formation, bone necrosis, fibroblast proliferation, and edema formation. The total score was expressed by its mean (± SD) for each group of animals at the respective week of sacrifice after implementation of the calcium cement with teicoplanin. Comparisons between consecutive weeks were done using ANOVA (p < 0.05).

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RESULTS

Osteomyelitis was induced in all animals. The infection was documented clinically in all animals on the basis of signs of malaise, high fever, refusal of food, weight loss, difficulty walking, and formation of abscesses and fistulas, so that all animals had a clinical score of 5 before initiation of treatment. No death occurred as a result of sepsis.

Positive cultures for MRSA from the extracted needles were observed in 34 animals (94.4%) and from bone fragments in all animals (100%). Plain radiographs of the femur showed an increased thickness and sclerosis of the inner cortex in nine specimens (25%), multiple bone cysts at the proximal femur in nine specimens (25%), inflammatory involvement of the entire diaphysis in eight specimens (22.2%), and spontaneous fractures of the inner cortex in three specimens (8.3%).

Substantial clinical improvement was observed in all rabbits 4–5 days after injection of calcium phosphate and teicoplanin mixture as reflected in the changes of their clinical performance score shown in Figure 1. Scores of controls were 5, 5, 5, 4, 5, and 5 at 1, 2, 3, 4, 5, and 6 weeks after removal of the needle.

Fig 1.

Fig 1.

Bone cultures obtained from treated animals became sterile during the second week, whereas all cultures from control animals remained positive throughout the 6-week experiment. Changes of bacterial counts of the MRSA isolate with time after implementation of the mixture are shown in Figure 2. A decrease in bacterial counts of the MSRA isolate was seen 2 weeks after implementation of the mixture compared with the bacterial counts at 1 week (p < 0.0001). Log10 changes of controls were −0.53, +0.16, −0.19, −0.57, −0.01, and −0.03 at 1, 2, 3, 4, 5, and 6 weeks after removal of the needle.

Fig 2.

Fig 2.

Histologic examination of the femur showed progressive remission of the inflammatory process, intense development of new bone tissue around the area of antibiotic-cement injection, and a good degree of cement replacement by bone (Figs 3–6). However, prominent signs of active inflammation were confirmed in all control samples. Changes of total histologic scores with time after implementation of the mixture are given in Figure 7. Respective scores of control animals sacrificed at 1, 2, 3, 4, 5, and 6 weeks after removal of the needle were 4, 4, 4, 4, 8, and 4. Decreases of the total histologic scores were seen at 3 (p = 0.022), 4 (p = 0.006), 5 (p = 0.040), and 6 weeks (p < 0.0001) of sacrifice compared with the total histologic scores of animals sacrificed at 1 week.

Fig 3.

Fig 3.

Fig 4.

Fig 4.

Fig 5.

Fig 5.

Fig 6.

Fig 6.

Fig 7.

Fig 7.

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DISCUSSION

Despite the advances achieved in orthopaedic surgery and antimicrobial chemotherapy, osteomyelitis is a difficult disease to eradicate, mainly because of the specific morphologic features of the bone tissue that prevent achievement of effective concentrations of the drugs in the inflamed bone.21

The severity of the disease and the high rate of failure of systemic chemotherapy requires development of local delivery systems that offer an alternative therapeutic solution.5,15 The main advantages expected from such approaches are the prolonged release of high concentrations of the drug in the affected bone, the avoidance of side effects related to systemic chemotherapy, and the reduction in the number of days required for patient hospitalization.14

The introduction of antibiotic-impregnated PMMA cement in the 1970s increased the concentration of antibiotics in a local area, augmenting the use of systemic-parental antibiotics for bone infection therapy.26 However, this may necessitate a second procedure for removal of the cement for subsequent bone grafting.5,26,32 In addition, PMMA without an antibiotic has toxic effects on macrophages.19 Two studies have shown that antibiotic impregnation of PMMA does not prevent microbial adherence or colonization.7,36 Another problem with PMMA as an antibiotic carrier is the limited elution of the drug from the carrier, in amount and in time.18

Later drug delivery systems offered one-stage biodegradable materials or coatings that produced high local concentrations of antibiotics with low systemic levels and eliminated the need for secondary removal of the implants.26,41 However, they often left significant voids after resorption. Newer materials provide local antibiotic delivery and have osteoconductive properties, thereby eliminating the need for surgery to remove implants and to add autograft.

Products such as hydroxyapatite blocks and beads,27,44 apatite-wollastonite glass ceramic,23 hydroxyapatite void-fill cement,3 and calcium sulfate24,26 are osteoconductive scaffolds that fill dead space and encourage tissue ingrowth, leading to repair of osseous defects while eluting high concentrations of local antibiotics. The best biodegradable materials used for local release of antibiotics are the collagen sponges,38,43 cubes of ceramic apatite,22 cubes of hydroxyapatite,20,35 implants from glycolic acid,14 polymers from lactate,6 and polycaprolactone.17 All of these systems allow release of drugs at concentrations beyond the MIC, at least for the common pathogens responsible for chronic osteomyelitis. The antibiotics are not released in the systemic circulation and do not cause any systemic side effects.

We investigated the calcium phosphate and 3% teicoplanin delivery system for release of effective concentrations of antibiotics for the prolonged period required for eradication of chronic osteomyelitis attributable to MRSA. This delivery system proved successful for the treatment of bone infection attributable to MRSA. Bacterial counts of the MRSA isolate after implantation of the cement after the second week were significantly decreased compared with those achieved during the first week of treatment. Also, the total histology scores at 3, 4, 5, and 6 weeks of sacrifice were significantly decreased compared with the total histology score of the first week of sacrifice.

In our study, the mixture of calcium phosphate with teicoplanin allowed filling of bone defects by newly growing host bone. An in vivo experimental and clinical study showed the bone remodeling ability of calcium phosphate cement.13 The mechanics of this cement are characterized by high resistance to compression and low resistance to slashing (intersecting) forces, which renders the material ideal for filling bone gaps.16 Because of its spongiform structure, calcium phosphate cement allows double the amount of the antibiotic to be incorporated compared with the solid acrylic material. The minimal bactericidal concentration of the antibiotic is achieved for double the time of acrylic mixtures, therefore calcium phosphate cement is an optimal material for local antimicrobial chemotherapy.25 The chemical and crystal properties of the cement that define its excellent behavior regarding incorporation and progressive replacement by newly growing bone tissue are not affected by enrichment with antibiotics.2,10 Also, the mechanical properties of the cement are unaffected.

Teicoplanin is used commonly in Europe to treat osteomyelitis because of its favorable pharmacokinetics, long half-life, and three compartmental mode of distribution after administration. Teicoplanin used with calcium phosphate as a delivery system may meet all prerequisites for management of infection.

Although clinical improvement of the animals after implementation of the cement might be considered biased because of subjective observer evaluation, careful quantitative estimation of bacterial eradication and histologic improvement (Figs 2, 7) accentuate the significance of the current findings. However, relapse of infection is the only study limitation because it requires a larger number of animals than those in each of our subgroups.

We developed a biomaterial as a carrier for release of antibiotics using calcium phosphate cement enriched with teicoplanin. The system may be complementary to systemic therapy in the treatment of osteomyelitis, because it fulfills all the required properties that a carrier should have such as tissue compatibility, release of high drug concentrations, and flexibility to choose the dose and type of antibiotic. In patients with chronic osteomyelitis, calcium phosphate cement enriched with teicoplanin can be used instead of the acrylic cement to avoid reoperation for cement removal. The excellent pharmacologic and biomechanical behavior of the local chemotherapy system we developed needs additional testing in well-designed experimental and clinical trials for the treatment of osteomyelitis.

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