Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Podsumowanie

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

Streszczenie

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.

In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.

In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

Wprowadzenie

Bone infection usually results from bacteria or other microorganism invasion after trauma, bone fracture, or other bone diseases¹. Bone infection may induce a high level of inflammation and bone tissue destruction. In the clinic, Staphylococcus aureus (S. aureus) is the predominant causative agent of bone infection^2,3. The bone infection is painful, debilitating, and often takes a chronic course that is extremely difficult to treat⁴. At present, debridement of necrotic tissue and implanting of vancomycin-loaded calcium (VCS) beads have been confirmed as an efficient strategy for controlling local infection^5,6. However, 10% to 15% of patients experienced a prolonged bone repair process, delayed union, or non-union after anti-infection treatment⁷. The large segment of a bone defect is the most difficult issue for orthopedic surgeons. An autologous bone graft is considered the optimal bone replacement in bone non-union treatment^8,9.

To date, most of the studies on bone infection and autologous bone implantation have been conducted in various kinds of animal models, such as rats, rabbits, dogs, pigs and sheep^10,11. Rabbit models are most commonly used for bone infection studies, as first performed by Norden and Kennedy in 1970^12,13. In our previous study, we used rabbit models following Norden's method, and we found that the quantity of S. aureus injected into bone marrow could not be quantified accurately, as the blood leaking out of bone marrow led to bacteria solution overflow.

This article presents an improved surgical method for inducing bone infection on rabbits. At the end of the procedure, a blood biochemistry test, a bacteriological examination, and a histopathologic examination were performed to verify the bone infection model. Then, VCS was implanted to inhibit infection, and autogenous bone was implanted to promote bone regeneration.

Protokół

The rabbits used in the present study were treated in accordance with the Guide for the Care and Use of Laboratory Animals. All the experimental procedures were followed by the rules of the Bioethics Committee of Zhejiang Academy of Traditional Chinese Medicine.

1. Preparation of the Bacterial Suspension

1. Dissolve 0.5 mg of S. aureus freeze-drying powder (ATCC 6538) with 0.3 mL of Luria-Bertani culture medium. Mix suspension completely.
2. Streak the bacteria suspension onto tryptic soy agar plates and incubate the bacterial colonies at 37 °C for 16 h.
3. Select a single bacterial colony forming unit (CFU) and culture in tryptic soy broth tubes for 24 h. Perform a subculture for approximately 24 h at 37 °C, and obtain mid-logarithmic growth phase bacteria after 16 to 18 h, when the optical density (OD) value is 0.6 at 600 nm¹⁴.
4. Transfer 1 mL of bacteria suspension into a centrifuge tube. Centrifuge for 5 min at 825 x g and 4 °C, and discard the supernatant. Resuspend and wash the bacteria with 100 µL of phosphate buffered saline (PBS); repeat this step 3 times. Finally, resuspend bacteria with 3 mL of PBS.
5. Estimate the bacteria concentration using McFarland's turbidimetry¹⁵.
   1. Transfer 100 to 500 µL of bacteria suspension to a colorimetric tube until the turbidity is equivalent to a 0.5 McFarland standard.
   2. Assess turbidity by visual comparison to the 0.5 McFarland, when the content of bacteria reaches approximately 10⁸ CFU/mL.
      NOTE: Make sure the volume of bacteria suspension is sufficient for the following protocols. For every rabbit, the volume of bacteria suspension is less than 1 mL.
6. Transfer 0.2 mL of bacterial suspension to an agar plate and apply it evenly. Incubate the plate at 37 °C for 16 h. Count the bacteria colonies to verify the CFU of bacteria suspension.

2. Preparation of Bone Infection Models

1. Keep male New Zealand white rabbits, aged 3 months, in individual cages, under air-controlled conditions (20 ± 1 °C) and 12 h/12 h light-dark illumination cycles. Offer routine diet and tap water daily.
2. Ensure that at the time of surgery that the rabbit weighs more than 3 kg.
3. Anaesthetize rabbits by intraperitoneal injection with pentobarbital sodium (3 mg per 100 g of body weight). Make sure rabbits are fully anesthetized by a failure to respond to a paw pinch. Fix rabbits on the operating table during operation procedure.
   NOTE: Make sure that the modeling procedure duration is less than 1 h.
4. Shave the proximal tibia region using an electric shaver against the direction of hair growth. Disinfect the skin by applying a povidone-iodine solution.
5. Mark the upper end of the tibia and the drilling hole position for injection with S. aureus (the distance to the upper end of the tibia is 1.5 cm) with pen and ruler. Make sure the drilling hole positions are in the middle of the tibial plateau horizontally (Figure 1A).
6. Cut tibia skin using a No. 11 scalpel and make a 1 cm incision in the periosteum (Figure 1B,C). Punch a 2 mm diameter hole in the tibia using an electric bone drill unit (Figure 1D).
7. Press the 2 mm diameter holes in the tibial plateau with a cylinder of bone wax of 2 mm diameter and 2 mm height (Figure 1E). Remove the spare bone wax along the horizontal plane of the tibial plateau (Figure 1F). Check that the 2 mm hole is full of bone wax (Figure 1G).
   NOTE: Ensure that the holes are full of bone wax by checking the hole with or without blood overflow.
8. Sew up the periosteum and skin with absorbable surgical suture in a vertical mattress suture to prevent the animal from chewing the sutures (Figure 1H).
9. Inject 1 x 10⁸ CFU/mL of S. aureus solutions (30 µL per 100 g of body weight) with a 1 mL asepsis injector (Figure 1I). Make sure the needles penetrate the bone wax and inject the S. aureus solution into bone marrow slowly.
10. Keep the animal in warm and clean conditions to avoid heat loss after modelling. Monitor respiratory rate and heart rate. After waking up, house the rabbits in individual cages with free access to food and water.

3. Evaluation of Bone Infection Model

1. At days 7, 14, 21 and 28 after infection, place rabbits into the rabbit fixer with the head and ear outside of the fixer.
2. Draw 2 mL of blood from the auricular veins into a dipotassium ethylenediaminetetraacetic acid (EDTA-K2) anticoagulant blood container. Draw 1 mL of blood from a blood vessel into a blood container. Centrifuge the serum for 10 min with a speed of 651 x g at room temperature.
   1. Determine white blood cell count (WBC) in whole blood using a blood biochemical analyzer, and C-reactive protein (CRP) by an enzyme-linked immunosorbent assay (ELISA) method¹⁶.
3. At days 7, 14, 21 and 28 after infection, anaesthetize one model rabbit with pentobarbital sodium at the dosage of 3 mg per 100 g body weight. Cut tibia skin using a No. 11 scalpel and make a 2 cm incision in the periosteum (Figure 2A).
4. Clean bone wax. Debride necrotic bone by punching two adjacent 4.8 mm diameter holes using an electric bone drill unit (Figure 2B). Debride necrotic bone marrow and granulation tissue using a bone spoon (Figure 2C).
   NOTE: Clean bone tissue during the debridement to avoid bone tissue remaining in the bone marrow.
5. Scrape and clean the bone tissue between the two holes (Figure 2D).
6. Spread 1 mL of bone marrow onto sheep blood agar plates. Incubate plates overnight at 37 °C. Select plates of 30–300 colonies, and calculate the number of colonies.
7. At the end of day 28 after infection, extract tibia specimens along the edges of knee and ankle joints. Fix the tibia specimens in 4% paraformaldehyde for 24 h. Decalcify the tibia specimens in 10% EDTA for 8 weeks.
8. Dehydrate the tibia specimens in a graded series of ethanol dilutions, and then embed in paraffin wax. Cut 4 consecutive 5 µm sections from the coronal planes. Stain sections with a hematoxylin and eosin (H&E) staining kit.
9. Use a microscope to view the stained sections and record transmitted light images with standard software.

4. Preparation of VCS Beads

1. Add 1 g of vancomycin hydrochloride powder to 9.5 g of medical grade calcium sulphate, and then add 3 mL of normal saline to the mixed power. Mix them thoroughly with a spatula for 30 to 45 s.
2. Place the mixed product into a flexible silica gel mold (cylinder of 4.8 mm diameter and 4.8 mm height), and dry at room temperature for 15 min. Remove the VCS beads by flexing the mold.

5. Antibiotic Treatment and Implantation of Autogenous Bone

1. Anaesthetize model rabbits with pentobarbital sodium at the dosage of 3 mg per 100 g body weight on the 28^th day after infection. Shave the proximal tibia region using an electric shaver. Disinfect the skin by applying povidone-iodine solution.
   NOTE: Make sure that the modelling procedure is less than 1 h.
2. Shave the tail region using an electric shaver and disinfect the tail by applying povidone-iodine solution.
3. Cut down the tail using surgical scissors. Cut tail skin using a No. 11 scalpel and reveal the tail bone. Sew up the skin at the tail region with absorbable surgical sutures in a vertical mattress suture to prevent the animal from chewing the sutures.
4. Remove any muscle, soft tissue and periosteum. Detach the tail bone at each joint and transfer the bone fragment to a 100 mm plastic dish containing sterile saline.
5. Implant 4 pieces of VCS beads (cylinder of 4.8 mm diameter and 4.8 mm height, 1.25 mg vancomycin per piece of bead) into the marrow cavity using curved tweezers (Figure 2E).
6. Fill the bone defect with 8 pieces of autogenous bones (cylinder of 2 mm diameter and 4 mm height per each piece) using curved tweezers (Figure 2E).
7. Sew up the periosteum and skin with absorbable surgical sutures in a mattress suture manner (Figure 2F).
   NOTE: Keep the temperature at 25 °C during the surgery.
8. Keep the animal in warm and clean conditions to avoid heat loss after surgery. Monitor respiratory rate and heart rate. After waking up, house the rabbits in individual cages with free access to food and water.

6. Assessments of Antibiotic Activity

1. Put rabbits into a rabbit fixer, and place the head and ear outside of the fixer at 2, 4, 6 and 8 weeks after treatment.
2. Draw blood from auricular veins with EDTA-K2 anticoagulant blood vessel. Draw 1 mL of blood from a blood vessel into a blood container. Centrifuge the serum for 10 min with a speed of 651 x g at room temperature.
3. Determine the white blood cell count (WBC) in whole blood by using blood biochemical analyzer, and C-reactive protein (CRP) by an ELISA method¹⁶.

7. Assessments of Bone Regeneration

1. Euthanize rabbits by injecting with an over dosages of pentobarbital sodium, at the end of 8 or 12 weeks after treatment.
2. Extract tibia specimens, along the edges of knee and ankle joints. Debride muscles and fascial layers.
3. Analyze structure of tibia by using micro-computed tomography (micro-CT). Choose an oval area 4.8 mm diameter and 9.6 mm long as the region of interest (ROI). Reconstruct 3D model images using bitmap data.
4. Choose scores of the ratio of bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecula number (Tb.N) and trabecular separation (Tb.Sp)from the3D models to assess bone regeneration.

Wyniki

Evaluation of Bone Infection Model
After infection with S. aureus, the pathological manifestations of rabbits were similar to the representative symptom of chronic osteomyelitis in the clinic. In our study, 30 rabbits were infected, and subjected as a model group, and 10 rabbits were subjected as control animals. All the model rabbits have infected sinuses of the tibia local site, with white and yellow pus over flow from the sinuses (Fi...

Dyskusje

In the previous studies, various kinds of animal models were constructed to study both acute and chronic bone infection; however, the search for the ideal model still persists^17,18. In addition, the ideal bone infection model is expected to simulate the pathological characteristics of bone infection in clinical setting, while the modelling periods, remain low cost and easy to carry out. So far, the rabbit bone infection model is the most common model in inflammat...

Materiały

Name	Company	Catalog Number	Comments
absorbable surgical suture	Jinghuan	18S0604A
asepsis injector	Jinglong	20170501
bone wax	ETHICON	JH5CQLM
CCD camera	Olympus	DP72
EDTA-K2 anticoagulant blood vessel	XINGE	20170802
Electric bone drill unit	Bao Kang	BKZ-1
Electric shaver	Codos	3800
flexible silica gel mold	WRIGHT	1527745
Hematoxylin and Eosin Staining Kit	Beyotime	20170523
Luria-Bertani culture medium	Baisi Biothchnology	20170306
Medical-grade calcium sulphate	WRIGHT	1527745
microcomputed tomography (micro-CT)	Bruker	SkyScan 1172
Microscope	Olympus	CX41
New Zealand white rabbits	Zhejiang Experimental Animal Center	SCXK 2014-0047
No. 11 scalpel	Yuanlikang	20170604
normal saline	Mingsheng	20170903
PBS	TBD(Jingyi)	20170703-0592
pentobarbital sodium	Merk	2070124
povidone-iodinesolution	Lierkang	20170114
S. aureus freeze drying powder	China General Microbiological Culture Collection Center	ATCC 6538
sheep blood agar	HuanKai Microbial	3103210
tryptic soy agar plates	HuanKai Microbial	3105697
tryptic soy broth tubes	HuanKai Microbial	3104260
Vancomycin	Lilly	C599180