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

Summary

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.

Abstract

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.

Introduction

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 conf....

Protocol

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 (....

Results

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 (Figu.......

Discussion

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.......

Materials

List of materials used in this article

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