Name: treatment with vancomycin loaded calcium sulphate and autogenous bone in an improved rabbit model of bone infection
Uploaded: 2019-03-14T12:30:00
Description: Watch this Scientific Journal Video about Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection at JoVE.com

This work was supported by the National Natural Science Foundation of China (81803808, 81873062), Zhejiang Provincial Medical and Health Science and Technology Fund (2017KY271) and the Science and Technology Project of Zhejiang Province (2017C37181).

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
<ol>
	<li>Dissolve 0.5 mg of S. aureus freeze-drying powder (ATCC 6538) with 0.3 mL of Luria-Bertani culture medium. Mix suspension completely.</li>
	<li>Streak the bacteria suspens...

<ol>
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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 persists17,18. 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...

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 infection2,3. 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 infection5,6. 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 treatment8,9.
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 sheep10,11. Rabbit models are most commonly used for bone infection studies, as first performed by Norden and Kennedy in 197012,13. In our previous study, we used rabbit models following Norden&#39;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.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">absorbable surgical suture</td>
			<td style="width:264px;">Jinghuan</td>
			<td style="width:119px;">18S0604A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">asepsis injector</td>
			<td style="width:264px;">Jinglong</td>
			<td style="width:119px;">20170501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">bone wax</td>
			<td style="width:264px;">ETHICON</td>
			<td style="width:119px;">JH5CQLM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">CCD camera</td>
			<td style="width:264px;">Olympus</td>
			<td style="width:119px;">DP72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">EDTA-K2 anticoagulant blood vessel</td>
			<td style="width:264px;">XINGE</td>
			<td style="width:119px;">20170802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Electric bone drill unit</td>
			<td style="width:264px;">Bao Kang</td>
			<td style="width:119px;">BKZ-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Electric shaver</td>
			<td style="width:264px;">Codos</td>
			<td style="width:119px;">3800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">flexible silica gel mold&#160;</td>
			<td style="width:264px;">WRIGHT</td>
			<td style="width:119px;">1527745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hematoxylin and Eosin Staining Kit</td>
			<td style="width:264px;">Beyotime</td>
			<td style="width:119px;">20170523</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Luria-Bertani culture medium</td>
			<td style="width:264px;">Baisi Biothchnology</td>
			<td style="width:119px;">20170306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Medical-grade calcium sulphate</td>
			<td style="width:264px;">WRIGHT</td>
			<td style="width:119px;">1527745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">microcomputed tomography (micro-CT)</td>
			<td style="width:264px;">Bruker</td>
			<td style="width:119px;">SkyScan 1172&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Microscope</td>
			<td style="width:264px;">Olympus</td>
			<td style="width:119px;">CX41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">New Zealand white rabbits</td>
			<td style="width:264px;">Zhejiang Experimental Animal Center&#160;</td>
			<td style="width:119px;">SCXK 2014-0047</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:275px;">No. 11 scalpel&#160;</td>
			<td style="width:264px;">Yuanlikang</td>
			<td style="width:119px;">20170604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">normal saline</td>
			<td style="width:264px;">Mingsheng</td>
			<td style="width:119px;">20170903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">PBS</td>
			<td style="width:264px;">TBD(Jingyi)</td>
			<td style="width:119px;">20170703-0592</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">pentobarbital sodium</td>
			<td style="width:264px;">Merk</td>
			<td style="width:119px;">2070124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">povidone-iodinesolution</td>
			<td style="width:264px;">Lierkang</td>
			<td style="width:119px;">20170114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">S. aureus freeze drying powder</td>
			<td style="width:264px;">China General Microbiological Culture Collection Center</td>
			<td style="width:119px;">ATCC 6538</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">sheep blood agar</td>
			<td style="width:264px;">HuanKai Microbial</td>
			<td style="width:119px;">3103210</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:275px;">tryptic soy agar plates</td>
			<td style="width:264px;">HuanKai Microbial</td>
			<td style="width:119px;">3105697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">tryptic soy broth tubes</td>
			<td style="width:264px;">HuanKai Microbial</td>
			<td style="width:119px;">3104260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Vancomycin</td>
			<td style="width:264px;">Lilly</td>
			<td style="width:119px;">C599180</td>
			<td></td>
		</tr>
	</tbody>
</table>

treatment with vancomycin loaded calcium sulphate and autogenous bone in an improved rabbit model of bone infection

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.

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...

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

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.

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

A producible animal model is very important for the bone infection research. However, currently there is no rabbit model for which the infections status and the number of bacteria is consistent. Our bone infection rabbit model uses a consistent bacteria and is treated by vancomycin-loaded calcium sulfate and autogenous fragments.The technique could be extended toward the diagnosis of bone infection and bone nonunion therapy, and in understanding the pathogenesis of bone infection. This method could provide insight into other bone infection models in rats, mice, and pigs. It's difficult to load the same volume of bacterial suspension into bone marrow.Visual demonstration can help researchers to carry out the critical steps easily and accurately. To prepare the bacterial suspension, first dissolve 0.5 milligrams of S.aureus freeze-dried powder in 0.3 milliliters of LB culture medium. Mix the suspension thoroughly, and streak the bacteria onto tryptic soy agar plates for their incubation for 16 hours at 37 degrees Celsius.The next morning, pick a single bacterial colony-forming unit for culture in tryptic soy broth tubes for 24 hours at 37 degrees Celsius. When the bacteria are in the mid-logarithmic growth phase, transfer 1 milliliter of bacteria into a 1.5 milliliter tube, and collect the bacteria by centrifugation. Then, wash the bacterial cell pellet three times in 100 microliters of PBS per wash, resuspending the bacteria in 3 milliliters of fresh PBS after the final wash.To set up the bone infection mold, confirm a lack of response to paw pinch in a three month old three kilogram male New Zealand white rabbit, and use an electric shaver to remove the hair from the proximal tibial region against the direction of the hair growth. Disinfect the exposed skin with povidone iodine solution, and use a pen and a ruler to mark the upper end of the tibia and the drilling hole position for the S.Aureus injection, taking care that the drilling hole position is in the horizontal middle of the tibial plateau. Next, use a number 11 scalpel to make an incision in the skin, followed by a one centimeter incision in the peritoneum.Using an electric bone drill unit, punch a two millimeter diameter hole in the tibia, and press the hole with a two millimeter diameter two millimeter long cylinder of bone wax. Remove any spare bone wax along the horizontal plane of the tibial plateau, and confirm that the hole is full of bone wax. Then, use absorbable surgical sutures to sew up the periosteum and skin in a vertical mattress suture to prevent the animal from chewing the stitches.And use a one milliliter asepsis injector to slowly inject one times 10 to the eight colony-forming units per milliliter of S.Aureus solution into the drill hole. At day seven, 14, 21 and 28 after injection, place the infected rabbits into a rabbit restrainer with the head and ear outside of the restrainer, and collect blood samples from the auricular veins for white blood cells and C-reactive protein analysis. After anesthesia, use a number 11 scalpel to make centimeter tibial skin and peritoneal incisions, and clean the bone wax.Next, use an electric bone drill unit to punch two adjacent 4.8 millimeter diameter holes to debride the necrotic bone, and use a bone spoon to debride the necrotic bone marrow and granulation tissue. Then, scrape and clean the bone tissue between the two holes, and spread one milliliter of the debrided bone marrow onto sheep blood agar plates for an overnight incubation at 37 degrees Celsius. The next morning, select plates with 30 to 300 colonies, and calculate the number of colony-forming units per infected animal.On the 28th day after infection, add one gram of vancomycin hydrochloride powder to 9.5 grams of medical grade calcium sulfate, and use a spatula to stir the mixture with three milliliters of normal saline for 30 to 45 seconds. Add the mixed product into a flexible silica gel mold, and allow the product to dry at room temperature for 15 minutes. Then, flex the mold to remove the vancomycin loaded calcium sulfate, or VCS bead.For antibiotic treatment and implantation of the autogenous bone, first, re-expose the injured tibia before shaving the tail region, and disinfecting the exposed tail skin with povidone iodine solution. Use surgical scissors to cut down the tail, and use absorbable surgical sutures to close the skin in a vertical mattress suture to prevent the animal from chewing the stitches. Use a number 11 scalpel to cut the tail skin to reveal the tail bone.Remove any muscle, soft tissue, and periosteum, and detach the tailbone at each joint. Transfer the bone fragments to a 100 millimeter plastic dish containing sterile saline. Next, use curved scissors to implant four pieces of the VCS bead into the tibial marrow cavity, and use curved forceps to fill the bone defect with eight pieces of autogenous tailbone fragments.Then, close the periosteum and skin incisions with absorbable surgical sutures in a mattress suture manner. C-reactive protein and white blood cell levels are higher in the bone infection model group than in the control animals. Two, four, six, and eight weeks after VCS or VCS autogenous bone treatment, C-reactive protein and white blood cell levels are significantly reduced.Two dimensional reconstruction images indicate a progressive increase in the bone volume during the 12 week period after treatment with VCS or VCS and autogenous bone, while bone loss is significant in the untreated bone infection model group. To analyze the bone regeneration quantitative indexes, 3D model images can be reconstructed using bitmap data, and an oval 4.8 millimeter diameter by 9.6 millimeter length oval region of interest can be selected in each bone. The ratio of bone volume to tissue volume in the untreated bone infection model group are significantly lower than those measured in the VCS and the VCS autogenous bone groups.The trabecular number and thickness scores in the VCS autogenous bone group are significantly higher than those in the untreated bone infection model and VCS animals. Moreover, the trabecular separation scores in the VCS autogenous bone group are markedly lower than those measured in the untreated bone infection model and VCS groups. Take care to select rabbits with appropriate body weight.Block the Staphylococcus aureus solution with bone wax. Debride the necrotic bone completely, and punch the two adjacent 4.8 millimeter diameter holes. Following the procedure, other diagnostic measure method such as ultrasound, radiology and computer tomography can be performed to monitor the pathological processes of the bone infection and repair.This improved bone infection model and the combination VCS and autogenous bone treatment could be helpful in studying the underlining mechanism of bone infection and bone regeneration. As the Staphylococcus aureus freeze-dried powder is hazardous, the bacterial suspension should always be prepared in an isolated bacterial laboratory.

Research

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JoVE Core

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Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

SCIENTISTS IN ACTION

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