The evaluation of effect of intra-articular injection of three antibiotics on articular cartilage

امتیاز کاربران
ضعیفعالی 

The evaluation of delayed effect of intra-articular injection of three antibiotics of Cefazolin, Gentamicin and Vancomycin on articular cartilage; an experimental study in rabbit

Short title: The evaluation of effect of intra-articular injection of three antibiotics on articular cartilage

Authors: Hamidreza Yazdi, Reza Jamei Moayedi, Mohammadali Shokrgozar, Mohammadmehdi Dehghan , Tahmineh Mokhtari 

  1. MD, Knee surgeon, Department of knee surgery, Shafa rehabilitation hospital, school of medicine, Iran university of medical science, Tehran ,Iran
  2. MD, Resident of orthopedics, Department of knee surgery, Shafa rehabilitation hospital, school of medicine, Iran University of medical science, Tehran ,Iran
  3. Ph. D, National cell bank of Iran, Pasteur Institute of Iran, Tehran, Iran.
  4. DVM, veterinary  surgeon, School of veterinary , Tehran University of  Iran, Tehran, Iran]
  5. Ph. D student of anatomy, Department of anatomy, School of medicine, Tehran University of medical science, Tehran, Iran.

 ABSTRACT

Aim: Antibiotics are the most common additives used in irrigation solutions for open fractures; nonetheless a few studies surveyed effects of antibiotics on articular cartilage.

We hypothesized that according to the characteristics of antibiotic solutions, they may have potential toxicity on articular cartilage and this effect may happen as a delayed phenomenon because of changes in matrix and cell viability.

Methods: 20 rabbits were divided into 4 groups each containing 5 rabbits and injection of normal saline as control, Gentamicin, Vancomycin and cefazolin was performed in both knees of each rabbits. After 8 weeks, articular cartilage of distal femurs was harvested and analysis with confocal microscopy and Real time PCR was done.

Results: According to confocal microscopy, Gentamicin and Vancomycin had significant decrease in live chondrocyte cell number however cefazolin had no effect.

Conclusion: Quantitative analysis did not show any significant difference between antibiotics and control group. We concluded that cefazolin can be safely used for intra-articular injections but use of Vancomycin and Gentamicin should be with caution.

 Keywords: antibiotic, chondrocyte, confocal microscope, Real time PCR

 

 Introduction

The chondrocyte is the resident cell of cartilage that is a prominent tissue in the embryo acting as a template for the development of skeletal elements. In the adult, the distribution of permanent cartilage is much more restricted and is necessary for mechanical support, growth and movement(1).

The Cartilage tissue is in a strong relationship with the surrounding organic environment and is particularly sensitive to small alterations in features such as oxygen saturation heat and PH (2-6).Several studies have reported thepotential toxicity of different substances on articular cartilage(7-8).

Antibiotics are the most common additives used in irrigation solutions for open fractures(9) including open joint fructures; nonetheless a few studies surveyed effects of antibiotics on articular cartilage(10-11). All these were in-vitro or short-term in-vivo studies without considering potential recovery of chondrocytes.

The hypothesis of this study is that according to the characteristics of antibiotic solutions, they may have potential toxicity on articular cartilage because of cellular damage or matrix changes and this potential toxicity may occur as a late phenomenon.We conducted an animal study to survey this claim.

 

 Materials and methods

20male rabbits weighting 1900g to 2200g were used for this study. They were normal in appearance and activity. The rabbits were divided into four groups each containing of five rabbits (10 knee joints).

We injected 2ml of normal saline solution into both knee joints of groupone which was our control group.2ml of Gentamicin (0.08 mg/ml), Cefazolin (1mg/ml) and Vancomycin (1mg/ml) was injected into both knees of group two, three and four separately.

We used normal saline because it is safely used for intra-articular procedures such as arthroscopic procedures according to previous studies(8, 12). All injections were performed in a sterile condition by an experienced veterinarian surgeon without insulting articular.

Before injections the animals were all anesthetized by use of ketamine hydrochloride (4.0 mg/kg) and Xylazine hydrochloride (0.4 mg/kg) and injection was performed craniolateral to patellar tandon.

In a preliminary study in which contrast media was injected into rabbit’s knee of the same size we determined that a 2ml injection distended the joint and it was spread throughout the knee joint equally in radiographs.

All rabbits were killed at 8 weeksusing lethal doses of thiopental and articular surface of distal femurs were harvested in sterile condition .Gross examination of the cartilage at the time of harvesting was smooth, glass-like and glistening surface.

Histological analysis:

Articular cartilage of distal femurs were harvested by use of microtome 20 µm depths and then were placed into staining solution of acridine orange for staining live cells for ten minutes and then in propidium iodide for staining dead cells again for ten minutes.

The samples washed and imaged using confocal microscope (microscope: DMI 6000CS, scanner: TCS sps II, leica, Germany). Confocal microscopy was used to generate a three dimensional volume and we performed to determine the percentage of live and dead cells within this three dimensional reconstruction by manual image analysis.

 

 Quantitative analysis:

Punched cartilage pieces of freshly harvested distal femurs were preserved in a cryostat which was set at -196̊c for mRNA analysis. Then by using mRNA isolation kit (RNA-queous@-Midikit-The Ambion) the process of mRNA isolation was performed.

The concentration of mRNA from each sample was measured using spectrophotometer.Concentrated mRNA solutions were diluted further so that adding 1 µl of each sample’s mRNA solution would equal exactly 200 ng of that sample for a polymerase chain reaction. Prime Script RT Master Mix (Perfect Real Time) was used for cDNA synthesis.

Real-time polymerase chain reaction (RT-PCR) is the method used for comparative quantitative mRNA analysis.RT-PCR was found to be the best choice for mRNA quantification in cartilage and chondrocyte culture by Mc Alinden and his colleagues. SYBR Premix Ex Taq II (TliRNase H Plus)-TAKARA was used as Real-time Master Mix.

Gene primers were obtained for the cellular metabolism-regulating transcription factor for Aggrecan, collagen type 1 and collagen type 2 expressions. Normalizing was done by 2-∆∆Ct method. Each reaction took about 2 h to complete.

 Statistical analysis

The changes at metabolic parameters of each antibiotic group were compared with the control group (normal saline group) by GAPDH housekeeping gene and 2-∆∆Ct normalization method. The t-test was used for statistical analysis. Any value of p<0.05 was considered significant.


 Results

Chondrocyte viability:

In gentamicin and vancomycin groups there was a significant decrease in  live cells count; (p<0.034, p<0.004 respectively) but we didn’t observe any cell death in Cefazolin group (Figure 1 , Figure 2 and Tab 1).

                                               

Quantitative analysis:

By use of Real time PCR, we didn’t find any significant difference in gene expressions between various groups. (Table 2, 3 ,4 and Fig 3)

 Discussion

In an animal study using gross evaluation and histological scoring systems, Lescunet al. stated that continuous infusion of Gentamicin into the tarsocrural joint of horses for 5 days is an acceptable method for the treatment of septic arthritis(10).

It was a short term study by harvesting cartilage specimens at day 14. In another study according to general and local clinical findings, again in an animal study, AO research institute concluded that intra-articular administration of doxycycline in calves had excellent compatibility. Significant decreases in matrix metalloproteinase activity in doxycycline-treated joints may indicate a potential chondroprotective effect of doxycycline(11).

In 1996 Edin ML et al. performed an in-vitro study to determine the effect of Cefazolin and Vancomycin on osteoblast-like cells.Different concentrations of Cefazolin and Vancomycin at order of magnitude intervals between 0 and 10mg/ml were used.Cell number and 3H-thymidine incorporation at 0, 24, and 72 hours were determined.

Levels of Vancomycin of 1 mg/ml and less have little or no effect on osteoblast replication. Concentrations of Cefazolin of 0.1mg/ml and less have little or no effect on osteoblast replication and concentrations of more than 0.2 mg/ml significantly decrease cell replication(13).

The concentration of Vancomycin in our study was the same as this study but Cefazolin was tenfold concentrated.

In study of Rathbone CR et al. osteoblasts were treated with 21 different antibiotics over 8 concentrations from 0 to 5,000µg/ml. Osteoblast deoxyribonucleic acid content and alkaline phosphatase activity (ALP) were measured to determine cell number and osteogenic activity, respectively.Gentamicin was amongst antibiotics that their cell number and ALP were significantly less than control at drug concentrations ≤0.2 mg/ml(14).In our study Gentamicin concentration was lower than this (0.08 mg/ml).

In another invitro study by Antoci V JR et al. they asked what dosage of antibiotic would cause reductions in osteoblast and chondrocyte cell numbers. Vancomycin at doses greater than 2 mg/ml severely decreased cellular proliferation(15).

We studied molecular structure of three common antibiotics used in irrigation lavage (Gentamicin, Cefazolin and Vancomycin) and we also determined PH of these antibiotic solutions. All the antibiotic solutions had below PH of 7(acidic). According to molecular structures, all antibiotics tend to take positive electrical alloy (Figure 4), so all these antibiotics are the same regarding PH and electrical tendency.

  

In Gentamicin and Vancomycin groups according to confocal microscopy there was a significant decrease in chondrocyte viability but we didn’t find this significant decrease in gene expressions.

To explain this finding, it seems that although these antibiotics can cause cell necrosis, this is not enough to change gene expression quantitively. We recommend for further studies increasing the number of the samples and increasing the time since injections to harvest of the samples to determine the exact value of gene expression.

We concluded that the use of Gentamicin and Vancomycin at the given doses for intra-articular cases should be with caution.

In Cefazolin group, there was neither cell death in confocal microscopy nor significant decrease in gene expression compared with control group during molecular study. We concluded that Cefazolin is a safe antibiotic to be used in intra-articular cases.

 

Conclusion:

According to our study, Cefazolin can be safely used at doses equal or less than 1mg/ml for intraarticular injections and Gentamicin and Vancomycin may be harmful at doses of 0.08mg/ml and 1mg/ml respectively. Further in-vivo studies by different concentrations of these antibiotics are needed for fine tuning the exact safe dose of these antibiotics and to balance their benefits and potential detrimental effects.

 

REFENCES:

1.         Archer CW, Francis-West P. The chondrocyte. The international journal of biochemistry & cell biology. 2003;35(4):401-4.

2.         Cheng SC, Jou IM, Chern TC, Wang PH, Chen WC. The effect of normal saline irrigation at different temperatures on the surface of articular cartilage: an experimental study in the rat. Arthroscopy 2004;20:55–61.

3.         Gradinger R, Tra¨ger J, Klauser RJ. Influence of various irrigation fluids on articular cartilage. Arthroscopy 1995;11:263–269.

4.         Mah ET, Lee WK, Southwood RT, Carbone A, Leppard PJ. Effects of irrigation fluid on human menisci: an experimental comparison of water, normal saline, and glycine. Arthroscopy 1991;7:24–32.

5.         Yang CY, Cheng SC, Shen CL. Effect of irrigation fluids on the articular cartilage: a scanning electron microscope study. Arthroscopy 1993; 9:425–30.

6.         Akgun U, Kocaoglu B, Ergun S, Karahan M, Turkmen M. The effect of environmental pH change on bovine articular cartilage metabolism: implications for the use of buffered solution during arthroscopy? Knee Surgery, Sports Traumatology, Arthroscopy. 2013:1-6.

7.         Chu CR, Coyle CH, Chu CT, Szczodry M, Seshadri V, Karpie JC, et al. In vivo effects of single intra-articular injection of 0.5% bupivacaine on articular cartilage. The Journal of Bone & Joint Surgery. 2010;92(3):599-608.

8.         Yang C-Y, Cheng S-C, Shen C-L. Effect of irrigation fluids on the articular cartilage: a scanning electron microscope study. Arthroscopy: The Journal of Arthroscopic & Related Surgery. 1993;9(4):425-30.

9.         Anglen JO. Comparison of soap and antibiotic solutions for irrigation of lower-limb open fracture woundsA prospective, randomized study. The Journal of Bone & Joint Surgery. 2005;87(7):1415-22.

10.       Lescun TB, Adams SB, Wu CC, Bill RP, Sickle DCV. Effects of continuous intra-articular infusion of gentamicin on synovial membrane and articular cartilage in the tarsocrural joint of horses. American journal of veterinary research. 2002;63(5):683-7.

11.       Haerdi-Landerer MC, Suter MM, Steiner A. Intra-articular administration of doxycycline in calves. American journal of veterinary research. 2007;68(12):1324-31.

12.       Jurvelin J, Jurvelin J, Kiviranta I, Klauser R. Effects of different irrigation liquids and times on articular cartilage: an experimental, biomechanical study. Arthroscopy: The Journal of Arthroscopic & Related Surgery. 1994;10(6):667-72.

13.       Edin ML, Miclau T, Lester GE, Lindsey RW, Dahners LE. Effect of cefazolin and vancomycin on osteoblasts in vitro. Clinical orthopaedics and related research. 1996;333:245-51.

14.       Rathbone CR, Cross JD, Brown KV, Murray CK, Wenke JC. Effect of various concentrations of antibiotics on osteogenic cell viability and activity. Journal of Orthopaedic Research. 2011;29(7):1070-4.

15.       Antoci Jr V, Adams CS, Hickok NJ, Shapiro IM, Parvizi J. Antibiotics for local delivery systems cause skeletal cell toxicity in vitro. Clinical orthopaedics and related research. 2007;462:200-6.

 

 Figure 1: Chondrocyte viability (%) in, Gentamicin, Vancomycin, Cefazolin compared to normal saline

* Significant difference between Gentamicin and Normal saline

* Significant difference between Vancomycin and Normal saline



 

Figure 2: Effect of A: cytotoxic agent (xylen), B: Normal saline, C: Gentamicin, D: Vancomycin and E: Cefazolin on chondrocyte viability (live cells stained green and dead cells stained red, 100X)



 

Figure 3: Aggrecan, Col I and Col II gene expressions in Gentamicin, Vancomycin and Cefazolin groups in comparison to Normal saline



Figure 4: Molecular structure of different antibiotics


 

 

 

Tab1: Chondrocyte viability (%) in  Gentamicin, Vancomycin and Cefazolin and control group

 

Gentamicin

NS

p value

Vancomycin

NS

p value

Cefazolin

NS

p value

Mean

82

100

0.034

77

100

0.004

99

100

0.21

SD

2.2

0

 

2.3

0

 

1.3

0

 

 

 

 

Tab2: Aggrecan gene expression in Gentamicin, Vancomycin and Cefazolin and control group

 

Gentamicin

NS

p value

Vancomycin

NS

p value

Cefazolin

NS

p value

Mean

0.86

1

0.631

1.08

1

0.671

1.53

1

0.298

SD

0.081

0

 

0.075

0

 

0.05

0

 

 

 

 

Tab3: Col I gene expression in Gentamicin, Vancomycin , Cefazolin and control group

 

Gentamicin

NS

p value

Vancomycin

NS

p value

Cefazolin

NS

p value

Mean

0.88

1

0.48

1.04

1

0.725

2.14

1

0.198

SD

0.026

0

 

0.086

0

 

0.029

0

 


 

 

Tab 4:  COL II gene expression in Gentamicin, Vancomycin , Cefazolin and control group

 

Gentamicin

NS

p value

Vancomycin

NS

p value

Cefazolin

NS

p value

Mean

0.79

1

0.232

1.55

1

0.238

1.14

1

0.906

SD

0.061

0

 

0.056

0

 

0.074

0